U.S. patent application number 10/003514 was filed with the patent office on 2002-10-31 for reversing advanced glycosylation cross-links using heterocyclic-substitute- d thiazolium salts.
Invention is credited to Cerami, Anthony, Egan, John J., Fang, Sheng-Ding, Rho, Taikyun, Ulrich, Peter, Vasan, Sara, Wagle, Dilip R..
Application Number | 20020160992 10/003514 |
Document ID | / |
Family ID | 22696355 |
Filed Date | 2002-10-31 |
United States Patent
Application |
20020160992 |
Kind Code |
A1 |
Wagle, Dilip R. ; et
al. |
October 31, 2002 |
Reversing advanced glycosylation cross-links using
heterocyclic-substitute- d thiazolium salts
Abstract
The present invention relates to compositions and methods for
reversing advanced glycosylation end product-mediated cross-linking
and protein aging. Accordingly, compositions are described which
comprise thiazolium compounds substituted with heterocyclic groups
which are capable of reversing the formation of advanced
glycosylation end product cross-links. Both industrial and
therapeutic applications for the invention are disclosed, as food
spoilage and animal protein aging can be treated. Such compounds
have particular application in the treatment of protein aging such
as is responsible for the complications of aging and diabetes.
Inventors: |
Wagle, Dilip R.; (Nanuet,
NY) ; Fang, Sheng-Ding; (Mahwah, NJ) ; Rho,
Taikyun; (Saddle Brook, NJ) ; Egan, John J.;
(New York, NY) ; Vasan, Sara; (New York, NY)
; Ulrich, Peter; (Old Tappan, NJ) ; Cerami,
Anthony; (New York, NY) |
Correspondence
Address: |
Allen Bloom, Esq.
Dechert Price & Rhoads
P. O. Box 5218
Princeton
NJ
08543
US
|
Family ID: |
22696355 |
Appl. No.: |
10/003514 |
Filed: |
October 23, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10003514 |
Oct 23, 2001 |
|
|
|
09644024 |
Aug 23, 2000 |
|
|
|
6319934 |
|
|
|
|
09644024 |
Aug 23, 2000 |
|
|
|
09189200 |
Nov 10, 1998 |
|
|
|
6121300 |
|
|
|
|
Current U.S.
Class: |
514/210.2 ;
514/217.1; 514/227.8; 514/236.8; 514/254.02; 514/326; 514/365 |
Current CPC
Class: |
A61K 31/541 20130101;
A61K 31/496 20130101; A61P 13/12 20180101; A61K 31/4709 20130101;
A61K 31/427 20130101; A61K 31/55 20130101; A61K 31/5355 20130101;
A61K 31/454 20130101; C07D 277/22 20130101; A61P 17/00 20180101;
C07D 417/06 20130101; A61K 31/4439 20130101; C07D 277/44 20130101;
A61P 27/02 20180101; A61P 9/14 20180101; A61P 3/10 20180101 |
Class at
Publication: |
514/210.2 ;
514/217.1; 514/227.8; 514/236.8; 514/254.02; 514/326; 514/365 |
International
Class: |
A61K 031/541; A61K
031/5377; A61K 031/496; A61K 031/55; A61K 031/454; A61K
031/427 |
Claims
What is claimed is:
1. A composition for reversing pre-formed advanced glycosylation
crosslinks comprising an effective amount of a compound of the
structural formula: 5wherein R.sup.1 and R.sup.2 are independently
selected from the group consisting of hydrogen and an alkyl group
optionally substituted by a hydroxy group; Y is a group of the
formula --CH.sub.2C(.dbd.O)R wherein R is a heterocyclic group
other than alkylenedioxyaryl containing 4-10 ring members and 1-3
heteroatoms selected from the group consisting of oxygen, nitrogen
and sulfur; said heterocyclic group optionally substituted by one
or more substituents selected from the group consisting of alkyl,
oxo, alkoxycarbonylalkyl, aryl, and aralkyl groups; and said one or
more substituents optionally substituted by one or more alkyl or
alkoxy groups; or a group of the formula --CH.sub.2C(.dbd.O)--NHR'
wherein R' is a heterocyclic group containing 4-10 ring members and
1-3 heteroatoms selected from the group consisting of oxygen,
nitrogen, and sulfur; said heterocyclic group optionally
substituted by one or more alkoxycarbonylalkyl groups; and X is a
halide, tosylate, methanesulfonate or mesitylenesulfonate ion; and
a carrier therefor.
2. The composition of claim 1 wherein Y is a group of the formula
--CH.sub.2C(.dbd.O)R wherein R is a heterocyclic group other than
alkylenedioxyaryl containing 4-10 ring members and 1-3 heteroatoms
selected from the group consisting of oxygen, nitrogen and sulfur;
said heterocyclic group optionally substituted by one or more
substituents selected from the group consisting of alkyl, oxo,
alkoxycarbonylalkyl, aryl, and aralkyl groups; and said one or more
substituents optionally substituted by one or more alkyl or alkoxy
groups; and X is a halide, tosylate, methanesulfonate or
mesitylenesulfonate ion.
3. The composition of claim 2 wherein Y is selected from the group
consisting of 3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl];
3-(2-(4-morpholinyl)-2-oxoethyl);
3-[2-(2,6-dimethyl-4-morpholinyl)-2-oxo- ethyl];
3-(2-(1-piperidinyl)-2-oxoethyl); 3-(2-(2-furanyl)-2-oxoethyl;
3-[2-(2-oxo-1,2,3,4-tetrahydro-6-quinolinyl)-2-oxoethyl];
3-(2-(1-pyrrolidinyl)-2-oxoethyl;
3-[2-(3-methyl-2-thianaphthenyl)-2-oxoe- thyl];
3-[2-(4-phenyl-1-piperazinyl)-2-oxoethyl;
3-(2-(2-thienyl)-2-oxoeth- yl); 3-(2-(2-thienyl)-2-oxoethyl);
3-(2-(4-thiomorpholinyl)-2-oxoethyl);
3-(2-(hexahydro-1-azepinyl)-2-oxoethyl);
3-[2-(4-[2-methoxyphenyl]-1-pipe- razinyl)-2-oxoethyl;
3-(2-(octahydro-1-azocinyl)-2-oxoethyl);
3-(2-(2-pyridinyl)-2-oxoethyl;
3-[2-(2-methyl-1-piperidinyl)-2-oxoethyl];
3-[2-(2,6-dimethyl-1-piperidinyl)-2-oxoethyl;
3-[2-(4-benzyl-1-piperidiny- l)-2-oxoethyl]; and
3-[2-(4-benzyl-1-piperazinyl)-2-oxoethyl].
4. The composition of claim 3 wherein said compound is
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-thiazolium bromide;
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-4,5-dimethylthiazolium
bromide;
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-4-methyl-5-(2-hydroxyethyl)-thia-
zolium bromide;
3-(2-(4-morpholinyl)-2-oxoethyl)-4,5-dimethylthiazolium bromide;
3-[2-(2,6-dimethyl-4-morpholinyl)-2-oxoethyl]-4,5-dimethylthiazo-
lium bromide;
3-(2(1-piperidinyl)-2-oxoethyl)-4,5-dimethylthiazolium bromide;
3-(2-(2-furanyl)-2-oxoethyl)-4,5-dimethylthiazolium bromide;
3-[2-(2-oxo-1,2,3,4-tetrahydro-6-quinolinyl)-2-oxoethyl]-4,5-dimethylthia-
zolium bromide;
3-(2-(1-pyrrolidinyl)-2-oxoethyl)-4,5-dimethyl-thiazolium bromide;
3-[2-(3-methyl-2-thianaphthenyl)-2-oxoethyl]-4,5-dimethylthiazol-
ium bromide;
3-[2-(4-phenyl-1-piperazinyl)-2-oxoethyl]-4,5-dimethylthiazol- ium
bromide; 3-(2-(2-thienyl)-2-oxoethyl)-4,5-dimethyl-thiazolium
bromide;
3-(2-(2-thienyl)-2-oxoethyl)-4-methyl-5-hydroxyethylthiazolium
bromide;
3-(2-(4-thiomorpholinyl)-2-oxoethyl)-4,5-dimethylthiazolium
bromide;
3-(2-(hexahydro-1-azepinyl)-2-oxoethyl)-4,5-dimethyl-thiazolium
bromide;
3-[2-(4-[2-methoxyphenyl]-1-piperazinyl)-2-oxoethyl]-4,5-dimethylthiazoli-
um chloride;
3-(2-(octahydro-1-azocinyl)-2-oxoethyl)-4,5-dimethyl-thiazoli- um
bromide; 3-(2-(2-pyridinyl)-2-oxoethyl)-4,5-dimethyl-thiazolium
bromide;
3-[2-(2-methyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthiazolium
chloride;
3-[2-(2,6-dimethyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthiaz-
olium chloride;
3-[2-(4-benzyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthia- zolium
chloride;
3-[2-(4-benzyl-1-piperazinyl)-2-oxoethyl]-4,5-dimethylthi- azolium
chloride; or another biologically acceptable salt thereof.
5. The composition of claim 1 wherein said compound has the formula
wherein Y is a group of the formula --CH.sub.2C(.dbd.O)--NHR'
wherein R' is a heterocyclic group containing 4-10 ring members and
1-3 heteroatoms selected from the group consisting of oxygen,
nitrogen, and sulfur; said heterocyclic group optionally
substituted by one or more alkoxycarbonylalkyl groups; and X is a
halide, tosylate, methanesulfonate or mesitylenesulfonate ion.
6. The composition of claim 5 wherein said compound is
3-[2-[4-(2-ethoxy-2-oxoethyl)-2-thiazolyl]amino-2-oxoethyl]-4,5-dimethylt-
hiazolium chloride or another biologically acceptable salt
thereof.
7. A pharmaceutical composition for administration to an animal to
reverse pre-formed advanced glycosylation crosslinks within said
animal, comprising a pharmaceutically effective amount of a
compound selected from the group consisting of compounds of the
formula: 6wherein R.sup.1 and R.sup.2 are independently selected
from the group consisting of hydrogen and a alkyl group optionally
substituted by a hydroxy group; Y is a group of the formula
--CH.sub.2C(.dbd.O)R wherein R is a heterocyclic group other than
alkylenedioxyaryl containing 4-10 ring members and 1-3 heteroatoms
selected from the group consisting of oxygen, nitrogen and sulfur;
said heterocyclic group optionally substituted by one or more
substituents selected from the group consisting of alkyl, oxo
alkoxycarbonylalkyl, aryl, and aralkyl groups; and said one or more
substituents optionally substituted by one or more alkyl or alkoxy
groups; or a group of the formula --CH.sub.2C(.dbd.O)--NHR' wherein
R' is a heterocyclic group containing 4-10 ring members and 1-3
heteroatoms selected from the group consisting of oxygen, nitrogen,
and sulfur; said heterocyclic group optionally substituted by one
or more alkoxycarbonylalkyl groups; and X is a halide, tosylate,
methanesulfonate or mesitylenesulfonate ion, and a carrier
therefor.
