U.S. patent application number 15/181211 was filed with the patent office on 2016-12-08 for inhibitors of cognitive decline.
The applicant listed for this patent is Cognition Therapeutics, Inc.. Invention is credited to Susan Catalano, Gilbert M. Rishton.
Application Number | 20160355458 15/181211 |
Document ID | / |
Family ID | 42936539 |
Filed Date | 2016-12-08 |
United States Patent
Application |
20160355458 |
Kind Code |
A1 |
Rishton; Gilbert M. ; et
al. |
December 8, 2016 |
INHIBITORS OF COGNITIVE DECLINE
Abstract
Compounds that are central nervous system drug candidates for
the treatment of cognitive decline and, more particularly,
Alzheimer's disease are provided. Methods of treating, inhibiting,
and/or abatement of cognitive decline and/or Alzheimer's disease
with a compound or pharmaceutically acceptable salt of the
invention are also provided. Also provided are methods of preparing
the compounds/compositions of the invention.
Inventors: |
Rishton; Gilbert M.;
(Malibu, CA) ; Catalano; Susan; (Pittsburgh,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cognition Therapeutics, Inc. |
Pittsburgh |
PA |
US |
|
|
Family ID: |
42936539 |
Appl. No.: |
15/181211 |
Filed: |
June 13, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14293755 |
Jun 2, 2014 |
9365491 |
|
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15181211 |
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13263162 |
Dec 20, 2011 |
8765816 |
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PCT/US2010/030130 |
Apr 6, 2010 |
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14293755 |
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61309091 |
Mar 1, 2010 |
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61308667 |
Feb 26, 2010 |
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61167984 |
Apr 9, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 211/22 20130101;
A61P 43/00 20180101; C07C 211/27 20130101; C07C 217/62 20130101;
C07D 207/08 20130101; A61P 25/28 20180101 |
International
Class: |
C07C 217/62 20060101
C07C217/62; C07D 207/08 20060101 C07D207/08; C07D 211/22 20060101
C07D211/22 |
Claims
1. A compound of Formula I: ##STR00018## or pharmaceutically
acceptable salt thereof, wherein: R.sup.1 is selected from (A1) and
(A2): ##STR00019## R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6
are each, independently, selected from H, OH, C.sub.1-6 alkoxy,
C.sub.1-6 haloalkoxy, halo, CN, NO.sub.2, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.3-7 cycloalkyl, NH.sub.2, NH(C.sub.1-4
alkyl), NH(C.sub.3-7 cycloalkyl), N(C.sub.1-4 alkyl).sub.2,
NHC(OX)(C.sub.1-4 alkyl), SH, S(C.sub.1-6 alkyl), C(O)OR.sup.a,
C(O)R.sup.b, C(O)NR.sup.cR.sup.d, OC(O)R.sup.b,
OC(O)NR.sup.cR.sup.d, NR.sup.cR.sup.d, NR.sup.cC(O)R.sup.b,
NR.sup.cC(O)OR.sup.a, NR.sup.cS(O).sub.2R.sup.b,
NR.sup.cS(O).sub.2NR.sup.cR.sup.d, S(O)R.sup.b, S(O).sub.2R.sup.b,
and S(O).sub.2NR.sup.cR.sup.d; R.sup.7 is H, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, or C.sub.3-7 cycloalkyl; R.sup.8 is C.sub.1-6
alkyl, C.sub.1-6 haloalkyl, or C.sub.3-7 cycloalkyl; R.sup.9 is H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or C.sub.3-7 cycloalkyl;
R.sup.10 is H; R.sup.11 is H; R.sup.12, R.sup.13, R.sup.14,
R.sup.15, and R.sup.16 are each, independently, selected from H,
OH, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, halo, CN, NO.sub.2,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.3-7 cycloalkyl,
NH.sub.2, NH(C.sub.1-4 alkyl), NH(C.sub.3-7 cycloalkyl),
N(C.sub.1-4 alkyl).sub.2, NHC(O)(C.sub.1-4 alkyl), SH, S(C.sub.1-6
alkyl), C(O)OR.sup.a1, C(O)R.sup.b1, C(O)NR.sup.c1 R.sup.d1,
OC(O)R.sup.b1, OC(O)NR.sup.c1 R.sup.d1, NR.sup.c1 R.sup.d1,
NR.sup.c1C(O)R.sup.b1, NR.sup.c1C(O)OR.sup.a1,
NR.sup.c1S(O).sub.2R.sup.b1, NR.sup.c1S(O).sub.2NR.sup.c1 R.sup.d1,
S(O)R.sup.b1, S(O).sub.2R.sup.b1, and S(O).sub.2NR.sup.c1 R.sup.d1;
each R.sup.a is independently selected from H, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl,
heterocycloalkylalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl,
cycloalkyl, heteroaryl and heterocycloalkyl, wherein each of the
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, arylalkyl, heteroarylalkyl,
cycloalkylalkyl, heterocycloalkylalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, aryl, cycloalkyl, heteroaryl and
heterocycloalkyl is optionally substituted with 1, 2, 3, 4, or 5
substituents independently selected from OH, CN, amino, halo,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6
haloalkoxy, aryl, arylalkyl, heteroaryl, heteroarylalkyl,
cycloalkyl and heterocycloalkyl; each R.sup.b is independently
selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, arylalkyl,
heteroarylalkyl, cycloalkylalkyl, heterocycloalkylalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl, heteroaryl,
heterocycloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl and
heterocycloalkylalkyl, wherein each of the C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl,
heterocycloalkylalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl,
cycloalkyl, heteroaryl, heterocycloalkyl, arylalkyl,
heteroarylalkyl, cycloalkylalkyl and heterocycloalkylalkyl is
optionally substituted with 1, 2, 3, 4, or 5 substituents
independently selected from OH, amino, halo, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.1-4 alkoxy, C.sub.1-6 haloalkoxy, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl and
heterocycloalkyl; R.sup.c and R.sup.d are independently selected
from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, arylalkyl,
heteroarylalkyl, cycloalkylalkyl, heterocycloalkylalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, aryl, heteroaryl, cycloalkyl,
heterocycloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl and
heterocycloalkylalkyl, wherein each of the C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl,
heterocycloalkylalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl,
heteroaryl, cycloalkyl, heterocycloalkyl, arylalkyl,
heteroarylalkyl, cycloalkylalkyl and heterocycloalkylalkyl is
optionally substituted with 1, 2, 3, 4, or 5 substituents
independently selected from OH, amino, halo, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl and
heterocycloalkyl; or R.sup.c and R.sup.d together with the N atom
to which they are attached form a 4-, 5-, 6- or 7-membered
heterocycloalkyl group that is optionally substituted with 1, 2, 3,
4, or 5 substituents independently selected from OH, amino, halo,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6
haloalkoxy, aryl, arylalkyl, heteroaryl, heteroarylalkyl,
cycloalkyl, and heterocycloalkyl; each R.sup.a1 is independently
selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, arylalkyl,
heteroarylalkyl, cycloalkylalkyl, heterocycloalkylalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl, heteroaryl and
heterocycloalkyl, wherein each of the C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl,
heterocycloalkylalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl,
cycloalkyl, heteroaryl and heterocycloalkyl is optionally
substituted with 1, 2, 3, 4, or 5 substituents independently
selected from OH, CN, amino, halo, C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, aryl, arylalkyl,
heteroaryl, heteroarylalkyl, cycloalkyl, and heterocycloalkyl; each
R.sup.b1 is independently selected from H, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl,
heterocycloalkylalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl,
cycloalkyl, heteroaryl, heterocycloalkyl, arylalkyl,
heteroarylalkyl, cycloalkylalkyl and heterocycloalkylalkyl, wherein
each of the C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, arylalkyl,
heteroarylalkyl, cycloalkylalkyl, heterocycloalkylalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl, heteroaryl,
heterocycloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl and
heterocycloalkylalkyl is optionally substituted with 1, 2, 3, 4, or
5 substituents independently selected from OH, amino, halo,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy,
C.sub.1-6haloalkoxy, aryl, arylalkyl, heteroaryl, heteroarylalkyl,
cycloalkyl, and heterocycloalkyl; R.sup.c1 and R.sup.d2 are
independently selected from H, C.sub.1-10 alkyl, C.sub.1-6
haloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl,
heterocycloalkylalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl,
heteroaryl, cycloalkyl, heterocycloalkyl, arylalkyl,
heteroarylalkyl, cycloalkylalkyl and heterocycloalkylalkyl, wherein
each of the C.sub.1-10 alkyl, C.sub.1-6 haloalkyl, arylalkyl,
heteroarylalkyl, cycloalkylalkyl, heterocycloalkylalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, aryl, heteroaryl, cycloalkyl,
heterocycloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl and
heterocycloalkylalkyl is optionally substituted with 1, 2, 3, 4, or
5 substituents independently selected from OH, amino, halo,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, d-.sub.6
haloalkoxy, aryl, arylalkyl, heteroaryl, heteroarylalkyl,
cycloalkyl, and heterocycloalkyl; or R.sup.c1 and R.sup.d1 together
with the N atom to which they are attached form a 4-, 5-, 6- or
7-membered heterocycloalkyl group that is optionally substituted
with 1, 2, 3, 4, or 5 substituents independently selected from OH,
amino, halo, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.1-6
alkoxy, C.sub.1-6 haloalkoxy, aryl, arylalkyl, heteroaryl,
heteroarylalkyl, cycloalkyl, and heterocycloalkyl; and m is 0, 1,
or 2, with the proviso that (a) when R.sup.1 is a moiety of (A1),
then two of R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are
independently selected from OH, C.sub.1-6 alkoxy, and C.sub.1-6
haloalkoxy; and (b) when R.sup.1 is a moiety of (A1), then at least
one of R.sup.12, R.sup.13, R.sup.14, R.sup.15, and R.sup.16 is
other than H.
2. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein two of R.sup.2, R.sup.3, R.sup.4, R.sup.5, and
R.sup.6 are independently selected from OH, C.sub.1-6 alkoxy, and
C.sub.1-6 haloalkoxy.
3. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6
are each, independently, selected from H, OH, C.sub.1-6 alkoxy,
C.sub.1-6 haloalkoxy, halo, CN, NO.sub.2, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.3-7 cycloalkyl, NH.sub.2, NH(C.sub.1-4
alkyl), NH(C.sub.3-7 cycloalkyl), N(C.sub.1-4 alkyl).sub.2, NHC(OX
C.sub.1-6 alkyl), SH, S(C.sub.1-6 alkyl), C(O)OH, C(O)O(C.sub.1-4
alkyl), C(O)(C.sub.1-4 alkyl), and C(O)NH(C.sub.1-4 alkyl).
4. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein: one of R.sup.2, R.sup.3, R.sup.4, R.sup.5, and
R.sup.6 is OH; and one of R.sup.2, R.sup.3, R.sup.4, R.sup.5, and
R.sup.6 is OH, C.sub.1-6 alkoxy, or C.sub.1-6haloalkoxy.
5. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein: one of R.sup.2, R.sup.3, R.sup.4, R.sup.5, and
R.sup.6 is OH; and one of R.sup.2, R.sup.3, R.sup.4, R.sup.5, and
R.sup.6 is C.sub.1-3 alkoxy or C.sub.1-3 haloalkoxy.
6. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein: one of R.sup.2, R.sup.3, R.sup.4, R.sup.5, and
R.sup.6 is OH; and one of R.sup.2, R.sup.3, R.sup.4, R.sup.5, and
R.sup.6 is methoxy or trihalomethoxy.
7. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein: one of R.sup.2, R.sup.3, R.sup.4, R.sup.5, and
R.sup.6 is OH; and one of R.sup.2, R.sup.3, R.sup.4, R.sup.5, and
R.sup.6 is methoxy.
8. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein: R.sup.4 is OH; and R.sup.5 is methoxy.
9. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein: R.sup.4 is OH; R.sup.5 is methoxy; and R.sup.2,
R.sup.3, and R.sup.6 are each H.
10. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein R.sup.7 is H or C.sub.1-6 alkyl.
11. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein R.sup.7 is H or C.sub.1-3 alkyl.
12. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein R.sup.7 is C.sub.1-3 alkyl.
13. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein R.sup.7 is H.
14. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein R.sup.8 is C.sub.1-6 alkyl.
15. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein R.sup.8 is C.sub.1-3 alkyl.
16. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein R.sup.8 is methyl.
17. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein R.sup.9 is H or C.sub.1-6 alkyl.
18. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein R.sup.9 is H or C.sub.1-6 alkyl.
19. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein R.sup.9 is H.
20. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein R.sup.9 is C.sub.1-3 alkyl.
21. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein R.sup.10 is H or C.sub.1-6 alkyl.
22. (canceled)
23. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein R.sup.10 is H.
24. (canceled)
25. (canceled)
26. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein R.sup.11 is H.
27. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein at least one of R.sup.2, R.sup.3, R.sup.4,
R.sup.15, and R.sup.16 is other than H.
28. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein R.sup.12, R.sup.13, R.sup.14, R.sup.15, and
R.sup.16 are each, independently, selected from H, OH, C.sub.1-6
alkoxy, C.sub.1-6 haloalkoxy, halo, CN, NO.sub.2, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.3-7 cycloalkyl, NH.sub.2, NH(C.sub.1-4
alkyl), NH(C.sub.3-7 cycloalkyl), N(C.sub.1-4 alkyl).sub.2,
NHC(O)(C.sub.1-6 alkyl), SH, S(C.sub.1-6 alkyl), C(O)OH,
C(O)O(C.sub.1-6 alkyl), C(O)(C.sub.1-4 alkyl), and C(O)NH(C.sub.1-4
alkyl).
29. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein R.sup.12, R.sup.13, R.sup.14, R.sup.15, and
R.sup.16 are each, independently, selected from H, halo, CN,
NO.sub.2, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.3-7
cycloalkyl, C(O)O(C.sub.1-4 alkyl), C(O)(C.sub.1-4 alkyl), and
C(O)NH(C.sub.1-4 alkyl).
30. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein at least one of R.sup.12, R.sup.13, R.sup.14,
R.sup.15, and R.sup.16 is selected from halo, CN, NO.sub.2,
C.sub.1-6 haloalkyl, C(O)O(C.sub.1-6 alkyl), C(O)(C.sub.1-4 alkyl),
and C(O)NH(C.sub.1-4 alkyl).
31. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein at least one of R.sup.12, R.sup.13, R.sup.14,
R.sup.15, and R.sup.16 is selected from halo and C.sub.1-6
haloalkyl.
32. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein R.sup.14 is halo.
33. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein R.sup.14 is C.sub.1-6 haloalkyl.
34. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein R.sup.14 and R.sup.15 are independently halo.
35. (canceled)
36. The compound of claim 35 or pharmaceutically acceptable salt
thereof, wherein the compound is a compound of Formula IIa:
##STR00020##
37. The compound of claim 35 or pharmaceutically acceptable salt
thereof, wherein the compound is a compound of Formula IIb:
##STR00021##
38. The compound of claim 1 or pharmaceutically acceptable salt
thereof, wherein the compound is a compound of Formula III:
##STR00022##
39. The compound of claim 38 or pharmaceutically acceptable salt
thereof, wherein m is 1.
40. The compound of claim 38 or pharmaceutically acceptable salt
thereof, wherein m is 0.
41. (canceled)
42. (canceled)
43. A pharmaceutical composition comprising a compound of claim 1
or pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier.
44. (canceled)
45. (canceled)
46. (canceled)
47. (canceled)
48. (canceled)
49. (canceled)
50. (canceled)
51. (canceled)
52. (canceled)
53. A method of inhibiting, treating, and/or abatement of cognitive
decline and/or inhibiting Alzheimer's disease in a patient
comprising administrating to the patient a compound of claim 1 or a
pharmaceutically acceptable salt thereof.
54. The method of claim 53 wherein the method comprises inhibiting,
treating, or abatement of one or more symptoms of cognitive decline
selected from the group consisting of memory loss, confusion,
impaired judgment, personality changes, disorientation, and loss of
language skills.
55. The method of claim 53 wherein the method comprises one or more
of: (i) restoration of long term potentiation; and/or (ii)
inhibiting, treating, and/or abatement of neurodegeneration; and/or
(iii) inhibiting, treating, and/or abatement of general
amyloidosis; and/or (iv) inhibiting, treating, and/or abatement of
one or more of amyloid production, amyloid assembly, amyloid
aggregation, amyloid oligomer binding, and amyloid deposition;
and/or (v) inhibiting, treating, and/or abatement of the
activity/effect of one or more of Abeta oligomers on a neuron
cell.
56. The method of claim 53 wherein the method comprises inhibiting,
treating, and/or abatement of one or more of amyloid production,
amyloid assembly, the activity/effect of one or more of Abeta
oligomers on a neuron cell, amyloid aggregation, amyloid binding,
and amyloid deposition.
57. The method of claim 53 wherein the method comprises inhibiting,
treating, and/or abatement of one or more of the activity/effect of
one or more of Abeta oligomers on a neuron cell, amyloid
aggregation, amyloid binding, and amyloid deposition.
58. A method of inhibiting, treating, and/or abatement of cognitive
decline and/or inhibiting Alzheimer's disease in a patient
comprising administrating to the patient a compound of claim 1 or a
pharmaceutically acceptable salt thereof.
59. The method of claim 58 wherein the method comprises inhibiting,
treating, or abatement of one or more symptoms of cognitive decline
selected from the group consisting of memory loss, confusion,
impaired judgment, personality changes, disorientation, and loss of
language skills.
60. The method of claim 58 wherein the method one or more of: (i)
restoration of long term potentiation; and/or (ii) inhibiting,
treating, and/or abatement of neurodegeneration; and/or (iii)
inhibiting, treating, and/or abatement of general amyloidosis;
and/or (iv) inhibiting, treating, and/or abatement of one or more
of amyloid production, amyloid assembly, amyloid aggregation,
amyloid oligomer binding, and amyloid deposition; and/or (v)
inhibiting, treating, and/or abatement of the activity/effect of
one or more of Abeta oligomers on a neuron cell.
61. The method of claim 58 wherein the method comprises inhibiting,
treating, and/or abatement of one or more of amyloid production,
amyloid assembly, the activity/effect of one or more of Abeta
oligomers on a neuron cell, amyloid aggregation, amyloid binding,
and amyloid deposition.
62. The method of claim 58 wherein the method comprises inhibiting,
treating, and/or abatement of one or more of the activity/effect of
one or more of Abeta oligomers on a neuron cell, amyloid
aggregation, amyloid binding, and amyloid deposition.
Description
[0001] This application claims benefit of priority to U.S.
provisional patent application Ser. No. 61/167,984 filed on Apr. 9,
2009, U.S. provisional patent application Ser. No. 61/308,667 filed
on Feb. 26, 2010, and U.S. provisional patent application Ser. No.
