U.S. patent application number 13/141035 was filed with the patent office on 2013-05-09 for plasmalogen compounds, pharmaceutical compositions containing the same and methods for treating diseases of the aging.
This patent application is currently assigned to PHENOMENOME DISCOVERIES INC.. The applicant listed for this patent is Pearson AHIAHONU, Dayan GOODENOWE, M. KHAN, Rishikesh MANKIDY, Paul WOOD. Invention is credited to Pearson AHIAHONU, Dayan GOODENOWE, M. KHAN, Rishikesh MANKIDY, Paul WOOD.
Application Number | 20130116312 13/141035 |
Document ID | / |
Family ID | 42286823 |
Filed Date | 2013-05-09 |
United States Patent
Application |
20130116312 |
Kind Code |
A2 |
KHAN; M. ; et al. |
May 9, 2013 |
PLASMALOGEN COMPOUNDS, PHARMACEUTICAL COMPOSITIONS CONTAINING THE
SAME AND METHODS FOR TREATING DISEASES OF THE AGING
Abstract
Described herein are routes of synthesis and therapeutic uses of
1-alkyl, 2-acyl glycerol derivatives of formula I: which when
administered to mammalian biological systems result in increased
cellular concentrations of specific sn-2 substituted ethanolamine
plasmalogens independent of the ether lipid synthesis capacity of
the system. Elevating levels of the specific sn-2 substituted
species in this way can cause lowering of membrane cholesterol
levels and the lowering of amyloid secretion. These compounds can
be used for the treatment or prevention of diseases of aging
associated with increased membrane cholesterol, increased amyloid,
and decreased plasmalogen levels, such as neurodegeneration
(including Alzheimer's disease, Parkinson's disease and age-related
macular degeneration), cognitive impairment, dementia, cancer (e.g.
prostate, lung, breast, ovarian, and kidney cancers), osteoporosis,
bipolar disorder and vascular diseases (such as atherosclerosis,
hypercholesterolemia).
Inventors: |
KHAN; M.; (Morgan Hill,
CA) ; WOOD; Paul; (Harrogate, TN) ; GOODENOWE;
Dayan; (Saskatoon, CA) ; MANKIDY; Rishikesh;
(Saskatoon, CA) ; AHIAHONU; Pearson; (Saskatoon,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KHAN; M.
WOOD; Paul
GOODENOWE; Dayan
MANKIDY; Rishikesh
AHIAHONU; Pearson |
Morgan Hill
Harrogate
Saskatoon
Saskatoon
Saskatoon |
CA
TN |
US
US
CA
CA
CA |
|
|
Assignee: |
PHENOMENOME DISCOVERIES
INC.
Saskatoon
SK
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20120035250 A1 |
February 9, 2012 |
|
|
Family ID: |
42286823 |
Appl. No.: |
13/141035 |
Filed: |
December 18, 2009 |
PCT Filed: |
December 18, 2009 |
PCT NO: |
PCT/CA2009/001853 PCKC 00 |
371 Date: |
October 27, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61139695 |
Dec 22, 2008 |
|
|
|
Current U.S.
Class: |
514/440 ;
514/547; 549/39; 554/110; 554/224 |
Current CPC
Class: |
C07C 229/22 20130101;
C07F 9/106 20130101; A61P 9/00 20180101; A61P 27/02 20180101; A61P
35/00 20180101; A61P 25/00 20180101; A61P 25/16 20180101; C07C
323/59 20130101; C07D 339/04 20130101; A61P 25/28 20180101; C07C
279/14 20130101; A61P 3/06 20180101; A61P 9/10 20180101 |
Class at
Publication: |
514/440 ;
554/110; 514/547; 554/224; 549/39 |
International
Class: |
A61K 31/232 20060101
A61K031/232; C07C 69/587 20060101 C07C069/587; C07C 227/18 20060101
C07C227/18; C07D 339/04 20060101 C07D339/04; C07C 231/12 20060101
C07C231/12; C07C 233/47 20060101 C07C233/47; A61P 25/28 20060101
A61P025/28; A61P 25/16 20060101 A61P025/16; A61P 27/02 20060101
A61P027/02; C07C 229/30 20060101 C07C229/30; A61K 31/385 20060101
A61K031/385 |
Claims
1. A compound of formula I: ##STR00042## (I) wherein: R.sub.1 and
R.sub.2 are the same or different and selected from an alkyl or
alkenyl hydrocarbon chain selected from the group consisting of:
CH.sub.3(CH.sub.2).sub.3--, CH.sub.3(CH.sub.2).sub.5--,
CH.sub.3(CH.sub.2).sub.7--, CH.sub.3(CH.sub.2).sub.9--,
CH.sub.3(CH.sub.2).sub.11--, CH.sub.3(CH.sub.2).sub.13--,
CH.sub.3(CH.sub.2).sub.15--, CH.sub.3(CH.sub.2).sub.17--,
CH.sub.3(CH.sub.2).sub.19--, CH.sub.3(CH.sub.2).sub.21--,
CH.sub.3(CH.sub.2).sub.23--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.4CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3CH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub-
.7--, CH.sub.3CH.sub.2(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.7(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.9(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.11(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.13(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.15(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.17(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.19(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.21(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.4CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.5(CH.dbd-
.CH),
CH.sub.3CH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2-
).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.4(CH.dbd.CHCH.sub.2).sub.2(CH.sub.2).sub.6--,
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.3(CH.sub.2).sub.6--,
CH.sub.3(CH.sub.2).sub.4(CH.dbd.CHCH.sub.2).sub.4(CH.sub.2).sub.2--,
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.5(CH.sub.2).sub.2--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.11, and
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.6CH.sub.2--; R.sub.3 is
selected from the group consisting of carnitine,
acetyl-D/L-carnitine, thiocarnitine, acetyl-D/L-thiocarnitine,
creatine, norcarnitine, lipoic acid, dihydrolipoic acid,
N-acetylcysteine, substituted or unsubstituted amino acid groups
and groups of the structures shown below: ##STR00043## R.sub.4 and
R.sub.5 are independently hydrogen or lower alkyl; R.sub.6 is
hydrogen or lower alkyl; and R.sub.7 and R.sub.8 are independently
hydrogen or lower alkyl, including racemates or isolated
stereoisomers and pharmaceutically acceptable salts or esters
thereof.
2. The compound of claim 1, wherein R.sub.2 is
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.6CH.sub.2--.
3. The compound of claim 1, wherein said compound is selected from
the group consisting of
2-acetoxy-4-(2-((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoylo-
xy)-3-(hexadecyloxy)propoxy)-N,N,N-trimethyl-4-oxobutan-1-aminium
(PPI-1009),
(4Z,7Z,10Z,13Z,16Z,19Z)-1-(5-((R)-1,2-dithiolan-3-yl)pentanoyloxy)-3-(hex-
adecyloxy)propan-2-yl docosa-4,7,10,13,16,19-hexaenoate (PPI-1011)
and
(4Z,7Z,10Z,13Z,16Z,19Z)-1-(2-acetamido-3-mercaptopropanoyloxy)-3-(hexadec-
yloxy)propan-2-yl docosa-4,7,10,13,16,19-hexaenoate (PPI-1014).
4. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a compound as defined in claim 1.
5. The pharmaceutical composition of claim 4, wherein R.sub.2 is
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.6CH.sub.2--.
6. The pharmaceutical composition of claim 4, wherein the compound
is selected from the group consisting of
2-acetoxy-4-(2-((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoylo-
xy)-3-(hexadecyloxy)propoxy)-N,N,N-trimethyl-4-oxobutan-1-aminium
(PPI-1009),
(4Z,7Z,10Z,13Z,16Z,19Z)-1-(5-((R)-1,2-dithiolan-3-yl)pentanoyloxy)-3-(hex-
adecyloxy)propan-2-yl docosa-4,7,10,13,16,19-hexaenoate (PPI-1011)
and
(4Z,7Z,10Z,13Z,16Z,19Z)-1-(2-acetamido-3-mercaptopropanoyloxy)-3-(hexadec-
yloxy)propan-2-yl docosa-4,7,10,13,16,19-hexaenoate (PPI-1014).
7. A method of treating or preventing diseases of aging associated
with increased membrane cholesterol, increased amyloid or decreased
plasmalogen levels, comprising administering to a patient in need
thereof an effective amount of a compound as defined in claim 1, or
a pharmaceutical composition comprising said compound admixed with
a pharmaceutically acceptable carrier.
8. The method of claim 7, wherein R.sub.2 is
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.6CH.sub.2--.
9. The method of claim 7, wherein the compound is selected from the
group consisting of
2-acetoxy-4-(2-((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoylo-
xy)-3-(hexadecyloxy)propoxy)-N,N,N-trimethyl-4-oxobutan-1-aminium
(PPI-1009),
(4Z,7Z,10Z,13Z,16Z,19Z)-1-(5-((R)-1,2-dithiolan-3-yl)pentanoyloxy)-3-(hex-
adecyloxy)propan-2-yl docosa-4,7,10,13,16,19-hexaenoate (PPI-1011)
and
(4Z,7Z,10Z,13Z,16Z,19Z)-1-(2-acetamido-3-mercaptopropanoyloxy)-3-(hexadec-
yloxy)propan-2-yl docosa-4,7,10,13,16,19-hexaenoate (PPI-1014).
10. The method of claim 7, wherein the diseases of aging are
selected from the group consisting of: neurodegeneration, cognitive
impairment, dementia, cancer, and vascular disease.
11. The method of claim 10, wherein the neurodegeneration disease
is selected from the group consisting of: Alzheimer's disease,
Parkinson's disease and age-related macular degeneration.
12. The method of claim 10, wherein the cancer is selected from the
group consisting of: prostate, lung, breast, ovarian, and kidney
cancer.
13. The method of claim 10, wherein the vascular disease is
selected from the group consisting of: atherosclerosis and
hypercholesterolemia.
14. A method of treating or preventing diseases of the aging
mediated by plasmalogen deficiency, by increasing PlsEtn levels to
above control levels in either PlsEtn deficient or PlsEtn
sufficient systems by administering to a patient in need thereof an
effective amount of a compound of formula I: ##STR00044## (I)
wherein R.sub.1 and R.sub.2 are the same or different and selected
from an alkyl or alkenyl hydrocarbon chain selected from the group
consisting of: CH.sub.3(CH.sub.2).sub.3--,
CH.sub.3(CH.sub.2).sub.5--, CH.sub.3(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.9--, CH.sub.3(CH.sub.2).sub.11--,
CH.sub.3(CH.sub.2).sub.13--, CH.sub.3(CH.sub.2).sub.15--,
CH.sub.3(CH.sub.2).sub.17--, CH.sub.3(CH.sub.2).sub.19--,
CH.sub.3(CH.sub.2).sub.21--, CH.sub.3(CH.sub.2).sub.23--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.4CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3CH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub-
.7--, CH.sub.3CH.sub.2(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.7(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.9(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.11(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.13(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.15(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.17(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.19(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.21(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.4CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.5(CH.dbd-
.CH),
CH.sub.3CH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2-
).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.4(CH.dbd.CHCH.sub.2).sub.2(CH.sub.2).sub.6--,
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.3(CH.sub.2).sub.6--,
CH.sub.3(CH.sub.2).sub.4(CH.dbd.CHCH.sub.2).sub.4(CH.sub.2).sub.2--,
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.5(CH.sub.2).sub.2--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.11, and
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.6CH.sub.2--; R.sub.3 is
selected from the group consisting of fatty acids, carnitine,
acetyl-D/L-carnitine, thiocarnitine, acetyl-D/L-thiocarnitine,
creatine, norcarnitine, phosphocholine, lipoic acid, dihydrolipoic
acid, phosphoethanolamine, phosphoserine, N-acetylcysteine,
substituted or unsubstituted amino acid groups and groups of the
structures shown below: ##STR00045## R.sub.4 and R.sub.5 are
independently hydrogen or lower alkyl; R.sub.6 is hydrogen or lower
alkyl; and R.sub.7 and R.sub.8 are independently hydrogen or lower
alkyl, including racemates or isolated stereoisomers and
pharmaceutically acceptable salts or esters thereof, or a
pharmaceutical composition comprising said compound admixed with a
pharmaceutically acceptable carrier.
15. The method of claim 14, wherein R.sub.2 is
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.6CH.sub.2--.
16. A method of treating or preventing a disorder resulting from
abnormal genetic expression of cholesterol transport proteins
comprising administering to a patient in need thereof an effective
amount of a compound of formula I: ##STR00046## (I) wherein R.sub.1
and R.sub.2 are the same or different and selected from an alkyl or
alkenyl hydrocarbon chain selected from the group consisting of:
CH.sub.3(CH.sub.2).sub.3--, CH.sub.3(CH.sub.2).sub.5--,
CH.sub.3(CH.sub.2).sub.7--, CH.sub.3(CH.sub.2).sub.9--,
CH.sub.3(CH.sub.2).sub.11--, CH.sub.3(CH.sub.2).sub.13--,
CH.sub.3(CH.sub.2).sub.15--, CH.sub.3(CH.sub.2).sub.17--,
CH.sub.3(CH.sub.2).sub.19--, CH.sub.3(CH.sub.2).sub.21--,
CH.sub.3(CH.sub.2).sub.23--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.4CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3CH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub-
.7--, CH.sub.3CH.sub.2(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.7(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.9(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.11(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.13(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.15(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.17(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.19(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.21(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.4CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.5(CH.dbd-
.CH),
CH.sub.3CH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2-
).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.4(CH.dbd.CHCH.sub.2).sub.2(CH.sub.2).sub.6--,
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.3(CH.sub.2).sub.6--,
CH.sub.3(CH.sub.2).sub.4(CH.dbd.CHCH.sub.2).sub.4(CH.sub.2).sub.2--,
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.5(CH.sub.2).sub.2--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.11, and
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.6CH.sub.2--; R.sub.3 is
selected from the group consisting of fatty acids, carnitine,
acetyl-D/L-carnitine, thiocarnitine, acetyl-D/L-thiocarnitine,
creatine, norcarnitine, phosphocholine, lipoic acid, dihydrolipoic
acid, phosphoethanolamine, phosphoserine, N-acetylcysteine,
substituted or unsubstituted amino acid groups and groups of the
structures shown below: ##STR00047## R.sub.4 and R.sub.5 are
independently hydrogen or lower alkyl; R.sub.6 is hydrogen or lower
alkyl; and R.sub.7 and R.sub.8 are independently hydrogen or lower
alkyl, including racemates or isolated stereoisomers and
pharmaceutically acceptable salts or esters thereof, or a
pharmaceutical composition comprising said compound admixed with a
pharmaceutically acceptable carrier.