8. The composition of claim 7 wherein Y is a group of the formula
--CH.sub.2C(.dbd.O)R wherein R is a heterocyclic group other than
alkylenedioxyaryl containing 4-10 ring members and 1-3 heteroatoms
selected from the group consisting of oxygen, nitrogen and sulfur;
said heterocyclic group optionally substituted by one or more
substituents selected from the group consisting of alkyl, oxo,
alkoxycarbonylalkyl, aryl, and aralkyl groups; and said one or more
substituents optionally substituted by one or more alkyl or alkoxy
groups; and X is a halide, tosylate, methanesulfonate or
mesitylenesulfonate ion.
9. The composition of claim 8 wherein Y is selected from the group
consisting of 3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl];
3-(2-(4-morpholinyl)-2-oxoethyl);
3-[2-(2,6-dimethyl-4-morpholinyl)-2-oxo- ethyl];
3-(2-(1-piperidinyl)-2-oxoethyl); 3-(2-(2-furanyl)-2-oxoethyl;
3-[2-(2-oxo-1,2,3,4-tetrahydro-6-quinolinyl)-2-oxoethyl];
3-(2-(1-pyrrolidinyl)-2-oxoethyl;
3-[2-(3-methyl-2-thianaphthenyl)-2-oxoe- thyl];
3-[2-(4-phenyl-1-piperazinyl)-2-oxoethyl;
3-(2-(2-thienyl)-2-oxoeth- yl); 3-(2-(2-thienyl)-2-oxoethyl);
3-(2-(4-thiomorpholinyl)-2-oxoethyl);
3-(2-(hexahydro-1-azepinyl)-2-oxoethyl);
3-[2-(4-[2-methoxyphenyl]-1-pipe- razinyl)-2-oxoethyl;
3-(2-(octahydro-1-azocinyl)-2-oxoethyl);
3-(2-(2-pyridinyl)-2-oxoethyl;
3-[2-(2-methyl-1-piperidinyl)-2-oxoethyl];
3-[2-(2,6-dimethyl-1-piperidinyl)-2-oxoethyl;
3-[2-(4-benzyl-1-piperidiny- l)-2-oxoethyl]; and
3-[2-(4-benzyl-1-piperazinyl)-2-oxoethyl].
10. The composition of claim 9 wherein said compound is
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-thiazolium bromide;
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-4,5-dimethylthiazolium
bromide;
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-4-methyl-5-(2-hydroxyethyl)-thia-
zolium bromide;
3-(2-(4-morpholinyl)-2-oxoethyl)-4,5-dimethylthiazolium bromide;
3-[2-(2,6-dimethyl-4-morpholinyl)-2-oxoethyl]-4,5-dimethylthiazo-
lium bromide;
3-(2-(1-piperidinyl)-2-oxoethyl)-4,5-dimethylthiazolium bromide;
3-(2-(2-furanyl)-2-oxoethyl)-4,5-dimethylthiazolium bromide;
3-[2-(2-oxo-1,2,3,4-tetrahydro-6-quinolinyl)-2-oxoethyl]-4,5-dimethylthia-
zolium bromide;
3-(2-(1-pyrrolidinyl)-2-oxoethyl)-4,5-dimethyl-thiazolium bromide;
3-[2-(3-methyl-2-thianaphthenyl)-2-oxoethyl]-4,5-dimethylthiazol-
ium bromide;
3-[2-(4-phenyl-1-piperazinyl)-2-oxoethyl]-4,5-dimethylthiazol- ium
bromide; 3-(2-(2-thienyl)-2-oxoethyl)-4,5-dimethyl-thiazolium
bromide;
3-(2-(2-thienyl)-2-oxoethyl)-4-methyl-5-hydroxyethylthiazolium
bromide;
3-(2-(4-thiomorpholinyl)-2-oxoethyl)-4,5-dimethylthiazolium
bromide;
3-(2-(hexahydro-1-azepinyl)-2-oxoethyl)-4,5-dimethyl-thiazolium
bromide;
3-[2-(4-[2-methoxyphenyl]-1-piperazinyl)-2-oxoethyl]-4,5-dimethylthiazoli-
um chloride;
3-(2-(octahydro-1-azocinyl)-2-oxoethyl)-4,5-dimethyl-thiazoli- um
bromide; 3-(2-(2-pyridinyl)-2-oxoethyl)-4,5-dimethyl-thiazolium
bromide;
3-[2-(2-methyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthiazolium
chloride;
3-[2-(2,6-dimethyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthiaz-
olium chloride;
3-[2-(4-benzyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthia- zolium
chloride;
3-[2-(4-benzyl-1-piperazinyl)-2-oxoethyl]-4,5-dimethylthi- azolium
chloride; or another biologically acceptable salt thereof.
11. The composition of claim 7 wherein said compound has the
formula wherein Y is group of the formula --CH.sub.2C(.dbd.O)--NHR'
and R' is a heterocyclic group containing 4-10 ring members and 1-3
heteroatoms selected from the group consisting of oxygen, nitrogen,
and sulfur; said heterocyclic group optionally substituted by one
or more alkoxycarbonylalkyl groups; and X is a halide, tosylate,
methanesulfonate or mesitylenesulfonate ion.
12. The composition of claim 11 wherein said compound is
3-[2-[4-(2-ethoxy-2-oxoethyl)-2-thiazolyl]amino-2-oxoethyl]-4,5-dimethyl--
thiazolium chloride or another biologically acceptable salt
thereof.
13. A method for reversing pre-formed advanced glycosylation
crosslinks, comprising contacting the target protein with an
effective amount of composition comprising a compound selected from
the group consisting of compounds of the formula 7wherein R.sup.1
and R.sup.2 are independently selected from the group consisting of
hydrogen and an alkyl group optionally substituted by a hydroxy
group; Y is a group of the formula --CH.sub.2C(.dbd.O)R wherein R
is a heterocyclic group other than alkylenedioxyaryl containing
4-10 ring members and 1-3 heteroatoms selected from the group
consisting of oxygen, nitrogen and sulfur; said heterocyclic group
optionally substituted by one or more substituents selected from
the group consisting of alkyl, oxo, alkoxycarbonylalkyl, aryl, and
aralkyl groups; and said one or more substituents optionally
substituted by one or more alkyl or alkoxy groups; or a group of
the formula --CH.sub.2C(.dbd.O)--NHR' wherein R' is a heterocyclic
group containing 4-10 ring members and 1-3 heteroatoms selected
from the group consisting of oxygen, nitrogen, and sulfur; said
heterocyclic group optionally substituted by one or more
alkoxycarbonylalkyl groups; and X is a halide, tosylate,
methanesulfonate or mesitylenesulfonate ion; and a carrier
therefor.
14. The composition of claim 13 wherein Y is a group of the formula
--CH.sub.2C(.dbd.O)R wherein R is a heterocyclic group other than
alkylenedioxyaryl containing 4-10 ring members and 1-3 heteroatoms
selected from the group consisting of oxygen, nitrogen and sulfur;
said heterocyclic group optionally substituted by one or more
substituents selected from the group consisting of alkyl, oxo,
alkoxycarbonylalkyl, aryl, and aralkyl groups; and said one or more
substituents optionally substituted by one or more alkyl or alkoxy
groups; and X is a halide, tosylate, methanesulfonate or
mesitylenesulfonate ion.
15. The composition of claim 14 wherein Y is selected from the
group consisting of 3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl];
3-(2-(4-morpholinyl)-2-oxoethyl);
3-[2-(2,6-dimethyl-4-morpholinyl)-2-oxo- ethyl];
3-(2-(1-piperidinyl)-2-oxoethyl); 3-(2-(2-furanyl)-2-oxoethyl;
3-[2-(2-oxo-1,2,3,4-tetrahydro-6-quinolinyl)-2-oxoethyl];
3-(2-(1-pyrrolidinyl)-2-oxoethyl;
3-[2-(3-methyl-2-thianaphthenyl)-2-oxoe- thyl];
3-[2-(4-phenyl-1-piperazinyl)-2-oxoethyl;
3-(2-(2-thienyl)-2-oxoeth- yl); 3-(2-(2-thienyl)-2-oxoethyl);
3-(2-(4-thiomorpholinyl)-2-oxoethyl);
3-(2-(hexahydro-1-azepinyl)-2-oxoethyl);
3-[2-(4-[2-methoxyphenyl]-1-pipe- razinyl)-2-oxoethyl;
3-(2-(octahydro-1-azocinyl)-2-oxoethyl);
3-(2-(2-pyridinyl)-2-oxoethyl;
3-[2-(2-methyl-1-piperidinyl)-2-oxoethyl];
3-[2-(2,6-dimethyl-1-piperidinyl)-2-oxoethyl;
3-[2-(4-benzyl-1-piperidiny- l)-2-oxoethyl]; and
3-[2-(4-benzyl-1-piperazinyl)-2-oxoethyl].
16. The composition of claim 15 wherein said compound is
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-thiazolium bromide;
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-4,5-dimethylthiazolium
bromide;
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-4-methyl-5-(2-hydroxyethyl)-thia-
zolium bromide;
3-(2-(4-morpholinyl)-2-oxoethyl)-4,5-dimethylthiazolium bromide;
3-[2-(2,6-dimethyl-4-morpholinyl)-2-oxoethyl]-4,5-dimethylthiazo-
lium bromide;
3-(2-(1-piperidinyl)-2-oxoethyl)-4,5-dimethylthiazolium bromide;
3-(2-(2-furanyl)-2-oxoethyl)-4,5-dimethylthiazolium bromide;
3-[2-(2-oxo-1,2,3,4-tetrahydro-6-quinolinyl)-2-oxoethyl]-4,5-dimethylthia-
zolium bromide;
3-(2-(-1-pyrrolidinyl)-2-oxoethyl)-4,5-dimethyl-thiazolium bromide;
3-[2-(3-methyl-2-thianaphthenyl)-2-oxoethyl]-4,5-dimethylthiazol-
ium bromide;
3-[2-(4-phenyl-1-piperazinyl)-2-oxoethyl]-4,5-dimethylthiazol- ium
bromide; 3-(2(2-thienyl)-2-oxoethyl)-4,5-dimethyl-thiazolium
bromide; 3-(2-
(2-thienyl)-2-oxoethyl)-4-dimethyl-5-hydroxyethylthiazolium
bromide; 3-(2-(4thiomorpholinyl)-2-oxoethyl)-4,5-dimethylthiazolium
bromide;
3-(2-(hexahydro-1-azepinyl)-2-oxoethyl)-4,5-dimethyl-thiazolium
bromide;
3-[2-(4-[2-methoxyphenyl]-1-piperazinyl)-2-oxoethyl]-4,5-dimethy-
lthiazolium chloride;
3-(2-(octahydro-1-azocinyl)-2-oxoethyl)-4,5-dimethyl- -thiazolium
bromide; 3-(2-(2-pyridinyl)-2-oxoethyl)-4,5-dimethyl-thiazoliu- m
bromide;
3-[2-(2-methyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthiazoliu- m
chloride;
3-[2-(2,6-dimethyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthia-
zolium chloride;
3-[2-(4-benzyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthi- azolium
chloride; 3-[2-(4-benzyl-1-piperazinyl)-2-oxoethyl]-4,5-dimethylth-
iazolium chloride; or another biologically acceptable salt
thereof.