61/309,091 filed on Mar. 1, 2010, each of which is hereby
incorporated by reference in its entirety.
SUMMARY
[0002] The present invention provides, inter alia, compounds of
Formula I, II, or III:
##STR00001##
or pharmaceutically acceptable salts, wherein constituent members
are provided below.
[0003] The present invention further provides pharmaceutical
compositions comprising a compound of Formula I, II, or III, or
pharmaceutically acceptable salt thereof, and at least one
pharmaceutically acceptable carrier.
[0004] The present invention further provides methods of
inhibiting, treating, and/or abating cognitive decline and/or
Alzheimer's disease with a compound of Formula I, II, or III, or
pharmaceutically acceptable salt of the same.
[0005] The present invention further provides methods of
inhibiting, treating, or abatement of cognitive decline with a
compound of Formula I, II, or III, or pharmaceutically acceptable
salt of the same.
[0006] The present invention further provides methods of
inhibiting, treating, or abatement of one or more of amyloid
production, amyloid assembly, amyloid aggregation, amyloid binding
(to cells in the brain such as neuron cells), the activity/effect
of Abeta oligomers on neurons, and amyloid deposition (on cells in
the brain such as neuron cells) with a compound of Formula I, II,
or III, or pharmaceutically acceptable salt of the same.
[0007] The present invention further provides compounds of Formula
I, II, or III, or pharmaceutically acceptable salts thereof, for
use in therapy.
[0008] The present invention further provides use of the compounds
of Formula I, II, or III, or pharmaceutically acceptable salts
thereof, for the manufacture/preparation of a medicament for use in
therapy.
[0009] In some embodiments, methods for preparation of compounds
useful for inhibiting, treating, or abatement of cognitive decline
are provided. In a method called "chemical conditioning", certain
compounds of the present invention are derived from naturally
occurring compounds, such as those found in medicinal plants, like
ginger. The chemical conditioning process described herein is
applicable to a large variety of biological extracts and may be
used to create compound arrays for screening for potential new drug
candidates. Further, in general, compounds derived by the chemical
conditioning process are chemically stable and structurally
diverse, and good candidates for use in drug screenings for
pharmaceutical activity. In some embodiments, compounds derived
from ginger oil are provided. According to some embodiments of the
invention, compounds derived from ginger oil by the chemical
conditioning process described herein are provided. In another
embodiment, the invention provides a method of preparing an array
of chemical compounds from ginger oil.
[0010] In some embodiments, the compounds of present invention
inhibit, treat, or abate (partially inhibit) binding of the amyloid
(including Abeta oligomers) to neurons (such as neurons in the
brain) and are useful for the inhibition, treatment, and abatement
of cognitive decline and/or Alzheimer's disease. In some
embodiments, the compounds of present invention inhibit, treat, or
abate one or more of amyloid aggregation, amyloid binding, and
amyloid deposition. In some embodiments, the compounds of present
invention inhibit, treat, or abate amyloid aggregation. In some
embodiments, the compounds of present invention inhibit, treat, or
abate amyloid binding. In some embodiments, the compounds of
present invention inhibit, treat, or abate amyloid deposition. In
some embodiments, the compounds of present invention inhibit,
treat, or abate the activity/effect of Abeta oligomers on neurons.
In some embodiments, the compounds show activity in a
beta-secretase assay and are potentially useful for the inhibition,
treatment, and abatement of cognitive decline and Alzheimer's
disease. In some embodiments the derivative of ginger oil is a
compound in purified and isolated form (for example, with a purity
of greater than 80%, 85%, 90%, 95%, 98%, or 99% by weight). The
compounds and methods described herein may be used to treat one or
more symptoms of cognitive decline and/or Alzheimer's disease such
as memory loss, confusion, impaired judgment, personality changes,
disorientation, and loss of language skills. Further, the compounds
and methods described herein may be useful in inhibiting, treating,
and/or abating cognitive decline and/or Alzheimer's disease by
restoring long term potentiation, and/or inhibiting, treating, or
abatement of one or both of neurodegeneration and general
amyloidosis, more specifically, by inhibiting, treating, or
abatement of one or more of amyloid production, amyloid assembly,
amyloid aggregation, amyloid binding, and amyloid deposition.
DESCRIPTION OF DRAWINGS
[0011] FIG. 1 shows results of an MTT assay in the presence and
absence of a processed product of amyloid precursor protein.
[0012] FIG. 2 shows inhibition of processed product of amyloid
precursor protein-mediated membrane trafficking effect by Compound
Example 2.
[0013] FIG. 3 shows Compound Example 2 inhibiting the memory loss
effects of a processed product of amyloid precursor protein.
[0014] FIG. 4 shows Compound Example 2 Inhibiting the membrane
trafficking effects of Abeta assemblies isolated from AD
patients.
DETAILED DESCRIPTION
[0015] Cognitive decline, such as memory loss, confusion, impaired
judgment, personality changes, disorientation, and loss of language
skills occurs in much of the population as they age, in varying
degree. The most common, severe and irreversible form of cognitive
decline is Alzheimer's disease, which, at present, is always
fatal.
[0016] The symptoms of cognitive decline and Alzheimer's disease
are thought to stem from the formation of amyloid plaques and
neurofibrillary tangles, which are thought to contribute to the
degradation of the neurons (nerve cells) in the brain and the
subsequent onset of symptoms. Amyloid is a general term for protein
fragments that the body produces normally. Beta-amyloid is a
fragment of a protein that is snipped from another protein called
amyloid precursor protein (APP). In a healthy brain, beta-amyloid
protein fragments are broken down and eliminated. In individuals
with Alzheimer's disease and other forms of cognitive decline, the
fragments accumulate to form hard, insoluble plaques.
Neurofibrillary tangles are insoluble twisted fibers that are found
inside of the brain's cells. The protein contained in
neurofibrillary tangles, i.e., the tau protein, forms a
microtubule, which helps transport nutrients and other important
substances from one part of the nerve cell to another. In
Alzheimer's disease the tau protein is abnormal and the microtubule
structures collapse.
[0017] Beta-secretase is the enzyme in the human brain responsible
for the production of Beta-amyloid, the pathogenic substance
responsible for the formation of brain plaques and tangles in the
Alzheimer's diseased brain. Beta-amyloid and its oligomers
(beta-amyloid oligomers or Abeta oligomers) are also believed to be
responsible for early cognitive decline in the pre-Alzheimer's
diseased brain. Inhibition of beta-secretase would be expected to
lessen beta-amyloid burden in the brain and thus slow cognitive
decline, block the formation of amyloid oligomers, the production
of plaques and tangles, halt neurodegeneration, and to potentially
treat mild cognitive impairment and more serious forms of cognitive
impairment such as Alzheimer's disease.
[0018] The gingerols are a series of natural small molecules
isolated from ginger, Zingiber officinale, and are classified
according to their alkyl chain length e.g., [6]-gingerol,
[8]-gingerol. Gingerols are known to be relatively unstable under
both chemical and biological conditions, forming Inactive
substances. For example, the beta-hydroxycarbonyl function of the
gingerols is vulnerable to oxidation or dehydration to form
inactive products, and the gingerols are particularly prone to
rapid dehydration under acidic conditions, such that even the pure
substance is difficult to store for long periods. Accordingly,
simple oral dosing of the gingerols for medicinal action might not
be possible due to the acidic environment of the stomach and upper
intestinal tract. Further, chemical and biological instability is
also likely to be a serious problem for intravenous doses.
Accordingly, there is strong need to discover inhibitors of
cognitive decline, and in particular, compounds that are useful in
the treatment and abatement of cognitive decline and Alzheimer's
disease, by methods such as inhibiting amyloid (including Abeta
oligomers) production, amyloid (including Abeta oligomers)
aggregation, and/or amyloid (including Abets oligomers) deposition
(i.e., plaqing), inhibiting neuorodegeneration, and/or restoring
long term potentiation, and/or Inhibiting the activity/effect of
Abeta oligomers on neurons. There is also a need for inhibitors of
cognitive decline that are chemically and biologically stable.
[0019] Plants have attracted relatively little attention as
potentially valuable resources for drug discovery in the area of
cognitive decline and Alzheimer's disease. The use of plant
extracts to produce unnatural derivatives of compounds of medicinal
interest is not generally used. Accordingly, there is also a need
for a method of producing compounds of medicinal interest from
plant extracts and extracts from other biological sources. In
particular, there is also a need to produce and identify compounds
derived from plant extracts that are useful in the treatment and
abatement of cognitive decline and Alzheimer's disease.
[0020] The compounds, compositions, and methods described herein
are directed toward these needs and other ends.
[0021] Embodiments of the present invention provides, inter alia,
compounds of Formula I:
##STR00002##
or pharmaceutically acceptable salts thereof wherein:
[0022] R.sup.1 is selected from (A1) and (A2):
##STR00003##
[0023] R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are each,
independently, selected from H, OH, C.sub.1-6 alkoxy, C.sub.1-6
haloalkoxy, halo, CN, NO.sub.2, C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.3-7cycloalkyl, NH.sub.2, NH(C.sub.1-4 alkyl),
NH(C.sub.3-7 cycloalkyl), N(C.sub.1-4 alkyl).sub.2,
NHC(O)(C.sub.1-4 alkyl), SH, S(C.sub.1-6 alkyl), C(O)OR.sup.a,
C(O)R.sup.b, C(O)NR.sup.cR.sup.d, OC(O)R.sup.b,
OC(O)NR.sup.cR.sup.d, NR.sup.cR.sup.d, NR.sup.cC(O)R.sup.b,
NR.sup.cC(O)OR.sup.a, NR.sup.cS(O).sub.2R.sup.b,
NR.sup.cS(O).sub.2NR.sup.cR.sup.d, S(O)R.sup.b, S(O).sub.2R.sup.b,
and S(O).sub.2NR.sup.cR.sup.d;
[0024] R.sup.7 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or
C.sub.3-7 cycloalkyl;
[0025] R.sup.8 is C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or
C.sub.3-7 cycloalkyl;
[0026] R.sup.9 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or
C.sub.3-7 cycloalkyl;
[0027] R.sup.10 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or
C.sub.3-7 cycloalkyl;
[0028] R.sup.11 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, or
C.sub.3-7 cycloalkyl;
[0029] R.sup.12, R.sup.13, R.sup.14, R.sup.15, and R.sup.16 are
each, independently, selected from H, OH, C.sub.1-6 alkoxy,
C.sub.1-6 haloalkoxy, halo, CN, NO.sub.2, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.3-7 cycloalkyl, NH.sub.2, NH(C.sub.1-4
alkyl), NH(C.sub.3-7 cycloalkyl), N(C.sub.1-4 alkyl).sub.2,
NHC(O)(C.sub.1-4 alkyl), SH, S(C.sub.1-6 alkyl), C(O)OR.sup.a1,
C(O)R.sup.b1, C(O)NR.sup.c1R.sup.d1, OC(O)R.sup.b1,
OC(O)NR.sup.c1R.sup.d1, NR.sup.e1R.sup.d1, NR.sup.c1C(O)R.sup.b1,
NR.sup.c1C(O)OR.sup.a1, NR.sup.c1S(O).sub.2R.sup.b1,
NR.sup.c1S(O).sub.2NR.sup.c1R.sup.d1, S(O)R.sup.b1, S(O)R.sup.b1,
and S(O).sub.2NR.sup.c1R.sup.d1;
[0030] each R.sup.a is independently selected from H, C.sub.1-6
alkyl, C.sub.1-6 haloalkyl, arylalkyl, heteroarylalkyl,
cycloalkylalkyl, heterocycloalkylalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, aryl, cycloalkyl, heteroaryl and
heterocycloalkyl, wherein each of the C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl,
heterocycloalkylalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl,
cycloalkyl, heteroaryl and heterocycloalkyl is optionally
substituted with 1, 2, 3, 4, or 5 substituents independently
selected from OH, CN, amino, halo, C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, aryl, arylalkyl,
heteroaryl, heteroarylalkyl, cycloalkyl and heterocycloalkyl;
[0031] each R.sup.b is independently selected from H, C.sub.1-6
alkyl, C.sub.1-6 haloalkyl, arylalkyl, heteroarylalkyl,
cycloalkylalkyl, heterocycloalkylalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl,
arylalkyl, heteroarylalkyl, cycloalkylalkyl and
heterocycloalkylalkyl, wherein each of the C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl,
heterocycloalkylalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl,
cycloalkyl, heteroaryl, heterocycloalkyl, arylalkyl,
heteroarylalkyl, cycloalkylalkyl and heterocycloalkylalkyl is
optionally substituted with 1, 2, 3, 4, or 5 substituents
independently selected from OH, amino, halo, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl and
heterocycloalkyl;
[0032] R.sup.c and R.sup.d are independently selected from H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, arylalkyl, heteroarylalkyl,
cycloalkylalkyl, heterocycloalkylalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl,
arylalkyl, heteroarylalkyl, cycloalkylalkyl and
heterocycloalkylalkyl, wherein each of the C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl,
heterocycloalkylalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl,
heteroaryl, cycloalkyl, heterocycloalkyl, arylalkyl,
heteroarylalkyl, cycloalkylalkyl and heterocycloalkylalkyl is
optionally substituted with 1, 2, 3, 4, or 5 substituents
independently selected from OH, amino, halo, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl and
heterocycloalkyl;
[0033] or R.sup.c and R.sup.d together with the N atom to which
they are attached form a 4-, 5-, 6- or 7-membered heterocycloalkyl
group that is optionally substituted with 1, 2, 3, 4, or 5
substituents independently selected from OH, amino, halo, C.sub.1-6
alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy,
aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, and
heterocycloalkyl; [0034] each R.sup.a1 is independently selected
from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, arylalkyl,
heteroarylalkyl, cycloalkylalkyl, heterocycloalkylalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, aryl, cycloalkyl, heteroaryl and
heterocycloalkyl, wherein each of the C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl,
heterocycloalkylalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl,
cycloalkyl, heteroaryl and heterocycloalkyl is optionally
substituted with 1, 2, 3, 4, or 5 substituents independently
selected from OH, CN, amino, halo, C.sub.1-6 alkyl, C.sub.1-6
haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, aryl, arylalkyl,
heteroaryl, heteroarylalkyl, cycloalkyl, and heterocycloalkyl;
[0035] each R.sup.b1 is independently selected from H, C.sub.1-6
alkyl, C.sub.1-6 haloalkyl, arylalkyl, heteroarylalkyl,
cycloalkylalkyl, heterocycloalkylalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl,
arylalkyl, heteroarylalkyl, cycloalkylalkyl and
heterocycloalkylalkyl, wherein each of the C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl,
heterocycloalkylalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl,
cycloalkyl, heteroaryl, heterocycloalkyl, arylalkyl,
heteroarylalkyl, cycloalkylalkyl and heterocycloalkylalkyl is
optionally substituted with 1, 2, 3, 4, or 5 substituents
independently selected from OH, amino, halo, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, and
heterocycloalkyl;
[0036] R.sup.c1 and R.sup.d2 are independently selected from H,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, arylalkyl, heteroarylalkyl,
cycloalkylalkyl, heterocycloalkylalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl,
arylalkyl, heteroarylalkyl, cycloalkylalkyl and
heterocycloalkylalkyl, wherein each of the C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkyl,
heterocycloalkylalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl,
heteroaryl, cycloalkyl, heterocycloalkyl, arylalkyl,
heteroarylalkyl, cycloalkylalkyl and heterocycloalkylalkyl is
optionally substituted with 1, 2, 3, 4, or 5 substituents
Independently selected from OH, amino, halo, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, aryl,
arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, and
heterocycloalkyl; [0037] or R.sup.c1 and R.sup.d1 together with the
N atom to which they are attached form a 4-, 5-, 6- or 7-membered
heterocycloalkyl group that is optionally substituted with 1, 2, 3,
4, or 5 substituents independently selected from OH, amino, halo,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, C.sub.1-6
haloalkoxy, aryl, arylalkyl, heteroaryl, heteroarylalkyl,
cycloalkyl, and heterocycloalkyl; and
[0038] m is 0, 1, or 2.
[0039] In some embodiments, when R.sup.1 is a moiety of (A1), then
two of R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are
independently selected from OH, C.sub.1-6 alkoxy, and C.sub.1-6
haloalkoxy.
[0040] In some embodiments, when R.sup.1 is a moiety of (A1), then
at least one of R.sup.12, R.sup.13, R.sup.14, R.sup.15, and
R.sup.16 is other than H.
[0041] In some embodiments, two of R.sup.2, R.sup.3, R.sup.4,
R.sup.5, and R.sup.6 are independently selected from OH, C.sub.1-6
alkoxy, and C.sub.1-6 haloalkoxy. In some further embodiments, each
of the rest of R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 is
H.
[0042] In some embodiments, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and
R.sup.6 are each, independently, selected from H, OH, C.sub.1-6
alkoxy, C.sub.1-6 haloalkoxy, halo, CN, NO.sub.2, C.sub.1-6 alkyl,
C.sub.1-6 haloalkyl, C.sub.3-7 cycloalkyl, NH.sub.2, NH(C.sub.1-4
alkyl), NH(C.sub.3-7 cycloalkyl), N(C.sub.1-4 alkyl).sub.2,
NHC(O)(C.sub.1-4 alkyl), SH, S(C.sub.1-6 alkyl), C(O)OH,
C(O)O(C.sub.1-4 alkyl), C(O)(C.sub.1-4 alkyl), and C(O)NH(C.sub.1-4
alkyl).
[0043] In some embodiments, one of R.sup.2, R.sup.3, R.sup.4,
R.sup.5, and R.sup.6 is OH; and one of R.sup.2, R.sup.3, R.sup.4,
R.sup.5, and R.sup.6 is OH, C.sub.1-6 alkoxy, or C.sub.1-6
haloalkoxy. In some further embodiments, each of the rest of
R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 is H.
[0044] In some embodiments, one of R.sup.2, R.sup.3, R.sup.4,
R.sup.5, and R.sup.6 is OH; and one of R.sup.2, R.sup.3, R.sup.4,
R.sup.5, and R.sup.6 is C.sub.1-3 alkoxy or C.sub.1-3 haloalkoxy
(In some further embodiments, each of the rest of R.sup.2, R.sup.3,
R.sup.4, R.sup.5, and R.sup.6 is H.). In some further embodiments,
one of R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 is OH; and
one of R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 is methoxy
or trihalomethoxy (In some further embodiments, each of the rest of
R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 is H.). In still
further embodiments, one of R.sup.2, R.sup.3, R.sup.4, R.sup.5, and
R.sup.6 is OH; and one of R.sup.2, R.sup.3, R.sup.4, R.sup.5, and
R.sup.6 is methoxy (In some further embodiments, each of the rest
of R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 is H.).
[0045] In some embodiments, R.sup.4 is OH; and R.sup.5 is methoxy.