17. The method of claim 16, wherein R.sub.2 is
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.6CH.sub.2--.
18. The method of claim 16, wherein the cholesterol transport
protein is apolipoprotein E.
19-30. (canceled)
31. A process for preparing a compound according to the structure
of formula II: ##STR00048## wherein R.sub.1 is an alkyl or alkenyl
hydrocarbon chain selected from the group consisting of:
CH.sub.3(CH.sub.2).sub.3--, CH.sub.3(CH.sub.2).sub.5--,
CH.sub.3(CH.sub.2).sub.7--, CH.sub.3(CH.sub.2).sub.9--,
CH.sub.3(CH.sub.2).sub.11--, CH.sub.3(CH.sub.2).sub.13--,
CH.sub.3(CH.sub.2).sub.15--, CH.sub.3(CH.sub.2).sub.17--,
CH.sub.3(CH.sub.2).sub.19--, CH.sub.3(CH.sub.2).sub.21--,
CH.sub.3(CH.sub.2).sub.23--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.4CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3CH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub-
.7--, CH.sub.3CH.sub.2(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.7(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.9(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.11(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.13(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.15(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.17(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.19(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.21(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.4CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.5(CH.dbd-
.CH),
CH.sub.3CH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2-
).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.4(CH.dbd.CHCH.sub.2).sub.2(CH.sub.2).sub.6--,
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.3(CH.sub.2).sub.6--,
CH.sub.3(CH.sub.2).sub.4(CH.dbd.CHCH.sub.2).sub.4(CH.sub.2).sub.2--,
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.5(CH.sub.2).sub.2--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.11, and
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.6CH.sub.2--; R.sub.4 and
R.sub.5 are independently hydrogen or lower alkyl; R.sub.6 is
hydrogen or lower alkyl; and R.sub.7 and R.sub.8 are independently
hydrogen or lower alkyl, including racemates or isolated
stereoisomers and pharmaceutically acceptable salts or esters
thereof, said process comprising reacting a compound of formula
III: ##STR00049## wherein R.sub.9 is a blocking group, in a
suitable solvent with tetrabutylammonium fluoride (TBAF) under
conditions to form said compound of formula II, and optionally
purifying said compound of formula II.
32. The process of claim 31, wherein said blocking group is a
silane blocking group.
33. The process of claim 32, wherein the silane blocking group is a
tert-butyldimethylsilyl halide.
34. The process of claim 31, wherein said solvent comprises
dimethylformamide (DMF).
35. The process of claim 31, wherein said reaction is carried out
at a temperature of 0 to 26.degree. C.
36. The process of claim 35, wherein said reaction is carried out
at about 20.degree. C.
37. The process of claim 35, wherein said reaction is allowed to
proceed for up to 20 hours.
38. A compound according to the structure of formula II:
##STR00050## wherein R.sub.1 is an alkyl or alkenyl hydrocarbon
chain selected from the group consisting of:
CH.sub.3(CH.sub.2).sub.3--, CH.sub.3(CH.sub.2).sub.5--,
CH.sub.3(CH.sub.2).sub.7--, CH.sub.3(CH.sub.2).sub.9--,
CH.sub.3(CH.sub.2).sub.11--, CH.sub.3(CH.sub.2).sub.13--,
CH.sub.3(CH.sub.2).sub.15--, CH.sub.3(CH.sub.2).sub.17--,
CH.sub.3(CH.sub.2).sub.19--, CH.sub.3(CH.sub.2).sub.21--,
CH.sub.3(CH.sub.2).sub.23--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.7--CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.4CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3CH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub-
.7--, CH.sub.3CH.sub.2(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.7(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.9(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.11(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.13(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.15(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.17(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.19(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.21(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.4CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.5(CH.dbd-
.CH),
CH.sub.3CH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2-
).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.4(CH.dbd.CHCH.sub.2).sub.2(CH.sub.2).sub.6--,
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.3(CH.sub.2).sub.6--,
CH.sub.3(CH.sub.2).sub.4(CH.dbd.CHCH.sub.2).sub.4(CH.sub.2).sub.2--,
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.5(CH.sub.2).sub.2--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.11, and
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.6CH.sub.2--; R.sub.4 and
R.sub.5 are independently hydrogen or lower alkyl; R.sub.6 is
hydrogen or lower alkyl; and R.sub.7 and R.sub.8 are independently
hydrogen or lower alkyl, including racemates or isolated
stereoisomers and pharmaceutically acceptable salts or esters
thereof.
39. A method of treating or preventing a neurodegeneration disease
selected from the group consisting of Alzheimer's disease,
Parkinson's disease and age-related macular degeneration, the
method comprising administering to a patient in need thereof, an
effective amount of a compound of formula I: ##STR00051## wherein
R.sub.1 and R.sub.2 are the same or different and selected from an
alkyl or alkenyl hydrocarbon chain selected from the group
consisting of: CH.sub.3(CH.sub.2).sub.3--,
CH.sub.3(CH.sub.2).sub.5--, CH.sub.3(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.9--, CH.sub.3(CH.sub.2).sub.11--,
CH.sub.3(CH.sub.2).sub.13--, CH.sub.3(CH.sub.2).sub.15--,
CH.sub.3(CH.sub.2).sub.17--, CH.sub.3(CH.sub.2).sub.19--,
CH.sub.3(CH.sub.2).sub.21--, CH.sub.3(CH.sub.2).sub.23--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.4CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3CH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub-
.7--, CH.sub.3CH.sub.2(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.7(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.9(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.11(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.13(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.15(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.17(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.19(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.21(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.4CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.5(CH.dbd-
.CH),
CH.sub.3CH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2-
).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.4(CH.dbd.CHCH.sub.2).sub.2(CH.sub.2).sub.6--,
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.3(CH.sub.2).sub.6--,
CH.sub.3(CH.sub.2).sub.4(CH.dbd.CHCH.sub.2).sub.4(CH.sub.2).sub.2--,
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.5(CH.sub.2).sub.2--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.11, and
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.6CH.sub.2--; R.sub.3 is
selected from the group consisting of fatty acids, carnitine,
acetyl-D/L-carnitine, thiocarnitine, acetyl-D/L-thiocarnitine,
creatine, norcarnitine, phosphocholine, lipoic acid, dihydrolipoic
acid, phosphoethanolamine, phosphoserine, N-acetylcysteine,
substituted or unsubstituted amino acid groups and groups of the
structures shown below: ##STR00052## R.sub.4 and R.sub.5 are
independently hydrogen or lower alkyl; R.sub.6 is hydrogen or lower
alkyl; and R.sub.7 and R.sub.8 are independently hydrogen or lower
alkyl, including racemates or isolated stereoisomers and
pharmaceutically acceptable salts or esters thereof.
40. The method of claim 39, wherein R.sub.2 is
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.6CH.sub.2--.
41. The method of claim 39, wherein the compound is selected from
the group consisting of
2-acetoxy-4-(2-((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoylo-
xy)-3-(hexadecyloxy)propoxy)-N,N,N-trimethyl-4-oxobutan-1-aminium
(PPI-1009),
(4Z,7Z,10Z,13Z,16Z,19Z)-1-(5-((R)-1,2-dithiolan-3-yl)pentanoyloxy)-3-(hex-
adecyloxy)propan-2-yl docosa-4,7,10,13,16,19-hexaenoate (PPI-1011)
and
(4Z,7Z,10Z,13Z,16Z,19Z)-1-(2-acetamido-3-mercaptopropanoyloxy)-3-(hexadec-
yloxy)propan-2-yl docosa-4,7,10,13,16,19-hexaenoate (PPI-1014).
42. A method of treating or preventing a vascular disease selected
from the group consisting of atherosclerosis and
hypercholesterolemia, the method comprising administering to a
patient in need thereof, an effective amount of a compound of
formula I: ##STR00053## wherein R.sub.1 and R.sub.2 are the same or
different and selected from an alkyl or alkenyl hydrocarbon chain
selected from the group consisting of: CH.sub.3(CH.sub.2).sub.3--,
CH.sub.3(CH.sub.2).sub.5--, CH.sub.3(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.9--, CH.sub.3(CH.sub.2).sub.11--,
CH.sub.3(CH.sub.2).sub.13--, CH.sub.3(CH.sub.2).sub.15--,
CH.sub.3(CH.sub.2).sub.17--, CH.sub.3(CH.sub.2).sub.19--,
CH.sub.3(CH.sub.2).sub.21--, CH.sub.3(CH.sub.2).sub.23--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.4CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3CH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub-
.7--, CH.sub.3CH.sub.2(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.7(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.9(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.11(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.13(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.15(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.17(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.19(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.21(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.4CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.5(CH.dbd-
.CH),
CH.sub.3CH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2-
).sub.5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.4(CH.dbd.CHCH.sub.2).sub.2(CH.sub.2).sub.6--,
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.3(CH.sub.2).sub.6--,
CH.sub.3(CH.sub.2).sub.4(CH.dbd.CHCH.sub.2).sub.4(CH.sub.2).sub.2--,
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.5(CH.sub.2).sub.2--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.11, and
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.6CH.sub.2--; R.sub.3 is
selected from the group consisting of fatty acids, carnitine,
acetyl-D/L-carnitine, thiocarnitine, acetyl-D/L-thiocarnitine,
creatine, norcarnitine, phosphocholine, lipoic acid, dihydrolipoic
acid, phosphoethanolamine, phosphoserine, N-acetylcysteine,
substituted or unsubstituted amino acid groups and groups of the
structures shown below: ##STR00054## R.sub.4 and R.sub.5 are
independently hydrogen or lower alkyl; R.sub.6 is hydrogen or lower
alkyl; and R.sub.7 and R.sub.8 are independently hydrogen or lower
alkyl, including racemates or isolated stereoisomers and
pharmaceutically acceptable salts or esters thereof.
43. The method of claim 42, wherein R.sub.2 is
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.6CH.sub.2--.
44. The method of claim 42, wherein the compound is selected from
the group consisting of
2-acetoxy-4-(2-((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoylo-
xy)-3-(hexadecyloxy)propoxy)-N,N,N-trimethyl-4-oxobutan-1-aminium
(PPI-1009),
(4Z,7Z,10Z,13Z,16Z,19Z)-1-(5-((R)-1,2-dithiolan-3-yl)pentanoyloxy)-3-(hex-
adecyloxy)propan-2-yl docosa-4,7,10,13,16,19-hexaenoate (PPI-1011)
and
(4Z,7Z,10Z,13Z,16Z,19Z)-1-(2-acetamido-3-mercaptopropanoyloxy)-3-(hexadec-
yloxy)propan-2-yl docosa-4,7,10,13,16,19-hexaenoate (PPI-1014).
45-50. (canceled)
Description
[0001] This application is a national stage application under 35
U.S.C. .sctn.371 from PCT Application No. PCT/CA2009/001853, filed
Dec. 18, 2009, which claims priority benefit of U.S. Provisional
Application No. 61/139,695, filed Dec. 22, 2008.
FIELD OF INVENTION
[0002] The present invention relates to the synthesis and utility
of novel chemical entities with useful biochemical, physiochemical,
and clinical properties. More specifically, a series of 1-alkyl,
2-acyl glycerol derivatives are provided which can be used for the
treatment or prevention of disease. The invention also relates to
pharmaceutical compositions and kits incorporating such
compounds.
BACKGROUND OF THE INVENTION
[0003] It is well known that many diverse human diseases such as
cancer, dementia, or decreased cognitive functioning increase in
incidence with age. From an epidemiological and statistical
perspective, these diseases often look very similar. However, from
a clinical perspective, each of the cancers, dementias, and
decreased cognitive functioning are very different. Currently, the
largest risk factor for these disorders is the subject's age.
Furthermore, it is well established that most cancers, dementias,
and decreased cognitive functioning have a long prodromal phase
(5-15 years) in which the disease is present but at a sub-clinical
manifestation. Age-associated increases in membrane
cholesterol.sup.1-3 and increased mitochondrial membrane
cholesterol.sup.4-6 have been reported. These increases in membrane
cholesterol result in decreased membrane fluidity.sup.2, decreased
ion channel function.sup.6-8, decreased activities of some
membrane-bound enzymes like 5'-nucleotidase.sup.9 and
.alpha.-secretase.sup.10, and altered diffusion properties for
signaling molecules like nitric oxide.sup.11.
[0004] Subjects suffering from increased membrane cholesterol
demonstrate an increased prevalence of neurodegenerative diseases
(e.g. Alzheimer's, Parkinson's, multiple sclerosis and age-related
macular degeneration), cognitive impairment, dementia, cancers
(e.g. prostate, lung, breast, ovarian, and kidney), osteoporosis,
bipolar disorder and vascular diseases (atherosclerosis,
hypercholesterolemia).
[0005] With respect to specific diseases, cholesterol accumulates
in the brain membranes of Alzheimer's patients in a
severity-dependent manner.sup.12-14. In this regard, lowering
membrane cholesterol has been shown to decrease the activities of
beta- and gamma-secretases, blocking the pathogenic processing of
beta-amyloid.sup.15-16. At the molecular level, cholesterol binds
to the transmembrane domain of amyloid precursor protein (APP),
activating the trafficking of APP to cholesterol-rich membrane
domains rich in beta- and gamma-secretases, resulting in
amyloid-beta production.sup.17. Synaptic membrane changes resulting
from increased cholesterol may also be an important factor in the
utilization of membrane phospholipids to support cholinergic
neurotransmission (autocannibalism concept).sup.18. Early
utilization of statins have also been suggested to reduce the
incidence or delay the onset of Alzheimer's and Parkinson's
diseases.sup.19. Cholesterol accumulation also occurs in the drusen
associated with age-related macular degeneration.sup.20. Membrane
cholesterol is also increased in cancer.sup.21 and increases in
mitochondrial membrane cholesterol have been hypothesized to be the
defect that leads to the Warburg effect that is associated with
most cancer cells.sup.22. The Warburg effect is a defining feature
of cancer cells in that, unlike normal cells, which rely almost
entirely upon respiration for energy, cancer cells can utilize both
respiration and glycolysis for energy.
[0006] In addition to complex negative membrane effects of
cholesterol accumulation, there is also increased oxysterol
production.sup.23. These oxysterols are cytotoxic (apoptosis and
necrosis), pro-inflammatory, deplete GSH and induce
phospholipidosis.sup.23-26. Diseases in which these toxic
oxysterols might be involved include neurodegeneration (both
neuronal and demyelinating), osteoporosis, age-related macular
degeneration and cardiovascular diseases, particularly
atherosclerosis.sup.23.
[0007] Current clinical therapies to reduce cholesterol consist
mainly of inhibiting cholesterol synthesis with statins or blocking
cholesterol absorption from the gastrointestinal tract with
ezetimibe. To the inventors' knowledge, there are currently no
drugs to mobilize cholesterol trafficking out of membranes.