17. The composition of claim 13 wherein said compound has the
formula wherein Y is a group of the formula
--CH.sub.2C(.dbd.O)--NHR' wherein R' is a heterocyclic group
containing 4-10 ring members and 1-3 heteroatoms selected from the
group consisting of oxygen, nitrogen, and sulfur; said heterocyclic
group optionally substituted by one or more alkoxycarbonylalkyl
groups; and X is a halide, tosylate, methanesulfonate or
mesitylenesulfonate ion.
18. The composition of claim 17 wherein said compound is
3-[2-[4-(2-ethoxy-2-oxoethyl)-2-thiazolyl]amino-2-oxoethyl]-4,5-dimethyl--
thiazolium chloride or another biologically acceptable salt
thereof.
19. A method for treating an animal to reverse pre-formed advanced
glycosylation crosslinks of a target protein within said animal,
said method comprising administering an effective amount of a
pharmaceutical composition, said pharmaceutical composition
comprising a compound selected from the group consisting of
compounds of the formula 8wherein R.sup.1 and R.sup.2 are
independently selected from the group consisting of hydrogen and an
alkyl group optionally substituted by a hydroxy group; Y is a group
of the formula --CH.sub.2C(.dbd.O)R wherein R is a heterocyclic
group other than alkylenedioxyaryl containing 4-10 ring members and
1-3 heteroatoms selected from the group consisting of oxygen,
nitrogen and sulfur; said heterocyclic group optionally substituted
by one or more substituents selected from the group consisting of
alkyl, oxo, alkoxycarbonylalkyl aryl, and aralkyl groups; and said
one or more substituents optionally substituted by one or more
alkyl or alkoxy groups; or a group of the formula
--CH.sub.2C(.dbd.O)--NHR' wherein R' is a heterocyclic group
containing 4-10 ring members and 1-3 heteroatoms selected from the
group consisting of oxygen, nitrogen, and sulfur; said heterocyclic
group optionally substituted by one or more alkoxycarbonylalkyl
groups; and X is a halide, tosylate, methanesulfonate or
mesitylenesulfonate ion; and a carrier therefor.
20. The composition of claim 19 wherein Y is a group of the formula
--CH.sub.2C(.dbd.O)R wherein R is a heterocyclic group other than
alkylenedioxyaryl containing 4-10 ring members and 1-3 heteroatoms
selected from the group consisting of oxygen, nitrogen and sulfur;
said heterocyclic group optionally substituted by one or more
substituents selected from the group consisting of alkyl, oxo,
alkoxycarbonylalkyl, aryl, and aralkyl groups; and said one or more
substituents optionally substituted by one or more alkyl or alkoxy
groups; and X is a halide, tosylate, methanesulfonate or
mesitylenesulfonate ion.
21. The composition of claim 20 wherein Y is selected from the
group consisting of 3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl];
3-(2-(4-morpholinyl)-2-oxoethyl);
3-[2-(2,6-dimethyl-4-morpholinyl)-2-oxo- ethyl];
3-(2-(1-piperidinyl)-2-oxoethyl); 3-(2-(2-furanyl)-2-oxoethyl;
3-[2-(2-oxo-1,2,3,4-tetrahydro-6-quinolinyl)-2-oxoethyl];
3-(2-(1-pyrrolidinyl)-2-oxoethyl;
3-[2-(3-methyl-2-thianaphthenyl)-2-oxoe- thyl];
3-[2-(4-phenyl-1-piperazinyl)-2-oxoethyl;
3-(2-(2-thienyl)-2-oxoeth- yl); 3-(2-(2-thienyl)-2-oxoethyl);
3-(2-(4-thiomorpholinyl)-2-oxoethyl);
3-(2-(hexahydro-1-azepinyl)-2-oxoethyl);
3-[2-(4-[2-methoxyphenyl]-1-pipe- razinyl)-2-oxoethyl;
3-(2-(octahydro-1-azocinyl)-2-oxoethyl);
3-(2-(2-pyridinyl)-2-oxoethyl;
3-[2-(2-methyl-1-piperidinyl)-2-oxoethyl];
3-[2-(2,6-dimethyl-1-piperidinyl)-2-oxoethyl;
3-[2-(4-benzyl-1-piperidiny- l)-2-oxoethyl]; and
3-[2-(4-benzyl-1-piperazinyl)-2-oxoethyl].
22. The composition of claim 21 wherein said compound is
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-thiazolium bromide;
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-4,5-dimethylthiazolium
bromide;
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-4-methyl-5-(2-hydroxyethyl)-thia-
zolium bromide;
3-(2-(4-morpholinyl)-2-oxoethyl)-4,5-dimethylthiazolium bromide;
3-[2-(2,6-dimethyl-4-morpholinyl)-2-oxoethyl]-4,5-dimethylthiazo-
lium bromide;
3-(2-(1-piperidinyl)-2-oxoethyl)-4,5-dimethylthiazolium bromide;
3-(2-(2-furanyl)-2-oxoethyl)-4,5-dimethylthiazolium bromide;
3-[2-(2-oxo-1,2,3,4-tetrahydro-6-quinolinyl)-2-oxoethyl-]-4,5-dimethylthi-
azolium bromide;
3-(2-(1-pyrrolidinyl)-2-oxoethyl)-4,5-dimethyl-thiazolium bromide;
3-[2-(3-methyl-2-thianaphthenyl)-2-oxoethyl]-4,5-dimethylthiazol-
ium bromide;
3-[2-(4-phenyl-1-piperazinyl)-2-oxoethyl]-4,5-dimethylthiazol- ium
bromide; 3-(2-(2-thienyl)-2-oxoethyl)-4,5-dimethyl-thiazolium
bromide;
3-(2-(2-thienyl)-2-oxoethyl)-4-methyl-5-hydroxyethylthiazolium
bromide;
3-(2-(4-thiomorpholinyl)-2-oxoethyl)-4,5-dimethylthiazolium
bromide;
3-(2-(hexahydro-1-azepinyl)-2-oxoethyl)-4,5-dimethyl-thiazolium
bromide;
3-[2-(4-[2-methoxyphenyl]-1-piperazinyl)-2-oxoethyl]-4,5-dimethylthiazoli-
um chloride;
3-(2-(octahydro-1-azocinyl)-2-oxoethyl)-4,5-dimethyl-thiazoli- um
bromide; 3-(2-(2-pyridinyl)-2-oxoethyl)-4,5-dimethyl-thiazolium
bromide;
3-[2-(2-methyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthiazolium
chloride;
3-[2-(2,6-dimethyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthiaz-
olium chloride;
3-[2-(4-benzyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthia- zolium
chloride;
3-[2-(4-benzyl-1-piperazinyl)-2-oxoethyl]-4,5-dimethylthi- azolium
chloride; or another biologically acceptable salt thereof.
23. The composition of claim 19 wherein said compound has the
formula wherein Y is a group of the formula
--CH.sub.2C(.dbd.O)--NHR' wherein R' is a heterocyclic group
containing 4-10 ring members and 1-3 heteroatoms selected from the
group consisting of oxygen, nitrogen, and sulfur; said heterocyclic
group optionally substituted by one or more alkoxycarbonylalkyl
groups; and X is a halide, tosylate, methanesulfonate or
mesitylenesulfonate ion.
24. The composition of claim 23 wherein said compound is
3-[2-[4-(2-ethoxy-2-oxoethyl)-2-thiazolyl]amino-2-oxoethyl]-4,5-dimethyl--
thiazolium chloride or another biologically acceptable salt
thereof.
25. A compound of the formula 9wherein R.sup.1 and R.sup.2 are
independently selected from the group consisting of hydrogen and an
alkyl group optionally substituted by a hydroxy group; Y is a group
of the formula --CH.sub.2C(.dbd.O)R wherein R is a heterocyclic
group other than alkylenedioxyaryl containing 4-10 ring members and
one heteroatom selected from the group consisting of nitrogen and
sulfur, or 2-3 heteroatoms selected from the group consisting of
oxygen, nitrogen and sulfur; said heterocyclic group optionally
substituted by one or more substituents selected from the group
consisting of alkyl, oxo, alkoxycarbonylalkyl, aryl, and aralkyl
groups; and said one or more substituents optionally substituted by
one or more alkyl or alkoxy groups; or a group of the formula
--CH.sub.2C(.dbd.O)--NHR' wherein R' is a heterocyclic group
containing 4-10 ring members and 1-3 heteroatoms selected from the
group consisting of oxygen. nitrogen, and sulfur; said heterocyclic
group optionally substituted by one or more alkoxycarbonylalkyl
groups; and X is a halide, tosylate, methanesulfonate or
mesitylenesulfonate ion.
26. The compound of claim 25 wherein said compound has the formula
wherein Y is group of the formula --CH.sub.2C(.dbd.O)R wherein R is
a heterocyclic group other than alkylenedioxyaryl containing 4-10
ring members and one heteroatom selected from the group consisting
of nitrogen and sulfur, or 2-3 heteroatoms selected from the group
consisting of oxygen, nitrogen and sulfur; said heterocyclic group
optionally substituted by one or more substituents selected from
the group consisting of alkyl, oxo, alkoxycarbonylalkyl, aryl, and
aralkyl groups; and said one or more substituents optionally
substituted by one or more alkyl or alkoxy groups; and X is a
halide, tosylate, methanesulfonate or mesitylenesulfonate ion.
27. The composition of claim 26 wherein Y is selected from the
group consisting of3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl];
3-(2-(4-morpholinyl)-2-oxoethyl);
3-[2-(2,6-dimethyl-4-morpholinyl)-2-oxo- ethyl];
3-(2-(1-piperidinyl)-2-oxoethyl); 3-[2-(2-oxo-1,2,3,4-tetrahydro-6-
-quinolinyl)-2-oxoethyl]; 3-(2-(1-pyrrolidinyl)-2-oxoethyl;
3-[2-(3-methyl-2-thianaphthenyl)-2-oxoethyl];
3-[2-(4-phenyl-1-piperaziny- l)-2-oxoethyl;
3-(2-(2-thienyl)-2-oxoethyl); 3-(2-(2-thienyl)-2-oxoethyl);
3-(2-(4-thiomorpholinyl)-2-oxoethyl);
3-(2-(hexahydro-1-azepinyl)-2-oxoet- hyl);
3-[2-(4-[2-methoxyphenyl]-1-piperazinyl)-2-oxoethyl;
3-(2-(octahydro-1-azocinyl)-2-oxoethyl);
3-(2-(2-pyridinyl)-2-oxoethyl;
3-[2-(2-methyl-1-piperidinyl)-2-oxoethyl];
3-[2-(2,6-dimethyl-1-piperidin- yl)-2-oxoethyl;
3-[2-(4-benzyl-1-piperidinyl)-2-oxoethyl]; and
3-[2-(4-benzyl-1-piperazinyl)-2-oxoethyl].