In some further embodiments, R.sup.4 is OH; R.sup.5 is methoxy; and
R.sup.2, R.sup.3, and R.sup.6 are each H.
[0046] In some embodiments, R.sup.7 is H or C.sub.1-6 alkyl. In
some further embodiments, R.sup.7 is H or C.sub.1-3 alkyl.
[0047] In some embodiments, R.sup.7 is C.sub.1-3 alkyl. In some
further embodiments, R.sup.7 is methyl or ethyl. In still further
embodiments, R.sup.7 is methyl.
[0048] In some embodiments, R.sup.7 is H.
[0049] In some embodiments, R.sup.8 is C.sub.1-6 alkyl. In some
further embodiments, R.sup.8 is C.sub.1-3 alkyl. In still further
embodiments, R.sup.8 is methyl.
[0050] In some embodiments, R.sup.9 is H or C.sub.1-6 alkyl. In
some further embodiments, R.sup.9 is H or C.sub.1-3 alkyl.
[0051] In some embodiments, R.sup.9 is H.
[0052] In some embodiments, R.sup.9 is C.sub.1-3 alkyl.
[0053] In some embodiments, R.sup.10 is H or C.sub.1-6 alkyl. In
some further embodiments, R.sup.10 is H or C.sub.1-3 alkyl. In
still further embodiments, R.sup.10 is H. In other embodiments,
R.sup.10 is C.sub.1-3 alkyl.
[0054] In some embodiments, R.sup.11 is H or C.sub.1-6 alkyl. In
some further embodiments, R.sup.11 is H or C.sub.1-3 alkyl. In
still further embodiments. R.sup.11 is H. In other embodiments,
R.sup.11 is C.sub.1-3 alkyl.
[0055] In some embodiments, at least one of R.sup.12, R.sup.13,
R.sup.14, R.sup.15, and R.sup.16 is other than H.
[0056] In some embodiments, R.sup.12, R.sup.13, R.sup.14, R.sup.15,
and R.sup.16 are each, independently, selected from H, OH,
C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, halo, CN, NO.sub.2,
C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.3-7 cycloalkyl,
NH.sub.2, NH(C.sub.1-4 alkyl), NH(C.sub.3-7cycloalkyl), N(C.sub.1-4
alkyl).sub.2, NHC(O)(C.sub.1-4 alkyl), SH, S(C.sub.1-6 alkyl),
C(O)OH, C(O)O(C.sub.1-4 alkyl), C(O)(C.sub.1-4 alkyl), and
C(O)NH(C.sub.1-4 alkyl).
[0057] In some embodiments, R.sup.12, R.sup.13, R.sup.14, R.sup.15,
and R.sup.16 are each, independently, selected from H, halo, CN,
NO.sub.2, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.3-7
cycloalkyl, C(O)O(C.sub.1-4 alkyl), C(O)(C.sub.1-4 alkyl), and
C(O)NH(C.sub.1-4 alkyl).
[0058] In some embodiments, at least one of R.sup.12, R.sup.13,
R.sup.14, R.sup.15, and R.sup.16 is selected from halo, CN,
NO.sub.2, C.sub.1-6 haloalkyl, C(O)O(C.sub.1-4 alkyl),
C(O)(C.sub.1-4 alkyl), and C(O)NH(C.sub.1-4 alkyl).
[0059] In some embodiments, at least one of R.sup.12, R.sup.13,
R.sup.14, R.sup.15, and R.sup.16 is selected from halo and
C.sub.1-6 haloalkyl. In some further embodiments, at least one of
R.sup.12, R.sup.13, R.sup.14, R.sup.15, and R.sup.16 is selected
from halo and C.sub.1-6 haloalkyl, and each of the rest is of
R.sup.12, R.sup.13, R.sup.14, R.sup.15, and R.sup.16 is H. In yet
further embodiments, one or two of R.sup.12, R.sup.13, R.sup.14,
R.sup.15, and R.sup.16 are selected from halo and C.sub.1-6
haloalkyl, and each of the rest is of R.sup.12, R.sup.13, R.sup.14,
R.sup.15, and R.sup.16 is H. In still further embodiments, one of
R.sup.12, R.sup.13, R.sup.14, R.sup.15, and R.sup.18 is selected
from halo and C.sub.1-6 haloalkyl, and each of the rest is of
R.sup.12, R.sup.13, R.sup.14, R.sup.15, and R.sup.16 is H.
[0060] In some embodiments, R.sup.14 is halo or C.sub.1-6 haloalkyl
(In some further embodiments, each of R.sup.12, R.sup.13, R.sup.15,
and R.sup.16 is H.). In some further embodiments, R.sup.14 is halo
or C.sub.1-3 haloalkyl (In some further embodiments, each of
R.sup.12, R.sup.13, R.sup.15, and R.sup.16 is H.). In still further
embodiments, R.sup.14 is halo or C.sub.1 haloalkyl (In some further
embodiments, each of R.sup.12, R.sup.13, R.sup.15, and R.sup.16 is
H.).
[0061] In some embodiments, R.sup.14 is halo (In some further
embodiments, each of R.sup.12, R.sup.13, R.sup.15, and R.sup.16 is
H.). In some embodiments, R.sup.14 is Cl or F. In some embodiments,
R.sup.14 is Cl. In some embodiments, R.sup.14 is F.
[0062] In some embodiments, R.sup.14 is C.sub.1-6 haloalkyl (In
some further embodiments, each of R.sup.12, R.sup.13, R.sup.15, and
R.sup.16 is H.). In some further embodiments, R.sup.14 is C.sub.1-3
haloalkyl. In still further embodiments, R.sup.14 is C.sub.1
haloalkyl. In yet further embodiments, R.sup.14 is CF.sub.3.
[0063] In some embodiments, R.sup.15 is halo or C.sub.1-6 haloalkyl
(In some further embodiments, each of R.sup.12, R.sup.13, R.sup.14,
and R.sup.16 is H.). In some further embodiments, R.sup.15 is halo
or C.sub.1-3 haloalkyl (In some further embodiments, each of
R.sup.12, R.sup.13, R.sup.14, and R.sup.16 is H.). In still further
embodiments, R.sup.15 is halo or C.sub.1 haloalkyl (In some further
embodiments, each of R.sup.12, R.sup.13, R.sup.14, and R.sup.16 is
H.).
[0064] In some embodiments, R.sup.15 is halo. In some embodiments,
R.sup.15 is Cl or F. In some embodiments, R.sup.15 is Cl. In some
embodiments, R.sup.15 is F.
[0065] In some embodiments, R.sup.14 is C.sub.1-6 haloalkyl. In
some further embodiments, R.sup.15 is C.sub.1-3 haloalkyl. In still
further embodiments, R.sup.15 is C.sub.1 haloalkyl. In yet further
embodiments, R.sup.15 is CF.sub.3.
[0066] In some embodiments, R.sup.14 and R.sup.15 are each
independently halo or C.sub.1-3 haloalkyl (In some further
embodiments, each of R.sup.12, R.sup.13, and R.sup.16 is H.). In
some further embodiments, R.sup.14 and R.sup.15 are each
independently halo or C.sub.1 haloalkyl.
[0067] In some embodiments, R.sup.14 and R.sup.15 are each
independently halo.
[0068] In some embodiments, the compound of Formula I is a compound
of Formula II:
##STR00004##
[0069] In some embodiments, the compound of Formula II or
pharmaceutically acceptable suit thereof is a compound of Formula
IIa or IIb:
##STR00005##
or pharmaceutically acceptable salt thereof.
[0070] In some embodiments, the compound of Formula II is a
compound of Formula IIa. In some further embodiments, R.sup.10 and
R.sup.11 are each, independently, selected from H and C.sub.1-3
alkyl. In yet further embodiments, R.sup.10 and R.sup.11 are each,
independently, selected from H and methyl. In still further
embodiments, R.sup.10 and R.sup.11 are each H.
[0071] In some embodiments of the compound of Formula IIa or
pharmaceutically acceptable salt thereof, one of R.sup.10 and
R.sup.11 is selected from H and C.sub.1-3 alkyl and the other is H.
In some further embodiments, one of R.sup.10 and R.sup.11 is
C.sub.1-3 alkyl. In yet further embodiments, one of R.sup.10 and
R.sup.11 is methyl.
[0072] In some embodiments of the compound of Formula IIa or
pharmaceutically acceptable salt thereof, both of R.sup.10 and
R.sup.11 are selected from C.sub.1-3 alkyl. In some further
embodiments, both R.sup.10 and R.sup.11 are methyl.
[0073] In some embodiments of the compound of Formula IIa or
pharmaceutically acceptable salt thereof, R.sup.12, R.sup.13,
R.sup.14, R.sup.15, and R.sup.16 are each, independently, selected
from H, OH, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, halo, CN,
NO.sub.2, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.3-7
cycloalkyl, NH.sub.2, NH(C.sub.1-4 alkyl), NH(C.sub.3-7cycloalkyl),
N(C.sub.1-4 alkyl).sub.2, NHC(O)(C.sub.1-4 alkyl), SH, S(C.sub.1-6
alkyl), C(O)OH, C(O)O(C.sub.1-4 alkyl), C(O)(C.sub.1-4 alkyl), and
C(O)NH(C.sub.1-4 alkyl).
[0074] In some embodiments of the compound of Formula IIa or
pharmaceutically acceptable salt thereof, R.sup.12, R.sup.13,
R.sup.14, R.sup.15, and R.sup.16 are each, independently, selected
from H, halo, CN, NO.sub.2, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl,
C.sub.3-7 cycloalkyl, C(O)O(C.sub.1-4 alkyl), C(O)(C.sub.1-4
alkyl), and C(O)NH(C.sub.1-4 alkyl).
[0075] In some embodiments of the compound of Formula IIa or
pharmaceutically acceptable salt thereof, R.sup.12, R.sup.13,
R.sup.14, R.sup.15, and R.sup.16 are each, independently, selected
from H, halo, CN, NO.sub.2, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl,
and C.sub.3-7 cycloalkyl. In some further embodiments, R.sup.12,
R.sup.13, R.sup.14, R.sup.15, and R.sup.16 are each, independently,
selected from H, halo, CN, C.sub.1-6 alkyl, and C.sub.1-6
haloalkyl. In yet further embodiments, R.sup.12, R.sup.13,
R.sup.14, R.sup.15, and R.sup.16 are each, independently, selected
from H, halo, C.sub.1-6 alkyl, and C.sub.1-6 haloalkyl.
[0076] In some embodiments of the compound of Formula IIa or
pharmaceutically acceptable salt thereof, at least one of R.sup.12,
R.sup.13, R.sup.14, R.sup.15, and R.sup.16 is selected from halo
and C.sub.1-6 haloalkyl, and each of the rest is of R.sup.12,
R.sup.13, R.sup.14, R.sup.15, and R.sup.16 is H. In some further
embodiments, one or two of R.sup.12, R.sup.13, R.sup.14, R.sup.15,
and R.sup.16 are selected from halo and C.sub.1-6 haloalkyl, and
each of the rest is of R.sup.12, R.sup.13, R.sup.14, R.sup.15, and
R.sup.16 is H. In yet further embodiments, one of R.sup.12,
R.sup.13, R.sup.14, R.sup.15, and R.sup.16 is selected from halo
and C.sub.1-6 haloalkyl, and each of the rest is of R.sup.12,
R.sup.13, R.sup.14, R.sup.15, and R.sup.16 is H.
[0077] In some embodiments of the compound of Formula IIa or
pharmaceutically acceptable salt thereof, at least one of R.sup.12,
R.sup.13, R.sup.14, R.sup.13, and R.sup.11 is selected from halo,
CN, NO.sub.2, C.sub.1-6 haloalkyl, C(O)O(C.sub.1-4 alkyl),
C(O)(C.sub.1-4 alkyl), and C(O)NH(C.sub.1-4 alkyl).
[0078] In some embodiments of the compound of Formula IIa, at least
one of R.sup.12, R.sup.13, R.sup.14, R.sup.15, and R.sup.16 is
selected from halo and C.sub.1-6 haloalkyl.
[0079] In some embodiments of the compound of Formula IIa or
pharmaceutically acceptable salt thereof, at least one of R.sup.12,
R.sup.13, R.sup.14, R.sup.15, and R.sup.16 is selected from halo
and C.sub.1-3 haloalkyl. In some further embodiments, at least one
of R.sup.12, R.sup.13, R.sup.14, R.sup.15, and R.sup.16 is selected
from halo and C.sub.1 haloalkyl.
[0080] In some embodiments of the compound of Formula IIa or
pharmaceutically acceptable salt thereof, R.sup.14 is halo or
C.sub.1-6 haloalkyl. In some further embodiments, R.sup.14 is halo
or C.sub.1-3 haloalkyl. In still further embodiments, R.sup.14 is
halo or C.sub.1 haloalkyl.
[0081] In some embodiments of the compound of Formula IIa or
pharmaceutically acceptable salt thereof, R.sup.14 is halo (In some
further embodiments, each of R.sup.12, R.sup.13, R.sup.15, and
R.sup.16 is H.). In some embodiments, R.sup.14 is Cl or F. In some
embodiments, R.sup.14 is Cl. In some embodiments, R.sup.14 is
F.
[0082] In some embodiments of the compound of Formula IIa, R.sup.14
is C.sub.1-6 haloalkyl (In some further embodiments, each of
R.sup.12, R.sup.13, R.sup.15, and R.sup.16 is H.). In some further
embodiments, R.sup.14 is C.sub.1-3 haloalkyl. In still further
embodiments, R.sup.14 is C.sub.1 haloalkyl. In yet further
embodiments, R.sup.14 is CF.sub.3.
[0083] In some embodiments of the compound of Formula IIa or
pharmaceutically acceptable salt thereof, R.sup.14 is halo or
C.sub.1-6 haloalkyl and each of R.sup.12, R.sup.13, R.sup.15, and
R.sup.16 is H.
[0084] In some embodiments of the compound of Formula IIa or
pharmaceutically acceptable salt thereof, R.sup.15 is halo or
C.sub.1-6 haloalkyl (In some further embodiments, each of R.sup.12,
R.sup.13, R.sup.14, and R.sup.6 is H.). In some further
embodiments, R.sup.15 is halo or C.sub.1-3 haloalkyl. In still
further embodiments, R.sup.15 is halo or C.sub.1 haloalkyl.
[0085] In some embodiments of the compound of Formula IIa or
pharmaceutically acceptable salt thereof, R.sup.15 is halo. In some
embodiments, R.sup.15 is Cl or F. In some embodiments, R.sup.15 is
Cl. In some embodiments, R.sup.11 is F.
[0086] In some embodiments of the compound of Formula IIa or
pharmaceutically acceptable salt thereof, R.sup.14 is C.sub.1-6
haloalkyl. In some further embodiments, R.sup.15 is C.sub.1-3
haloalkyl. In still further embodiments, R.sup.15 is C.sub.1
haloalkyl. In yet further embodiments, R.sup.15 is CF.sub.3.
[0087] In some embodiments of the compound of Formula IIa or
pharmaceutically acceptable salt thereof, R.sup.14 and R.sup.15 are
each independently halo or C.sub.1-3 haloalkyl (In some further
embodiments, each of R.sup.12, R.sup.13, and R.sup.16 is H.). In
some further embodiments, R.sup.14 and R.sup.15 are each
independently halo or C.sub.1 haloalkyl. In yet further
embodiments, R.sup.14 and R.sup.15 are each independently halo.
[0088] In some embodiments, the compound of Formula II or
pharmaceutically acceptable salt thereof is a compound of Formula
IIb or pharmaceutically acceptable salt thereof.
[0089] In some embodiments of the compound of Formula IIb or
pharmaceutically acceptable salt thereof, R.sup.12, R.sup.13,
R.sup.14, R.sup.15, and R.sup.16 are each, independently, selected
from H, OH, C.sub.1-6 alkoxy, C.sub.1-6 haloalkoxy, halo, CN,
NO.sub.2, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl,
C.sub.3-7cycloalkyl, NH.sub.2, NH(C.sub.1-4 alkyl), NH(C.sub.1-4
cycloalkyl), N(C.sub.1-4 alkyl).sub.2, NHC(O)(C.sub.1-14 alkyl),
SH, S(C.sub.1-s alkyl), C(O)OH, C(O)O(C.sub.1-4 alkyl),
C(O)(C.sub.1-4 alkyl), and C(O)NH(C.sub.1-4 alkyl).
[0090] In some embodiments of the compound of Formula IIb or
pharmaceutically acceptable salt thereof, R.sup.12, R.sup.13,
R.sup.14, R.sup.15, and R.sup.16 are each, independently, selected
from H, halo, CN, NO.sub.2, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl,
C.sub.3-7 cycloalkyl, C(O)O(C.sub.1-4 alkyl), C(O)(C.sub.1-4
alkyl), and C(O)NH(C.sub.1-4 alkyl).
[0091] In some embodiments of the compound of Formula IIb or
pharmaceutically acceptable salt thereof, R.sup.12, R.sup.13,
R.sup.14, R.sup.15, and R.sup.16 are each, independently, selected
from H, halo, CN, NO.sub.2, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl,
and C.sub.3-7 cycloalkyl. In some further embodiments, R.sup.12,
R.sup.13, R.sup.14, R.sup.15, and R.sup.16 are each, independently,
selected from H, halo, CN, C.sub.1-6 alkyl, and C.sub.1-6
haloalkyl. In yet further embodiments, R.sup.12, R.sup.13,
R.sup.14, R.sup.15, and R.sup.16 are each, independently, selected
from H, halo, C.sub.1-6 alkyl, and C.sub.1-6 haloalkyl.
[0092] In some embodiments of the compound of Formula IIb or
pharmaceutically acceptable salt thereof, at least one of R.sup.12,
R.sup.13, R.sup.14, R.sup.15, and R.sup.16 is selected from halo
and C.sub.1-6 haloalkyl, and each of the rest is of R.sup.12,
R.sup.13, R.sup.14, R.sup.15, and R.sup.16 is H. In some further
embodiments, one or two of R.sup.12, R.sup.13, R.sup.14, R.sup.15,
and R.sup.16 are selected from halo and C.sub.1-6 haloalkyl, and
each of the rest is of R.sup.12, R.sup.13, R.sup.14, R.sup.15, and
R.sup.16 is H. In yet further embodiments, one of R.sup.12,
R.sup.13, R.sup.14, R.sup.15, and R.sup.16 is selected from halo
and C.sub.1-6 haloalkyl, and each of the rest is of R.sup.12,
R.sup.13, R.sup.14, R.sup.15, and R.sup.16 is H.
[0093] In some embodiments of the compound of Formula IIb or
pharmaceutically acceptable salt thereof, at least one of R.sup.12,
R.sup.13, R.sup.14, R.sup.15, and R.sup.16 is selected from halo,
CN, NO.sub.2, C.sub.1-6 haloalkyl, C(O)O(C.sub.1-4 alkyl),
C(O)(C.sub.1-4 alkyl), and C(O)NH(C.sub.1-4 alkyl).