SUMMARY OF THE INVENTION
[0008] The present invention relates to compounds and methods for
treating diseases of aging associated with abnormal membrane
cholesterol levels. The described compounds include novel
plasmalogen precursors which decrease membrane free cholesterol
levels and augment cholesterol esterification for transport from
cell membranes. These compounds therefore are useful for reducing
membrane cholesterol levels in subjects suffering from elevated
membrane cholesterol levels. The compounds can also be used to
treat or prevent diseases associated with increased membrane
cholesterol such as neurodegenerative diseases (including but not
limited to Alzheimer's disease, Parkinson's disease, multiple
sclerosis and age-related macular degeneration), cognitive
impairment, dementia, cancers (including but not limited to
prostate, lung, breast, ovarian, and kidney cancer), osteoporosis,
bipolar disorder and vascular diseases (including but not limited
to atherosclerosis and hypercholesterolemia). Furthermore, these
compounds are useful in the treatment of disorders resulting from
abnormal genetic expression of cholesterol transport proteins, such
as apolipoprotein E.
[0009] These plasmalogen precursors contain a glycerol backbone
with an alkyl or alkenyl lipid substitution at sn-1 and an acyl
lipid substitution at sn-2. A polar substituent is provided at sn-3
to improve pharmaceutical properties (such as to improve stability
and/or bioavailability, or for formulation as a salt).
[0010] Without wishing to be bound by theory, it is believed that
in certain embodiments the sn-3 substituent is cleaved by lipases
and the resulting 1-alkyl, 2-acyl glycerol or 1-alkenyl, 2-acyl
glycerol is then converted to plasmalogens in the endoplasmic
reticulum, thereby bypassing the peroxisomal compartment which can
demonstrate decreased function with ageing.
[0011] Accordingly, with respect to composition, there are provided
compounds of formula I:
##STR00001## [0012] wherein: [0013] R.sub.1 and R.sub.2 can be the
same or different; and are an alkyl or alkenyl hydrocarbon chain
selected from Table 1 or 2;
TABLE-US-00001 [0013] TABLE 1 Alkyl and alkenyl hydrocarbon groups
No. Chemical Structure Abbr. 1 CH3(CH2)3-- C4:0 2 CH3(CH2)5-- C6:0
3 CH3(CH2)7-- C8:0 4 CH3(CH2)9-- C10:0 5 CH3(CH2)11-- C12:0 6
CH3(CH2)13-- C14:0 7 CH3(CH2)15-- C16:0 8 CH3(CH2)17-- C18:0 9
CH3(CH2)19-- C20:0 10 CH3(CH2)21-- C22:0 11 CH3(CH2)23-- C24:0 12
CH3(CH2)3CH.dbd.CH(CH2)7-- C14:1 13 CH3(CH2)5CH.dbd.CH(CH2)7--
C16:1 14 CH3(CH2)7CH.dbd.CH(CH2)7-- C18:1 15
CH3(CH2)4CH.dbd.CHCH2CH.dbd.CH(CH2)7-- C18:2 16
CH3CH2CH.dbd.CHCH2CH.dbd.CHCH2CH.dbd.CH(CH2)7-- C18:3 17
CH3CH2(CH.dbd.CH)-- C4:1 18 CH3(CH2)3(CH.dbd.CH)-- C6:1 19
CH3(CH2)5(CH.dbd.CH)-- C8:1 20 CH3(CH2)7(CH.dbd.CH)-- C10:1 21
CH3(CH2)9(CH.dbd.CH)-- C12:1 22 CH3(CH2)11(CH.dbd.CH)-- C14:1 23
CH3(CH2)13(CH.dbd.CH)-- C16:1 24 CH3(CH2)15(CH.dbd.CH)-- C18:1 25
CH3(CH2)17(CH.dbd.CH)-- C20:1 26 CH3(CH2)19(CH.dbd.CH)-- C22:1 27
CH3(CH2)21(CH.dbd.CH)-- C24:1 28
CH3(CH2)3CH.dbd.CH(CH2)5(CH.dbd.CH)-- C14:2 29
CH3(CH2)5CH.dbd.CH(CH2)5(CH.dbd.CH)-- C16:2 30
CH3(CH2)7CH.dbd.CH(CH2)5(CH.dbd.CH)-- C18:2 31
CH3(CH2)4CH.dbd.CHCH2CH.dbd.CH(CH2)5(CH.dbd.CH)-- C18:3 32
CH3CH2CH.dbd.CHCH2CH.dbd.CHCH2CH.dbd.CH(CH2)5(CH.dbd.CH)--
C18:4
TABLE-US-00002 TABLE 2 Unsaturated cis fatty acid side chains No.
Name of acid Chemical Structure 1 Myristoleic (14:1)
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.7-- 2 Palmitoleic
(16:1) CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.7-- 3 Oleic
(18:1) CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7-- 4
Linoleic (18:2)
CH.sub.3(CH.sub.2).sub.4(CH.dbd.CHCH.sub.2).sub.2(CH.sub.2).sub.6--
5 Linolenic (18:3)
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.3(CH.sub.2).sub.6-- 6
Arachidonic (20:4)
CH.sub.3(CH.sub.2).sub.4(CH.dbd.CHCH.sub.2).sub.4(CH.sub.2).sub.2--
7 Eicosapentaenoic (20:5)
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.5(CH.sub.2).sub.2-- 8
Erucic (22:1) CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.11--
9 Docosahexaenoic (22:6)
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.6CH.sub.2--
[0014] R.sub.3 is a group selected from fatty acids, carnitine,
acetyl-D/L-carnitine, thiocarnitine, acetyl-D/L-thiocarnitine,
creatine, norcarnitine, phosphocholine, lipoic acid, dihydrolipoic
acid, phosphoethanolamine, phosphoserine, N-acetylcysteine,
substituted or unsubstituted amino acids and groups of the
structures shown below in Table 3.
TABLE-US-00003 [0014] TABLE 3 ##STR00002## 1 ##STR00003## 2
##STR00004## 3 ##STR00005## 4 ##STR00006## 5 ##STR00007## 6
##STR00008## 7 ##STR00009## 8
[0015] R.sub.4 and R.sub.5 are either the same or different and may
be hydrogen or lower alkyl, for example methyl or ethyl; [0016]
R.sub.6 is hydrogen or lower alkyl, for example methyl or ethyl;
and [0017] R.sub.7 and R.sub.8 are either the same or different and
may be hydrogen or lower alkyl, for example methyl and ethyl,
[0018] and also including racemates or isolated stereoisomers and
pharmaceutically acceptable salts or esters thereof.
[0019] Another aspect of this invention is directed to
pharmaceutical compositions comprising a pharmaceutically
acceptable carrier and a compound as described above.
[0020] In one embodiment of the present invention R.sub.2 may be a
docosahexaenoic acid (DHA) side chain, or
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.6CH.sub.2--.
[0021] In another embodiment, the compound can be
2-acetoxy-4-(2-((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoylo-
xy)-3-(hexadecyloxy)propoxy)-N,N,N-trimethyl-4-oxobutan-1-aminium.
##STR00010##
[0022] In other embodiments the compound may be
(4Z,7Z,10Z,13Z,16Z,19Z)-1-(5-((R)-1,2-dithiolan-3-yl)pentanoyloxy)-3-(hex-
adecyloxy)propan-2-yl docosa-4,7,10,13,16,19-hexaenoate:
##STR00011##
or
(4Z,7Z,10Z,13Z,16Z,19Z)-1-(2-acetamido-3-mercaptopropanoyloxy)-3-(hexa-
decyloxy)propan-2-yl docosa-4,7,10,13,16,19-hexaenoate:
##STR00012##
[0023] The invention also includes pharmaceutical compositions
comprising PPI-1009, PPI-1011, PPI-1014, or combinations
thereof.
[0024] Without wishing to be bound by theory, certain embodiments
of the compounds described herein are believed to increase
plasmalogen levels and the hydrolysis of acetyl-L-carnitine from
sn-3 position, and may participate in potential molecular
mechanisms that include: (i) acetylation of --NH.sub.2 and --OH
functional groups in amino acids and N-terminal amino acids in
peptides and proteins resulting in modification of their structure,
dynamic, function and turnover; and/or (ii) acting as a molecular
chaperone to larger molecules resulting in a change in the
structure, molecular dynamics, and function of the larger
molecule.
[0025] Carnitine is important in the beta oxidation of fatty acids
and the acetyl moiety can be used to maintain acetyl-CoA levels.
Acetyl-L-carnitine (ALCAR) actions include modulation of: (i) brain
energy and phospholipid metabolism; (iii) cellular macromolecules,
including neurotrophic factors and hormones; (iii) synaptic
morphology; and (iv) synaptic transmission of multiple
neurotransmitters.
[0026] According to a further aspect of the present invention there
is provided a method of treating or preventing diseases of aging
mediated by plasmalogen deficiency, comprising administering to a
patient in need thereof an effective amount of a compound or
composition as described above.
[0027] In a further aspect of the present invention there is
provided a method of treating diseases of aging associated with
increased membrane cholesterol, increased amyloid and decreased
plasmalogen levels comprising administering to a patient in need
thereof, an effective amount of a compound or composition as
described above.
[0028] The invention also relates to a process for preparing a
compound according to the structure of formula II:
##STR00013##
wherein R.sub.1 and R.sub.4 to R.sub.8 are as described above and
R.sub.9 is a blocking group, and including racemates or isolated
stereoisomers and pharmaceutically acceptable salts or esters
thereof. The process involves reacting a compound of formula
III:
##STR00014##
in a suitable solvent with a nucleophilic catalyst, a base and a
blocking agent under conditions to form said compound of formula
II, and optionally purifying the resulting compound.
[0029] In certain non-limiting embodiments the blocking agent may
be a silane blocking agent, for instance a tert-butyldimethylsilyl
halide which then gives rise to R.sub.9 being a
tert-butyldimethylsilyl group.
[0030] In other non-limiting embodiments the nucleophilic catalyst
may be DMAP, the base may be triethylamine, and the solvent may
comprise dimethylformamide (DMF) and CH.sub.2Cl.sub.2.
[0031] Without wishing to be limiting, it may in certain
embodiments be preferred to prepare a solution including the
compound of formula III, the nucleophilic catalyst, and the base in
the appropriate solvent at from 0 to 5.degree. C. prior to addition
of the blocking agent, followed by addition of the blocking agent
and then carrying out the reaction at a temperature of from about
15 to about 25.degree. C., for example at about 20.degree. C. The
reaction will then preferably be allowed to proceed to completion,
which may be up to 20 hours.
[0032] Also provided is an intermediate compound according to the
structure of formula II:
##STR00015##
wherein R.sub.1 and R.sub.4 to R.sub.9 are as described above,
including racemates or isolated stereoisomers and pharmaceutically
acceptable salts or esters thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] These and other features of the invention will become more
apparent from the following description in which reference is made
to the appended drawings wherein:
[0034] FIG. 1 shows the general experimental procedure for
preparation of PPI-1009:
2-acetoxy-4-(2-((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19--
hexaenoyloxy)-3-(hexadecyloxy)propoxy)-N,N,N-trimethyl-4-oxobutan-1-aminiu-
m, in accordance with an embodiment of the present invention.
[0035] FIG. 2 shows decreased levels of total DHA plasmalogens in
N-Rel cells, relative to control CHO cells, are restored in a
concentration-dependent manner by PPI-1005 (R.sub.1=16:0;
R.sub.2=DHA; R.sub.3=OH). 72 hr incubation. * p<0/05 vs. vehicle
(veh).
[0036] FIG. 3 shows (A) a time course of PPI-1009 (10 .mu.M)
incorporation into plasmalogens and lack of effect on cellular
levels of chimyl alcohol or the associated alkenyl-acyl-glycerol
(B) of N-Rel cells (0, 6, 12, 24, 48 and 72 hr). Cellular
plasmalogens and docosahexaenoic (DHA) acid were quantitated by
LC-MS/MS while chimyl alcohol was quantitated by GC-MS.
[0037] FIG. 4 shows (A) the concentration-dependent (72 hr)
increase of DHA plasmalogens in CHO cells with PPI-1005 and
PPI-1009. While chimyl alcohol increased DHA-plasmalogens in N-Rel
cells there was no effect in CHO cells (B). Cellular plasmalogens
were quantitated by LC-MS and expressed relative to CHO controls or
N-Rel controls.
[0038] FIG. 5 shows the concentration response for the decrease in
membrane cholesterol in N-Rel cells after a 48 hr incubation with
16:0(sn-1 alkyl)/DHA(sn-2 acyl) glycerol (PLM-05). Note that
cholesterol levels are significantly elevated (p<0.05) in NRel
cells relative to CHO cells and that at 20 .mu.M (PLM-05) decreases
membrane free cholesterol and increases membrane cholesterol esters
(p<0.05).
[0039] FIG. 6 shows structure-specific and concentration-dependent
incorporation (72 hr) into the associated plasmalogen
(R.sub.1=16:0, R.sub.2=22:6, R.sub.3=phosphoethanolamine) of N-Rel
cells with PPI-1005 (A; 1 and 5 .mu.M) and PPI-1009 (B; 0.5, 1, 2,
3 & 10 .mu.M). The fatty acid substitutions at sn-2 also
underwent deacylation and reacylation to form the associated
plasmalogens with 20:4, 18:3, 18:2 and 18:1 at sn-2. Cellular
plasmalogens were quantitated by LC-MS/MS and normalized relative
to N-Rel cells treated with vehicle.
[0040] FIG. 7 shows that membrane cholesterol is increased in
mutant N-Rel cells relative to control CHO cells. Membrane
cholesterol in N-Rel cells is decreased (p<0.05) after a 48 hr
incubation (20 .mu.M) with plasmalogen precursors possessing either
palmitic (16:0) or stearic (18:0) acid at sn-1 in combination with
unsaturated fatty acids, particularly DHA, at sn-2. At 20 .mu.M,
free DHA was ineffective in altering membrane free cholesterol
levels. In contrast to the activity of analogs with an alkyl
linkage at sn-1, the diacyl analog (16:0*:DHA glycerol) was
inactive. PPI-1009 (16:0/DHA/ALCAR) produced the most robust
decreases in free cholesterol and augmentation of esterified
cholesterol. V, vehicle.
[0041] FIG. 8 shows the decrease (p<0.04) in membrane
cholesterol in HEK293 cells after a 48 hr incubation with 20 .mu.M
16:0(sn-1 alkyl)/DHA(sn-2 acyl) glycerol, 18:0/DHA glycerol, or
16:0/18:3 glycerol. At 20 .mu.M, 16:0/18:1 glycerol, 16:0/18:2
glycerol, 16:0/20:4 glycerol and free DHA were ineffective in
altering membrane free cholesterol levels. In contrast to the
activity of analogs with an alkyl linkage at sn-1, the diacyl
analog (16:0*:DHA glycerol) was inactive.