28. The composition of claim 27 wherein said compound is
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-thiazolium bromide;
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-4,5-dimethylthiazolium
bromide;
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-4-methyl-5-(2-hydroxyethyl)-thia-
zolium bromide;
3-(2-(4-morpholinyl)-2-oxoethyl)-4,5-dimethylthiazolium bromide;
3-[2-(2,6-dimethyl-4-morpholinyl)-2-oxoethyl]-4,5-dimethylthiazo-
lium bromide;
3-(2-(1-piperidinyl)-2-oxoethyl)-4,5-dimethylthiazolium bromide;
3-[2-(2-oxo-1,2,3,4-tetrahydro-6-quinolinyl)-2-oxoethyl]-4,5-dim-
ethylthiazolium bromide;
3-(2-(1-pyrrolidinyl)-2-oxoethyl)-4,5-dimethyl-th- iazolium
bromide; 3-[2-(3-methyl-2-thianaphthenyl)-2-oxoethyl]-4,5-dimethy-
lthiazolium bromide;
3-[2-(4-phenyl-1-piperazinyl)-2-oxoethyl]-4,5-dimethy- lthiazolium
bromide; 3-(2-(2-thienyl)-2-oxoethyl)-4,5-dimethyl-thiazolium
bromide;
3-(2-(2-thienyl)-2-oxoethyl)-4-methyl-5-hydroxyethylthiazolium
bromide;
3-(2-(4-thiomorpholinyl)-2-oxoethyl)-4,5-dimethylthiazolium
bromide;
3-(2-(hexahydro-1-azepinyl)-2-oxoethyl)-4,5-dimethylthiazolium
bromide;
3-[2-(4-[2-methoxyphenyl]-1-piperazinyl)-2-oxoethyl]-4,5-dimethy-
lthiazolium chloride;
3-(2-(octahydro-1-azocinyl)-2-oxoethyl)-4,5-dimethyl- -thiazolium
bromide; 3-(2-(2-pyridinyl)-2-oxoethyl)-4,5-dimethyl-thiazoliu- m
bromide;
3-[2-(2-methyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthiazoliu- m
chloride;
3-[2-(2,6-dimethyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthia-
zolium chloride;
3-[2-(4-benzyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthi- azolium
chloride; 3-[2-(4-benzyl-1-piperazinyl)-2-oxoethyl]-4,5-dimethylth-
iazolium chloride; or another biologically acceptable salt
thereof.
29. The composition of claim 25 wherein said compound has the
formula wherein Y is group of the formula --CH.sub.2C(.dbd.O)--NHR'
and R' is a heterocyclic group containing 4-10 ring members and 1-3
heteroatoms selected from the group consisting of oxygen, nitrogen,
and sulfur; said heterocyclic group optionally substituted by one
or more alkoxycarbonylalkyl groups; and X is a halide, tosylate,
methanesulfonate or mesitylenesulfonate ion.
30. The composition of claim 29 wherein said compound is
3-[2-(2'-amino-5'-carboethoxymethylene-thiazolyl)-2-oxoethyl]-4,5-dimethy-
l-thiazolium chloride or another biologically acceptable salt
thereof.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates generally to the aging of
proteins resulting from their reaction with glucose and other
reducing sugars, and more particularly to the reversing or cleavage
of cross-links formed as a consequence of the formation of advanced
glycosylation (glycation) end products.
[0002] The reaction between glucose and proteins has been known for
some time. Its earliest manifestation was in the appearance of
brown pigments during the cooking of food, which was identified by
Maillard in 1912, who observed that glucose or other reducing
sugars react with amino acids to form adducts that undergo a series
of dehydrations and rearrangements to form stable brown pigments.
Further studies have suggested that stored and heat treated foods
undergo nonenzymatic browning as a result of the reaction between
glucose and the polypeptide chain, and that the proteins are
resultantly cross-linked and correspondingly exhibit decreased
bioavailability. As described in copending application Ser. No.
08/588,249, incorporated herein by reference, these reactions have
a parallel in vivo, and have been found to occur with a variety of
other body proteins, such as lens crystallins, collagen and nerve
proteins. These reactions are accelerated in the presence of
elevated glucose levels, as occur in individuals with diabetes
mellitus, but still occur in vivo at normal glucose levels. Termed
advanced glycosylation (or glycation) end products (AGEs), the
cross-linked products involving structural and other proteins
within the body leads not only to aberrant physico-chemical
properties of, for example, connective tissue, but also results in
the formation of new chemical structures which are recognized by
specific receptors on various cell types and as a consequence of
their recognition, initiate pathogenetic mechanisms leading to the
complications of diabetes and aging.
[0003] Several successful therapeutic approaches have been achieved
based upon intervening in the accumulation of AGEs in vivo. One
approach, exemplified in U.S. Pat. No. 4,758,583, incorporated
herein by reference, concerns the inhibition of the formation of
AGEs from its precursors, by the administration of agents such as
aminoguanidine and related compounds. By reacting with an early
glycosylation product that results from the original reaction
between the target protein and glucose, these agents block the
formation of AGEs and further formation of AGEs and cross-links in
tissues is inhibited. Efficacy of this approach has been
demonstrated in numerous animal models of diabetes and aging,
including positive effects on macrovascular, renal, retinal, and
neural pathology. These data have been reviewed by Vlassara et al.,
1994, "Biology of Diseases. Pathogenic effects of advanced
glycosylation: biochemical, biologic and clinical implications for
diabetes and aging," Laboratory Investigation 70:138-151; Brownlee,
1995, "The pathological implications of protein glycation," Clin.
Invest. Med., 18:275-281; and Brownlee, 1995, "Advanced protein
glycosylation in diabetes and aging," Ann. Rev. Med. 46:223-34.
[0004] In another pharmacological approach to controlling levels of
AGEs in tissues, especially in those tissues in which AGE
cross-links have already accumulated to levels which are
responsible for subclinical or clinical pathology, administration
of agents that reverse or break AGE cross-links has proven
successful. As described in U.S. Pat. No. 5,656,261 and copending
U.S. application Ser. Nos. 08/583,249 and 08/848,776, all of which
are incorporated herein by reference in their entireties, agents
and methods are disclosed which reverse (also termed cleave or
break) existing AGE cross-links in vitro and in vivo. Studies
demonstrate positive effects of such agents on cardiovascular
complications related to aging which are accelerated in
experimental diabetes (see Wolffenbuttel et al., 1998, "Breakers of
Advanced Glycation End Products Restores Large Artery Properties in
Experimental Diabetes," Proc. Nat. Acad. Sci. U.S.A. 95:4630-4634).
In these studies, rats diabetic for 9 weeks followed by 1 to 3
weeks administration of an AGE breaker compound resulted in
reversal of diabetes-induced increases in large artery stiffness.
Parameters that were improved included cardiac output, peripheral
resistance, systemic arterial compliance, input impedance of the
aorta, and compliance of the carotid artery.
[0005] It is toward the identification of additional agents capable
of reversing AGE cross-links that the present invention is
directed.
SUMMARY OF THE INVENTION
[0006] In accordance with the present invention, compounds and
method are disclosed for reversing AGE cross-linking. AGE
cross-linking caused by other reactive sugars present in vivo or in
foodstuffs, including ribose, galactose and fructose would also be
reversed by the methods and compositions of the present
invention.
[0007] The agents useful in the present invention are members of
the class of compounds known as thiazoliums, and in particular
thiazolium compounds substituted with heterocyclic groups.
[0008] The agents comprise compounds having the following
structural formula: 1
[0009] wherein
[0010] R.sup.1 and R.sup.2 are independently selected from the
group consisting of hydrogen and an alkyl group optionally
substituted by a hydroxy group;
[0011] Y is a group of the formula --CH.sub.2C(.dbd.O)R wherein R
is a heterocyclic group other than alkylenedioxyaryl containing
4-10 ring, members and 1-3 heteroatoms selected from the group
consisting of oxygen, nitrogen and sulfur; said heterocyclic group
optionally substituted by one or more substituents selected from
the group consisting of alkyl, oxo, alkoxycarbonylalkyl, aryl, and
aralkyl; and said one or more substituents optionally substituted
by one or more alkyl or alkoxy groups;
[0012] or a group of the formula --CH.sub.2C(.dbd.O)--NHR' wherein
R' is a heterocyclic group containing 4-10 ring members and 1-3
heteroatoms selected from the group consisting of oxygen. nitrogen,
and sulfur; said heterocyclic group optionally substituted by one
or more alkoxycarbonylalkyl groups;
[0013] and X is a halide, tosylate, methanesulfonate or
mesitylenesulfonate ion.
[0014] The ability to reverse already-formed advanced glycosylation
products carries with it significant implications in all
applications where advanced glycation and concomitant molecular
crosslinking is a serious detriment. In the area of food
technology, for instance, the cleavage of cross-links would confer
a reversal of the increased toughness resulting from the formation
of AGEs during storage. In a preferred embodiment, the application
of agents capable of reversal of the Maillard process has
particular benefit in vivo as AGE cross-linking adversely affects
several of the significant protein masses in the body, among them
collagen, elastin, lens proteins, and the kidney glomerular
basement membrane. These proteins deteriorate both with age (hence
the application of the term "protein aging") and more rapidly as a
consequence of diabetes. Accordingly, the ability to reverse the
cross-linking of these proteins and thus to reduce the amount of
cross-links present between advanced glycosylation end products and
other proteins in the body carries the promise for treatment of the
complications of diabetes and aging for instance, and thereby
improving the quality and, perhaps, duration of animal and human
life.
[0015] It is a yet further object of the present invention to
provide agents which reverse the advanced glycosylation end
products formed as a consequence of the aforesaid advanced
glycosylation reaction sequence by cleaving the
.alpha.-dicarbonyl-based protein crosslinks present in the advanced
glycosylation end products.
[0016] It is a still further object of the present invention to
provide therapeutic methods of treating the adverse consequences of
molecular or protein aging by resort to the aforesaid method and
agents to achieve the reversal or cleavage of cross-links derived
from advanced glycosylation reactions.
[0017] It is a still further object of the present invention to
provide compositions, including pharmaceutical compositions,
incorporating the agents of the present invention.