[0094] In some embodiments of the compound of Formula IIb, at least
one of R.sup.12, R.sup.13, R.sup.14, R.sup.15, and R.sup.16 is
selected from halo and C.sub.1-6 haloalkyl.
[0095] In some embodiments of the compound of Formula IIb or
pharmaceutically acceptable salt thereof, at least one of R.sup.12,
R.sup.13, R.sup.14, R.sup.15, and R.sup.16 is selected from halo
and C.sub.1-3 haloalkyl. In some further embodiments, at least one
of R.sup.12, R.sup.13, R.sup.14, R.sup.15, and R.sup.16 is selected
from halo and C.sub.1 haloalkyl.
[0096] In some embodiments of the compound of Formula IIb or
pharmaceutically acceptable salt thereof, R.sup.14 is halo or
C.sub.1-6 haloalkyl. In some further embodiments, R.sup.14 is halo
or C.sub.1-3 haloalkyl. In still further embodiments, R.sup.14 is
halo or C.sub.1 haloalkyl.
[0097] In some embodiments of the compound of Formula IIb or
pharmaceutically acceptable salt thereof, R.sup.14 is halo (In some
further embodiments, each of R.sup.12, R.sup.13, R.sup.15, and
R.sup.16 is H.). In some embodiments, R.sup.14 is Cl or F. In some
embodiments, R.sup.14 is Cl. In some embodiments, R.sup.14 is
F.
[0098] In some embodiments of the compound of Formula IIb, R.sup.14
is C.sub.1-6 haloalkyl (In some further embodiments, each of
R.sup.12, R.sup.13, R.sup.15, and R.sup.16 is H.). In some further
embodiments, R.sup.14 is C.sub.1-3 haloalkyl. In still further
embodiments, R.sup.14 is C.sub.1 haloalkyl. In yet further
embodiments, R.sup.14 is CF.sub.3.
[0099] In some embodiments of the compound of Formula IIb or
pharmaceutically acceptable salt thereof, R.sup.14 is halo or
C.sub.1-6 haloalkyl and each of R.sup.12, R.sup.13, R.sup.15, and
R.sup.16 is H.
[0100] In some embodiments of the compound of Formula IIb or
pharmaceutically acceptable salt thereof, R.sup.15 is halo or
C.sub.1-6 haloalkyl (In some further embodiments, each of R.sup.12,
R.sup.13, R.sup.14, and R.sup.16 is H.). In some further
embodiments, R.sup.15 is halo or C.sub.1-3 haloalkyl. In still
further embodiments, R.sup.15 is halo or Cl haloalkyl.
[0101] In some embodiments of the compound of Formula IIb or
pharmaceutically acceptable salt thereof, R.sup.15 is halo. In some
embodiments, R.sup.15 is Cl or F. In some embodiments, R.sup.15 is
Cl. In some embodiments, R.sup.15 is F.
[0102] In some embodiments of the compound of Formula IIb or
pharmaceutically acceptable salt thereof, R.sup.15 is C.sub.1-6
haloalkyl. In some further embodiments. R.sup.15 is C.sub.1-3
haloalkyl. In still further embodiments, R.sup.15 is C.sub.1
haloalkyl. In yet further embodiments, R.sup.15 is CF.sub.3.
[0103] In some embodiments of the compound of Formula IIb or
pharmaceutically acceptable salt thereof, R.sup.14 and R.sup.15 are
each independently halo or C.sub.1-3 haloalkyl (In some further
embodiments, each of R.sup.12, R.sup.13, and R.sup.16 is H.). In
some further embodiments, R.sup.14 and R.sup.15 are each
independently halo or C.sub.1 haloalkyl. In yet further
embodiments, R.sup.14 and R.sup.15 are each independently halo.
[0104] In some embodiments, the compound of Formula I is a compound
of Formula III:
##STR00006##
[0105] In some embodiments of compounds of Formula III or
pharmaceutically acceptable salt thereof, m is 1.
[0106] In some embodiments of compounds of Formula III or
pharmaceutically acceptable salt thereof, m is 0.
[0107] In some embodiments of compounds of Formula III or
pharmaceutically acceptable salt thereof, at least one of R.sup.2,
R.sup.3, R.sup.4, R.sup.5, and R.sup.6 is selected from OH,
C.sub.1-6 alkoxy, and C.sub.1-6 haloalkoxy.
[0108] In some embodiments of compounds of Formula III or
pharmaceutically acceptable salt thereof, at least two of R.sup.2,
R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are independently selected
from OH, C.sub.1-6 alkoxy, and C.sub.1-6 haloalkoxy.
[0109] In some embodiments of compounds of Formula III or
pharmaceutically acceptable salt thereof, at least one of R.sup.12,
R.sup.13, R.sup.14, R.sup.15, and R.sup.16 is other than H.
[0110] In some embodiments of the compound of Formula III or
pharmaceutically acceptable salt thereof, R.sup.14 and R.sup.15 are
each independently halo or C.sub.1-3 haloalkyl. In some further
embodiments, R.sup.14 and R.sup.15 are each independently halo or
C.sub.1 haloalkyl.
[0111] At various places in the present specification, substituents
of compounds of the invention are disclosed in groups or in ranges.
It is specifically Intended that embodiments the invention include
each and every individual subcombination of the members of such
groups and ranges. For example, the term "C.sub.1-6 alkyl" is
specifically intended to individually disclose methyl (C.sub.1
alkyl), ethyl (C.sub.2 alkyl), C.sub.3 alkyl, C.sub.4 alkyl,
C.sub.5 alkyl, and C.sub.6 alkyl.
[0112] For compounds of the invention in which a variable appears
more than once, each variable can be a different moiety selected
from the Markush group defining the variable. For example, where a
structure is described having two R groups that are simultaneously
present on the same compound, then the two R groups can represent
different moieties selected from the Markush group defined for
R.
[0113] It is further appreciated that certain features of the
invention, which are, for clarity, described in the context of
separate embodiments, can also be provided in combination in a
single embodiment. Conversely, various features of the invention
which are, for brevity, described in the context of a single
embodiment, can also be provided separately or in any suitable
subcombination.
[0114] The term "n-membered" where n is an integer typically
describes the number of ring-forming atoms in a moiety where the
number of ring-forming atoms is n. For example, pyridine is an
example of a 6-membered heteroaryl ring and thiophene is an example
of a 5-membered heteroaryl group.
[0115] As used herein, the term "alkyl" Is meant to refer to a
saturated hydrocarbon group which is straight-chained or branched.
Example alkyl groups include, but are not limited to, methyl (Me),
ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g.,
n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl,
neopentyl), and the like. An alkyl group can contain from 1 to
about 20, from 2 to about 20, from 1 to about 10, from 1 to about
8, from 1 to about 6, from 1 to about 4, or from 1 to about 3
carbon atoms. The term "alkylene" refers to a divalent alkyl
linking group. An example of alkylene is methylene (CH.sub.2).
[0116] As used herein, "alkenyl" refers to an alkyl group having
one or more double carbon-carbon bonds. Example alkenyl groups
include, but are not limited to, ethenyl, propenyl, cyclohexenyl,
and the like. The term "alkenylenyl" refers to a divalent linking
alkenyl group.
[0117] As used herein, "alkynyl" refers to an alkyl group having
one or more triple carbon-carbon bonds. Example alkynyl groups
include, but are not limited to, ethynyl, propynyl, and the like.
The term "alkynylenyl" refers to a divalent linking alkynyl
group.
[0118] As used herein, "haloalkyl" refers to an alkyl group having
one or more halogen substituents. Example haloalkyl groups include,
but are not limited to, CF.sub.3, C.sub.2F.sub.5, CHF.sub.2,
CCl.sub.3, CHCl.sub.2, C.sub.2Cl.sub.5, CH.sub.2CF.sub.3, and the
like.
[0119] As used herein, "aryl" refers to monocyclic or polycyclic
(e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as,
for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl,
indenyl, and the like. In some embodiments, aryl groups have from 6
to about 20 carbon atoms. In some embodiments, aryl groups have
from 6 to about 10 carbon atoms.
[0120] As used herein, "cycloalkyl" refers to non-aromatic cyclic
hydrocarbons including cyclized alkyl, alkenyl, and alkynyl groups
that contain up to 20 ring-forming carbon atoms. Cycloalkyl groups
can include mono- or polycyclic (e.g., having 2, 3 or 4 fused
rings) ring systems as well as spiro ring systems. A cycloalkyl
group can contain from 3 to about 15, from 3 to about 10, from 3 to
about 8, from 3 to about 6, from 4 to about 6, from 3 to about 5,
or from 5 to about 6 ring-forming carbon atoms. Ring-forming carbon
atoms of a cycloalkyl group can be optionally substituted by oxo or
sulfido. Example cycloalkyl groups include, but are not limited to,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl,
norbornyl, norpinyl, norcarnyl, adamantyl, and the like. Also
included in the definition of cycloalkyl are moieties that have one
or more aromatic rings fused (i.e., having a bond in common with)
to the cycloalkyl ring, for example, benzo or thienyl derivatives
of pentane, pentene, hexane, and the like (e.g.,
2,3-dihydro-1H-indene-1-yl, or 1H-inden-2(3H)-one-1-yl).
Preferably, "cycloalkyl" refers to cyclized alkyl groups that
contain up to 20 ring-forming carbon atoms. Examples of cycloalkyl
preferably Include cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, cycloheptyl, adamantyl, and the like.
[0121] As used herein, "heteroaryl" groups refer to an aromatic
heterocycle having up to 20 ring-forming atoms and having at least
one heteroatom ring member (ring-forming atom) such as sulfur,
oxygen, or nitrogen. In some embodiments, the heteroaryl group has
at least one or more heteroatom ring-forming atoms each
independently selected from sulfur, oxygen, and nitrogen.
Heteroaryl groups include monocyclic and polycyclic (e.g., having
2, 3 or 4 fused rings) systems. Examples of heteroaryl groups
include without limitation, pyridyl, pyrimidinyl, pyrazinyl,
pyridazinyl, triazinyl, furyl, quinolyl, Isoquinolyl, thienyl,
imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, benzofuryl,
benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl,
tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl,
benzothienyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, and
the like. In some embodiments, the heteroaryl group has from 1 to
about 20 carbon atoms, and in further embodiments from about 1 to
about 5, from about 1 to about 4, from about 1 to about 3, from
about 1 to about 2, carbon atoms as ring-forming atoms. In some
embodiments, the heteroaryl group contains 3 to about 14, 3 to
about 7, or 5 to 6 ring-forming atoms. In some embodiments, the
heteroaryl group has 1 to about 4, 1 to about 3, or 1 to 2
heteroatoms.
[0122] As used herein, "heterocycloalkyl" refers to non-aromatic
heterocycles having up to 20 ring-forming atoms including cyclized
alkyl, alkenyl, and alkynyl groups where one or more of the
ring-forming carbon atoms is replaced by a heteroatom such as an O,
N, or S atom. Hetercycloalkyl groups can be mono or polycyclic
(e.g., both fused and spiro systems). Example "heterocycloalkyl"
groups include morpholino, thiomorpholino, piperazinyl,
tetrahydrofuranyl, tetrahydrothienyl, 2,3-dihydrobenzofuryl,
1,3-benzodioxole, benzo-1,4-dioxane, piperidinyl, pyrrolidinyl,
isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl,
thiazolidinyl, Imidazolidinyl, pyrrolidin-2-one-3-yl, and the like.
Ring-forming carbon atoms and heteroatoms of a heterocycloalkyl
group can be optionally substituted by oxo or sulfido. For example,
a ring-forming S atom can be substituted by 1 or 2 oxo [i.e., form
a S(O) or S(O).sub.2]. For another example, a ring-forming C atom
can be substituted by oxo (I.e., form carbonyl). Also included in
the definition of heterocycloalkyl are moieties that have one or
more aromatic rings fused (i.e., having a bond in common with) to
the nonaromatic heterocyclic ring, for example pyridinyl,
thiophenyl, phthalimidyl, naphthalimidyl, and benzo derivatives of
heterocycles such as indolene, isoindolene, isoindolin-1-one-3-yl,
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-5-yl,
5,6-dihydrothieno[2,3-c]pyridin-7(4H)-one-5-yl, and
3,4-dihydroisoquinolin-1 (2H)-one-3yl groups. Ring-forming carbon
atoms and heteroatoms of the heterocycloalkyl group can be
optionally substituted by oxo or sulfido. In some embodiments, the
heterocycloalkyl group has from 1 to about 20 carbon atoms, and in
further embodiments from about 3 to about 20 carbon atoms. In some
embodiments, the heterocycloalkyl group contains 3 to about 14, 3
to about 7, or 5 to 6 ring-forming atoms. In some embodiments, the
heterocycloalkyl group has 1 to about 4, 1 to about 3, or 1 to 2
heteroatoms. In some embodiments, the heterocycloalkyl group
contains 0 to 3 double bonds. In some embodiments, the
heterocycloalkyl group contains 0 to 2 triple bonds.
[0123] As used herein, "halo" or "halogen" includes fluoro, chloro,
bromo, and iodo.
[0124] As used herein, "alkoxy" refers to an --O-alkyl group.
Example alkoxy groups include methoxy, ethoxy, propoxy (e.g.,
n-propoxy and isopropoxy), t-butoxy, and the like.
[0125] As used herein, "haloalkoxy" refers to an --O-haloalkyl
group. An example haloalkoxy group is OCF.sub.3. As used herein,
"trihalomethoxy" refers to a methoxy group having three halogen
substituents. Examples of trihalomethoxy groups include, but are
not limited to, --OCF.sub.3, --OCClF.sub.2, --OCCl.sub.3, and the
like.
[0126] As used herein, "arylalkyl" refers to a C.sub.1-6 alkyl
substituted by aryl and "cycloalkylalkyl" refers to C.sub.1-6 alkyl
substituted by cycloalkyl.
[0127] As used herein, "heteroarylalkyl" refers to a C.sub.1-8
alkyl group substituted by a heteroaryl group, and
"heterocycloalkylalkyl" refers to a C.sub.1-6 alkyl substituted by
heterocycloalkyl.
[0128] As used herein, "amino" refers to NH.sub.2.
[0129] As used herein, "alkylamino" refers to an amino group
substituted by an alkyl group.
[0130] As used herein, "dialkylamino" refers to an amino group
substituted by two alkyl groups.
[0131] As used here, C(O) refers to C(.dbd.O).
[0132] As used here, C(S) refers to C(.dbd.S).
[0133] As used here, S(O) refers to S(.dbd.O).
[0134] As used here, S(O).sub.2 refers to S(.dbd.O).sub.2.
[0135] As used herein, the term "optionally substituted" means that
substitution is optional and therefore includes both unsubstituted
and substituted atoms and moieties. A "substituted" atom or moiety
indicates that any hydrogen on the designated atom or moiety can be
replaced with a selection from the indicated substituent group,
provided that the normal valency of the designated atom or moiety
is not exceeded, and that the substitution results in a stable
compound. For example, if a methyl group (i.e., CH.sub.3) is
optionally substituted, then 3 hydrogen atoms on the carbon atom
can be replaced with substituent groups.
[0136] As used herein, "about" in connection with a numerical value
means that the numerical value is approximate and small variations
would not significantly affect the practice of the disclosed
embodiments. Where a numerical limitation is used, unless indicated
otherwise by the context, "about" means the numerical value can
vary by .+-.10% and remain within the scope of the disclosed
embodiments.
[0137] The compounds described in the embodiments herein can be
asymmetric (e.g., having one or more stereocenters). All
stereoisomers, such as enantiomers and diastereomers, are intended
unless otherwise indicated. Compounds of the present invention that
contain asymmetrically substituted carbon atoms can be isolated in
optically active or racemic forms. Methods on how to prepare
optically active forms from optically active starting materials are
known in the art, such as by resolution of racemic mixtures or by
stereoselective synthesis. Many geometric isomers of olefins,
C.dbd.N double bonds, and the like can also be present in the
compounds described herein, and all such stable isomers are
contemplated in the present invention. Cis and trans geometric
isomers of the compounds of the present invention are described and
may be Isolated as a mixture of isomers or as separated isomeric
forms. Where a compound capable of stereoisomerism or geometric
isomerism is designated in its structure or name without reference
to specific R/S or cis/trans configurations, it is intended that
all such isomers are contemplated.
[0138] Resolution of racemic mixtures of compounds can be carried
out by any of numerous methods known in the art. An example method
includes fractional recrystallization using a chiral resolving acid
which is an optically active, salt-forming organic acid. Suitable
resolving agents for fractional recrystallization methods are, for
example, optically active acids, such as the D and L forms of
tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid,
mandelic acid, malic acid, lactic acid or the various optically
active camphorsulfonic acids such as 3-camphorsulfonic acid. Other
resolving agents suitable for fractional crystallization methods
include stereoisomerically pure forms of .alpha.-methylbenzylamine
(e.g., Sand R forms, or diastereomerically pure forms),
2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine,
cyclohexylethylamine, 1,2-diaminocyclohexane, and the like.
[0139] Resolution of racemic mixtures can also be carried out by
elution on a column packed with an optically active resolving agent
(e.g., dinitrobenzoylphenylglycine). Suitable elution solvent
composition can be determined by one skilled in the art.
[0140] Compounds of embodiments the invention also include
tautomeric forms. Tautomeric forms result from the swapping of a
single bond with an adjacent double bond together with the
concomitant migration of a proton. Tautomeric forms include
prototropic tautomers which are isomeric protonation states having
the same empirical formula and total charge. Example prototropic
tautomers include ketone--enol pairs, amide--imidic acid pairs,
lactam--lactim pairs, amide--imdic acid pairs, enamine--Imine
pairs, and annular forms where a proton can occupy two or more
positions of a heterocyclic system, for example, 1H- and
3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole,
and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or
sterically locked into one form by appropriate substitution.
[0141] Compounds of embodiments the invention further include
hydrates and solvates, as well as anhydrous and non-solvated
forms.
[0142] The term, "compound," as used herein is meant to include all
stereoisomers, geometric iosomers, tautomers, and isotopes of the
structures depicted.
[0143] All compounds and pharmaceutically acceptable salts thereof,
can be prepared or present together with other substances such as
water and solvents (e.g. hydrates and solvates) or can be
isolated.
[0144] Compounds of embodiments the invention can also include all
isotopes of atoms occurring in the intermediates or final
compounds. Isotopes Include those atoms having the same atomic
number but different mass numbers. For example, isotopes of
hydrogen include tritium and deuterium.