[0042] FIG. 9 shows the concentration response for the decrease in
membrane cholesterol in HEK293 cells after a 48 hr incubation with
16:0(sn-1 alkyl)/DHA(sn-2 acyl)/acetyl-L-carnitine (sn-3 acyl)
glycerol (PPI-1009).
[0043] FIG. 10 shows PPI-1005 (5a, 20 .mu.M) decreases basal and
cholesterol (25.8 .mu.M)-stimulated A.beta.42 secretion by HEK293
cells (A). PPI-1009 (PLM09, 10 .mu.M) acted in a similar manner
(B).
[0044] FIG. 11 illustrates the effect of PPI-1011 on plasmalogens
in rabbit plasma. PPI-1011 was incorporated into plasma
ethanolamine plasmalogens (PlsEtn) and phosphatidylethanolamines
(PtdEtn) 1, 3, 6, and 12 hours after a 200 mg/kg dose orally in a
gelatin capsule. The release of DHA (free 22:6) from sn-2, via
deacylases, was also monitored. Groups consisted of 3 to 5
rabbits.
[0045] FIG. 12 shows a plot of the timecourse of incorporation of
pasmalogen precursor PPI-1011 into circulating Pls 16:0/22:6, DHA,
Pls 18:0/22:6, Pls 18:1/22:6 and Ptd 16:0/22:6. Incorporation of
PPI-1011 in plasma ethanolamine plasmalogens (Pls) and
phosphatidylethanolamines (Ptd) was measured 1, 3, 6, 12, 18, 24
and 48 hours after a 200 mg/kg dose orally in a gelatin capsule.
The release of DHA from sn-2, via deacylases, was also monitored.
Groups consisted of 3 to S rabbits except the 12 hour timepoint
which includes 7 rabbits from 2 separate experiments.
[0046] FIG. 13 illustrates the dose-dependent incorporation of
PPI-1011 into plasma plasmalogens and phosphatidylethanolamines.
Incorporation of PPI-1011 in plasma ethanolamine plasmalogens (Pls)
and phosphatidylethanolamines (Ptd) was measured 6 hours after
doses of 10, 75, 200, 500, and 1000 mg/kg orally in a gelatin
capsule. The release of DHA from sn-2, via deacylases, was also
monitored. Groups consisted of 3 to 5 rabbits.
[0047] FIG. 14 shows augmentation of tissue plasmalogens and DHA by
PPI-1011 in rabbit kidney. Incorporation of PPI-1011 in kidney
ethanolamine plasmalogens (PlsEtn) and phosphatidylethanolamines
(PtdEtn) was measured 1, 3, 6, and 12 hours after a 200 mg/kg dose
orally in a gelatin capsule. The release of DHA (free 22:6) from
sn-2, via deacylases, was also monitored. Groups consisted of 3 to
5 rabbits.
[0048] FIG. 15 shows a timecourse of the tissue plasmalogens and
DHA augmentation by PPI-1011 in rabbit kidney. Incorporation of
PPI-1011 in kidney ethanolamine plasmalogen (16:0/22:6) was
measured 1, 3, 6, 12, 18, 24 and 48 hours after a 200 mg/kg dose
orally in a gelatin capsule. Groups consisted of 3 to 5
rabbits.
[0049] FIG. 16 shows a timecourse of the tissue plasmalogens and
DHA augmentation by PPI-1011 in rabbit liver. Incorporation of
PPI-1011 in liver ethanolamine plasmalogen (16:0/22:6) was measured
12, 18, 24 and 48 hours after a 200 mg/kg dose orally in a gelatin
capsule. Groups consisted of 3 to 5 rabbits.
[0050] FIG. 17 shows structure-specific and concentration-dependent
incorporation of PPI-1014 in N-Rel ethanolamine plasmalogens
(PlsEtn) and phosphatidylethanolamines (PtdEtn) after 72 hours (5,
10 and 20 .mu.M). Cellular plasmalogens were quantitated by
LC-MS/MS and normalized relative to N-Rel cells treated with
vehicle. Groups consisted of three 10 cm.sup.2 plates.
[0051] FIG. 18 shows (A) effects of increasing PPI-1005
concentration on membrane resident proteins in cholesterol loaded
HEK293 cells, (B) effects of PPI-1005 on membrane resident proteins
in wild-type HEK293 cells, and (C) effects of pravastatin on
membrane-resident proteins ADAM10 and SOAT1. .beta.-actin was used
as a loading control.
DETAILED DESCRIPTION
[0052] Described herein are compounds according to structure of
formula I:
##STR00016## [0053] wherein: [0054] R.sub.1 and R.sub.2 are the
same or different and selected from an alkyl or alkenyl hydrocarbon
chain selected from the group consisting of: CH3(CH2)3-,
CH3(CH2)5-, CH3(CH2)7-, CH3(CH2)9-, CH3(CH2)11-, CH3(CH2)13-,
CH3(CH2)15-, CH3(CH2)17-, CH3(CH2)19-, CH3(CH2)21-, CH3(CH2)23-,
CH3(CH2)3CH.dbd.CH(CH2)7-, CH3(CH2)5CH.dbd.CH(CH2)7-,
CH3(CH2)7CH.dbd.CH(CH2)7-, CH3(CH2)4-CH.dbd.CHCH2CH.dbd.CH(CH2)7-,
CH3CH2CH.dbd.CHCH2CH.dbd.CHCH2CH.dbd.CH(CH2)7-,
CH3CH2(CH.dbd.CH)--, CH3(CH2)3(CH.dbd.CH)--,
CH3(CH2)5(CH.dbd.CH)--, CH3(CH2)7(CH.dbd.CH)--,
CH3(CH2)9(CH.dbd.CH)--, CH3(CH2)11(CH.dbd.CH)--,
CH3(CH2)13(CH.dbd.CH)--, CH3(CH2)15(CH.dbd.CH)--,
CH3(CH2)17(CH.dbd.CH)--, CH3(CH2)19(CH.dbd.CH)--,
CH3(CH2)21(CH.dbd.CH)--, CH3(CH2)3CH.dbd.CH(CH2)5(CH.dbd.CH)--,
CH3(CH2)5CH.dbd.CH(CH2)5(CH.dbd.CH)--,
CH3(CH2)7CH.dbd.CH(CH2)5(CH.dbd.CH)--,
CH3(CH2)4-CH.dbd.CHCH2CH.dbd.CH(CH2)5(CH.dbd.CH),
CH3CH2CH.dbd.CHCH2CH.dbd.CHCH2CH.dbd.CH(CH2)5(CH.dbd.CH)--,
CH.sub.3(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.5CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7--,
CH.sub.3(CH.sub.2).sub.4(CH.dbd.CHCH.sub.2).sub.2(CH.sub.2).sub.6--,
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.3(CH.sub.2).sub.6--,
CH.sub.3(CH.sub.2).sub.4(CH.dbd.CHCH.sub.2).sub.4(CH.sub.2).sub.2--,
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.5(CH.sub.2).sub.2--,
CH.sub.3(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.11, and
CH.sub.3CH.sub.2(CH.dbd.CHCH.sub.2).sub.6CH.sub.2--; [0055] R.sub.3
is a group selected from fatty acids, carnitine,
acetyl-D/L-carnitine, thiocarnitine, acetyl-D/L-thiocarnitine,
creatine, norcarnitine, phosphocholine, lipoic acid, dihydrolipoic
acid, phosphoethanolamine, phosphoserine, N-acetylcysteine,
substituted or unsubstituted amino acids and groups of the
structures shown below:
[0055] ##STR00017## [0056] R.sub.4 and R.sub.5 are independently
hydrogen or lower alkyl; [0057] R.sub.6 is hydrogen or lower alkyl;
and [0058] R.sub.7 and R.sub.8 are independently hydrogen or lower
alkyl, and [0059] including racemates or isolated stereoisomers and
pharmaceutically acceptable salts or esters thereof.
[0060] Such compounds are useful for treating or preventing
diseases of aging associated with increased membrane cholesterol,
increased amyloid or decreased plasmalogen levels.
[0061] Such compounds are also useful for treating or preventing
diseases of aging mediated by plasmalogen deficiency.
[0062] Such compounds can also be used to treat neurodegenerative
diseases (including but not limited to Alzheimer's disease,
Parkinson's disease and age-related macular degeneration),
cognitive impairment, dementia, cancer (including but not limited
to prostate, lung, breast, ovarian, and kidney cancers),
osteoporosis, bipolar disorder and vascular diseases (including but
not limited to atherosclerosis and hypercholesterolemia).
[0063] For the purposes of this invention, the hydroxy groups at
the sn-1, sn-2, and sn-3 positions of the glycerol back-bone of the
compounds of formula I are named using conventional plasmalogen
nomenclature, i.e., the oxygen atom of the glycerol bonded to the
carbonyl group --C.dbd.O (acetyl), --C (form ether bond) and --P
(Phosphoryl) is designated in the formula I.
[0064] In certain non-limiting embodiments, compounds as described
herein may contain one or more chiral centers. Typically, such
compounds will be prepared as a racemic mixture. If desired,
however, such compounds can be prepared or isolated as pure
stereoisomers, i.e., as individual enantiomers or diastereomers, or
as stereoisomer-enriched mixtures. All such stereoisomers (and
enriched mixtures) of the compounds of formula I are included
within the scope of this invention. Pure stereoisomers (or enriched
mixtures) may be prepared using, for example, optically active
starting materials or stereoselective reagents well known in the
art. Alternatively, racemic mixtures of such compounds can be
separated using, for example, chiral column chromatography, chiral
resolving agents and the like.
DEFINITIONS
[0065] When describing the alkyl/acyl fatty acids (Table 1, Table
2) and biologically active compounds (Table 3), pharmaceutical
compositions and methods of this invention, the following terms
have the following meanings unless otherwise specified.
[0066] "Fatty acids" are aliphatic monocarboxylic acids, derived
from, or contained in esterified form in an animal or vegetable
fat, oil or wax. Natural fatty acids commonly have a chain of 4 to
28 carbons (usually unbranched and even numbered), which may be
saturated or unsaturated. These are known as acyclic aliphatic
carboxylic acids.
[0067] Within the meaning of saturated fatty acids, the term
"saturated" refers to carbons (apart from the initial carboxylic
[--COOH] group) containing as many hydrogens as possible. In other
words, the omega (.omega.)) end contains 3 hydrogen atoms
(CH.sub.3--), and carbon within the chain contains 2 hydrogen
atoms.
[0068] Unsaturated fatty acids (including but not limited to the
examples described in Table 2) are of similar form to saturated
fatty acids, except that one or more alkenyl functional groups
exist along the chain, with each alkene substituting a single
bonded --CH2-CH2- part of the chain with a double-bonded
--CH.dbd.CH-- portion (that is a carbon double-bonded to another
carbon). These are named as CIS/TRANS and C:D where C is known as
number of carbon atoms and D known as double bond.
[0069] "Unsubstituted and substituted amino acids" refers to an
optionally substituted amino acid moiety containing an amino group,
a carboxylic acid group and a variable side chain, which side chain
may include common amino acid side chains, i.e., those used in
forming proteins, or others as are known in the art. Protein
forming amino acid moieties are particularly preferred, and include
alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,
glutamic acid, glycine, histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, proline, serine, threonine, tryptophan,
tyrosine and valine amino acid groups. Substitutions on the amino
acid moieties are also possible, including substitutions with
functional groups including but not limited to lower alkyl,
acetate, phosphate, lipids and carbohydrates.]
[0070] The term "lower alkyl" refers to a cyclic, branched or
straight chain monovalent alkyl radical of one to seven carbon
atoms (C.sub.1-C.sub.7), and in certain non-limiting embodiments
from one to four carbon atoms (C.sub.1-C.sub.4). This term is
further exemplified by such radicals as methyl, ethyl, n-propyl,
i-propyl, n-butyl, t-butyl, i-butyl (or 2-methylpropyl),
cyclopropylmethyl, i-amyl, n-amyl, hexyl and heptyl. Lower alkyl
groups can also be unsubstituted or substituted, where a specific
example of a substituted alkyl is 1,1-dimethyl heptyl.
[0071] "Hydroxyl" refers to --OH.
[0072] "Pharmaceutically-acceptable salt" refers to any salt of a
compound of this invention which retains its biological properties
and which is not biologically or otherwise undesirable. Such salts
may be derived from a variety of organic and inorganic counter-ions
well known in the art and include, by way of example, sodium,
potassium, calcium, magnesium, ammonium, tetraalkylammonium, and
the like; and when the molecule contains a basic functionality,
salts of organic or inorganic acids, such as hydrochloride,
hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the
like. The term "pharmaceutically-acceptable cation" refers to a
pharmaceutically acceptable cationic counter-ion of an acidic
functional group. Such cations are exemplified by sodium,
potassium, calcium, magnesium, ammonium, tetraalkylammonium
cations, and the like.
[0073] "Pharmaceutically acceptable ester" refers to a
conventionally esterified compound of Formula I having a carboxyl
group, which esters retain the biological effectiveness and
properties of the compounds of Formula I and are cleaved in vivo
(in the organism) to the corresponding active carboxylic acid.
Information concerning esters and the use of esters for the
delivery of pharmaceutical compounds is available in Design of
Prodrugs. Bundgaard H ed. (Elsevier 1985). See also, H. Ansel et.
al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed.
1995) at pp. 108-109; Krogsgaard-Larsen, et. al. Textbook of Drug
Design and Development (2d Ed. 1996) at pp. 152-191.
[0074] A "pharmaceutical agent" or "drug" refers to a chemical
compound or composition capable of inducing a desired therapeutic
or prophylactic effect when properly administered to a subject.
[0075] The term "effective amount" means that amount of a drug or
pharmaceutical agent that will elicit the biological or medical
response of a tissue, system, animal, or human that is being
sought, for instance, by a researcher or clinician. Furthermore,
the term "therapeutically effective amount" means any amount which,
as compared to a corresponding subject who has not received such
amount, results in improved treatment, healing, prevention, or
amelioration of a disease, disorder, or side effect, or a decrease
in the rate of advancement of a disease or disorder. The term also
includes within its scope amounts effective to enhance normal
physiological function.
[0076] All chemical compounds include both the (+) and (-)
stereoisomers, as well as either the (+) or (-) stereoisomer.
[0077] Other chemistry terms herein are used according to
conventional usage in the art, as exemplified by The McGraw-Hill
Dictionary of Chemical Terms (1985) and The Condensed Chemical
Dictionary (1981).