[0018] Other objects and advantages will become apparent to those
skilled in the art from a consideration of the ensuing
description.
DETAILED DESCRIPTION OF THE INVENTION
[0019] In accordance with the present invention, agents,
compositions including pharmaceutical compositions containing said
agents and associated methods are described which reverse
already-formed advanced glycosylation end product-derived
cross-links (AGE cross-links). Useful agents, for instance,
comprise compounds having, the structural formula: 2
[0020] wherein
[0021] R.sup.1 and R.sup.2 are independently selected from the
group consisting of hydrogen and an alkyl group optionally
substituted by a hydroxy group;
[0022] Y is a group of the formula --CH.sub.2C(.dbd.O)R wherein R
is a heterocyclic group other than alkylenedioxyaryl containing
4-10 ring members and 1-3 heteroatoms selected from the group
consisting of oxygen, nitrogen and sulfur; said heterocyclic group
optionally substituted by one or more substituents selected from
the group consisting of alkyl, oxo, alkoxycarbonylalkyl aryl, and
aralkyl groups; and said one or more substituents optionally
substituted by one or more alkyl or alkoxy groups;
[0023] or a group of the formula --CH.sub.2C(.dbd.O)--NHR' wherein
R' is a heterocyclic group containing 4-10 ring members and 1-3
heteroatoms selected from the group consisting of oxygen, nitrogen,
and sulfur; said heterocyclic group optionally substituted by one
or more alkoxycarbonylalkyl groups;
[0024] and X is a halide, tosylate, methanesulfonate or
mesitylenesulfonate ion.
[0025] The heterocyclic groups referred to herein include 4-8
membered rings having at least one and up to 3 heteroatoms, e.g.,
oxygen, nitrogen, or sulfur, therein; and including various degrees
of unsaturation. Representatives of such heterocyclic groups are
those such as isoxazolyl, phenylisoxazolyl, furanyl, morpholino,
thiomorpholino, pyrimidinyl, piperidino, homopiperidino,
piperazino, methylpiperazino, hexamethyleneimino,
tetrahydroquinolyl, pyridyl, methylpyridyl, imidazolyl,
pyrrolidinyl, 2,6-dimethylmorpholino, furfuryl, 1,2,4-triazoylyl,
thiazolyl, thiophenyl, thiazolinyl, methylthiazolyl, and the like.
Excluded are alkylenedioxyaryl substituents, which are described in
copending application Ser. No. 08/588,249, incorporated herein by
reference. The heterocyclic groups of the present invention may be
further substituted, for example, by an oxo group, to form, for
example, a 2-oxo-tetrahydroquinolinyl group, or substituted by one
or more alkyl, alkoxycarbonylalkyl, aryl, or aralkyl groups, and
such substituents may be further substituted by one or more alkyl
or alkoxy groups.
[0026] Examples of Y groups of the compounds of the present include
but are not limited to: 3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl];
3-(2-(4-morpholinyl)-2-oxoethyl);
3-[2-(2,6-dimethyl-4-morpholinyl)-2-oxo- ethyl];
3-2-(1-piperidinyl)-2-oxoethyl); 3-[2-(2-oxo-1,2,3,4-tetrahydro-6--
quinolinyl)-2-oxoethyl]; 3-(2-(1-pyrrolidinyl)-2-oxoethyl;
3-[2-(3-methyl-2-thianaphthenyl)-2-oxoethyl];
3-[2-(4-phenyl-1-piperaziny- l)-2-oxoethyl;
3-(2-(2-thienyl)-2-oxoethyl); 3-(2-(2-thienyl)-2-oxoethyl);
3-(2-(4-thiomorpholinyl)-2-oxoethyl);
3-(2-(hexahydro-1-azepinyl)-2-oxoet- hyl);
3-[2-(4-[2-methoxyphenyl]-1-piperazinyl)-2-oxoethyl;
3-(2-(octahydro-1-azocinyl)-2oxoethyl);
3-(2-(2-pyridinyl)-2-oxoethyl;
3-[2-(2-methyl-1-piperidinyl)-2-oxoethyl];
3-[2-(2,6-dimethyl-1-piperidin- yl)-2-oxoethyl;
3-[2-(4-benzyl-1-piperidinyl)-2-oxoethyl]; and
3-[2-(4-benzyl-1-piperazinyl)-2-oxoethyl].
[0027] The alkyl groups referred to above contain one to about
eighteen carbon atoms and include, for example, methyl, ethyl,
propyl, butyl, pentyl, hexyl, octyl, decyl, dodecyl, and octadecyl,
and the corresponding branched-chain isomers thereof. Lower alkyl
groups, of one to about six carbon atoms, are preferred. The alkyl
groups optionally substituted by hydroxy groups include alkyl
groups as hereinbefore defined substituted with a hydroxy group at
any position, such as but not limited to the following examples:
hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl,
2-hydroxypropyl, 3-hydroxypropyl, 6-hydroxyhexyl, and the like.
Hydroxy-substituted lower alkyl groups as defined above are
preferred. Similarly, the alkoxy groups contain from one to about
eighteen carbon atoms, and include, for example, methoxy, ethoxy,
propoxy, butoxy, pentoxy, hexoxy, decoxy, and octadecoxy, and the
corresponding branched-chain isomers thereof. Lower alkoxy groups
of one to about 6 carbons, are preferred. The alkoxycarbonylalkyl
groups encompassed by the above formula include those wherein the
alkoxy portion contain from one to about eighteen carbon atoms and
the alkyl portion contains from 1 to about eighteen carbon atoms.
Typical alkoxycarbonyl portions are those such as acetoxy or
ethanoyloxy, propanoyloxy, butanoyloxy, pentanoyloxy, hexanoyloxy,
decanoyloxy, and octadecanoyloxy, and the corresponding branched
chain isomers thereof. The preferred alkyl portions of these
molecules have from one to about six carbon atoms.
[0028] The aryl groups encompassed by the above formula are those
containing 6-10 carbon atoms, such as naphthyl, phenyl and alkyl or
alkoxy substituted-phenyl, e.g., toluyl and xylyl.
[0029] For the purposes of this invention, the compounds of the
present invention are formed as biologically and pharmaceutically
acceptable salts. Useful salt forms are the halides, particularly
the bromide and chloride, tosylate, methanesulfonate, and
mesitylenesulfonate salts. Other related salts can be formed using
similarly non-toxic, and biologically and pharmaceutically
acceptable anions.
[0030] Of the compounds encompassed herein, certain substituents
are preferred. For instance, the compounds wherein R.sub.1 or
R.sub.2 are hydrogen or alkyl groups are preferred. Also highly
preferred are the compounds wherein Y is a 2-oxoethyl group with a
heterocyclic group of thiophenyl, thiomorpholinyl, furanyl,
2-oxo-tetrahydroquinolinyl, and pyrrolidinyl.
[0031] As described in the formula above, the heterocyclic group
may be represented by the R group of the formula
--CH.sub.2C(.dbd.O)--R, or it may represent the R' group of the
formula --CH.sub.2C(.dbd.O)NHR'. Representative, non-limiting
examples of compounds of the present invention are:
[0032] 3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-thiazolium
bromide
[0033]
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-4,5-dimethylthiazolium
bromide
[0034]
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-4-methyl-5-(2-hydroxyethyl-
)-thiazolium bromide
[0035]
3-[2-[4-(2-ethoxy-2-oxoethyl)-2-thiazolyl]amino-2-oxoethyl]-4,5-dim-
ethyl-thiazolium chloride
[0036]
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-4-methyl-5-(6-hydroxyhexyl-
)-thiazolium bromide
[0037]
3-[2-(2,6-dimethyl-4-morpholinyl)-2-oxoethyl]-4,5-dimethylthiazoliu-
m bromide
[0038] 3-(2-(1-piperidinyl)-2-oxoethyl)-4,5-dimethylthiazolium
bromide
[0039] 3-(2-(2-furanyl)-2-oxoethyl)-4,5-dimethylthiazolium
bromide
[0040] 3-(2-(2-furanyl)-2-oxoethyl)-4-(2-hydroxypentyl)thiazolium
bromide
[0041]
3-[2-(2-oxo-1,2,3,4-tetrahydro-6-quinolinyl)-2-oxoethyl]-4,5-dimeth-
ylthiazolium bromide
[0042] 3-(2-(1-pyrrolidinyl)-2-oxoethyl)-4,5-dimethyl-thiazolium
bromide
[0043]
3-[2-(3-methyl-2-thianaphthenyl)-2-oxoethyl]-4,5-dimethylthiazolium
bromide
[0044]
3-[2-(4-phenyl-1-piperazinyl)-2-oxoethyl]-4,5-dimethylthiazolium
bromide
[0045] 3-(2-(2-thienyl)-2-oxoethyl)-4,5-dimethyl-thiazolium
bromide
[0046]
3-(2-(2-thienyl)-2-oxoethyl)-4-methyl-5-hydroxyethylthiazolium
bromide
[0047] 3-(2-(4-thiomorpholinyl)-2-oxoethyl)-4,5-dimethylthiazolium
bromide
[0048]
3-(2-(hexahydro-1-azepinyl)-2-oxoethyl)-4,5-dimethylthiazolium
bromide
[0049]
3-[2-(4-[2-methoxyphenyl]-1-piperazinyl)-2-oxoethyl]-4,5-dimethylth-
iazolium chloride
[0050]
3-(2-(octahydro-1-azocinyl)-2-oxoethyl)-4,5-dimethyl-thiazolium
bromide
[0051] 3-(2-(2-pyridinyl)-2-oxoethyl)-4,5-dimethyl-thiazolium
bromide
[0052]
3-[2-(2-methyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthiazolium
chloride
[0053]
3-[2-(2,6-dimethyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthiazoliu-
m chloride
[0054]
3-[2-(4-benzyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthiazolium
chloride
[0055]
3-[2-(4-benzyl-1-piperazinyl)-2-oxoethyl]-4,5-dimethylthiazolium
chloride
[0056] 3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-4-octylthiazolium
bromide
[0057] 3-(2-(4-morpholinyl)-2-oxoethyl)-4,5-dimethylthiazolium
bromide
[0058]
3-[2-[4-(2-ethoxy-2-oxoethyl)-2-thiazolyl]amino-2-oxoethyl]-4,5-dip-
ropylthiazolium chloride
[0059] 3-(2-(4-morpholinyl)-2-oxoethyl)-4,5-dioctadecylthiazolium
bromide
[0060]
3-[2-(2,6-dimethyl-4-morpholinyl)-2-oxoethyl]-4,5-dipentylthiazoliu-
m bromide
[0061] 3-(2-(1-piperidinyl)-2-oxoethyl)-4,5-didodecylthiazolium
bromide
[0062] 3-(2-(2-furanyl)-2-oxoethyl)-5-decylthiazolium bromide
[0063]
3-[2-(2-oxo-1,2,3,4-tetrahydro-6-quinolinyl)-2-oxoethyl]-4,5-diocty-
lthiazolium bromide
[0064] 3-(2-(1-pyrrolidinyl)-2-oxoethyl)-4,5-diethylthiazolium
bromide
[0065]
3-(2-(3-methyl-2-thianaphthenyl)-2oxoethyl]-4,5-dipentylthiazolium
bromide
[0066] 3-[2-(4-phenyl-1-piperazinyl)-2-oxoethyl]-thiazolium
bromide
[0067] 3-(2-(2-thienyl)-2-oxoethyl)-thiazolium bromide
[0068]
3-(2-(2-thienyl)-2-oxoethyl)-4-methyl-5-(6-hydroxyhexyl)thiazolium
bromide
[0069] 3-(2-(4-thiomorpholinyl)-2-oxoethyl)thiazolium bromide
[0070]
3-(2-(hexahydro-1-azepinyl)-2-oxoethyl)-4,5-dioctylthiazolium
bromide
[0071]
3-(2-(octahydro-1-azocinyl)-2-oxoethyl)-4,5-didecylthiazolium
bromide
[0072] 3-(2-(2-pyridinyl )-2-oxoethyl)-4,5-dioctylthiazolium
bromide
[0073]
3-[2-(2-methyl-1-piperidinyl)-2-oxoethyl]-4,5-dipropylthiazolium
chloride
[0074]
3-[2-(2,6-dimethyl-1-piperidinyl)-2-oxoethyl]-4-methylthiazolium
chloride
[0075] 3-[2-(4-benzyl-1-piperidinyl)-2-oxoethyl]-5-methylthiazolium
chloride
[0076] 3-[2-(4-benzyl-1-piperazinyl)-2-oxoethyl]-4-octylthiazolium
chloride
[0077] The above compounds are capable of reversing already-formed
advanced glycosylation end products on proteins. The cross-linking
of proteins by formation of advanced glycosylation end products
contributes to the entrapment of other proteins and results in the
development in vivo of conditions such as reduced elasticity and
wrinkling of the skin, certain kidney diseases, atherosclerosis,
osteoarthritis and the like. Similarly, foodstuffs including plant
and animal material that undergoes nonenzymatic browning
deteriorates and becomes spoiled or toughened and, consequently,
inedible, unpalatable or non-nutritious. Thus, the compounds
employed in accordance with this invention reduce the level of the
advanced glycosylation end product-associated cross-links already
present in the protein material.