[0145] In some embodiments, the compounds of the invention, or
salts thereof, are substantially isolated. By "substantially
isolated" is meant that the compound is at least partially or
substantially separated from the environment in which is formed or
detected. Partial separation can include, for example, a
composition enriched in the compound of the invention. Substantial
separation can include compositions containing at least about 50%,
at least about 60%, at least about 70%, at least about 80%, at
least about 90%, at least about 95%, at least about 97%, or at
least about 99% by weight of the compound of the invention, or salt
thereof. Methods for isolating compounds and their salts are
routine in the art.
[0146] Compounds of embodiments the invention are Intended to
Include compounds with stable structures. As used herein, "stable
compound" and "stable structure" are meant to indicate a compound
that is sufficiently robust to survive isolation to a useful degree
of purity from a reaction mixture, and formulation into an
efficacious therapeutic agent.
[0147] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0148] The expressions, "ambient temperature" and "room
temperature," as used herein, are understood in the art, and refer
generally to a temperature, e.g. a reaction temperature, that is
about the temperature of the room in which the reaction Is carried
out, for example, a temperature from about 20.degree. C. to about
30.degree. C.
[0149] Embodiments of the present invention also includes
pharmaceutically acceptable salts of the compounds described
herein. As used herein, "pharmaceutically acceptable salts" refers
to derivatives of the disclosed compounds wherein the parent
compound is modified by converting an existing acid or base moiety
to its salt form. Examples of pharmaceutically acceptable salts
include, but are not limited to, mineral or organic acid salts of
basic residues such as amines; alkali or organic salts of acidic
residues such as carboxylic acids; and the like. The
pharmaceutically acceptable salts of the present Invention include
the conventional non-toxic salts of the parent compound formed, for
example, from non-toxic inorganic or organic acids. The
pharmaceutically acceptable salts of the present invention can be
synthesized from the parent compound which contains a basic or
acidic moiety by conventional chemical methods. Generally, such
salts can be prepared by reacting the free acid or base forms of
these compounds with a stoichiometric amount of the appropriate
base or acid in water or in an organic solvent, or in a mixture of
the two; generally, nonaqueous media like ether, ethyl acetate,
ethanol, isopropanol, or acetonitrile (ACN) are preferred. Lists of
suitable salts are found in Remington's Pharmaceutical Sciences,
17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and
Journal of Pharmaceutical Science, 66, 2 (1977), each of which is
incorporated herein by reference in its entirety.
Synthesis
[0150] Compounds of embodiments the invention, including salts
thereof, can be prepared using known organic synthesis techniques
and can be synthesized according to any of numerous possible
synthetic routes.
[0151] The reactions for preparing compounds of the Invention can
be carried out in suitable solvents which can be readily selected
by one of skill in the art of organic synthesis. Suitable solvents
can be substantially non-reactive with the starting materials
(reactants), the intermediates, or products at the temperatures at
which the reactions are carried out, e.g., temperatures which can
range from the solvent's freezing temperature to the solvent's
boiling temperature. A given reaction can be carried out in one
solvent or a mixture of more than one solvent. Depending on the
particular reaction step, suitable solvents for a particular
reaction step can be selected by the skilled artisan.
[0152] Preparation of compounds of the invention can involve the
protection and deprotection of various chemical groups. The need
for protection and deprotection, and the selection of appropriate
protecting groups, can be readily determined by one skilled in the
art. The chemistry of protecting groups can be found, for example,
in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic
Synthesis, 3.sup.rd Ed., Wiley & Sons, Inc., New York (1999),
which is incorporated herein by reference in its entirety.
[0153] Reactions can be monitored according to any suitable method
known in the art. For example, product formation can be monitored
by spectroscopic means, such as nuclear magnetic resonance
spectroscopy (e.g., .sup.1H or .sup.13C), infrared spectroscopy,
spectrophotometry (e.g., UV-visible), mass spectrometry, or by
chromatographic methods such as high performance liquid
chromatography (HPLC) or thin layer chromatography (TLC).
[0154] The compounds of embodiments of the invention can be
prepared, for example, according to the reaction pathways,
synthetic procedures, and techniques described below.
[0155] As shown in Scheme 1, benzaldehyde derivative 1-1 can be
reacted with acetaldehyde in the presence of either an acid or a
base catalyst to afford cinnamic aldehyde 1-2. Reaction of cinnamic
aldehyde 1-2 with an organometallic compound such as a Grignard
reagent R.sup.8MgX.sup.1 [wherein X.sup.1 is halo such as Cl or
Br], followed by oxidation of the intermediate alcohol to ketone
and by reduction of the C.dbd.C bond to a C--C single bond under a
hydrogenation condition (for example, in the presence of Pd/C
catalyst), affords ketone 1-3. Reaction of ketone 1-3 with amine
1-4 under reductive amination condition (such as in the presence of
a borohydride reducing reagent) affords compound 1-5.
##STR00007##
[0156] As shown in Scheme 2, Ketone 2-1 can be reacted with an
organometallic compound such as a Grignard reagent R.sup.7MgX.sup.2
[wherein R.sup.7 can be C.sub.1-6 alkyl or C.sub.3-7cycloalkyl; and
X.sup.2 can be halo such as Cl or Br] to afford alcohol 2-2a.
Reduction of Ketone 2-1 such as in the presence of a borohydride
reducing reagent affords alcohol 2-2b. The OH group of alcohol 2-2
(wherein R.sup.7 can be H, C.sub.1-6 alkyl, or C.sub.3-7
cycloalkyl) can be converted to a better leaving group Lg.sup.1
such as OMs [mesylate or CH.sub.3S(O).sub.2O--] or OTf [triflate or
CF.sub.3S(O).sub.2O--], followed by reaction with NH.sub.3 to
afford amine 2-4. Ketone 2-1 can also undergo reductive amination
with NH.sub.3 to afford amine 2-5.
##STR00008##
[0157] As shown in Scheme 3, amine 3-1 (wherein R.sup.7 can be,
e.g., H, C.sub.1-6 alkyl, or C.sub.3-7 cycloalkyl) can be reacted
with compound 3-2 [Lg.sup.2 can be a leaving group such as triflate
group (--OTf) or halo (e.g. Cl or Br)] to afford compound 3-3.
##STR00009##
[0158] As shown in Scheme 4, benzaldehyde derivative 4-1 can be
reacted with a methylmetalic compound such as a Grignard reagent
MeMgX.sup.1 [wherein X.sup.1 is halo such as CI or Br], followed by
oxidation of the intermediate alcohol to ketone to afford ketone
4-2. Ester compound 4-3 [wherein R' can be alkyl (e.g. methyl or
ethyl) or arylalkyl; and Pg.sup.1 can be an amine protecting group
(such as tert-butyloxycarbonyl or Boc; benzyloxycarbonyl or Cbz; or
benzyl)] can be reduced to an alcohol (in the presence of a
reducing reagent such as Lithium aluminium hydride or LAH),
followed by conversion of the OH group to a better leaving group
Lg.sup.3 such as OMs [mesylate or CH.sub.3S(O).sub.2O--] or OTf
[triflate or CF.sub.3S(O).sub.2O--], to afford compound 4-4.
[0159] Reaction of compound 4-2 with compound 4-4 in the presence
of a strong base (such as lithium diisopropylamide or LDA),
followed by hydrogenation (such as in the presence of a Pd/C
catalyst) to reduce the C(O) to CH.sub.2 and by removal of the
protecting group Pg.sup.1 under suitable conditions (for example, a
benzyl group can be removed under hydrogenation condition in the
presence of Pd/C; or Boc group can be removed under acidic
condition), affords amine 4-5. Amine 4-5 can be reacted with
compound 4-6 [wherein Lg.sup.2 can be a leaving group such as
triflate group (--OTf) or halo (e.g. Cl or Br)] to afford compound
4-7.
##STR00010##
[0160] Those skilled in the art can recognize that in all of the
schemes described herein, if there are functional (reactive) groups
present on a substituent group such as R.sup.1, R.sup.2, R.sup.3,
R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10,
R.sup.11, R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16, etc.,
further modification can be made if appropriate and/or desired. For
example, a CN group can be hydrolyzed to afford an amide group; a
carboxylic acid can be converted to an amide; a carboxylic acid can
be converted to an ester, which in turn can be reduced to an
alcohol, which in turn can be further modified. For another
example, an OH group can be converted into a better leaving group
such as mesylate, which in turn is suitable for nucleophilic
substitution, such as by CN. For another example, an --S-- can be
oxidized to --S(O)-- and/or --S(O).sub.2--. For yet another
example, unsaturated bond such as C.dbd.C or C.ident.C can be
reduced to saturated bond by hydrogenation. In some embodiments, a
primary amine or a secondary amine moiety (present on a substituent
group such as R.sup.1-R.sup.16, etc.) can be converted to amide,
sulfonamide, urea, or thiourea moiety by reacting it with an
appropriate reagent such as an acid chloride, a sulfonyl chloride,
an isocyanate, or a thioisocyanate compound. Thus, a compound of
Formula I (such as compound 3-3 of Scheme 3) having a substituent
which contains a functional group can be converted to another
compound of Formula I having a different substituent group.
[0161] As used herein, the term "reacting" refers to the bringing
together of designated chemical reactants such that a chemical
transformation takes place generating a compound different from any
initially introduced into the system. Reacting can take place in
the presence or absence of solvent.
Chemical Conditioning
[0162] In some embodiments, a method of preparing an array of
chemical compounds from a biological extract such as ginger oil is
provided.
[0163] The method of the invention, termed "chemical conditioning"
is generally applicable to all biological extracts, in particular,
natural plant extracts, common or medicinal. See e.g. US20080193574
and WO2008042755, each of which is incorporated herein by reference
in its entirety. Chemical conditioning is a method which produces
novel unnatural drug-like compounds from readily available natural
materials. In general, the "chemical conditioning" of natural
extracts coupled with pre-fractionation of the chemically
conditioned extracts facilitates successful biochemical screening
of extracts by destroying reactive natural compounds that generate
false positive results in biochemical assays. Chemical conditioning
produces novel lead-like and drug-like compounds and, the reductive
amination protocol described here can produce structurally diverse
nitrogen-containing products that are particularly lead-like and
drug-like.
[0164] In certain embodiments of the present invention, a method of
preparing chemical compounds from a biological extract is
exemplified in Scheme 5a below. According to the method, first, a
biological extract, e.g., a plant extract is provided, the
biological extract has one or more biological compounds, each
biological compound having one or more reactive electrophilic
groups. Next, the biological compounds in the biological extract
are reacted with an amine to incorporate the amine into the
biological compounds. Next, the biological compounds having the
incorporated amine are reacted with a reducing agent to reduce the
intermediate imine and enamine compounds and form one or more
nitrogen-containing chemical compounds. Thus, the resultant
nitrogen-containing chemical compounds are derivatives of the
biological compounds in the biological extract. In some
embodiments, the biological compounds in the biological extract are
compounds having ketones and aldehydes that are reacted with
various amines. This reaction is followed by hydride reduction of
the intermediate imines and enamines to provide secondary and
tertiary amines. The reaction of ketones and aldehydes with amines,
followed by reduction to form imines and enamines is known in the
art.
##STR00011##
R' and R'' represent a variety of substituents that make up a
biological compound; and R* represents a variety of substituent(s)
that, together with the nitrogen, make up an amine compound.
[0165] The chemical conditioning method described herein employs a
biological extract, using many different reagents, to efficiently
produce an array of nitrogen-containing chemical compounds. The
ready commercial availability of many low molecular weight amines
for use as inputs in the reductive amination sequence enables the
development of many different and structurally diverse central
nervous system druglike mixtures from the same natural extract.
Suitable amines for use in the present method are selected from the
group consisting of primary amines, secondary amines, cyclic
amines, pyrollidine, and amino acids. Suitable reducing agents for
use in the present method are selected from the group of hydride
reducing agents including but not limited to sodium borohydride,
sodium triacetoxyborohydride, and lithium aluminum hydride.
[0166] The method may further comprise quenching the reaction a
quenching agent, wherein the quenching agent is selected from but
not limited to the group consisting of sodium bicarbonate, sodium
carbonate, sodium sulfate, sodium sulfate decahydrate. The method
may also further comprise isolating one or more of the
nitrogen-containing chemical compounds, in a purified or unpurified
form. The resultant nitrogen-containing chemical may then be
screened or tested for biological activity.
[0167] The process of chemical conditioning by reductive amination
described herein destroys reactive electrophiles in the natural
extract, including ketones, as in the gingerols, and converts them
to chemically stable compounds such as amines. The resulting
conditioned extracts contain both natural compounds and novel
unnatural nitrogen-containing amine products that are potential
drug candidates. In the case of the extracts of gingerol, the
nitrogen-containing amine products are potential central nervous
system drugs.
[0168] For the purpose of this disclosure, the following terms have
the following meanings.
[0169] The term "biological compound" as used herein refers to a
chemical compound that occurs in nature.
[0170] The term "biological extract" as used herein refers to an
extract from a biological sample, such as a plant extract, or other
extract from organic matter, containing chemical compounds that
occur in nature.
[0171] The term "reactive electrophilic group" as used herein
refers to an atom or group of atoms that has the ability to react
with a nucleophile.
[0172] The term "nitrogen-containing derivative" as used herein
represents those derivatives containing a nitrogen atom, where the
nitrogen atom is a substitution another atom, such as oxygen in the
parent compound.
[0173] In one embodiment, a specific example of the chemical
conditioning process is shown in Scheme 5 below. Scheme 5 shows the
two-step reductive amination chemical conditioning protocol
performed on ginger oil and ginger oleoresin in accordance with one
embodiment of the method, wherein ginger oil or gingerol comprising
ketone 5-1 are converted to amine 5-4. According to the method
shown in Scheme 5, ginger oil (an extract of ginger containing
ketone 5-1 and other molecules occurring in natural ginger) is
reacted with amine 5-2 to form compound 5-3. Then, the resultant
compound 5-3 is then reduced, with a reducing agent such as a
borohydride, to from the nitrogen-containing compound 5-4 (the
reaction crude product also include other chemical compounds).
##STR00012##
[0174] In the next step of the method, amine 5-4 is
isolated/purified from the extract (the crude reaction product of
the 2-step reductive amination). The conditioned extracts can be
fractionated by flash chromatography. The fraction that contains
amine 5-4 can undergo further purification/isolation according to
the methods known to those in the art. Further isolation and
characterization of the fraction that contains amine 5-4 may
follow. The isolated amine 5-4 is tested for its biological
activity such as by those methods described hereinwith.
[0175] Some examples of benzylamine 5-2 used in the chemical
conditioning process of the invention shown in Scheme 5
include:
##STR00013##
[0176] New lead compounds generated by this chemical conditioning
method can also be prepared to the synthetic methods described
hereinwith.
[0177] In some embodiments, the derivatives of ginger oil such as
amine 5-4 possess beta-secretase inhibitory activity, and/or
inhibit amyloid production, amyloid assembly, the activity/effect
of Abeta oligomers on neurons (such as neurons in the brain),
amyloid aggregation, amyloid (including amyloid oligomer) binding,
or amyloid deposition. These compounds are useful therapeutic
agents for the treatment and prevention of cognitive decline,
amyloid production, neurodegeneration, and Alzhelmer's disease.
Methods
[0178] In some embodiments, the compounds of present invention
inhibit, treat, or abate (partially inhibit) binding of amyloid
(including Abeta oligomers) to neurons (such as neurons in the
brain) and are useful for the inhibition, treatment, and abatement
of cognitive decline and/or Alzheimer's disease. In some
embodiments, the compounds of present invention inhibit, treat, or
abate (partially inhibit) one or more of amyloid aggregation,
amyloid oligomer binding, and amyloid deposition. In some
embodiments, the compounds of present invention inhibit, treat, or
abate (partially inhibit) amyloid deposition. In some embodiments,
the compounds of present invention inhibit, treat, or abate
(partially inhibit) the activity/effect of Abeta oligomers on
neurons (such as neurons in the brain) and are useful for the
inhibition, treatment, and abatement of cognitive decline and/or
Alzheimer's disease. In some embodiments, the compounds of present
invention inhibit, treat, or abate (partially inhibit) the
activity/effect of Abeta oligomers on neurons (such as neurons in
the brain) via disruption of Abeta oligomers, inhibition of Abeta
oligomer binding to neurons, and/or counteraction of signal
transduction mechanisms of action initiated by Abeta oligomer
binding.
[0179] In some embodiments, the compounds show activity in a
beta-secretase assay and are useful for the inhibition, treatment,
and abatement of cognitive decline and Alzheimer's disease. In some
embodiments the derivative of ginger oil is a compound in purified
and Isolated form (for example, with a purity of greater than 80%,
85%, 90%, 95%, 98%, or 99% by weight). The compounds and methods
described herein may be used to treat one or more symptoms of
cognitive decline and/or Alzheimer's disease such as memory loss,
confusion, Impaired judgment, personality changes, disorientation,
and loss of language skills. Further, the compounds and methods
described herein may be useful in inhibiting, treating, and/or
abating cognitive decline and/or Alzheimer's disease by restoring
long term potentiation, and/or inhibiting, treating, or abatement
of one or both of neurodegeneration and general amyloidosis, more
specifically, by inhibiting, treating, or abatement of one or more
of amyloid production, amyloid assembly, amyloid aggregation,
amyloid (including amyloid oligomer) binding, and amyloid
deposition.
[0180] In some embodiments, compounds of the invention can inhibit,
treat, or abate one or more of amyloid production, amyloid
assembly, amyloid aggregation, amyloid oligomer binding, and
amyloid deposition. In some embodiments, compounds of the invention
can restore long term potentiation, inhibit, treat, or abate one or
both of neurodegeneration and general amyloidosis.
[0181] In some embodiments, compounds of present invention inhibit,
treat, or abate (partially inhibit) one or more of amyloid
aggregation, amyloid oligomer binding, and amyloid deposition. In
some embodiments, the compounds of present invention inhibit (or
partially inhibit) amyloid deposition. In some embodiments, the
compounds of present invention Inhibit, treat, or abate (partially
inhibit) binding of amyloid (including Abeta oligomers) to neurons
(such as neurons in the brain). In some embodiments, the compounds
of present invention are useful for the inhibition, treatment, and
abatement of cognitive decline and/or Alzheimer's disease.
[0182] In some embodiments, compounds of the invention can inhibit
activity of beta-secretase. In some embodiments, compounds of the
invention can be used in methods of inhibiting activity of
beta-secretase by contacting the beta-secretase with any one or
more of the compounds or compositions described herein.
[0183] Another aspect of the present invention pertains to methods
of treating cognitive decline and/or Alzheimer's disease in an
individual (e.g., patient) by administering to the individual a
therapeutically effective amount or dose of a compound of the
present invention or a pharmaceutical composition thereof.
[0184] Treatment of the diseases/disorders herein includes treating
one or more symptoms associated with the diseases/disorders, for
example, symptoms of cognitive decline and/or Alzheimer's
disease.