[0078] The compounds described herein, which include plasmalogen
precursors derived from a glycerol back-bone with substitution at
sn-1 and sn-2 with fatty acids, and sn-3 with fatty acids or
endogenous metabolic intermediate compounds, can be prepared from
readily available starting materials using the following general
methods and procedures shown in FIG. 1. It will be appreciated that
where typical or preferred process conditions (i.e., reaction
temperatures, times, mole ratios of reactants, solvents, pressures,
etc.) are given, other process conditions can also be used unless
otherwise stated. Optimum reaction conditions may vary with the
particular reactants or solvent used, but such conditions can be
determined by one skilled in the art by routine optimization
procedures.
[0079] Additionally, as will be apparent to those skilled in the
art, conventional protecting groups may be necessary to prevent
certain functional groups from undergoing undesired reactions. The
choice of a suitable protecting group for a particular functional
group as well as suitable conditions for protection and
de-protection are well known in the art. For example, numerous
protecting groups, and their introduction and removal, are
described in T. W. Greene and G. M. Wuts, Protecting Groups in
Organic Synthesis, Second Edition, Wiley, New York, 1991, and
references cited therein.
[0080] In a preferred method of synthesis of the compounds
described herein, which include glycerol substitutions at sn-1 and
sn-2 with fatty acids and sn-3 with fatty acids or endogenous
metabolic intermediate compounds as described herein, are prepared
by protection/deprotection of hydroxyl groups of the glycerol
back-bone with suitable protecting groups, followed by O-alkylation
and O-acylation of the compound; for example PPI-1009, PPI-1011 and
PPI-1014.
[0081] When employed as pharmaceuticals, compounds as described
herein are typically administered in the form of a pharmaceutical
composition. Such compositions can be prepared using procedures
well known in the pharmaceutical art and comprise at least one
active compound.
[0082] Generally the compounds of this invention are administered
in a pharmaceutically effective amount. The amount of the compound
actually administered will typically be determined by a physician,
in the light of the relevant circumstances, including the condition
to be treated, the chosen route of administration, the actual
compound administered, the age, weight, and response of the
individual patient, the severity of the patient's symptoms, and the
like.
[0083] The compounds and compositions described herein can be
administered to a subject, preferably a mammal, more preferably a
human, to treat and/or prevent disease by any suitable routes
including, by way of illustration, oral, topical, rectal,
transdermal, subcutaneous, intravenous, intramuscular, intranasal,
and the like. Depending on the intended route of delivery, the
compounds of this invention are preferably formulated as either
oral, topical or injectable compositions.
[0084] Pharmaceutical compositions for oral administration can take
the form of bulk liquid solutions or suspensions, or bulk powders.
More commonly, however, such compositions are presented in unit
dosage forms to facilitate accurate dosing. The term "unit dosage
forms" refers to physically discrete units suitable as unitary
dosages for human subjects and other mammals, each unit containing
a predetermined quantity of active material calculated to produce
the desired therapeutic effect, in association with a suitable
pharmaceutical excipient. Typical unit dosage forms include
prefilled, premeasured ampoules or syringes of the liquid
compositions or pills, tablets, capsules or the like in the case of
solid compositions.
[0085] Liquid forms suitable for oral administration may include a
suitable aqueous or non aqueous vehicle with buffers, suspending
and dispensing agents, colorants, flavors and the like. Solid forms
may include, for example, any of the following ingredients, or
compounds of a similar nature: a binder such as microcrystalline
cellulose, gum tragacanth or gelatin; an excipient such as starch
or lactose, a disintegrating agent such as alginic acid, Primogel,
or corn starch; a lubricant such as magnesium stearate; a glidant
such as colloidal silicon dioxide; a sweetening agent such as
sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
[0086] Topical compositions are typically formulated as a topical
ointment or cream containing the active ingredient(s), generally in
an amount ranging from about 0.01 to about 20% by weight,
preferably from about 0.1 to about 10% by weight, and more
preferably from about 0.5 to about 15% by weight. When formulated
as an ointment, the active ingredients will typically be combined
with either a paraffinic or a water-miscible ointment base.
Alternatively, the active ingredients may be formulated in a cream
with, for example, an oil-in-water cream base. Such topical
formulations are well-known in the art and generally include
additional ingredients to enhance the dermal penetration or
stability of the active ingredients or the formulation. All such
known topical formulations and ingredients are included within the
scope of this invention.
[0087] The compounds of this invention can also be administered by
a transdermal device. Accordingly, topical administration can be
accomplished using a patch either of the reservoir or porous
membrane type or of a solid matrix variety.
[0088] Injectable compositions are typically based upon injectable
sterile saline or phosphate-buffered saline or other injectable
carriers known in the art. As before, the alkyl nitrone compound in
such compositions is typically a minor component, often being from
about 0.05 to 10% by weight with the remainder being the injectable
carrier and the like.
[0089] The above-described components for orally and topically
administrable or injectable compositions are merely representative.
Other materials as well as processing techniques and the like are
set forth in Part 8 of Remington's Pharmaceutical Sciences, 18th
edition, 1990, Mack Publishing Company, Easton, Pa., 18042, which
is incorporated herein by reference.
[0090] The compounds of this invention can also be administered in
sustained release forms or from sustained release drug delivery
systems. A description of representative sustained release
materials can be found in the incorporated materials in Remington's
Pharmaceutical Sciences.
[0091] The pharmaceutical compositions of this invention can be
formulated into tablets, capsules, liquid, injection formulation,
or an ointment. The present invention, however, is not limited to
the following pharmaceutical compositions. For example, yet without
wishing to be limiting in any way, the compound of formula I can be
dissolved in a buffered sterile saline injectable aqueous medium to
a appropriate concentration of approximately 5 mg/mL.
[0092] The invention also encompasses kits that can simplify the
administration of a pharmaceutically active agent to an animal. A
typical kit of the invention comprises a unit dosage form of a
pharmaceutical composition according to the invention. In one
embodiment, the unit dosage form is a container (such as a vial, a
pouch, a tube, a syringe, or the like), which can advantageously be
sterile, containing a pharmaceutical composition of the invention.
The kit can further comprise a label or printed instructions
instructing the use of the pharmaceutically active agent to treat
or prevent a condition. In another embodiment, the kit comprises a
unit dosage form of a pharmaceutical composition of the invention
and a dropper, syringe, or other applicator for administering the
pharmaceutical composition. Typically, the components of the kit,
for example, the unit dosage form and instructions, are contained
within a suitable packaging material.
[0093] Herein it is shown that bioavailable plasmalogen precursors
with docosahexaenoic acid (22:6) substitution at sn-2 decreased
membrane cholesterol levels. In contrast, stearic acid (18:0),
oleic acid (18:1), linoeic acid (18:2), arachidonic acid (20:4) or
linolenic (18:3), at sn-2, were much less active and free DHA was
inactive. The fatty acid substitution at sn-1 demonstrated an
absolute requirement for an alkenyl linkage, with an acyl linkage
completely eliminating cholesterol-lowering activity. The alkenyl
linkage can be generated in the endoplasmic reticulum from the
alkyl precursor (e.g. PPI-1009); however, the synthetic alkenyl
form could also be a potential therapeutic molecule. The
pharmaceutical properties of these targeted plasmalogen precursors
can also be improved with the addition of a polar substituent to
sn-3. This substitution provides improved pharmaceutical properties
including: i) stabilization of the sn-2 substitution from migration
to sn-3.sup.27-28 ii) the ability to generate a
pharmaceutically-acceptable salt to improve formulation and drug
dissolution and absorption; and iii) the ability of sn-3
substituents to be readily removed by lipases.sup.29 such that the
precursor can be converted to the corresponding endogenous
plasmalogen.
[0094] Accordingly, administering compounds of the present
invention to mammalian biological systems results in increased
cellular concentrations of specific sn-2 substituted ethanolamine
plasmalogens independent of the ether lipid synthesis capacity of
the system. The elevated levels of these specific sn-2 substituted
species give rise to lowering of membrane cholesterol levels and
lowering of amyloid secretion, therefore making these compounds
useful for the treatment or prevention of diseases of aging
associated with increased membrane cholesterol, increased amyloid,
and decreased plasmalogen levels.
[0095] Without wishing to be bound by theory, it is believed that
the compounds described herein are capable of bypassing peroxisomal
ether lipid biosynthesis pathways enabling both the restoration of
plasmalogen levels in plasmalogen deficient subjects, as well as
the delivery of pharmaceutically effective cholesterol lowering
levels of specific cholesterol lowering plasmalogens. Accordingly,
these molecules can be used to treat or prevent diseases associated
with either decreased levels of plasmalogens, increased levels of
membrane cholesterol or increased amyloid levels. These factors are
believed to be causal in a wide variety of human diseases such as
neurodegeneration (including without limitation Alzheimer's
disease, Parkinson's disease and age-related macular degeneration),
cognitive impairment, dementia, cancer (including without
limitation prostate, lung, breast, ovarian, and kidney cancer),
osteoporosis, bipolar disorder and vascular diseases (including but
not limited to atherosclerosis and hypercholesterolemia).
Accordingly, the present invention relates to the treatment of
these diseases using the described plasmalogen precursors.
Furthermore, these derivatives have utility in the treatment of
disorders resulting from abnormal genetic expression of cholesterol
transport proteins such as apolipoprotein E.
[0096] It is also shown herein that the administration of
1-alkyl-2-alkyl glycerols result in the stereoselective elevation
of PlsEtn levels in both PlsEtn normal and PlsEtn deficient
systems. These data also demonstrate for the first time that
1-alkyl, 2-acylglycerols reduce membrane cholesterol levels and
amyloid levels and that these effects require specific fatty acid
substitutions at the sn-2 position.
[0097] The inventors also demonstrate herein that: [0098] 1) the
ether bond at sn-1 is stable and is further processed by a
desaturase (endoplasmic reticulum) to generate the essential
alkenyl bond at sn-1 characteristic of plasmalogens but that this
desaturation occurs after the addition of phosphoethanolamine by
CDP-ethanolamine transferase (endoplasmic reticulum); [0099] 2) the
charged substitution at sn-3 stabilizes the fatty acid substitution
at sn-2 from migrating.sup.27-28 to sn-3; [0100] 3) the charged
substitution at sn-3 is readily cleaved by cellular lipases and
provides the free hydroxyl group for the addition of
phosphoethanolamine by CDP-ethanolamine transferase (endoplasmic
reticulum); [0101] 4) the fatty acid substituent at sn-2 is able to
undergo deacylation and reacylation by other fatty acids in cells;
[0102] 5) DHA substitution at sn-2 is optimal for lowering membrane
cholesterol, and [0103] 6) the charged substitution at sn-3
improves the pharmaceutical properties (stability, bioavailability
and formulation as a salt) of the novel presented plasmalogen
precursors.
[0104] The compounds of the present invention are effectively
converted to PlsEtn species in cells with impaired plasmalogen
biosynthesis capacity and in cells with unimpaired plasmalogen
biosynthesis capacity. These results are in direct contrast to the
prior art regarding other plasmalogen precursors. 1-alkyl,
2-hydroxy glycerols (chimyl, batyl, salachyl alcohols) have been
shown to increase PlsEtn levels in PlsEtn deficient systems to
control levels but not to above control levels in either PlsEtn
deficient or PlsEtn sufficient systems. Therefore, the compounds of
the present invention can be useful in the prevention of diseases
of the aging mediated by plasmalogen deficiency, but increasing
PlsEtn levels to above control levels in either PlsEtn deficient or
PlsEtn sufficient systems.
[0105] As discussed above, the compounds described herein are
suitable for use in a variety of drug delivery systems; however,
without wishing to be limiting, the compounds are especially useful
for oral delivery in a capsule or tablet. In such embodiments the
maximum total dose is not expected to exceed 2 g/day for a 40 to 80
kg human patient.
[0106] In the following examples the following abbreviations have
the following meanings. Abbreviations not defined below have their
generally accepted meaning.
bd=broad doublet bs=broad singlet d=doublet dd=doublet of doublets
dec=decomposed dH.sub.2O=distilled water ELISA=enzyme-linked
immuno-sorbent assay EtOAc=ethyl acetate EtOH=ethanol g=grams
h=hours Hz=hertz ip=intraperitoneal L=liter m=multiplet min=minutes
M=molar MeOH=methanol mg=milligram MHz=megahertz mL=milliliter
mmol=millimole m.p.=melting point N=normal po=per os, oral
q=quartet quint.=quintet s=singlet t=triplet THF=tetrahydrofuran
tlc=thin layer chromatography .mu.g=microgram .mu.L=microliter
UV=ultraviolet
[0107] In the examples below, all temperatures are in degrees
Celsius unless otherwise indicated.
[0108] The following synthetic and biological examples are offered
to illustrate this invention and are not to be construed in any way
as limiting the scope of this invention.
EXAMPLES
Example 1
Chemical Synthesis
[0109] PPI-1009:
2-acetoxy-4-(2-((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoylo-
xy)-3-(hexadecyloxy)propoxy)-N,N,N-trimethyl-4-oxobutan-1-aminium
was prepared according to the following general experimental
procedure.
Step-1
[0110] At 0.degree. C., to a solution of unlabelled PPI-1001 (50 g,
158 mmol) in dry DMF (20 mL), was added imidazole (21.5 g, 316
mmol), the resulting mixture stirred for 10 min. A solution of
TBDMS-Cl (26.2 g, 174 mmol) in DMF (50 mL) was added drop wise and
the resulting solution was stirred at rt for 4 h. The reaction
mixture was diluted with water (50 ml), extracted with EtOAc
(2.times.250 mL). The organic layer washed with ice water
(2.times.100 mL), brine (50 mL), dried (Na.sub.2SO.sub.4) and
evaporated to obtain crude compound 4 that was purified by column
chromatography (neutral alumina, EtOAc-Pet ether (0.5:9.5) to
obtain compound 3 (45 g, 66%). R.sub.f=0.65 (EtOAc-Pet ether
(1-9))
Step-2
[0111] At 0.degree. C., to a solution of compound 3 (75 g, 174
mmol) in DMF (750 mL) NaH (60% dispersion in oil, 5 g, 209 mmol)
was added portion wise, stirred for 30 min, benzyl bromide (44.8 g,
262 mmol) added drop wise over a period of 1 h, slowly allowed to
rt and stirred overnight until complete consumption of compound 3
as evidenced by tlc analysis. The reaction mixture was cooled to
0.degree. C., methanol (5 mL), ice cold water (50 mL) added,
extracted with Et.sub.2O (2.times.250 mL), organic layer washed
with water (2.times.50 mL), brine (50 mL), dried (Na.sub.2SO.sub.4)
and evaporated to obtain crude compound 4 (90 g) as yellow oil
(R.sub.f=0.7; EtOAc-Pet ether [5-95]), that was used as such for
the next step without further purification.