[0078] The present methods and compositions hold the promise for
reversing the aging of key proteins both in animals and plants, and
concomitantly, conferring both economic and medical benefits as a
result thereof. In the instance of foodstuffs, the administration
of the present composition holds the promise for reversing the
physico-chemical changes imparted to foodstuffs on storage, such as
the increased toughness of meats that occurs during aging or
storage.
[0079] The therapeutic implications of the present invention relate
to the reversal of the aging process which has, as indicated
earlier, been identified and exemplified in the aging of key
proteins by advanced glycosylation and cross-linking. Thus, body
proteins, and particularly structural body proteins, such as
collagen, elastin, lens proteins, nerve proteins, kidney glomerular
basement membranes and other extravascular matrix components would
all benefit in their longevity and operation from the practice of
the present invention. The present invention thus reduces the
incidence of pathologies involving the entrapment of proteins by
cross-linked target proteins, such as retinopathy, cataracts,
diabetic kidney disease, glomerulosclerosis, peripheral vascular
disease, arteriosclerosis obliterans, peripheral neuropathy,
stroke, hypertension, atherosclerosis, osteoarthritis,
periarticular rigidity, loss of elasticity and wrinkling of skin,
stiffening of joints, glomerulonephritis, etc. Likewise, all of
these conditions are in evidence and tend to occur at an
accelerated rate in patients afflicted with diabetes mellitus as a
consequence of their hyperglycemia. Thus, the present therapeutic
method is relevant to treatment of these and related conditions in
patients either of advanced age or those suffering from one of the
mentioned pathologies.
[0080] Molecular cross-linking through advanced glycosylation
product formation can decrease solubility of structural proteins
such as collagen in vessel walls and can also trap serum proteins,
such as lipoproteins to the collagen. Also, the presence of
advanced glycosylation end products and associated cross-links may
result in increased permeability of the endothelium and
consequently covalent trapping of extravasated plasma proteins in
subendothelial matrix, as well as a reduction in susceptibility of
both plasma and matrix proteins to physiologic degradation by
enzymes. For these reasons, the progressive occlusion of diabetic
vessels induced by chronic hyperglycemia has been hypothesized to
result from excessive formation of sugar-derived and particularly,
glucose-derived cross-links. Such diabetic microvascular changes
and microvascular occlusion can be effectively reversed by chemical
reversal or cleavage of the advanced glycosylation product
cross-link formation utilizing a composition and the methods of the
present invention.
[0081] Certain of the compounds disclosed herein are novel: 3
[0082] wherein
[0083] R.sup.1 and R.sup.2 are independently selected from the
group consisting of hydrogen and an alkyl group, optionally
substituted by a hydroxy group;
[0084] Y is a group of the formula --CH.sub.2C(.dbd.O)R wherein R
is a heterocyclic group other than alkylenedioxyaryl containing
4-10 ring members and one heteroatom selected from the group
consisting of sulfur and nitrogen, or 2-3 heteroatoms selected from
the group consisting of oxygen, nitrogen and sulfur; said
heterocyclic group optionally substituted by one or more
substituents selected from the group consisting of alkyl, oxo,
alkoxycarbonylalkyl, aryl, and aralkyl groups; and said one or more
substituents optionally substituted by one or more alkyl or alkoxy
groups;
[0085] or a group of the formula --CH.sub.2C(.dbd.O)--NHR' wherein
R' is a heterocyclic group containing 4-10 ring members and 1-3
heteroatoms selected from the group consisting of oxygen, nitrogen,
and sulfur; said heterocyclic group optionally substituted by one
or more alkoxycarbonylalkyl groups;
[0086] and X is a halide, tosylate, methanesulfonate or
mesitylenesulfonate ion.
[0087] The heterocyclic groups referred to herein include 4-8
membered rings having at least one nitrogen or sulfur atom, or 4-8
membered rings having 2-3 heteroatoms, e.g., oxygen, nitrogen, or
sulfur, therein; and including various degrees of unsaturation.
Representatives of such heterocyclic groups are those such as
isoxazolyl, phenylisoxazolyl, morpholino, thiomorpholino,
pyrimidinyl, piperidino, homopiperidino, piperazino,
methylpiperazino, hexamethyleneimino, tetrahydroquinolyl, pyridyl,
methylpyridyl, imidazolyl, pyrrolidinyl, 2,6-dimethylmorpholino,
furfuryl, 1,2,4-triazoylyl, thiazolyl, thiophenyl, thiazolinyl,
methylthiazolyl, and the like. Excluded are alkylenedioxyaryl
substituents, which are described in co-pending application Ser.
No. 08/588,249, incorporated herein by reference. The heterocyclic
groups of the present invention may be further substituted, for
example, by an oxo group, to form, for example, a
2-oxo-tetrahydroquinolinyl group, or substituted by one or more
alkyl, alkoxycarbonylalkyl, aryl, or aralkyl groups, and such
substituents may be further substituted by one or more alkyl or
alkoxy groups.
[0088] The alkyl groups, alkoxy groups, alkoxycarbonylalkyl groups,
aryl groups, and salts are as described hereinabove.
[0089] As described in the formula above, the heterocyclic group
may be represented by the R group of the formula
--CH.sub.2C(.dbd.O)--R, or it may represent the R' group of the
formula --CH.sub.2C(.dbd.O)NHR'. Representative examples of novel
compounds of the present invention are:
[0090] 3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]thiazolium
bromide
[0091]
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-4,5-dimethylthiazolium
bromide
[0092]
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-4-methyl-5-(2-hydroxyethyl-
)thiazolium bromide
[0093]
3-[2-[4-(2-ethoxy-2-oxoethyl)-2-thiazolyl]amino-2-oxoethyl]-4,5-dim-
ethylthiazolium chloride
[0094] 3-(2-(4-morpholinyl)-2-oxoethyl)-4,5-dimethylthiazolium
bromide
[0095]
3-[2-(2,6-dimethyl-4-morpholinyl)-2-oxoethyl]-4,5-dimethylthiazoliu-
m bromide
[0096] 3-(2-(1-piperidinyl)-2-oxoethyl)-4,5-dimethylthiazolium
bromide
[0097]
3-[2-(2-oxo-1,2,3,4-tetrahydro-6-quinolinyl)-2-oxoethyl]-4,5-dimeth-
ylthiazolium bromide
[0098] 3-(2-(1-pyrrolidinyl)-2-oxoethyl)-4,5-dimethylthiazolium
bromide
[0099]
3-[2-(3-methyl-2-thianaphthenyl)-2-oxoethyl]-4,5-dimethylthiazolium
bromide
[0100]
3-[2-(4-phenyl-1-piperazinyl)-2-oxoethyl]-4,5-dimethylthiazolium
bromide
[0101] 3-(2-(2-thienyl)-2-oxoethyl)-4,5-dimethylthiazolium
bromide
[0102]
3-(2-(2-thienyl)-2-oxoethyl)-4-methyl-5-hydroxyethylthiazolium
bromide
[0103] 3-(2-(4-thiomorpholinyl)-2-oxoethyl)-4,5-dimethylthiazolium
bromide
[0104]
3-(2-(hexahydro-1-azepinyl)-2-oxoethyl)-4,5-dimethyl-thiazolium
bromide
[0105]
3-[2-(4-[2-methoxyphenyl]-1-piperazinyl)-2-oxoethyl]-4,5-dimethylth-
iazolium chloride
[0106]
3-(2-(octahydro-1-azocinyl)-2-oxoethyl)-4,5-dimethylthiazolium
bromide
[0107] 3-(2-(2-pyridinyl)-2-oxoethyl)-4,5-dimethylthiazolium
bromide
[0108]
3-[2-(2-methyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthiazolium
chloride
[0109]
3-[2-(2,6-dimethyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthiazoliu-
m chloride
[0110]
3-[2-(4-benzyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthiazolium
chloride
[0111]
3-[2-(4-benzyl-1-piperazinyl)-2-oxoethyl]-4,5-dimethylthiazolium
chloride
[0112] As described in the Background section above, studies on
other AGE cross-link cleavage agents have shown the ability to
reverse cardiovascular damage in an experimental model of diabetes.
As diabetes is often considered to be a model of accelerated aging,
especially with respect to macrovascular complications, the agents
of the present invention are suitable for therapeutic use in such
complications of aging, examples of which are described above.