[0185] As used herein, the term "contacting" refers to the bringing
together of indicated moieties in an in vitro system or an in vivo
system. For example, "contacting" a beta-secretase or a neuron cell
(or a neuron cell in the presence of one or more of beta-amyloid
oligomers) with a compound of the invention includes the
administration of a compound of the present invention to an
individual or patient, such as a human, having a beta-secretase or
a neuron cell, as well as, for example, introducing a compound of
the invention into a sample containing a cellular or purified
preparation containing the a beta-secretase or a neuron cell (or a
neuron cell in the presence of one or more of beta-amyloid
oligomers).
[0186] As used herein, the term "individual" or "patient," used
interchangeably, refers to any animal, including mammals,
preferably mice, rats, other rodents, rabbits, dogs, cats, swine,
cattle, sheep, horses, or primates, and most preferably humans.
[0187] As used herein, the phrase "therapeutically effective
amount" refers to the amount of active compound or pharmaceutical
agent that elicits the biological or medicinal response that is
being sought in a tissue, system, animal, individual or human by a
researcher, veterinarian, medical doctor or other clinician.
[0188] As used herein, the term "treating" or "treatment" refers to
one or more of (1) preventing the disease; for example, preventing
a disease, condition or disorder in an individual who may be
predisposed to the disease, condition or disorder but does not yet
experience or display the pathology or symptomotology of the
disease; (2) inhibiting/retarding the disease; for example,
inhibiting/retarding a disease, condition or disorder in an
individual who is experiencing or displaying the pathology or
symptomotology of the disease, condition or disorder; and (3)
ameliorating the disease; for example, ameliorating a disease,
condition or disorder in an individual who is experiencing or
displaying the pathology or symptomotology of the disease,
condition or disorder (i.e., reversing the pathology and/or
symptomotology) such as decreasing the severity of disease or
completely eliminating/curing the disease. As used herein, treating
a disease further includes treating one or more symptoms associated
with the disease.
Combination Therapies
[0189] In certain embodiments, one or more additional
pharmaceutical agents for treatment of cognitive decline and/or
Alzheimer's disease can be used in combination with the compounds
of the present invention for treatment of cognitive decline and/or
Alzheimer's disease. The one or more additional pharmaceutical
agents can be administered to a patient simultaneously or
sequentially.
Pharmaceutical Formulations and Dosage Forms
[0190] In certain embodiments, the compounds of the Invention can
be administered in the form of pharmaceutical compositions. These
compositions can be prepared in a manner well known in the
pharmaceutical art, and can be administered by a variety of routes,
depending upon whether local or systemic treatment Is desired and
upon the area to be treated. Administration may be topical
(including transdermal, epidermal, ophthalmic and to mucous
membranes including intranasal, vaginal and rectal delivery),
pulmonary (e.g., by inhalation or insufflation of powders or
aerosols, including by nebulizer; intratracheal or intranasal),
oral or parenteral. Parenteral administration includes intravenous,
intraarterial, subcutaneous, intraperitoneal intramuscular or
injection or infusion; or intracranial, e.g., intrathecal or
intraventricular, administration. Parenteral administration can be
in the form of a single bolus dose, or may be, for example, by a
continuous perfusion pump. Pharmaceutical compositions and
formulations for topical administration may include transdermal
patches, ointments, lotions, creams, gels, drops, suppositories,
sprays, liquids and powders. Conventional pharmaceutical carriers,
aqueous, powder or oily bases, thickeners and the like may be
necessary or desirable. Coated condoms, gloves and the like may
also be useful.
[0191] Embodiments of this invention also include pharmaceutical
compositions which contain, as the active ingredient, one or more
of the compounds of the invention above in combination with one or
more pharmaceutically acceptable carriers (excipients). In making
the compositions of the Invention, the active ingredient is
typically mixed with an excipient, diluted by an excipient or
enclosed within such a carrier in the form of, for example, a
capsule, sachet, paper, or other container. When the excipient
serves as a diluent, it can be a solid, semi-solid, or liquid
material, which acts as a vehicle, carrier or medium for the active
ingredient. Thus, the compositions can be in the form of tablets,
pills, powders, lozenges, sachets, cachets, elixirs, suspensions,
emulsions, solutions, syrups, aerosols (as a solid or in a liquid
medium), ointments containing, for example, up to 10% by weight of
the active compound, soft and hard gelatin capsules, suppositories,
sterile injectable solutions, and sterile packaged powders.
[0192] In preparing a formulation, the active compound can be
milled to provide the appropriate particle size prior to combining
with the other ingredients. If the active compound is substantially
insoluble, it can be milled to a particle size of less than 200
mesh. If the active compound is substantially water soluble, the
particle size can be adjusted by milling to provide a substantially
uniform distribution in the formulation, e.g. about 40 mesh.
[0193] The compounds of the invention may be milled using known
milling procedures such as wet milling to obtain a particle size
appropriate for tablet formation and for other formulation types.
Finely divided (nano particulate) preparations of the compounds of
the invention can be prepared by processes known in the art, for
example see International Patent Application No. WO
2002/000196.
[0194] Some examples of suitable excipients include lactose,
dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,
calcium phosphate, alginates, tragacanth, gelatin, calcium
silicate, microcrystalline cellulose, polyvinylpyrrolidone,
cellulose, water, syrup, and methyl cellulose. The formulations can
additionally include: lubricating agents such as talc, magnesium
stearate, and mineral oil; wetting agents; emulsifying and
suspending agents; preserving agents such as methyl- and
propylhydroxy-benzoates; sweetening agents; and flavoring agents.
The compositions of the invention can be formulated so as to
provide quick, sustained or delayed release of the active
ingredient after administration to the patient by employing
procedures known in the art.
[0195] The compositions can be formulated in a unit dosage form,
each dosage containing from about 5 to about 1000 mg (1 g), more
usually about 100 to about 500 mg, of the active ingredient. 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.
[0196] The active compound can be effective over a wide dosage
range and can be generally administered in a pharmaceutically
effective amount. For example, the dosage of the active compounds
of the invention as employed for the treatment of a patient in need
thereof (such as an adult human) may range from 0.1 to 3000 mg per
day, depending on the route and frequency of administration. Such a
dosage corresponds to 0.001 to 50 mg/kg per day. In some
embodiments, the dosage of the active compounds of the invention as
employed for the treatment of a patient in need thereof (such as an
adult human) may range from 1 to 2000 mg per day, from 1 to 1000 mg
per day, from 10 to 1000 mg per day, or from to 500 mg per day. It
will be understood, however, that the amount of the compound
actually administered will usually be determined by a physician,
according to 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.
[0197] For preparing solid compositions such as tablets, the
principal active ingredient can be mixed with a pharmaceutical
excipient to form a solid pre-formulation composition containing a
homogeneous mixture of a compound of the present invention. When
referring to these pre-formulation compositions as homogeneous, the
active ingredient is typically dispersed evenly throughout the
composition so that the composition can be readily subdivided into
equally effective unit dosage forms such as tablets, pills and
capsules. This solid pre-formulation is then subdivided into unit
dosage forms of the type described above containing from, for
example, about 0.1 to about 1000 mg of the active ingredient of the
present invention.
[0198] The tablets or pills of the present invention can be coated
or otherwise compounded to provide a dosage form affording the
advantage of prolonged action. For example, the tablet or pill can
comprise an inner dosage and an outer dosage component, the latter
being in the form of an envelope over the former. The two
components can be separated by an enteric layer which serves to
resist disintegration in the stomach and permit the inner component
to pass intact into the duodenum or to be delayed in release. A
variety of materials can be used for such enteric layers or
coatings, such materials including a number of polymeric acids and
mixtures of polymeric acids with such materials as shellac, cetyl
alcohol, and cellulose acetate.
[0199] The liquid forms in which the compounds and compositions of
the present invention can be incorporated for administration orally
or by injection include aqueous solutions, suitably flavored
syrups, aqueous or oil suspensions, and flavored emulsions with
edible oils such as cottonseed oil, sesame oil, coconut oil, or
peanut oil, as well as elixirs and similar pharmaceutical
vehicles.
[0200] Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable, aqueous
or organic solvents, or mixtures thereof, and powders. The liquid
or solid compositions may contain suitable pharmaceutically
acceptable excipients as described supra. In some embodiments, the
compositions are administered by the oral or nasal respiratory
route for local or systemic effect. Compositions in can be
nebulized by use of inert gases. Nebulized solutions may be
breathed directly from the nebulizing device or the nebulizing
device can be attached to a face masks tent, or intermittent
positive pressure breathing machine. Solution, suspension, or
powder compositions can be administered orally or nasally from
devices which deliver the formulation in an appropriate manner.
[0201] The amount of compound or composition administered to a
patient will vary depending upon what is being administered, the
purpose of the administration, such as prophylaxis or therapy, the
state of the patient, the manner of administration, and the like.
In therapeutic applications, compositions can be administered to a
patient already suffering from a disease in an amount sufficient to
cure or at least partially arrest the symptoms of the disease and
its complications. Effective doses will depend on the disease
condition being treated as well as by the judgment of the attending
clinician depending upon factors such as the severity of the
disease, the age, weight and general condition of the patient, and
the like.
[0202] The compositions administered to a patient can be in the
form of pharmaceutical compositions described above. These
compositions can be sterilized by conventional sterilization
techniques, or may be sterile filtered. Aqueous solutions can be
packaged for use as is, or lyophilized, the lyophilized preparation
being combined with a sterile aqueous carrier prior to
administration. The pH of the compound preparations typically will
be between 3 and 11, more preferably from 5 to 9 and most
preferably from 7 to 8. It will be understood that use of certain
of the foregoing excipients, carriers, or stabilizers will result
in the formation of pharmaceutical salts.
[0203] The therapeutic dosage of the compounds of the present
invention can vary according to, for example, the particular use
for which the treatment is made, the manner of administration of
the compound, the health and condition of the patient, and the
judgment of the prescribing physician. The proportion or
concentration of a compound of the invention in a pharmaceutical
composition can vary depending upon a number of factors including
dosage, chemical characteristics (e.g., hydrophobicity), and the
route of administration. For example, the compounds of the
invention can be provided in an aqueous physiological buffer
solution containing about 0.1 to about 10% w/v of the compound for
parenteral administration. Some typical dose ranges are from about
1 g/kg to about 1 g/kg of body weight per day. In some embodiments,
the dose range is from about 0.01 mg/kg to about 100 mg/kg of body
weight per day. The dosage is likely to depend on such variables as
the type and extent of progression of the disease or disorder, the
overall health status of the particular patient, the relative
biological efficacy of the compound selected, formulation of the
excipient, and its route of administration. Effective doses can be
extrapolated from dose-response curves derived from in vitro or
animal model test systems.
[0204] The compositions of the invention can further include one or
more additional pharmaceutical agents such as a chemotherapeutic,
steroid, anti-inflammatory compound, or immunosuppressant, examples
of which are listed hereinabove.
Labeled Compounds and Assay Methods
[0205] Another aspect of the present invention relates to labeled
compounds of the invention (radio-labeled, fluorescent-labeled,
etc.) that would be useful not only in radio-imaging but also in
assays, both in vitro and in vivo, for localizing and quantitating
the enzyme in tissue samples, including human, and for identifying
ligands by inhibition binding of a labeled compound. Accordingly,
the present invention includes enzyme assays that contain such
labeled compounds.
[0206] Embodiments of the present invention further includes
isotopically-labeled compounds of the invention. An "isotopically"
or "radio-labeled" compound is a compound of the invention where
one or more atoms are replaced or substituted by an atom having an
atomic mass or mass number different from the atomic mass or mass
number typically found in nature (i.e., naturally occurring).
Suitable radionuclides that may be incorporated in compounds of the
present invention include but are not limited to .sup.2H (also
written as D for deuterium), .sup.3H (also written as T for
tritium), .sup.11C, .sup.13C, .sup.14C, .sup.13N, .sup.15N
.sup.15O, .sup.17O, .sup.18O, .sup.18F, .sup.35S, .sup.36Cl,
.sup.82Br, .sup.75Br, .sup.76Br, .sup.77Br, .sup.123I, .sup.124I,
.sup.125I and .sup.131I. The radionuclide that is incorporated in
the radio-labeled compounds will depend on the specific application
of that radio-labeled compound. For example, for in vitro receptor
labeling and competition assays, compounds that incorporate
.sup.3H, .sup.14C, .sup.82Br, .sup.125I, .sup.131I, .sup.35S or
will generally be most useful. For radio-imaging applications
.sup.11C, .sup.18F, .sup.125I, .sup.123I, .sup.124I, .sup.131I,
.sup.75Br, .sup.76Br or .sup.77Br will generally be most
useful.
[0207] It is understood that a "radio-labeled compound" is a
compound that has incorporated at least one radionuclide. In some
embodiments the radionuclide is selected from .sup.3H, .sup.14C,
.sup.125I, .sup.35S and .sup.82Br.
[0208] In some embodiments, the labeled compounds of the present
invention contain a fluorescent label.
[0209] Synthetic methods for incorporating radio-isotopes and
fluorescent labels into organic compounds are well known in the
art.
[0210] A labeled compound of the invention (radio-labeled,
fluorescent-labeled, etc.) can be used in a screening assay to
identify/evaluate compounds. For example, a newly synthesized or
identified compound (i.e., test compound) which is labeled can be
evaluated for its ability to bind a beta-secretase or a neuron cell
(or a neuron cell in the presence of one or more of beta-amyloid
oligomers) by monitoring its concentration variation when
contacting with the beta-secretase or the neuron cell (or the
neuron cell in the presence of one or more of beta-amyloid
oligomers), through tracking the labeling. For another example, a
test compound (labeled) can be evaluated for its ability to reduce
binding of another compound which is known to bind to
beta-secretase or neuron cell (i.e., standard compound).
Accordingly, the ability of a test compound to compete with the
standard compound for binding to the beta-secretase or the neuron
cell directly correlates to its binding affinity. Conversely, in
some other screening assays, the standard compound is labeled and
test compounds are unlabeled. Accordingly, the concentration of the
labeled standard compound is monitored in order to evaluate the
competition between the standard compound and the test compound,
and the relative binding affinity of the test compound is thus
ascertained.
Kits
[0211] Embodiments of the present invention also includes
pharmaceutical kits useful, for example, in the treatment or
prevention of cognitive decline and/or Alzheimer's disease which
include one or more containers containing a pharmaceutical
composition comprising a therapeutically effective amount of a
compound of the invention. Such kits can further include, if
desired, one or more of various conventional pharmaceutical kit
components, such as, for example, containers with one or more
pharmaceutically acceptable carriers, additional containers, etc.,
as will be readily apparent to those skilled in the art.
Instructions, either as inserts or as labels, indicating quantities
of the components to be administered, guidelines for
administration, and/or guidelines for mixing the components, can
also be included in the kit.
[0212] The invention will be described in greater detail by way of
specific examples. The following examples are offered for
illustrative purposes, and are not intended to limit the invention
in any manner. Those of skill in the art will readily recognize a
variety of non-critical parameters which can be changed or modified
to yield essentially the same results. Certain compounds of the
Examples were found to be inhibit, treat, or abate one or more of
amyloid production, amyloid assembly, the activity/effect of Abeta
oligomers on neurons (such as neurons in the brain), amyloid
aggregation, amyloid oligomer binding, and amyloid deposition
according to one or more of the assays provided herein. In some
further embodiments, certain compounds of the Examples were found
to be Inhibit, treat, or abate one or more of the activity/effect
of Abeta oligomers on neurons (such as neurons in the brain),
amyloid aggregation, amyloid (including amyloid oligomer) binding,
and amyloid deposition according to one or more of the assays
provided herein.
[0213] In some embodiments, the compound of invention has an
IC.sub.50 value of less than 100 .mu.M, 50 .mu.M, 20 .mu.M, 15
.mu.M, 10 .mu.M, 5 .mu.M, 1 .mu.M, 500 nM, 100 nM, 50 nM, or 10 nM
with respect to inhibition of one or more of the activity/effect of
Abeta oligomers on neurons (such as neurons in the brain), amyloid
aggregation, amyloid (including amyloid oligomer) binding, and
amyloid deposition. In some embodiments, the compound of invention
has an IC.sub.50 value of less than 100 .mu.M, 50 .mu.M, 20 .mu.M,
15 .mu.M, 10 .mu.M, 5 .mu.M, 1 .mu.M, 500 nM, 100 nM, 50 nM, or 10
nM with respect to inhibition the activity/effect of Abeta
oligomers on neurons (such as neurons in the brain).
[0214] In some embodiments, percentage inhibition of the compound
of invention to one or more of the activity/effect of Abeta
oligomers on neurons (such as neurons in the brain), amyloid
aggregation, amyloid (including amyloid oligomer) binding, and
amyloid deposition was measured at a concentration of from 10 nM to
10 .mu.M. In some embodiments, the percentage inhibition measured
is about 1% to about 20%, about 20% to about 50%, about 1% to about
50%, or about 1% to about 80%.
[0215] The invention may be appreciated in certain aspects with
reference to the following examples, offered by way of
illustration, not by way of limitation. Materials, reagents and the
like to which reference is made in the following examples are
obtainable from commercial sources, unless otherwise noted.
EXAMPLES
Materials And Methods
Ginger Oil
[0216] The light oil extract from ginger root was obtained by
supercritical CO.sub.2 extraction.
Ginger Oleoresin
[0217] The heavy remainder oil was obtained following extraction of
ginger root by supercritical CO.sub.2 extraction.
Example 1
A. Conditioned Extraction of Ginger Oil: Reaction of Ginger Oil
with 4-Chlorobenzylamine Followed by Reduction with Sodium
Borohydride in Methanol and by Fractioning Using Column
Chromatography
[0218] Ginger oil (10 g) was dissolved in toluene (250 mL) and
4-chlorobenzylamine (3.4 g) was added. The mixture was maintained
under an atmosphere of nitrogen and heated at reflux with removal
of water by Dean-Stark distillation for 16 hours. At this time the
Dean-Stark trap was removed and the reaction mixture was cooled to
00.degree. C. on an ice bath. A solution of sodium borohydride (10
g) in methanol (100 mL) was added portion-wise over 30 minutes with
vigorous stirring. When the addition was complete the mixture was
heated to reflux for 16 hours. At this time the reaction mixture
was cooled to room temperature and poured into saturated aqueous
sodium bicarbonate solution (300 mL). The resulting mixture was
concentrated by rotary evaporation and the aqueous residue was
partitioned between water and chloroform. The chloroform layer was
dried over anhydrous sodium sulfate and then filtered and
concentrated. The products were then fractionated using silica gel
column chromatography employing a gradient from 100% chloroform to
chloroform:methanol (5:1). The product was detected in the
relatively polar fractions by thin layer chromatography (TLC).