Step-3
[0112] At -15.degree. C., to a solution of compound 4 (1.7 g, 3.26
mmol) in dry THF (15 mL), a solution of TBAF (1.7 g, 6.53 mmol) in
dry THF (5 mL) added and the reaction mixture slowly warmed to rt
and stirred overnight until complete consumption of compound 4 as
evidenced by tlc analysis. The reaction mixture was quenched with
water (10 mL), extracted with EtOAc (2.times.50 mL), organic layer
washed with brine (20 mL), dried (Na.sub.2SO.sub.4) and evaporated
to obtain crude compound 6 that was purified by flash column
chromatography (100-200 mesh silica gel, EtOAc-Pet ether (3:7) to
obtain compound 5 (850 mg, 65%) as a light yellow oil. R.sub.f=0.54
(EtOAc-Pet ether (3:7).
Step-4
[0113] At 0.degree. C., to a solution of compound 7 (8 g, 39 mmol)
in dry CH.sub.2Cl.sub.2(50 mL)-DMF (3 drops) oxalyl chloride (4.2
mL, 40 mmol) was added and resulting mixture slowly warmed to rt
stirred for 5 h. Excess oxalyl chloride was evaporated and residue
dissolved in toluene and solvent evaporated to obtain compound 6. A
solution of compound 6 in dry CH.sub.2Cl.sub.2 (25 mL) was added
dropwise to a solution of compound 5 (11 g, 20 mmol) in dry
CH.sub.2Cl.sub.2 (50 mL) and the resulting solution purged with
Argon gas till complete consumption of compound 5 as evidenced by
tlc analysis. The reaction mixture was concentrated to obtain crude
compound 8 (14 g) that was used as such for the next step without
further purification. R.sub.f=0.45 (MeOH--CHCl.sub.3 (1:4)).
Step-5
[0114] A solution of compound 8 (14 g crude) in EtOH (50 mL), was
hydrogenated over 10% Pd/C at 40 psi, until complete consumption of
compound 8 as indicated by TLC analysis. The reaction mixture was
filtered and evaporated to obtain crude compound 9, which was
purified by column chromatography over neutral alumina to give
compound 9 (6 g, 6 g, 61% over two steps) as a light brown oil
R.sub.f=0.25 (MeOH--CHCl.sub.3 (2:3)).
Step-6
[0115] At 0.degree. C., to a solution of compound 9 (8.2 mg, 16
mmol) in THF (150 mL), catalytic amount of DMAP (1.9 g, 20 mmol),
pyridine (7.6 mL, 90 mmol) were added and stirred at rt for an
hour. To this solution, a solution of DHA-Cl (prepared by addition
of a solution of oxalyl chloride (2.5 mL, 28 mmol) in dry
CH.sub.2Cl.sub.2 at 0.degree. C. to DHA-acid (7.7 g, 20 mmol),
evaporating excess oxalyl chloride to give DHA-Cl) in
CH.sub.2Cl.sub.2 (50 mL) was added drop wise to the reaction
mixture at 0.degree. C. and stirred for 0.5 h. The reaction mixture
was slowly warmed to rt and stirred for 24 h. The reaction mixture
was diluted with water (50 mL), extracted with EtOAc (2.times.200
mL), washed with brine (50 mL) dried (Na.sub.2SO.sub.4) and
evaporated to obtain crude that was purified by column
chromatography (neutral alumina MeOH--CHCl.sub.3(5:95)) to obtain
PPI-1009 (1.2 g, .about.10%, HPLC purity 97% along with 1.7 g of
impure material) as a light brown semisolid R.sub.f=0.45
(MeOH--CHCl.sub.3 (15:85)).
Example 2
Biological Testing
[0116] Chinese hamster ovary (CHO), N-Rel.sup.30 (a mutant CHO cell
line deficient in the peroxisomal enzyme dihydroxyacetonephosphate
acyltransferase), and human embryonic kidney (HEK293) cells were
grown in 10 cm.sup.2 dishes in DMEM/Ham's F12 (1:1) containing 10%
FBS. Cells were incubated with plasmalogen precursors dissolved in
ethanol (final ethanol concentration of 0.1%) and harvested for
plasmalogen analysis by LC-MS-MS.sup.31, and cholesterol and
cholesterol ester analyses utilizing a commercial colorimetric kit
(BioVision #K613).
[0117] 1-alkyl, 2-acyl glycerols with 16:0 at sn-1, DHA at sn-2 and
either OH or acetyl-L-carnitine at sn-3, PPI-1005 or PPI-1009,
respectively were effectively converted to PlsEtn species in cells
with impaired plasmalogen biosynthesis capacity (N-Rel, FIGS. 2,
3A) and in cells with unimpaired plasmalogen biosynthesis capacity
(CHO, FIG. 4A). These results are in direct contrast to the prior
art regarding other plasmalogen precursors. 1-alkyl, 2-hydroxy
glycerols (chimyl, batyl, salachyl alcohols) has been shown to
increase PlsEtn levels in PlsEtn deficient systems to control
levels but not to above control levels in either PlsEtn deficient
or PlsEtn sufficient systems (FIG. 4B). In addition the
bioconversion of the 1-alkyl, 2-acyl glycerols described in this
invention did not result in increased levels of either chimyl
alcohol or 1-alkenyl, 2-acyl glycerols (FIG. 3B) indicating that
neither chimyl alcohol nor 1-alkenyl, 2-acyl glycerols are
intermediates in the biotransformation pathway of the described
molecules to PlsEtn.
[0118] The compound PPI-005 is described in applicant's co-pending
application PCT/CA2007/001472, and is shown below:
##STR00018##
[0119] Treatment of cells with 1-alkyl, 2-acyl glycerols with 16:0
at sn-1, DHA at sn-2 and either OH or acetyl-L-carnitine at sn-3
resulted in the structure-specific enrichment of PlsEtn with DHA at
sn-2 (FIG. 6). This is the first description of the
structure-specific enrichment of PlsEtn.
[0120] PPI-1009 is a plasmalogen precursor with improved
pharmaceutical properties. This molecule exists at room
temperatures as a salt and represents the first non-oil based
plasmalogen precursor ever reported.
[0121] Biological systems with a pre-existing deficiency in
plasmalogen synthesis and subsequent low plasmalogen levels (N-Rel
vs. CHO) (FIG. 1) have elevated membrane cholesterol levels (FIG.
5). Elevation of DHA-PlsEtn to 80% of control values by PPI-1005
(FIG. 2) resulted in a significant reduction in membrane
cholesterol (FIG. 5).
[0122] The cholesterol lowering effect of elevated PlsEtn levels
was discovered to be dependent upon the sn-2 substituent (FIGS. 7
and 8). Only polyunsaturated fatty acids (DHA, 18:3) were observed
to have cholesterol lowering activity in HEK293 cells whereas
saturated, mono and diunsaturated fatty acids had no effect. Only
the restoration of DHA-PlsEtn levels resulted in a robust
attenuation of the elevated membrane cholesterol levels observed in
N-Rel cells as a result of decreased plasmalogen levels (FIG. 7).
These results indicate that poly-unsaturated fatty acid containing
PlsEtn are selectively involved in membrane cholesterol
homeostasis. PPI-1009 was also demonstrated to
concentration-dependently decrease membrane cholesterol levels in
NRel (FIG. 7) and HEK293 cells (FIG. 9). These results indicate
that the membrane lowering effect of elevated PlsEtn levels
requires the administration of plasmalogen precursor capable of
selectively elevating PlsEtn levels with specific sn-2
substitutions.
[0123] The treatment of HEK293 cells with either PPI-1005 or
PPI-1009 resulted in decreased levels of both AB-40 and AB-42 in
both normal cells and cholesterol loaded cells (FIG. 10). These
results further exemplify the functional utility of the plasmalogen
precursors by their ability to positively modulate membrane protein
function. In this regard, decreased membrane cholesterol levels are
known to negatively modulate amyloid peptide production.sup.15. In
addition to lowering membrane cholesterol in HEK293 cells, PPI-1005
and PPI-1009 were also observed to decrease the secretion of
amyloid peptides (FIG. 10).
[0124] A structure-specific and concentration-dependent
incorporation of PPI-1014 in N-Rel ethanolamine plasmalogens
(PlsEtn) and phosphatidylethanolamines (PtdEtn) was also observed
after 72 hours using 5, 10 and 20 .mu.M concentrations of PPI-1014
(FIG. 17).
Example 3
Preparation of PPI-1011
##STR00019##
[0125] Synthetic Scheme:
##STR00020##
[0126] Synthetic Scheme for PPI-1011
##STR00021##
[0127] Brief Procedure:
[0128] A solution of compound 1 (2.0 Kg, 15.15 mol) in 10% NaOH
solution (10.0 L) was stirred at 80.degree. C. for 1 h, TBAB (992.0
g, 3.03 mol) added and stirred for 15 min. Cetyl bromide (5.5 kg,
18.18 mol) was added slowly and the reaction mixture was stirred at
80.degree. C. for 20 h. (The reaction progress was monitored by
diluting small aliquots with water, extracting with ethyl acetate
and spotting over an analytical silica gel TLC plate (30% Ethyl
acetate in pet-ether) and visualizing respective spots using Mo
stain and KMnO.sub.4 solution). The following are the R.sub.fs of
the components of the mixture: compound 1 (0.1), compound 2
(0.7).
Work Up and Purification:
[0129] The reaction mixture was cooled to RT, extracted with
CH.sub.2Cl.sub.2 (3.times.3.0 L). The combined CH.sub.2Cl.sub.2
layer was washed with NaHCO.sub.3 solution (1.0 L), water
(2.times.2.0 L), brine solution (1.0 L), dried over anhydrous
Na.sub.2SO.sub.4 and concentrated to afford crude compound 2 (2.9
Kg, crude) as pale yellow liquid, which was used in next step.
Reaction Step:
##STR00022##
[0130] Brief Procedure:
[0131] A solution of compound 2 (2.9 Kg, 81.23 mol) in 20% aq HCl
(14.5 L) was stirred at 85.degree. C. for 16 h. (The reaction
progress was monitored by diluting small aliquots with water,
extracting with ethyl acetate and spotting over an analytical
silica gel TLC plate (40% ethyl acetate in pet ether) and
visualizing spots using Mo stain). The following are the R.sub.fs
of the components of the mixture: compound 2 (0.8), compound 3
(0.2).
Work Up and Purification:
[0132] The reaction mixture was cooled to RT, extracted with
CH.sub.2Cl.sub.2 (3.times.4.0 L). The combined organic layer was
washed with NaHCO.sub.3 solution (1.0 L), water (2.times.2.0 L),
brine solution (1.0 L), dried over anhydrous Na.sub.2SO.sub.4 and
concentrated to afford crude compound, which was triturated with 5%
ethyl acetate in pet-ether (2.times.1.0 L) and dried to afford
compound 3 (2.0 Kg, 42% from 2-steps) obtained as off white
solid.
Reaction Step:
##STR00023##
[0133] Brief Procedure:
[0134] To a cooled solution of compound 3 (1.0 Kg, 3.16 mol) in DMF
(640.0 mL) and CH.sub.2Cl.sub.2 (400.0 mL) at 0.degree. C., added
DMAP (38.6 g, 0.32 mol) followed by triethylamine (735.0 g, 7.27
mol). After addition the reaction mixture was stirred at 0.degree.
C. for 30 min and added TBSCl (572.0 g, 3.79 mol) in equal portions
(3 portions) for 1 h and the reaction mixture was stirred for 20 h
at RT. (The reaction progress was monitored by quenching small
aliquots with water, extracting with CH.sub.2Cl.sub.2 and spotting
over an analytical silica gel TLC plate (20% Ethyl acetate in pet
ether) and visualizing spots using Mo stain and KMnO.sub.4). The
following are the R.sub.fs of the components of the mixture:
compound 3 (0.15), compound 4(0.7).
Work Up and Purification:
[0135] The reaction mixture was diluted with CH.sub.2Cl.sub.2 (2.0
L), washed with water (3.times.3.0 L), brine solution (1.0 L),
dried over anhydrous Na.sub.2SO.sub.4 and concentrated. The
obtained crude compound was purified by column chromatography
(silica gel 100-200 mesh) using 5% ethyl acetate in pet ether as
eluent to afford compound 4 (850 g, 90%) obtained as pale yellow
oil.
Reaction Step:
##STR00024##
[0136] Brief Procedure:
[0137] To a cooled solution of compound 5 (160.0 g, 0.487 mol), DMF
(1.0 mL) in CH.sub.2Cl.sub.2 (500.0 mL) at 0.degree. C., Oxalyl
chloride (105.0 g, 0.828 mol) was added slowly for 30 min. After
addition the reaction mixture was stirred at 26.degree. C. for 4 h.
(The reaction progress was monitored by quenching small aliquots
with MeOH and spotting over an analytical silica gel TLC plate (10%
Ethyl acetate in pet ether) and visualizing spots using KMnO.sub.4
solution). The following are the R.sub.fs of the components of the
mixture: compound 5 (0.3), compound 6 (0.8).
Work Up and Purification:
[0138] The reaction mixture was concentrated under N.sub.2
atmosphere to afford crude compound 6 (175 g, crude).
Reaction Step:
##STR00025##
[0139] Brief Procedure:
[0140] To a cooled solution of compound 4 (150.0 g, 0.348 mol) in
Toluene (1.25 L) at 0.degree. C., pyridine (110.0 g, 1.39 mol) was
added followed by DMAP (122.2 g, 0.348 mol) and stirred for 10 min.
Added a solution of crude compound 6 (175.0 g, 0.504 mol) in
Toluene (250.0 mL) for 15 min. After addition the reaction mixture
was stirred at RT for 20 h. (The reaction progress was monitored by
quenching small aliquots with water, extracting with EtOAc and
spotting over an analytical silica gel TLC plate (5% Ethyl acetate
in pet ether) and visualizing spots using KMnO.sub.4 solution). The
following are the R.sub.fs of the components of the mixture:
compound 4 (0.2), compound 7 (0.5).
Work Up and Purification:
[0141] The reaction mixture was diluted with ethyl acetate (3.0 L),
washed with water (1.0 L), 0.05 N HCl (500.0 mL), water
(2.times.1.0 L), brine solution (500.0 mL) dried over anhydrous
Na.sub.2SO.sub.4 and concentrated to afford crude compound 7, which
was purified by column chromatography (silica gel 100-200 mesh)
using 2% ethyl acetate in pet ether as eluent to afford compound 7
(162 g, 62.7%) obtained as pale yellow oil.