[0113] As will be shown in the Examples below, and as cited above,
compounds capable of reversing AGE cross-links show significant
promise in the treatment of various diseases and complications
related to protein aging. Compounds active in an in vitro model of
AGE cross-linking in which the compounds are evaluated for their
ability to reverse the covalent cross-linking of IgG to red blood
cell membranes, or cleave collage cross-links, has corresponding
in-vivo activity in a model of protein aging. As described above,
such studies demonstrate positive effects of such agents on
cardiovascular complications related to aging which are accelerated
in experimental diabetes (see Wolffenbuttel et al., 1998. "Breakers
of Advanced Glycation End Products Restores Large Artery Properties
in Experimental Diabetes," Proc. Nat. Acad. Sci. U.S.A.
95:4630-4634). In these studies, rats diabetic for 9 weeks followed
by 1 to 3 weeks administration of an AGE breaker compound resulted
in reversal of diabetes-induced increases in large artery
stiffness. Parameters that were improved included cardiac output,
peripheral resistance, systemic arterial compliance, input
impedance of the aorta, and compliance of the carotid artery. Thus,
active AGE-reversing compounds of the present invention are
candidates for the treatment of the complications of protein
aging.
[0114] In the instance where the compositions of the present
invention are utilized for in vivo or therapeutic purposes, it may
be noted that the compounds or agents used therein are
biocompatible. Various biologically or pharmaceutically acceptable
salts may be used, such as halides, tosylate, etc. Pharmaceutical
compositions may be prepared with a therapeutically effective
quantity of the agents or compounds of the present invention and
may include a pharmaceutically acceptable carrier, selected from
known materials utilized for this purpose. Such compositions may be
prepared in a variety of forms, depending on the method of
administration. Also, various pharmaceutically acceptable addition
salts of the compounds of the present invention may be
utilized.
[0115] A liquid form would be utilized in the instance where
administration is by intravenous, intramuscular or intraperitoneal
injection. When appropriate, solid dosage forms such as tablets,
capsules, or liquid dosage formulations such as solutions and
suspensions, etc., may be prepared for oral administration. For
topical or dermal application to the skin or eye, a solution, a
lotion or ointment may be formulated with the agent in a suitable
vehicle such as water, ethanol, propylene glycol, perhaps including
a carrier to aid in penetration into the skin or eye. For example,
a topical preparation could include up to about 10% of the compound
of Formula I. Other suitable forms for administration to other body
tissues are also contemplated.
[0116] In the instance where the present method has therapeutic
application, the animal host intended for treatment may have
administered to it a quantity of one or more of the agents, in a
suitable pharmaceutical form. Administration may be accomplished by
known techniques, such as oral, topical and parenteral techniques
such as intradermal, subcutaneous, intravenous or intraperitoneal
injection, as well as by other conventional means. Administration
of the agents may take place over an extended period of time at a
dosage level of, for example, up to about 30 mg/kg.
[0117] The agent of the present invention is formulated in
compositions in an amount effective to inhibit and reverse the
formation of advanced glycosylation end products. This amount will,
of course, vary with the particular agent being utilized and the
particular dosage form, but typically is in the range of 0.01% to
1.0%, by weight, of the particular formulation.
[0118] The compounds of the present invention can be prepared
generally according to the methods described in Potts et al., 1976.
J. Org. Chem. 41:187, and Potts et al., 1977, J. Org. Chem.
42:1648, or as shown in the following, scheme wherein R is a
heterocyclic group, R.sup.1, R.sup.2, and Z are as described
hereinabove, and X is a halogen atom: 4
[0119] In the reaction scheme above, the appropriate substituted
thiazole compounds of formula I is reacted with the appropriate
halo compound of formula II, to afford the desired compound of the
present invention; all substituents are as hereinbefore
defined.
[0120] The halo reactant may be prepared by suitable techniques
known in the art. For example, for the preparation of
3-(2-thiophenyl-2-oxoethyl)-- 4,5-dimethyl-thiazolium bromide, the
reactant 2-bromothiophene is reacted with dimethylthiazole.
2-Bromothiophene may be prepared according to the method of King,
et al., 1964, J. Org. Chem. 29:3459, by the bromination of
2-acetylthiophene with copper (II) bromide. Specific methods are
described in the examples below.
[0121] The conditions for the reaction between the halo compound
and the thiazole derivative generally involve refluxing the mixture
at 110.degree. C. in an oil bath for 3-7 hours with a minimum
amount of solvent such as acetonitrile, or refluxing the mixture in
ethanol or acetonitrile for 3-5 hours. If the halo reactant
contains chlorine, the first condition is used. For a bromo
compound, the second condition is preferable.
[0122] The present invention may be better understood by reference
to the following non-limiting Examples, which are provided as
exemplary of the invention. The following examples are presented in
order to more fully illustrate the preferred embodiments of the
invention. They should in no way be construed, however, as limiting
the broad scope of the invention.
EXAMPLE 1
Preparation of 3-(2-(1-thienyl)-2-oxoethyl)-4,5-dimethylthiazolium
bromide
[0123] This compound was prepared by reacting 4,5-dimethylthiazole
with 2-bromoacetylthiophene. 2-Bromoacetylthiophene was prepared by
bromination of 2-acetylthiophene with copper (II) bromide according
to the method of King et al., 1964, J. Org. Chem. 29:3459.
2-Acetylthiophene (6 g, 47.6 mmol) was dissolved in chloroform (60
mL) and added to a slurry of copper (II) bromide (13.5 g, 60.44
mmol) in ethyl acetate (120 mL). The mixture was refluxed for 6
hours and then filtered while still hot through a celite pad. The
filtrate cake was washed with ethyl acetate and the combined
filtrate was evaporated to give 2-bromoacetylthiophene (9.5 g,
97.3%). The crude product was used directly in the next
reaction.
[0124] A solution of 4,5-dimethylthiazole (2.2 g, 19.4 mmol) and
2-bromoacetylthiophene (4 g, 19.4 mmol) in ethyl alcohol (20 mL)
was refluxed for 3 hours. It was cooled to room temperature and
t-butyl methyl ether (10 mL) was added. The reaction mixture was
left at room temperature overnight with stirring. The white product
separated and was filtered and dried. It was crystallized from
ethyl alcohol (4.42 g. 73%). m.p. 203-205.degree. C.
EXAMPLE 2
Synthesis of
3-(2-(1-pyrrolidinyl)-2-oxoethyl)-4,5-dimethylthiazolium
chloride
[0125] 3-(2-(1-pyrrolidinyl)-2-oxoethyl)-4,5-dimethyl-thiazolium
chloride was prepared from the reaction of
N-(chloroacetyl)pyrrolidine with 4,5-dimethylthiazole.
N-(chloroacetyl)pyrrolidine was prepared as follows. Pyrrolidine
(63.9 g, 0.9 mol) was taken in methylene chloride (640 mL) and
cooled to 0.degree. C. in a salt-ice bath. To the stirred mixture
was added chloroacetyl chloride (101.8 g in 450 mL of
CH.sub.2Cl.sub.2, 0.9 mol) dropwise keeping the inside temperature
below 15.degree. C. After adding the chloroacetyl chloride,, the
mixture was stirred for an hour at 5.degree. C. Sodium hydroxide
solution (7 M, 190 mL) was added with vigorous stirring such that
the inside temperature did not exceed 20.degree. C. The mixture was
stirred for 15 minutes and the aqueous layer was separated. The
organic layer was washed successively with saturated sodium
bicarbonate solutions (2.times.200 mL), water (1.times.200 mL) and
dried over anhydrous sodium sulfate. The solvent was removed in
vacuo and the residue was recrystallized from hexane to give 64.5 g
(48.6%) of white plate crystals, m.p. 43.degree. C.
[0126] A mixture of N-(chloroacetyl)pyrrolidine (37.3 g, 0.25 mol),
4,5-dimethylthiazole (28.69 g, 0.25 mol) and acetonitrile (17 mL)
was refluxed in an oil bath at 105-110.degree. C. for 7 hours. To
the reaction mixture was added acetonitrile (230 mL) and continued
to heat for 20 minutes, then t-butyl methyl ether (250 mL) was
added. The reaction mixture was kept at room temperature overnight.
The product was filtered and washed with a mixture of t-butyl
methyl ether and acetonitrile (1:1 v/v, 100 mL), and t-butyl methyl
ether (150 mL) to obtain 59.64 g (90.2% yield) of white solid. The
crude product (59.64 g) was dissolved in acetonitrile (350 mL) with
heating, filtered, t-butyl methyl ether (350 mL) added, and the
solution was allowed to cool at room temperature for 3 hours. The
product was filtered and washed with a mixture of t-butyl methyl
ether and acetonitrile (1:1 v/v, 300 mL), and t-butyl methyl ether
(300 mL) to yield 56.95 g (86.1% yield) of white crystals, m.p.
196-198.degree. C.
EXAMPLE 3
[0127] Using the procedures described above in Examples 1 and 2,
the following compounds were prepared using the corresponding
reactants. The melting points are indicated.
1 3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-thiazolium bromide
213-214.degree. C. 3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl-
]-4,5-dimethylthiazolium bromide 245-246.degree. C. (dec.)
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-4-methyl-5-(2-hydroxyethyl)-thia-
zolium bromide 209-210.degree. C. (dec.)
3-[2-[4-(2-ethoxy-2-oxoethyl)-2-thiazolyl]amino-2-oxoethyl]-4,5-dimethyl--
thiazolium 120-121.degree. C. chloride
3-(2-(4-morpholinyl)-2-oxoethyl)-4,5-dimethylthiazolium bromide
197-198.degree. C. (dec.) 3-[2-(2,6-dimethyl-4-morpholinyl-
)-2-oxoethyl]-4,5-dimethylthiazolium bromide 215-216.degree. C.
3-(2-(1-piperidinyl)-2-oxoethyl)-4,5-dimethylthiazolium bromide
240-242.degree. C. 3-(2-(2-furanyl)-2-oxoethyl)-4,5-dimethylthiazo-
lium bromide 195-196.degree. C. (dec.)
3-[2-(2-oxo-1,2,3,4-tetrahydro-6-quinolinyl)-2-oxoethyl]-4,5-dimethylthia-
zolium bromide 274-275.degree. C. (dec.)
3-(2-(1-pyrrolidinyl)-2-oxoethyl)-4,5-dimethyl-thiazolium chloride
189-198.degree. C. 3-[2-(3-methyl-2-thianaphthenyl)-2-oxoethyl]-4,-
5-dimethylthiazolium bromide 171-172.degree. C.
3-[2-(4-phenyl-1-piperazinyl)-2-oxoethyl]-4,5-dimethylthiazolium
bromide 150-152.degree. C.
3-(2-(2-thienyl)-2-oxoethyl)-4,5-dimethyl-thiaz- olium bromide
203-205.degree. C. 3-(2-(2-thienyl)-2-oxoethyl)-4-met-
hyl-5-hydroxyethylthiazolium bromide 175-176.degree. C.