Product-containing fractions were combined and concentrated then
dried under high vacuum overnight to provide a light brown oil
(0.672 g). .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 7.30-7.24 (m,
4H), 6.81 (d, J=7.8 Hz, 1H), 6.66-6.62 (m, 2H), 4.25 (br s, 2H),
3.82 (s, 3H), 3.82 (d, J=13.2 Hz, 1H), 3.72 (d, J=13.2 Hz, 1H),
2.73 (m, 1H), 2.66-2.51 (m, 1H), 1.86-1.78 (m, 1H), 1.72-1.63 (m,
1H), 1.62-1.51 (m, 1H), 1.17 (d, J=6.3 Hz, 3H). .sup.13C NMR (125
MHz, CDCl.sub.3) .delta.: 146.6, 143.8, 133.9 132.8, 129.9, 129.7,
128.6, 120.8, 114.5, 110.9, 55.8, 51.9, 50.2, 38.5, 31.9, 31.6,
29.7, 26.9, 22.6, 19.9. Analytic MS(M+H*): m/z 320.2.
[0219] .sup.1H NMR (500 MHz, CD.sub.3OD) .delta.: 7.10-7.30 (m,
4H), 6.63 (br s, 1H), 6.58 (m, 1H), 6.48 (m, 1H), 3.68 (s, 3H),
3.65 (m, 1H), 3.58 (m, 1H), 2.57 (m, 1H), 2.50 (m, 1H), 2.35 (m,
1H), 1.73 (m, 1H), 1.49 (m, 1H), 1.04 (d, 3H). .sup.13C NMR (125
MHz, CD.sub.3OD) .delta.: 147.5, 145.3, 137.2, 133.3 132.6, 129.9,
128.1, 120.4, 114.7, 111.6, 54.9, 51.3, 49.2, 37.7, 31.5, 18.0.
[0220] The weight ratio of Ginger oil to 4-chlorobenzylamine used
in the reductive amination is about 3:1 (from 2.7:1 to 3.3:1). The
chemical shift measure by .sup.1H NMR may vary, for example, up to
0.2 ppm. The chemical shift measure by .sup.13H NMR may vary, for
example, up to 0.5 ppm. The analytical Mass Spectrum may have an
experimental error of +/-0.3.
Purity Determination
[0221] The purity of the product was measure by HPLC. The major
peak of retention time of 2.22 minutes indicating greater than
about 80%, 85%, 90, or 95% of purity. The HPLC conditions used are
as follows.
HPLC Conditions:
[0222] Mobile Phase A: 13.3 mM ammonium formate/6.7 mM formic acid
in water
[0223] Mobile Phase B: 6 mM ammonium formate/3 mM formic acid in
water/CH.sub.3CN (1/9, v/v)
[0224] Column: Synergi Fusion-RP 100A Mercury, 2.times.20 mm, 2.5
micron [0225] (Phenomenex Part No 00M-4423-B0_CE)
[0226] Gradient Program: RT=2.22 minutes
TABLE-US-00001 Time, minute % Phase B Flow rate, ml/min 0 100 0.5 1
100 0.5 2.5 40 0.5 3.4 40 0.5 3.5 100 0.5 4.5 100 0.5
[0227] The purity of the product was also measure by .sup.1H NMR
indicating it to be a single compound of a purity of greater than
90% or 95%.
[0228] The structure of Compound Example 1 (or Example Compound 1)
Is determined to be as follows.
##STR00014##
Compound Example 1
4-(3-(4-chlorobenzylamino)butyl)-2-methoxyphenol
B. Synthesis by Reductive Amination
##STR00015##
[0230] Vanillylacetone (5.00 g, 25.7 mmol) was dissolved in toluene
(250 mL) and 4-chlorobenzylamine (3.82 g, 27.0 mmol) was added. The
mixture was maintained under an atmosphere of nitrogen and heated
at reflux with removal of water by Dean-Stark distillation for 16
hours. At this time the Dean-Stark trap was removed and the
reaction mixture was cooled to 0.degree. C. on an ice bath. A
solution of sodium borohydride (5 g) in methanol (100 mL) was added
portion-wise over 30 minutes with vigorous stirring. When the
addition was complete the mixture was heated at reflux for 16
hours. At this time the reaction mixture was cooled to room
temperature and poured into saturated aqueous sodium bicarbonate
solution (300 mL). The resulting mixture was concentrated by rotary
evaporation and the aqueous residue was partitioned between water
and chloroform. The chloroform layer was dried over anhydrous
sodium sulfate and then filtered and concentrated. The product was
then purified using silica gel column chromatography employing a
mobile phase of 5% ammonia-methanol in chloroform.
Product-containing fractions were combined and concentrated then
dried under high vacuum overnight to provide a light brown oil
(6.16 g, 75%). .sup.1H NMR, .sup.13C NMR, and Mass Spectrum of the
product were substantially the same as those in Example 1, A (made
by the Conditioned Extraction method).
Example 2
A. Conditioned Extraction of Ginger Oil: Reaction of Ginger Oil
with 4-Trifluoromethylbenzylamine Followed by Reduction with Sodium
Borohydride in Methanol and by Fractioning Using Column
Chromatography
[0231] Ginger oil (10 g) was dissolved in toluene (250 mL) and
4-trifluoromethylbenzylamine (3.5 g) was added. The mixture was
maintained under an atmosphere of nitrogen and heated at reflux
with removal of water by Dean-Stark distillation for 16 hours. At
this time the Dean-Stark trap was removed and the reaction mixture
was cooled to 0.degree. C. on an ice bath. A solution of sodium
borohydride (10 g) in methanol (100 mL) was added portion-wise over
30 minutes with vigorous stirring. When the addition was complete
the mixture was heated to reflux for 16 hours. At this time the
reaction mixture was cooled to room temperature and poured into
saturated aqueous sodium bicarbonate solution (300 mL). The
resulting mixture was concentrated by rotary evaporation and the
aqueous residue was partitioned between water and chloroform. The
chloroform layer was dried over anhydrous sodium sulfate and then
filtered and concentrated. The products were then fractionated
using silica gel column chromatography employing a gradient from
100% chloroform to chloroform:methanol (5:1). The product was
detected in the relatively polar fractions by thin layer
chromatography (TLC). Product-containing fractions were combined
and concentrated then dried under high vacuum overnight to provide
a light brown oil (0.761 g). .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta.: 7.57 (d, J=7.8 Hz, 2H), 7.43 (d, J=7.9 Hz, 2H), 6.82 (d,
J=7.3 Hz, 1H), 6.65 (m, 2H), 5.16-4.42 (br s, 2H), 3.90 (d, J=13.7
Hz, 1H), 3.84 (s, 3H), 3.80 (d, J=13.7 Hz, 1H), 2.76-2.70 (m, 1H),
2.67-2.55 (m, 2H), 1.84-1.77 (m, 1H), 1.69-1.63 (m, 1H), 1.17 (d,
J=6.3 Hz, 3H). .sup.13C NMR (125 MHz, CDCl.sub.3) .delta.: 146.7,
144.6, 143.9, 134.0, 129.1, 128.4, 127.5, 125.4, 125.3, 123.2,
120.8, 114.6, 111.0, 55.7, 52.1, 50.6, 38.8, 32.0, 20.1. MS (CI)
m/z 353 (M.sup.+).
[0232] The weight ratio of ginger oil to
4-trifluoromethylbenzylamine used in the reductive amination is
about 3:1 (from 2.7:1 to 3.3:1). The chemical shift measure by
.sup.1H NMR may vary, for example, up to 0.2 ppm. The chemical
shift measure by .sup.13H NMR may vary, for example, up to 0.5 ppm.
The analytical Mass Spectrum may have an experimental error of
+/-0.3.
Purity Determination
[0233] The purity of the product was measure by HPLC. The major
peak of retention time of 2.22 minutes indicating greater than
about 80%, 85%, 90, or 95% of purity. The HPLC conditions used are
as follows.
HPLC Conditions:
[0234] Mobile Phase A: 13.3 mM ammonium formate/6.7 mM formic acid
in water Mobile Phase B: 6 mM ammonium formate/3 mM formic acid in
water/CH.sub.3CN (1/9, v/v)
[0235] Column: Synergi Fusion-RP 100A Mercury, 2.times.20 mm, 2.5
micron [0236] (Phenomenex Part No 00M-4423-B0_CE)
[0237] Gradient Program: RT=2.22 minutes
TABLE-US-00002 Time, minute % Phase B Flow rate, ml/min 0 100 0.5 1
100 0.5 2.5 40 0.5 3.4 40 0.5 3.5 100 0.5 4.5 100 0.5
[0238] The purity of the product was also measure by .sup.1H NMR
indicating it to be a single compound of a purity of greater than
90% or 95%.
[0239] The structure of Compound Example 2 (or Example Compound 2)
is determined to be as follows.
##STR00016##
Compound Example 2
4-(3-(4-(trifluoromethyl)benzylamino)butyl)-2-methoxyphenol
B. Synthesis by Reductive Amination
##STR00017##
[0241] Vanillylacetone (5.00 g, 25.7 mmol) was dissolved in toluene
(250 mL) and 4-trifluoromethylbenzylamine (4.73 g, 27.0 mmol) was
added. The mixture was maintained under an atmosphere of nitrogen
and heated at reflux with removal of water by Dean-Stark
distillation for 16 hours. At this time the Dean-Stark trap was
removed and the reaction mixture was cooled to 0.degree. C. on an
ice bath. A solution of sodium borohydride (5 g) in methanol (100
mL) was added portion-wise over 30 minutes with vigorous stirring.
When the addition was complete the mixture was heated at reflux for
16 hours. At this time the reaction mixture was cooled to room
temperature and poured into saturated aqueous sodium bicarbonate
solution (300 mL). The resulting mixture was concentrated by rotary
evaporation and the aqueous residue was partitioned between water
and chloroform. The chloroform layer was dried over anhydrous
sodium sulfate and then filtered and concentrated. The product was
then purified using silica gel column chromatography employing a
mobile phase of 5% ammonia-methanol in chloroform.
Product-containing fractions were combined and concentrated then
dried under high vacuum overnight to provide a light brown oil
(6.72 g, 74%). .sup.1H NMR, .sup.13C NMR, and Mass Spectrum of the
product were substantially the same as those in Example 2, A (made
by the Conditioned Extraction method).
Example AA
Exocytosis Assay/MTT Assay
[0242] Primary neurons from E18 Sprague-Dawley rat embryos are
plated at optimized concentrations in 384 well plates in NB media
(Invitrogen). Neurons are maintained in cultures for 3 weeks, with
twice weekly feeding of NB media with N.sub.2 supplement
(Invitrogen). A test compound is added to cells, followed by
addition of Vehicle or Abeta oligomer preparations (1.5 .mu.M), and
incubated for 1 to 24 hr at 37.degree. C. in 5% CO.sub.2. MTT
reagent (3-(4,5-dimethyithizaol-2yl)-2,5diphenyl tetrazolium
bromide) (Roche Molecular Biochemicals) is reconstituted in
phosphate buffered saline to 5 mg/mL. 10 .mu.L of MTT labeling
reagent is added to each well and incubated at 37.degree. C. for 1
h, then imaged.
[0243] Each assay plate is formatted so that compounds are tested
with and without Abeta on each plate. This design eliminates toxic
or metabolically active compounds early on in the screening cascade
(at the level of the primary screen). Statistical performance of
the screening plate layout are assessed, screening will be
initiated if the current performance is maintained.
[0244] Similar procedures for exocytosis assays/MTT assays can be
found in the literature. See e.g., Liu Y, et. al., Detecting
bioactive amyloid beta peptide species in Alzheimer's disease. J
Neurochem. 2004 November; 91(3):648-56; Liu Y, and Schubert D.
"Cytotoxic amyloid peptides inhibit cellular
3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyitetrazolium bromide (MTT)
reduction by enhancing MTT formazan exocytosis." J Neurochem. 1997
December; 69(6):2285-93; and Liu Y, and Schubert D. "Treating
Alzheimer's disease by inactivating bioactive amyloid beta peptide"
Curr. Alzheimer Res. 2006 April; 3(2):129-35.
Experimental Controls:
[0245] Abeta 1-42 oligomers made according to published methods
methods [See e.g. Dahlgren et al., "Oligomeric and fibrillar
species of amyloid-beta peptides differentially affect neuronal
viability" J Biol Chem. 2002 Aug. 30; 277(35):32046-53. Epub 2002
Jun. 10; LeVine H 3rd. "Alzheimer's beta-peptide oligomer formation
at physiologic concentrations" Anal Biochem. 2004 Dec. 1;
335(1):81-90; Shrestha et. al, "Amyloid beta peptide adversely
affects spine number and motility in hippocampal neurons" Mol Cell
Neurosci. 2006 November; 33(3):274-82. Epub 2006 Sep. 8; Puzzo et
al., "Amyloid-beta peptide inhibits activation of the nitric
oxide/cGMP/cAMP-responsive element-binding protein pathway during
hippocampal synaptic plasticity" J Neurosci. 2005 Jul. 20;
25(29):6887-97; Barghorn et al., "Globular amyloid beta-peptide
oligomer--a homogenous and stable neuropathological protein in
Alzheimer's disease" J Neurochem. 2005 November; 95(3):834-47. Epub
2005 Aug. 31; Johansson et al., Physiochemical characterization of
the Alzheimer's disease-related peptides A beta 1-42 Arctic and A
beta 1-42 wt. FEBS J. 2006 June; 2 73(12):2618-30] as well as
brain-derived Abeta oligomers (See e.g. Walsh et al., Naturally
secreted oligomers of amyloid beta protein potently inhibit
hippocampal long-term potentiation in vivo. Nature (2002). 416,
535-539; Lesne et al., A specific amyloid-beta protein assembly in
the brain impairs memory. Nature. 2006 Mar. 16; 440(7082):352-7;
Shankar et al, Amyloid-beta protein dimers isolated directly from
Alzheimer's brains impair synaptic plasticity and memory. Nat Med.
2008 August; 14(8):837-42. Epub 2008 Jun. 22) constitute the
positive controls. Negative controls include vehicle-treated
neurons as well as neurons treated with 28 .mu.M concentrations of
memantine. Memantine produces 50% inhibition of oligomer effects at
this dose. These controls, on each plate, serve as normalization
tools to calibrate assay performance on a plate-by-plate basis.
Primary Neuronal Cultures
[0246] Optimal cell density is determined based on cellular
response to Abeta oligomers using the exocytosis assay as a
readout, and immunohistochemical analysis of the relative
proportion of glia to neurons in the cultures. Cultures are
monitored on a weekly basis with immunohistochemistry and image
processing-based quantification to monitor the percentage of the
cultures that are neurons vs. glia (Glial cells). Cultures
containing more than 20% glia (positive for GFAP) vs. neurons
(staining positively with antibodies directed against MAP2) at the
screening age of 21 days in vitro (21 DIV) are rejected.
Abeta Oligomer Preparations
[0247] Human amyloid peptide 1-42 is obtained from California
Peptide, with lot-choice contingent upon quality control analysis.
Quality controls of oligomer preparations consist of Westerns to
determine oligomer size ranges and relative concentrations, and the
MTT assay to confirm exocytosis acceleration without toxicity.
Toxicity is monitored in each image-based assay via quantification
of nuclear morphology visualized with the DNA binding dye DAPI
(Invitrogen). Nuclei that are fragmented are considered to be in
late stage apoptosis (Majno and Joris '95). Peptide lots producing
unusual peptide size ranges or significant toxicity at a standard
1.5 uM concentration on neurons are rejected. Plate-based
controls--The assay optimization will be complete when reformatted
plates achieve a minimum of statistically significant two-fold
separation between vehicle and Abeta oligomer-treated neurons
(p<0.01, Student's t-test, unequal variance) on a routine basis,
with no more than 10% CV between plates, equivalent to its current
performance.
Statistical Software and Analysis:
[0248] Data handling and analysis are accomplished by Cellomics VTI
image analysis software and STORE automated database software.
Because of the low dynamic range and neuronal well-to-well
variability after three weeks in culture, statistical comparisons
are made via pairwise Tukey-Kramer analysis to determine the
significance of the separation between compound+Abeta oligomers
from Abeta alone, and between compound alone from vehicle. These
statistics are more akin to what is seen in animal behavioral
testing than the z' statistic that has been used for the past two
decades in high throughput screening. The ability of mature primary
neurons to more closely approximate the electrophysiologically
mediated signal transduction network of the adult brain justifies
this screening strategy. Power analysis will be set for a number of
replicate screening wells that will minimize false negatives (e.g
N=4) and shift the burden of distinguishing false positives from
actual hits to dose-response confirmation screening. Rank ordering
of compounds is done on the basis of secondary assay mechanism of
action and physicochemical properties of the compound structures.
Certain test compounds significantly reverse the effects of Abeta
oligomers but not affect neuronal metabolism.
[0249] Compound Example 1 was dosed in the MTT assay and was shown
to block the Abeta oligomer-induced acceleration of exocytosis with
an EC.sub.50 of 10 .mu.M, indicating that Compound Example 1
blocks/abate the activity/effect of Abeta oligomer on neuron
cells.
Example BB
Binding Assay
[0250] Each test compound was added to a plate followed by an
addition of one or more of Abeta 1-42 Oligomers. The plates were
fixed with 3.7% paraformaldehyde in phosphate buffered saline (PBS)
for 15 min. The plate was then washed 3.times. with PBS for 5 min
each. The plates were blocked at room temperature for 1 hour in 5%
goat serum and 0.5% Triton X-100 (CAS number: 9002-93-1) in PBS.
Primary antibodies (anti-MAP 2 polyclonal, Millipore #AB5622 and
anti-Beta Amyloid 6E10 monoclonal, Convance #SIG-39300) were
diluted 1:1000 in 5% goat serum with PBS. Primary antibodies were
incubated either overnight at 4.degree. C. or 1 hour at RT. The
plate was then washed 3.times. with PBS for 5 min each. Secondary
antibodies (Alex Flor 488 polyclonal, Invitrogen # A11008 and Alexa
Flor 647 monoclonal, Invitrogen #A21235) were diluted 1:1000 in 5%
goat serum with PBS. Secondary antibodies were incubated at RT for
1 hr. The plates were washed once with PBS. DAPI
(4',6-diamidino-2-phenylindole, Invitrogen) was then applied at
0.03 .mu.g/.mu.L and incubated at RT for 5 min, then washed with
PBS. Image process was carried out for analysis.
[0251] Similar procedures for binding assays can be found in the
literature. See e.g., Look G C, et. al. Discovery of
ADDL--targeting small molecule drugs for Alzheimer's disease. Curr
Alzheimer Res. 2007 December; 4(5):562-7. Review.
Abeta Oligomer Preparations:
[0252] Human amyloid peptide 1-42 is obtained from California
Peptide, with lot-choice contingent upon quality control analysis.
Abeta 1-42 oligomers made according to published methods [See e.g.
Dahlgren et al., "Oligomeric and fibrillar species of amyloid-beta
peptides differentially affect neuronal viability" J Biol Chem.