Reaction Step:
##STR00026##
[0142] Brief Procedure:
[0143] To a cooled solution of compound 7 (160.0 g, 216.21 mmol) in
THF (10.0 mL) and acetic acid (52.0 g) at 0.degree. C., TBAF (226.0
g, 864.86 mmol) was added in equal portions for 30 min. After
addition the reaction mixture was stirred at RT for 6 h. (The
reaction progress was monitored by spotting over an analytical
silica gel TLC plate (20% Ethyl acetate in pet ether) and
visualizing spots using Mo stain and KMnO.sub.4 solution). The
following are the R.sub.fs of the components of the mixture:
compound 7 (0.9), compound PPI-1005 (0.4).
Work Up and Purification:
[0144] The reaction mixture was diluted with ethyl acetate (3.0 L),
washed with water (2.times.2.0 L), brine (500.0 mL), dried over
anhydrous Na.sub.2SO.sub.4 and concentrated to afford crude
compound, which was purified by column chromatography (silica gel
100-200 mesh) using 7% ethyl acetate in pet ether as eluent to
afford compound PPI-1005 (72 g, 53%) obtained as pale yellow
oil.
Reaction Step:
##STR00027##
[0145] Brief Procedure:
[0146] To a cooled solution of .alpha.-Lipoic acid 1 (12.0 g, 58.25
mmol) in THF (500.0 mL) at 0.degree. C., triethylamine (8.1 mL,
58.25 mmol) was added slowly for 10 min, followed by
2,4,6-Trichlorobenzoyl chloride (14.2 g, 58.25 mmol). After
addition the reaction mixture was allowed to RT and stirred for 18
h. (The reaction progress was monitored by spotting on an
analytical silica gel TLC plate (20% EtOAc in pet-ether), and
visualizing spots using 254 nm UV light and Hanessian's stain). The
following are the R.sub.fs of the components of the mixture:
compound 1 (0.2), intermediate (0.6).
[0147] The reaction mixture was filtered off, the solid was washed
with THF (25.0 mL), the combined filtrate was concentrated using
reduced pressure under N.sub.2 atmosphere to obtain the crude
anhydride, which was dissolved in benzene (500.0 mL, cooled to
0.degree. C. and added DMAP (7.1 g, 58.25 mmol), stirred for 10
min. To the reaction mixture added a solution of PPI-1005 (40.1 g,
64.07 mmol) in benzene (100.0 mL) slowly at 0.degree. C. After
addition the reaction mixture was allowed to RT and stirred for 24
h. (The reaction progress was monitored by quenching small aliquots
with H.sub.2O, extracting with ethyl acetate and spotting on an
analytical silica gel TLC plate (15% THF in pet-ether), and
visualizing spots using 254 nm UV light and Hanessian's stain). The
following are the R.sub.fs of the components of the mixture:
PPI-1005 (0.3), PPI-1011 (0.5).
Work Up and Purification:
[0148] The reaction mixture was diluted with ethyl acetate (2000
mL), washed with saturated NaHCO3 solution (1.times.400 mL), 0.05N
HCl (1.times.400 mL), water (1.times.400 mL), brine (1.times.400
mL) and dried over anhydrous Na.sub.2SO.sub.4 and concentrated to
obtain the crude compound, which was purified by column
chromatography (neutral silica gel 100-200 mesh) using 2.5 to 5%
THF in pet ether as eluent.
[0149] The title compound PPI-1011 (28.0 g, 54%) was obtained as
pale brown oil.
Example 4
Alternate Method for Preparation of PPI-1009
##STR00028##
[0150] Synthetic Scheme:
##STR00029##
[0151] Reaction Step:
##STR00030##
[0152] Procedure:
[0153] A solution of compound 1 (2.0 Kg, 15.15 mol, Alfa Aesar) in
10% NaOH solution (10.0 L) was stirred at 80.degree. C. for 1 h,
added TBAB (992.0 g, 3.03 mol, Rajdhani scientific) and stirred for
15 min. Added cetyl bromide (5.5 kg, 18.18 mol, Alfa Aesar) slowly
and the reaction mixture was stirred at 80.degree. C. for 20 h.
(The reaction progress was monitored by diluting small aliquots
with water, extracting with ethyl acetate and spotting over an
analytical silica gel TLC plate (30% Ethyl acetate in pet-ether)
and visualizing respective spots using Mo stain and KMnO.sub.4
solution). The following are the R.sub.fs of the components of the
mixture: compound 1 (0.1), compound 2 (0.7). The reaction mixture
was cooled to RT, extracted with CH.sub.2Cl.sub.2 (3.times.3.0 L).
The combined CH.sub.2Cl.sub.2 layer was washed with NaHCO.sub.3
solution (1.0 L), water (2.times.2.0 L), brine solution (1.0 L),
dried over anhydrous Na.sub.2SO.sub.4 and concentrated to afford
crude compound 2 (2.9 Kg, crude) as pale yellow liquid, which was
used in next step.
Reaction Step:
##STR00031##
[0154] Procedure:
[0155] A solution of compound 2 (2.9 Kg, 81.23 mol) in 20% aq HCl
(14.5 L) was stirred at 85.degree. C. for 16 h. (The reaction
progress was monitored by diluting small aliquots with water,
extracting with ethyl acetate and spotting over an analytical
silica gel TLC plate (40% ethyl acetate in pet ether) and
visualizing spots using Mo stain). The following are the R.sub.fs
of the components of the mixture: compound 2 (0.8), compound 3
(0.2). The reaction mixture was cooled to RT, extracted with
CH.sub.2Cl.sub.2 (3.times.4.0 L). The combined organic layer was
washed with NaHCO.sub.3 solution (1.0 L), water (2.times.2.0 L),
brine solution (1.0 L), dried over anhydrous Na.sub.2SO.sub.4 and
concentrated to afford crude compound, which was triturated with 5%
ethyl acetate in pet-ether (2.times.1.0 L) and dried to afford
compound 3 (2.0 Kg, 42% from 2-steps) obtained as off white
solid.
Reaction Step:
##STR00032##
[0156] Procedure:
[0157] To a cooled solution of compound 3 (1.0 Kg, 3.16 mol) in DMF
(640.0 mL) and CH.sub.2Cl.sub.2 (400.0 mL) at 0.degree. C., added
DMAP (38.6 g, 0.32 mol) followed by triethylamine (735.0 g, 7.27
mol, Rankem). After addition the reaction mixture was stirred at
0.degree. C. for 30 min and added TBSCl (572.0 g, 3.79 mol, Fluoro
chem 3.0 kg) in equal portions (3 portions) for 1 h and the
reaction mixture was stirred for 20 h at RT. (The reaction progress
was monitored by quenching small aliquots with water, extracting
with CH.sub.2Cl.sub.2 and spotting over an analytical silica gel
TLC plate (20% Ethyl acetate in pet ether) and visualizing spots
using Mo stain and KMnO.sub.4). The following are the R.sub.fs of
the components of the mixture: compound 3 (0.15), compound 4(0.7).
The reaction mixture was diluted with CH.sub.2Cl.sub.2 (2.0 L),
washed with water (3.times.3.0 L), brine solution (1.0 L), dried
over anhydrous Na.sub.2SO.sub.4 and concentrated. The obtained
crude compound was purified by column chromatography (silica gel
100-200 mesh) using 5% ethyl acetate in pet ether as eluent to
afford compound 4 (850 g, 90%) obtained as pale yellow oil.
Reaction Step:
##STR00033##
[0158] Procedure:
[0159] To a cooled solution of compound 4 (734 g, 1.706 mol) in DMF
(2.5 L) at 0.degree. C., added 60% NaH (204.0 g, 5.12 mol) in
portions for 30 min. After addition the reaction mixture was
stirred at 0.degree. C. for further 30 min and added benzyl bromide
(438.0 g, 2.56 mol, S.D fine chemicals) drop wise for 1 h. The
reaction mixture allowed to RT was stirred for 20 h. (The reaction
progress was monitored by quenching small aliquots with water,
extracting with EtOAc and spotting over an analytical silica gel
TLC plate (5% Ethyl acetate in pet ether) and visualizing spots
using Mo stain and KMnO.sub.4). The following are the R.sub.fs of
the components of the mixture: compound 4 (0.2), compound 5(0.5).
The reaction mixture was quenched with methanol (250 mL), cold
water (1500 mL) and extracted using ethyl acetate (2.times.2.0 L).
The combined ethyl acetate layer was washed with water (3.times.1.0
L), brine solution (1.0 L), dried over anhydrous Na.sub.2SO.sub.4
and concentrated. The obtained crude compound 5 (887 g) was used in
the next step without further purification.
Reaction Step:
##STR00034##
[0160] Procedure:
[0161] To a cold solution of crude compound 5 (887.0 g, 1.702 mol)
in THF (2.0 L) at 0.degree. C., a solution of TBAF (1.3 Kg, 5.107
mol, Chemrich fine chemicals) in THF (1.0 L) was added slowly for 1
h. After addition the reaction mixture was stirred at RT for 16 h.
(The reaction progress was monitored by quenching small aliquots
with water, extracting with EtOAc spotting over an analytical
silica gel TLC plate (30% Ethyl acetate in pet ether) and
visualizing spots using Mo stain and KMnO.sub.4 solution). The
following are the R.sub.fs of the components of the mixture:
compound 5 (0.9), compound 6 (0.3). The reaction mixture was
diluted with ethyl acetate (2.5 L), washed with water (2.times.1.0
L), brine (1.0 L), dried over anhydrous Na.sub.2SO.sub.4 and
concentrated to afford crude compound, which was purified by column
chromatography (silica gel 100-200 mesh) using 7% ethyl acetate in
pet ether as eluent to afford compound 6 (330 g, 48% from 2 steps)
obtained as pale yellow oil.
Reaction Step:
##STR00035##
[0162] Procedure:
[0163] To a cold solution of N-Acetyl carnitene 8 (239.0 g, 1.177
mol, Molecula life Sciences) in CH.sub.2Cl.sub.2 (500.0 mL) and DMF
(5.0 mL) at 0.degree. C., added oxalyl chloride (179.4 g, 1.412
mol) slowly for 30 min and stirred at RT for 4 h. The solvent from
the reaction mixture was removed by distillation under reduced
pressure and traces of oxalyl chloride by co-distilling with
CH.sub.2Cl.sub.2. The obtained crude compound 9 (250 g) was
dissolved in CH.sub.2Cl.sub.2 (500.0 mL) and added slowly to a cold
solution of compound 6 (334.0 g, 0.823 mol) in CH.sub.2Cl.sub.2
(500.0 mL) at 0.degree. C. with bubbling N.sub.2 gas. After
addition the reaction mixture was stirred for 20 h with continuous
bubbling of N.sub.2 at RT. (The reaction progress was monitored by
quenching small aliquots with water, extracting with
CH.sub.2Cl.sub.2 and spotting over an analytical silica gel TLC
plate (25% MeOH in chloroform) and visualizing spots using Mo stain
and KMnO.sub.4). The following are the R.sub.fs of the components
of the mixture: compound 6 (0.8) and compound 10 (0.3). The
reaction mixture was diluted with CH.sub.2Cl.sub.2 (2.0 L), washed
with brine solution (250 mL), dried over anhydrous Na.sub.2SO.sub.4
and concentrated to obtain crude compound which was purified by
column chromatography (silica gel 100-200 mesh) using 5% MeOH in
chloroform as eluent to afford compound 10 (153.0 g, 31.5%)
obtained as pale yellow oil.
Reaction Step:
##STR00036##
[0164] Brief Procedure:
[0165] To a suspension of 10% Pd/C (40.0 g, 25% w/w, AlfaAesar) in
ethanol (1.2 L), compound 10 (150 g, 0.298 mol) was added and
hydrogenated (H.sub.2, 40 psi pressure) at RT for 20 h. (The
reaction progress was monitored by spotting over an analytical
silica gel TLC plate (25% MeOH in chloroform) and visualizing spots
using Mo stain and Ninhydrin solution). The following are the
R.sub.fs of the components of the mixture: compound 10 (0.4),
compound 11(0.2). The reaction mixture was filtered off through
celite bed, washed the cake with ethanol (2.times.200 mL), the
combined filtrate was concentrated to obtain crude compound, was
purified by column chromatography (silica gel 100-200 mesh) using
10% Methanol in chloroform as eluent to afford compound 11 (75 g,
60%) obtained as pale yellow oil.
Reaction Step:
##STR00037##
[0166] Brief Procedure:
[0167] To a cooled solution of compound 5 (48.0 g, 0.146 mol,
Nu-Chek-Prep Inc), DMF (1.0 mL) in CH.sub.2Cl.sub.2 (300.0 mL) at
0.degree. C., Oxalyl chloride (22.3 g, 0.175 mol, Molecula
Lifesciences) was added slowly for 30 min. After addition the
reaction mixture was stirred at 26.degree. C. for 4 h. (The
reaction progress was monitored by quenching small aliquots with
MeOH and spotting over an analytical silica gel TLC plate (10%
Ethyl acetate in pet ether) and visualizing spots using KMnO.sub.4
solution). The following are the R.sub.fs of the components of the
mixture: compound 12 (0.3), compound 13 (0.8). The reaction mixture
was concentrated under N.sub.2 atmosphere to afford crude compound
6 (57 g, crude).
Reaction Step:
##STR00038##
[0168] Brief Procedure:
[0169] To a cooled solution of compound 11 (50.0 g, 0.102 mol) in
THF (1.0 L) at 0.degree. C., pyridine (32.3 g, 0.409 mol) was added
followed by DMAP (12.5 g, 0.102 mol) and stirred for 10 min. Added
a solution of crude compound 13 (57.0 g, 0.163 mol) in Toluene (1.0
mL) for 15 min. After addition the reaction mixture was stirred at
RT for 20 h. (The reaction progress was monitored by quenching
small aliquots with water, extracting with EtOAc and spotting over
an analytical silica gel TLC plate (20% Methanol in chloroform) and
visualizing spots using Mo stain and Ninhydrin solution). The
following are the R.sub.fs of the components of the mixture:
compound 11 (0.2) and PPI-1009 (0.5). The reaction mixture was
diluted with ethyl acetate (2.0 L), washed with 0.5N HCl (250 mL),
brine solution (250.0 mL) dried over anhydrous Na.sub.2SO.sub.4 and
concentrated to afford crude compound, which was purified by column
chromatography (silica gel 100-200 mesh) using 20% Methanol in
chloroform as eluent to afford compound PPI-1009 (27 g, 33.3%)
obtained as pale yellow oil.