3-(2-(4-thiomorpholinyl)-2-oxoethyl)-4,5-dimethylthiazolium
chloride 232-233.degree. C. (dec.)
3-(2-(hexahydro-1-azepinyl)-2-ox- oethyl)-4,5-dimethyl-thiazolium
chloride 218-219.degree. C. (dec.)
3-[2-(4-[2-methoxyphenyl]-1-piperazinyl)-2-oxoethyl]-4,5-dimethylt-
hiazolium chloride 215-216.degree. C. (dec.)
3-(2-(octahydro-1-azocinyl)-2-oxoethyl)-4,5-dimethyl-thiazolium
chloride 210-212.degree. C. (dec.)
3-(2-(2-pyridinyl)-2-oxoethyl)-4- ,5-dimethyl-thiazolium bromide
178-179.degree. C.
3-[2-(2-methyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthiazolium
chloride 220-221.degree. C. (dec.)
3-[2-(2,6-dimethyl-1-piperidinyl-
)-2-oxoethyl]-4,5-dimethylthiazolium chloride 248-249.degree. C.
(dec.) 3-[2-(4-benzyl-1-piperidinyl)-2-oxoethyl]-4,5-dimethylthiaz-
olium chloride 170-171.degree. C.
3-[2-(4-benzyl-1-piperazinyl)-2-o- xoethyl]-4,5-dimethylthiazolium
chloride 180-182.degree. C.
EXAMPLE 4
[0128] A typical pharmaceutical dosage form is prepared as
follows.
2 mg/tablet Compound of the present invention 50 Starch 50 Mannitol
75 Magnesium stearate 2 Stearic acid 5
[0129] The compound, a portion of the starch and the lactose are
combined and wet granulated with starch paste. The wet granulation
is placed on trays and allowed to dry overnight at a temperature of
45.degree. C. The dried granulation is comminuted in a comminutor
to a particle size of approximately 20 mesh. Magnesium stearate,
stearic acid and the balance of the starch are added and the entire
mix blended prior to compression on a suitable tablet press. The
tablets are compressed at a weight of 232 mg. using a {fraction
(11/32)}" punch with a hardness of 4 kg. These tablets will
disintegrate within a half hour according to the method described
in USP XVI.
EXAMPLE 5
[0130]
3 Lotion mg/g Compound of Formula I 1.0 Ethyl alcohol 400.0
Polyethylene glycol 400 300.0 Hydroxypropyl cellulose 5.0 Propylene
glycol to make 1.0 g
EXAMPLE 6
Reversing Age Cross-links
[0131] The activity of the compounds of the present invention to
reverse AGE cross-links was evaluated in an in-vitro model, as
follows. Blood was collected from streptozotocin-diabetic rats (6
to 8 wk diabetic) in heparinized tubes, centrifuged at 2500.times.g
for 15 min. at 40.degree. C., and the plasma and buffy-coat layers
aspirated off. The RBC were washed with PBS (ca. 5 ml PBS per ml
blood) three times.
[0132] Test compounds were dissolved in PBS and the pH adjusted to
7.0, if necessary. Two hundred microliters of washed RBC were added
to 2 mL of test compound solution, and the mixture was incubated
overnight at 37.degree. C. For a control, 200 .mu.l of RBC was
incubated in 2 mL of PBS.
[0133] After the overnight incubation, the reaction mixtures were
centrifuged and the RBC pellets were washed three times with PBS,
followed by diluting 1:30 in PBS. Immunoglobulin G (IgG) bound to
the surface of the washed RBCs was then determined.
[0134] Assay for RBC-IgG: The assay is performed in a
Multiscreen-HA. 0.45 .mu.m cellulose mixed esters membrane-sealed
96 well plate (Millipore MAHAS45 ). The membranes are first wet by
filling the wells with 100 uL PBS, and the wells emptied by
applying a vacuum to the Millititer vacuum manifold. Three hundred
uL of Superblock (pre-warmed to 37.degree. C.) is then added to
each well and incubated at 37.degree. C. for one hour. The
Superblock is then removed from the wells by application of vacuum,
then the wells are washed once with 300 ul of PBS-Tween (0.05%) and
three times with PBS. The vacuum is then turned off.
[0135] One hundred .mu.l of PBS is then added to each well. Each
RBC sample is gently vortexed, and 50 uL pipetted into the wells,
in sextuplicate. The wells labeled A1-A3 are left for a reagent
blank and wells A4-A6 reserved for an antibody blank. Vacuum is
then applied to remove the buffer and the RBCs are washed once with
PBS. Alkaline phosphatase-labeled rabbit-anti-rat IgG is diluted 1
to 25000 in PBS, and 50 .mu.l is added to triplicate wells of each
sample. PBS alone is added to the other three wells of each sample.
These will serve as sample blanks to adjust for any endogenous
alkaline phosphatase activity. The samples are allowed to stand at
room temperature for two hours. The solution is then removed by
application of vacuum and the red blood cells are washed with
PBS-Tween twice, PBS twice and with TBS twice. The bottom of the
plate is rinsed with distilled water and blotted dry with paper
towels. To each well is then added 100 .mu.l of p-nitrophenyl
phosphate substrate (1 mg/ml in diethanolamine buffer pH 9.5), and
the color is allowed to develop for two hours at 37.degree. C. The
solutions in each well are then transferred to a 96 well microtiter
plate inside the vacuum manifold. One hundred .mu.l of PBS to each
well and vacuum applied again to transfer any remaining solution
into the microtiter plate. The OD at 410 nm is then read in a
Dynatech Plate reader (Sample filter 1 and Ref. filter 4).
[0136] For data analysis, breaking activity (percent reversal) is
expressed as the percent decrease in O.D. caused by incubation of
RBC with the test compound compared to RBC incubated in PBS alone.
Using the above assay, the following data was generated on the
compounds of the present invention:
4 Percent Compound reversal
3-(2-(2-thienyl)-2-oxoethyl)-4-methyl-5-hydroxyethylthiazolium
bromide 24% 3-(2-(2-pyridinyl)-2-oxoethyl)-4,5-dimethyl-thiazolium
bromide 27% 3-(2-(1-piperidinyl)-2-oxoethyl)-4,5-dimethylthiazolium
bromide 37% 3-[2-(2,6-dimethyl-4-morpholinyl)-2-oxoethyl]-4,5-dime-
thylthiazolium bromide 38%
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]- -thiazolium bromide 38%
3-(2-(4-thiomorpholinyl)-2-oxoethyl)-4,5-di- methylthiazolium
bromide 47% 3-(2-(2-thienyl)-2-oxoethyl)-4,5-dimet- hyl-thiazolium
bromide 53% 3-(2-(1-pyrrolidinyl)-2-oxoethyl)-4,5-di-
methyl-thiazolium bromide 63%
3-(2-(2-furanyl)-2-oxoethyl)-4,5-dime- thylthiazolium bromide 76%
3-[2-(4-benzyl-1-piperidinyl)-2-oxoethyl- ]-4,5-dimethylthiazolium
chloride 70% 3-[2-(2-oxo-1,2,3,4-tetrahydr-
o-6-quinolinyl)-2-oxoethyl]-4,5-dimethylthiazolium 84% bromide
EXAMPLE 7
Reversing Collagen Cross-links
[0137] In a further evaluation of the ability of compounds of the
present invention to reverse AGE cross-links between a circulating
protein and collagen. AGE-BSA was cross-linked to rat tail tendon
collagen (type 1)-coated 96 well microtiter plates, and reversal of
cross-linking was determined.
[0138] AGE-BSA preparation. AGE-BSA is prepared by incubation of a
solution of BSA (400 mg/ml) in 0.4 M sodium phosphate buffer pH 7.4
with an equal volume of 400 mM glucose solution in the same buffer
at 37.degree. C. for 12 weeks. The pH of the incubation mixture is
monitored weekly and adjusted to pH 7.4 if necessary. After 12
weeks, the AGE-BSA solution is dialyzed against PBS for 48 hours
with 4 buffer changes. Protein concentration is determined by the
micro-Lowry method.
[0139] Assay protocol. A Biocoat (Collaborative Biomedical
Products) plate is blocked with Superblock (300 mL /well) at
37.degree. C. for 1 hr and washed with PBS-Tween three times.
AGE-BSA was diluted in PBS to a concentration required to obtain
maximum cross-linking as determined in a preliminary experiment.
One hundred .mu.l of the AGE-BSA working solution is added to test
wells and a similar concentration of BSA is added to blank wells.
The first three wells are left empty for the reagent blank. The
plate is incubated at 37.degree. C. for four hours and washed with
PBS-Tween three times. Test compounds are dissolved in PBS and pH
adjusted to 7.0 if necessary. One hundred .mu.l of a test compound
is added to triplicate wells. To measure maximum crosslinking 100
.mu.l of PBS is added to three to six wells. The test compounds and
PBS are also added to triplicate wells incubated with BSA, to
obtain the blank readings. The plate is then incubated overnight at
37.degree. C. The plate is washed with PBS-Tween, 50 mL of
Rabbit-anti-BSA antibody (1 to 4000 in PBS) is added to each well
and the plate is incubated at room temperature for 60 min. After
the plate is washed with PBS-Tween, 50 mL of horseradish
peroxidase-conjugated-goat-anti-rabbit IgG (1 to 4000 in PBS) is
added to each well except the first three wells. The plate is
incubated at room temperature for 30 min. and washed with
PBS-Tween. Two hundred .mu.l of ABTS substrate (prepared from
HRP-substrate buffer 10.times. (Sigma) diluted 1:10 in deionized
water and mixed with 50.times.ABTS reagent (Sigma)) is added to
each well and color developed at 37.degree. C. for 15 min.
[0140] Optical density is read at 410 nm with the sample filter set
to "1" and the reference filter set to "5" on the Dynatech ELISA
plate reader.
[0141] Data Analysis: The average optical density (OD) is
calculated for each triplicate determination: Corrected OD=(Average
of OD AGE-BSA wells-Average of OD BSA wells). Percent breaking by
test compounds is expressed as the percent decrease in OD of
TTC-AGE-BSA wells incubated with test compounds compared to
TTC-AGE-BSA wells incubated with PBS.
[0142] Using the procedure above, it was found that 3 mM
3-[2-(3-phenyl-5-isoxazolyl)-2-oxoethyl]-thiazolium bromide caused
a 15% reversal of AGE cross-links.
[0143] This invention may be embodied in other forms or carried out
in other ways without departing from the spirit or essential
characteristics thereof. The present disclosure is therefore to be
considered as in all respects illustrative and not restrictive, the
scope of the invention being indicated by the appended claims, and
all changes which come within the meaning and range of equivalency
are intended to be embraced therein.
[0144] Various citations to the literature are presented herein,
all of which are incorporated herein in their entireties.
* * * * *