2002 Aug. 30; 277(35):32046-53. Epub 2002 Jun. 10; LeVine H 3rd.
"Alzheimer's beta-peptide oligomer formation at physiologic
concentrations" Anal Biochem. 2004 Dec. 1; 335(1):81-90; Shrestha
et. al, "Amyloid beta peptide adversely affects spine number and
motility in hippocampal neurons" Mol Cell Neurosci. 2006 November;
33(3):274-82. Epub 2006 Sep. 8; Puzzo et al., "Amyloid-beta peptide
inhibits activation of the nitric oxide/cGMP/cAMP-responsive
element-binding protein pathway during hippocampal synaptic
plasticity" J Neurosci. 2005 Jul. 20; 25(29):6887-97; Barghorn et
al., "Globular amyloid beta-peptide oligomer--a homogenous and
stable neuropathological protein in Alzheimer's disease" J
Neurochem. 2005 November; 95(3):834-47. Epub 2005 Aug. 31;
Johansson et al., Physiochemical characterization of the
Alzheimer's disease-related peptides A beta 1-42 Arctic and A beta
1-42 wt. FEBS J. 2006 June; 2 73(12):2618-30] as well as
brain-derived Abeta oligomers (See e.g. Walsh et al., Naturally
secreted oligomers of amyloid beta protein potently inhibit
hippocampal long-term potentiation in vivo. Nature (2002). 416,
535-539; Lesne et al., A specific amyloid-beta protein assembly in
the brain impairs memory. Nature. 2006 Mar. 16; 440(7082):352-7;
Shankar et al, Amyloid-beta protein dimers isolated directly from
Alzheimer's brains impair synaptic plasticity and memory. Nat Med.
2008 August; 14(8):837-42. Epub 2008 Jun. 22) will serve as
positive controls. Quality controls of oligomer preparations
consist of Westerns to determine oligomer size ranges and relative
concentrations, and the MTT assay to confirm exocytosis
acceleration without toxicity. Toxicity is monitored in each
image-based assay via quantification of nuclear morphology
visualized with the DNA binding dye DAPI (Invitrogen). Nuclei that
are fragmented are considered to be in late stage apoptosis (Majno
and Joris Apoptosis, oncosis, and necrosis. An overview of cell
death. Am J Pathol 1995; 146:3-16). Peptide lots producing unusual
peptide size ranges or significant toxicity at standard
concentrations on neurons are rejected.
Image Processing
[0253] Images were captured and analyzed with the Cellomics VTI
automated microscope platform, using the Neuronal Profiling
algorithm. For statistical analysis, a Tukey-Kramer pair-wise
comparison with unequal variance was used.
Western Blots
[0254] Samples containing Abeta 1-42 were diluted (1:5) in
non-reducing lane marker sample buffer (Pierce #1859594). A 30
microliter (.mu.L) sample was loaded onto an eighteen well precast
4-15% Tris-HCl gel (BIORAD #345-0028). Electrophoresis was
performed in a BIO-RAD Criterian precast gel system using
Tris-Glycine buffer at 125 volt (V) for 90 minutes. The gels were
blotted onto 0.2 .mu.M nitrocellulose membranes in Tris-Glycine/10%
methanol buffer at 30V for 120 minutes. The membranes were boiled
for 5 minutes in a PBS solution and blocked over night with TBS/5%
milk solution at 4.degree. C. The membrane was probed with 6E10-HRP
(Covance #SIG-39345) diluted to 10 .mu.g/mL in TBS/1% milk solution
for one hour at room temperature. Membrane was washed three times
for 40 minutes each with a solution of TBS/0.05% tween-20 and
developed with ECL reagent (BIO-RAD #162-0112) for 5 minutes. Image
acquisition was performed on an Alpha Innotech FluorChem Q
quantitative imaging system and analyzed with AlphaView Q
software.
PK Studies:
[0255] PK studies are performed at CEREP Inc of Redmond Wash.,
according to their standard protocols: The plasma samples were
processed using acetonitrile precipitation and analyzed by HPLC-MS
or HPLC-MS/MS. Peak areas were recorded, and the concentrations of
the test compound in the unknown plasma samples were determined
using the respective calibration curve. The reportable linear range
of the assay was determined, along with the lower limit of
quantitation (LLQ).
NMR Spectroscopy and Mass Spectrometry:
[0256] Active fractions were analyzed by 1H NMR (Varian 500 MHz NMR
spectrometer) and purified compounds were characterized using a
combination 1D and 2D 1H NMR experiments and 13C NMR experiments.
Structure proof was obtained using these NMR techniques in
combination with low resolution mass spectrometry to determine
molecular weight and high resolution mass spectrometry (Thermo
Finnigan LCQ Ion trap) to determine composition-of-matter.
[0257] Compound Example 1 was shown to partially block binding of
the Abeta oligomer ligand to neurons by 24% according to the
binding assay (using imaging processing algorithm).
Example CC
Pharmacokinetic Studies
[0258] Pharmacokinetic studies were performed according to the
following protocols: The plasma samples were processed using
acetonitrile precipitation and analyzed by HPLC-MS or HPLC-MS/MS.
Peak areas were recorded, and the concentrations of the test
compound in the unknown plasma samples were determined using the
respective calibration curve. The reportable linear range of the
assay was determined, along with the lower limit of quantitation
(LLQ). For example, Compound Example 1 was determined to have a
half life of 46 minutes in the plasma of rats when injected
intravenously at 1 mg/Kg.
Example DD
A Primary Neuron-Based Functional Screening Assay to Detect Small
Molecule Abets Oligomer Blockers
[0259] Primary rat neurons grown for at least 3 weeks in vitro were
chosen as the basis for this screening assay. These neurons express
the full complement of synaptic proteins characteristic of neurons
in the mature brain, and exhibit a complex network of
activity-dependent electrical signaling. Neurons and glia in such
cultures have molecular signaling networks exhibiting excellent
registration with intact brain circuitry, and for this reason have
been used for over two decades as a model system for learning and
memory (See e.g. Kaech S, Banker G. Culturing hippocampal neurons.
Nat Protoc. 2006; 1(5):2406-15. Epub 2007 Jan. 11; See also Craig A
M, Graf E R, Linhoff M W. How to build a central synapse: clues
from cell culture. Trends Neurosci. 2006 Jan. 29(1):8-20. Epub 2005
Dec. 7. Review). More complex systems such as acute or organotypic
brain slices are very useful but not amenable to high throughput
screening. Immortalized or transformed neuronal cell lines are
amenable to high throughput screening, but do not replicate the
electrophysiological state-dependent signaling of primary neuronal
cultures and are unlikely to adequately model the subtle
alterations in this signaling that are caused by oligomers during
the earliest manifestations of the disease state (See e.g. Gortz P,
Flelscher W, Rosenbaum C, Otto F, Siebler M. Neuronal network
properties of human teratocarcinoma cell line-derived neurons.
Brain Res. 2004 Aug. 20; 1018(1):18-25). For this reason, primary
neuronal cultures were chosen because of their ability to be used
in high throughput screens and fidelity to what occurs in vivo.
[0260] Reduced formazan was first visible in intracellular vesicles
(FIG. 1A). Example of neurons filled with labeled vesicles
following endocytosis of dye and reduction to an insoluble purple
product. (Scale bar=20 microns in FIG. 1A). Eventual formazan
exocytosis was accelerated via Abeta oligomers in mature
hippocampal neurons in vitro (FIG. 1B). Example photomicrograph of
neurons covered with insoluble purple dye that have been extruded
via exocytosis. The dye precipitated in the aqueous environment of
the culture and formed needle-shaped crystals on the surface of the
neuron. (FIG. 1B). Endocytosis rate was altered in the presence of
Abeta oligomers. (FIG. 1C) Exocytosis rate was altered in the
presence of Abeta oligomers (FIG. 1D).
[0261] Since synaptic and memory deficits, and not widespread cell
death, predominate at the earliest stages of Alzheimer's disease,
assays that measure these changes can be used to discover small
molecule inhibitors of oligomer activity. The MTT assay can be used
as a measure of toxicity in cultures. Yellow tetrazolium salts were
endocytosed by cells and reduced to insoluble purple formazan in
the endosomal pathway. The level of purple formazan was a
reflection of the number of actively metabolizing cells in culture,
and reduction in the amount of formazan was taken as a measure of
cell death or metabolic toxicity in culture. When observed through
a microscope, the purple formazan was first visible in
intracellular vesicles that fill the cell (Figure. 1A). Over time,
the vesicles were exocytosed and the formazan precipitated as
needle-shaped crystals on the outer surface of the plasma membrane
as the insoluble formazan was exposed to the aqueous media
environment (FIG. 1B). Cells respond to sublethal levels of Abeta
oligomers by selectively accelerating the exocytosis rate of
reduced formazan, while leaving endocytosis rate unaffected, which
can be seen in mature primary neurons in vitro and quantified these
morphological shifts via automated microscopy and image processing.
At a given point in time following tetrazolium salt addition to the
culture well, vehicle-treated cells had the appearance of those in
FIG. 1A, while Abeta oligomer-treated cells had the appearance of
those in FIG. 1B. Under these circumstances, there was no overall
change in the total amount of reduced formazan, simply a shift in
its morphology. This assay is sensitive to low levels of oligomers
that do not cause cell death.
[0262] Evidence suggests that Abeta oligomer-mediated reduction in
neuronal surface receptor expression mediated by membrane
trafficking are the basis for oligomer inhibition of
electrophysiological measures of synaptic plasticity (LTP) and thus
learning and memory (See Kamenetz F, Tomita T, Hsieh H, Seabrook G,
Borchelt D, Iwatsubo T, Sisodia S. Malinow R. APP processing and
synaptic function. Neuron. 2003 Mar. 27; 37(6):925-37; and Hsieh H,
Boehm J, Sato C, Iwatsubo T, Tomita T, Sisodia S, Malinow R. AMPAR
removal underlies Abeta-induced synaptic depression and dendritic
spine loss. Neuron. 2006 Dec. 7; 52(5):831-43). Measuring membrane
trafficking rate changes induced by oligomers via formazan
morphological shifts has been used in cell lines to discover Abeta
oligomer-blocking drugs [Maezawa I, Hong H S, Wu H C, Battina S K,
Rana S, Iwamoto T, Radke G A, Pettersson E, Martin G M, Hua D H,
Jin L W. A novel tricyclic pyrone compound ameliorates cell death
associated with intracellular amyloid-beta oligomeric complexes. J
Neurochem. 2006 July; 98(1):57-67; Liu Y, Schubert D. Cytotoxic
amyloid peptides inhibit cellular
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
reduction by enhancing MTT formazan exocytosis. J Neurochem. 1997
December; 69(6):2285-93; Liu Y, Dargusch R, Banh C, Miller C A,
Schubert D. Detecting bioactive amyloid beta peptide species in
Alzheimer's disease. J Neurochem. 2004 November; 91(3):648-56; Liu
Y, Schubert D. Treating Alzheimer's disease by inactivating
bioactive amyloid beta peptide. Curr Alzheimer Res. 2006 April;
3(2):129-35; Rana S, Hong H S, Barrigan L, Jin L W, Hua D H.
Syntheses of tricyclic pyrones and pyridinones and protection of
Abeta-peptide induced MC65 neuronal cell death. Bioorg Med Chem
Lett. 2009 Feb. 1; 19(3):670-4. Epub 2008 Dec. 24; and Hong H S,
Maezawa I, Budamagunta M, Rana S, Shi A, Vassar R, Liu R, Lam K S,
Cheng R H, Hua D H, Voss J C, Jin L W. Candidate anti-Abeta
fluorene compounds selected from analogs of amyloid imaging agents.
Neurobiol Aging. 2008 Nov. 18. (Epub ahead of print)] that lower
Abeta brain levels in rodents in vivo [Hong H S, Rana S, Barrigan
L, Shi A, Zhang Y, Zhou F, Jin L W, Hua D H. Inhibition of
Alzheimer's amyloid toxicity with a tricyclic pyrone molecule in
vitro and in vivo. J Neurochem. 2009 February;
108(4):1097-1108].
[0263] The exocytosis assay was adapted for use with mature primary
neuronal cultures grown for 3 weeks in vitro. Abeta oligomers
caused a dose-dependent decrease in the amount of intracellular
vesicles (puncta) filled with reduced purple formazan (FIG. 2A,
squares; 3 .mu.M dose corresponds to image in FIG. 2C) as measured
via image processing using a Cellomics VTI automated microscopy
system. Increasing the amount of Abeta oligomers eventually
resulted in overt toxicity. Thus, the concentration of neuroactive
Abeta oligomers was much lower than that causing cell death. This
decrease can be blocked by adding stoichiometric amounts of
anti-Abeta monoclonal antibody 6E10 (IgG) to the cultures prior to
oligomer addition (FIG. 2A, circle; the circle corresponds to image
in FIG. 2D; antibody alone [down triangle] has no effect on the
neurons). Several compounds were tested that have been reported to
block the effects of Abeta oligomers, including the sugar alcohol
scyllo-inositol (AZD-103), the nAChR antagonist hexamethonium
bromide, and the NMDAR antagonists MK-801 and none were active
(Fenili et al., '07, Calabrese et al., '06, LeCor et al., '07).
[0264] The assay was optimized for performance in 384-well
microtiter plates with automated liquid handling robotics for
compound formatting and assay plate stamping, routinely achieving
statistically significant two-fold separation between vehicle and
Abeta oligomer-treated neurons (Student's t-test, unequal
variance). Compounds were added to neurons first, then oligomers
were added. When configured in this manner the assay was able to
detect compounds that act via disruption of oligomers, inhibition
of oligomer binding to neurons, or counteraction of signal
transduction mechanisms of action initiated by oligomer
binding.
[0265] Compounds were considered active if they significantly block
Abeta-mediated changes in membrane trafficking, but do not
significantly affect membrane trafficking when dosed on their own.
An example is shown in FIG. 2B; Compound Example 2 inhibits
oligomer effects on membrane trafficking with an EC50 of 7
.mu.M.
[0266] FIG. 2A shows dose-dependent decrease of intracellular
formazan-filled vesicles (puncta) caused by Abeta 42 oligomer
treatment acceleration of exocytosis (squares). Oligomer effects
were blocked by anti Abeta IgG (circle and up triangle; circle
refers to stoich amount of IgG, i.e., 3 .mu.M of A.beta. and 1.5
.mu.M of IgG; up triangle refers to substoich IgG, i.e., 3 .mu.M of
AB and 0.5 .mu.M of IgG). IgG itself (down triangle) has no effect.
FIG. 2B shows Example Compound 2, which inhibits oligomer effects
on membrane trafficking. FIG. 2C shows representative micrographs
of 21 DIV hippocampal neurons in vitro showing oligomer effects
membrane trafficking (corresponding to data point 3 .mu.M in FIG.
2A); and FIG. 2D shows blockade by anti-Abeta antibodies
(corresponding to the circle in FIG. 2A). Data were the average of
3 experiments. Scale bar=20 micron in FIG. 2D.
Example EE
Fear Conditioning Assay
[0267] Compound Example 2 was tested in an animal model of a
memory-dependent behavioral task known as fear conditioning. The
study protocol was designed based on published protocols (See e.g.
Puzzo D, Privitera L, Leznik E, Fa M, Staniszewski A, Palmeri A,
Arancio O. Picomolar amyloid-beta positively modulates synaptic
plasticity and memory in hippocampus. J Neurosci. 2008 Dec. 31;
28(53):14537-45.). The formation of contextual memories is
dependent upon the integrity of medial temporal lobe structures
such as the hippocampus. In this assay mice were trained to
remember that a particular salient context (conditioned stimulus;
CS) is associated with an aversive event, in this case a mild foot
shock (the unconditioned stimulus, US). Animals that show good
learning will express an increase in freezing behavior when placed
back into the same context. This freezing is absent in a novel
context. Increased freezing in the context indicates strong
hippocampal-dependent memory formation in animals. Memory tested in
Fear Conditioning is sensitive to elevations of soluble AD. FIG. 3
shows the results of administration of Abeta oligomers (bar labeled
with "a") during training results in memory deficits when animals
are tested 24 later, compared to vehicle administration (bar
labeled with "b"). Example Compound 2 was effective at stopping
Abeta oligomer mediated effects on membrane trafficking (FIG. 3).
When administered to animals prior to Abeta oligomer
administration, Example Compound 2 blocked oligomer effects on
memory in a dose-dependent manner. The compound completely blocked
oligomer-mediated memory deficits at the 2 pmol dose (FIG. 3, bar
labeled with "d"). This behavioral efficacy demonstrates that the
membrane trafficking assay is able to predict which compounds will
be efficacious in treating the behavioral memory loss caused by
oligomers. The fear condition model for memory was performed as
described herein.
[0268] FIG. 3 shows that Abeta produces significant deficits in
memory formation vs. vehicle (p<0.05) in the contextual fear
conditioning memory task. FIG. 3 shows that the 2 pmol dose of
Compound Example 2+Abeta (200 nM) completely blocked the effect of
Abeta on memory (p<0.05, one way ANOVA, post hoc comparison with
Bonferroni correction). No effect of compound alone was
observed(data not shown). No adverse behavioral changes were
observed at any dose.
Example FF
Membrane Trafficking Assay
[0269] Abeta assemblies were isolated from patients with
Alzheimer's Disease (AD) or from normal patients. The Abeta
assemblies were tested for their ability to modulate membrane
trafficking. HMW (>100 KDa) Abets assemblies isolated from AD
patients do not affect membrane trafficking (not shown). IMW
(10-100 KDa) Abeta assemblies isolated from AD patients
significantly affect membrane trafficking. (FIG. 4). IMW Abeta
assemblies isolated from Age-matched normal individuals do not
affect membrane trafficking (FIG. 4). Compound Example 2 has no
effect on Abeta assemblies isolated from Age-matched normal
individuals. (FIG. 4). Compound Example 2 significantly blocked the
trafficking effects of AD-brain derived Abeta aseemblies. (FIG.
4).
[0270] Although the present invention has been described in
considerable detail with reference to certain preferred versions
thereof, other versions are possible. Therefore, the spirit and
scope of the invention should not be limited to the description of
the preferred versions described herein.
[0271] All features disclosed in the specification, including the
abstract and drawings, and all the steps in any method or process
disclosed, may be combined in any combination, except combinations
where at least some of such features and/or steps are mutually
exclusive. Each feature disclosed in the specification, including
abstract and drawings, can be replaced by alternative features
serving the same, equivalent or similar purpose, unless expressly
stated otherwise. Thus, unless expressly stated otherwise, each
feature disclosed is one example only of a generic series of
equivalent or similar features. Various modifications of the
invention, in addition to those described herein, will be apparent
to those skilled in the art from the foregoing description. Such
modifications are also intended to fall within the scope of the
appended claims.
[0272] Each reference cited in the present application is herein
incorporated by reference in its entirety.
* * * * *