Example 5
Preparation of PPI-1014
Target Molecule
##STR00039##
[0170] Synthetic Scheme:
##STR00040##
[0171] Reaction Step:
##STR00041##
[0172] Brief Procedure:
[0173] To a cooled solution of PPI-1005 (12.0 g, 19.16 mmol) in THF
(600 mL), added Triphenyl phosphene (7.5 g, 28.75 mmol) and stirred
at 0.degree. C. for 10 min, followed by slow addition of DIEAD (5.8
g, 28.75 mmol). After stirring at 0.degree. C. for 30 min the
reaction mixture was added N-Acetyl cystine (4.6 g, 28.75 mmol) and
allowed to stir at RT for 16 h. (The reaction progress was
monitored by extracting with ethyl acetate and spotting on an
analytical silica gel TLC plate (30% Ethyl acetate in pet-ether),
and visualizing spots using Mo stain). The following are the
R.sub.fs of the components of the mixture: PPI-1005 (0.8), PPI-1014
(0.4).
Work Up and Purification:
[0174] The reaction mixture was diluted with water (75 mL) and
extracted using ethyl acetate (3.times.200 mL). The combined ethyl
acetate layer was washed with brine (50 mL), dried over anhydrous
Na.sub.2SO.sub.4 and concentrated to afford the crude compound,
which was purified by column chromatography (100-200 mesh silica
gel) using 0 to 13% ethyl acetate in pet ether as eluent.
[0175] The title compound PPI-1014 (3.2 g, 22%) was obtained as
pale yellow oil exhibited the following properties. .sup.1H NMR
(300 MHz, CDCl.sub.3): .delta. 6.35 (bs, 1H), 5.39-5.22 (m, 13H),
4.92-4.88 (m, 1H), 4.55-4.22 (m, 3H), 3.55-3.41 (m, 5H), 3.02-2.97
(m, 2H), 2.85-2.77 (m, 10H), 2.40 (s, 5H), 2.11-2.04 (m, 5H),
1.57-1.52 (m, 2H), 1.36-1.25 (m, 30H), 0.97 (t, J=7.66 Hz; 3H),
0.88 (t, J=6.63 Hz; 3H). Mass (M+H): 772.3, HPLC purity-93.68%.
Example 6
Animal Studies
[0176] Male New Zealand rabbits (1.8-2.5 kg) were orally dosed with
PPI-1011, neat in hard gelatin capsules (size 3). For time course
studies, rabbits were dosed with 200 mg/kg of PPI-1011 and animals
sacrificed via euthenol overdose at 1, 3, 6, 12, 18, 24 and 48 hr.
Blood was collected by cardiac puncture and plasma frozen at
-70.degree. C. for later analysis. Kidney and liver also were
harvested and stored at -70.degree. C. for later analysis. These
studies were conducted as 2 experiments with overlapping groups at
12 hrs (Exp. 1: 1, 3, 6, and 12 hrs; Exp. 2: 12, 18, 24 and 48
hrs). Controls were harvested at each timepoint. Plasmalogens and
lipids were extracted and analyzed by LC-MS/MS as previously
reported (Goodenowe et al., 2007).
[0177] As seen in FIG. 11, using an oral dose of 200 mg/kg the
plasmalogen precursor PPI-1011 was incorporated into circulating
plasmalogens. Deacylation at sn-2, releasing docosahexaenoic acid
(DHA), also was observed. The greatest incorporation was into the
16:0/22:6, 18:0/22:6 and 18:1/22:6 ethanolamine plasmalogens and
phosphatidylethanolamines. No change in the reference
phosphatidylethanolamine 16:0/18:0 was observed.
[0178] Further examination of the timecourse (FIG. 12) of
incorporation in plasma revealed that maximum incorporation was at
12 hours and that this level of incorporation was maintained over
the remaining observation period (48 hr). This was the case for
both the phosphatidylethanolamines and the ethanolamine
plasmalogens. In contrast, circulating DHA levels peaked at 6 hours
and fell to a lesser steady-state by 18 hours.
[0179] Studies of dose-dependent incorporation (FIG. 13) of
PPI-1011 into plasma plasmalogens and phosphatidylethanolamines
demonstrated that a new steady-state level in these circulating
phospholipids was attained in a dose-dependent manner from 10 to
200 mg/kg. However, further increases in dose did not increase the
steady-state levels of plasmalogens and phosphatidylethanolamines
above that obtained with 200 mg/kg. In contrast, the highest
steady-state levels of circulating DHA were obtained at 500 mg/kg
and did not further increase at a dose of 1000 mg/kg.
[0180] Augmentation of tissue plasmalogens and DHA was also
observed in kidney (FIGS. 4 and 5) and liver (FIG. 6) tissues.
[0181] These results indicate that PPI-1011 is orally bioavailable
in the rabbit and is converted to DHA-containing ethanolamine
plasmalogens and phosphatidylethanolamines via
deacylation/re-acylation reactions. In addition, the results
suggest that endogenous metabolic systems may limit the maximal
increase that that can be pharmacologically augmented.
Example 7
Modulating Membrane Protein Abundance In Vitro with Plasmalogen
Precursors
[0182] The following studies demonstrate the effectiveness of a
plasmalogen precursor (1-alkyl-2 acyl glycerol) in altering the
abundance of membrane-resident proteins. The cellular effects of
the compound are demonstrated in wild-type cells, as well as in
conditions of artificially elevated membrane cholesterol.
[0183] In wild-type cells, an elevation of amyloid precursor
protein (APP) modulating enzyme ADAM10 and cholesterol
esterification protein SOAT1 was observed with an increase in
plasmalogen precursor concentration. Similar effects were observed
in a cholesterol-loaded model. Additionally, a different APP
processing enzyme, BACE1, showed a decrease in abundance only in
cholesterol loaded cells. Without wishing to be bound by theory,
this evidence supports a method of reducing the amyloid load in the
context of Alzheimer's disease and simultaneously re-equilibrating
the membrane cholesterol content of the system, thereby offering
potential benefits in the treatment of diseases like
atherosclerosis and hypercholesterolemia, in addition to
Alzheimer's disease.
[0184] APP is predominantly processed by a canonical pathway
comprised of sequential cleavage by .gamma.-secretases (encoded by
presenilin1/2 genes) and .alpha.-secretase (encoded by ADAM10).
This non-pathological processing of APP results in the formation of
a neutotrophic peptide (sAPP.alpha.) which exhibits protection
against glutamate toxicity and hypoglycemia (Araki et al., 1991;
Mattson et al., 1993; Postina et al., 2004; Fahrenholz, 2007). An
alternate APP processing pathway manifests itself in Alzheimer's
disease, wherein APP is cleaved by .gamma.- and .beta.-secretases
in cholesterol-rich lipid rafts. This "non-canonical" pathway
results in the formation of A.beta. peptides 38-43 amino acids long
which tend to aggregate into plaques in the extracellular matrix, a
hallmark of AD (Selkoe, 2002; Walsh et al., 2002; Selkoe, 2003;
Meyer-Luehmann et al., 2008). While early-onset familial AD is
explained by genetic lesions in APP or APP processing enzymes
(PSEN1/2, BACE, ADAM), the underlying cause of late-onset sporadic
AD (switch from non-pathogenic to pathogenic APP processing)
remains unclear.
[0185] The importance of cholesterol homeostasis in the etiology of
AD has been investigated in humans (Corder et al., 1993; Saunders
et al., 1993; Blacker et al., 1997; Hofman et al., 1997) and in
animal models (Joyce et al., 2002; Singaraja et al., 2002; Van Eck
et al., 2006; Wahrle et al., 2008). Altering the cholesterol
content of the plasma membrane has been shown to affect the
function of membrane resident proteins (Scanlon et al., 2001; Lange
et al., 2004). Elevated brain cholesterol was shown in subjects
with AD (Mori et al., 2001), while rabbits fed on a
cholesterol-rich diet have been shown to develop plaques in the
brain (Ghribi et al., 2006). In vitro data showed that plasmalogen
deficient cells possess elevated free-cholesterol in the membrane,
while in humans, serum-plasmalogen deficiency has been shown to
correlate with a decline in cognition status (Goodenowe et al.,
2007). Based on these observations, we investigated the interplay
between cholesterol and plasmalogens, and identify how the balance
between the two affects the pathological manifestations of AD,
measured in terms of secreted A.beta..
Proposed Mode of Action: A Shift in APP Processing Via Membrane
Lipid Modulation
[0186] Human embryonic kidney (HEK293) cells express APP and the
membrane-bound machinery required to process APP, making it a good
model to study APP processing. The present in vitro investigation
corroborated previous studies in that modulating membrane fluidity
via cholesterol loading and/or plasmalogen precursor
supplementation altered the extracellular A.beta.42 content.
Cholesterol loading of HEK293 cells increases the amount of free
cholesterol by 17% (compared with Control) in the cells following a
48 hour incubation period. This elevation is accompanied with a
parallel and significant increase (p<0.05) in the A.beta.42
content in the conditioned medium compared with control. The 65%
increase in amyloid is primarily due to 22% increase in
.beta.-secretase concentration (FIG. 18A, lane 2); the basal APP
levels remain unchanged with cholesterol loading. Treatment of the
cholesterol loaded HEK293 system with plasmalogen precursor
PPI-1005 significantly reduced the free cholesterol content of the
cell membrane (p<0.05). The A.beta.42 content in the conditioned
medium dropped 70% below cholesterol-loaded levels at the 20 .mu.M
concentration (p=0.0001), while the sAPP.alpha. content in the
conditioned medium was elevated. The effects on APP processing do
not appear to be due to a change in APP expression, rather it
appears to be due to a shift in the APP processing pathway by
virtue of changes in the abundance of APP processing enzymes. While
.beta.-secretase was restored to normal levels at 20 .mu.M
concentration of PPI-1005, a 73% increase in sAPP.alpha. species
was detected in the conditioned medium. This elevation was
explained by 63% increase in ADAM10 (FIG. 18A, lane 3), the enzyme
responsible for sAPP.alpha. formation.
[0187] The change in cholesterol profile of the cells following
plasmalogen supplementation can be explained by the observation
that SOAT1, the enzyme responsible for esterification of free
cholesterol is approximately 25% upregulated (compared with the
cholesterol loaded condition) with increasing plasmalogen
concentration (FIG. 18A, lane 5).
[0188] In a separate study, the effects of PPI-1005 were
investigated in wild-type HEK293 cells, not loaded with
cholesterol. FIG. 18B shows a concentration-dependent increase in
abundance of ADAM10 (35%) and SOAT1 (50%); No change in BACE1 or
APP abundance was observed. Although when HEK293 cells were
depleted of cholesterol by HMGCoA reductase inhibition by
pravastatin, ADAM10 and SOAT1 protein levels remained constant
(FIG. 18C). While plasmalogen and pravastatin treatments both
significantly reduce the fraction of free cholesterol in the cells,
it is the plasmalogen treatment alone that alters ADAM10 and SOAT
levels in the cell. This suggests that the effects on ADAM10 and
SOAT1 abundance is an effect of membrane plasmalogen content rather
than membrane cholesterol content.
[0189] Accordingly, the abundance of membrane-resident proteins can
be altered in vitro by modulating the cellular plasmalogen content,
which is achieved by treating cells with plasmalogen precursors as
described herein.
Materials and Methods
Cholesterol Loading
[0190] HEK 293 cells, cultured in DMEM, 10% FBS at 37.degree. C.,
5% CO.sub.2, were seeded the day before the treatment. The
following day, cells membranes were loaded with exogenous
cholesterol at a concentration of 10 .mu.g/ml media using
methyl-.beta.-cyclodextrin as the carrier to deliver cholesterol as
described (Rong et al., 2003).
Cholesterol Assay
[0191] Cells were treated with the plasmalogen precursor PPI-1005
or with ethanol as the control. Cells were harvested after 48 hours
using Versene: TryPLe express cocktail, washed with PBS. Lipids
were extracted with chloroform containing 1% Triton X-100. The
organic fraction was recovered and dried under a stream of
nitrogen. The dried lipids were resuspended in cholesterol reaction
buffer (Biovision, Mountain View, Calif.), and the total, free and
esterified fractions of cholesterol were quantified using the
cholesterol quantification kit (Biovision, Mountain View, Calif.)
as per the manufacturer's recommendations. Cholesterol was
initially calculated as .mu.g/million cells, and reported as a
percentage of control conditions for each experiment.
Amyloid Assay
[0192] HEK293 cells were loaded with exogenous cholesterol as
described, and treated with a PPI 1005 or with ethanol as a
control. Conditioned media from the treated cells was collected at
the end of a 48 hour incubation period. For assaying
A.beta..sub.1-42 content, the conditioned media was enriched using
Amicon ultracentrifugal filter devices (Millipore, Billerica,
Mass.) prior to loading into the microplate. ELISA was carried out
as per the manufacturer's recommendations (Covance Labs, Princeton,
N.J.). The reactions were quenched 25 minutes after addition of the
substrate, and the absorbance was read at 495 nm. The experiment
was carried out in triplicate. Values were calculated as pg/ml of
conditioned media, and were normalized to the amount of A.beta.
detected in the conditioned media from untreated, cholesterol
loaded control HEK293 cells.
Immunoblotting and Immunoprecipitation
[0193] HEK293 cells were treated as described in the amyloid assay.
The cell pellet was washed in PBS and lysed in a RIPA buffer
containing a protease inhibitor cocktail (Sigma, St. Louis, Mo.).
Protein in the cell lysate was quantified using the Bio-Rad Protein
Assay (Bio-Rad, Hercules, Calif.). The following antibodies were
used for western analyses: APP (Calbiochem, Darmstadt, Germany),
BACE1 and ADAM10 (Millipore, Temecula, Calif.), sAPP.alpha. (IBL,
Gunma, Japan), SOAT1 (Santa Cruz Biotechnology Inc., CA), and
.beta.-actin (Sigma, St. Louis, Mich.). Immunoprecipitation was
carried out to estimate sAPP.alpha. in the conditioned medium.
Briefly, antibody to sAPP.alpha. was added to conditioned media and
incubated for 16 hours at 4.degree. C. IP was carried out by
incubating with protein A/G agarose beads for 6 hours at 4.degree.
C. Beads were washed with PBS and the eluted proteins were detected
by immunoblotting with anti-sAPP.alpha. antibody. Band intensities
were quantified using Image Processing and Analysis in Java
(ImageJ) software (National Institutes of Health, Bethesda,
Md.)
Statistical Analysis
[0194] Statistical Analysis of the data was performed using
Microsoft.TM. Office Excel 2007 and JMP version 8. Multiple
comparison Dunnett's tests were applied to analyze the differences
between the treatments and the control.
[0195] It will be apparent to persons skilled in the art that a
number of variations and modifications can be made without
departing from the scope of the invention as defined in the
claims.
[0196] The following references, as well as those references
provided above, are hereby incorporated by reference.
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