U.S. patent application number 12/308773 was filed with the patent office on 2011-06-09 for bicyclic compositions and methods for modulating a kinase cascade.
This patent application is currently assigned to Kinex Pharmaceuticals, LLC. Invention is credited to David G. Hangauer, JR..
Application Number | 20110136807 12/308773 |
Document ID | / |
Family ID | 38704923 |
Filed Date | 2011-06-09 |
United States Patent
Application |
20110136807 |
Kind Code |
A1 |
Hangauer, JR.; David G. |
June 9, 2011 |
Bicyclic compositions and methods for modulating a kinase
cascade
Abstract
The invention relates to compounds and methods for modulating
one or more components of a kinase cascade.
Inventors: |
Hangauer, JR.; David G.;
(Lancaster, NY) |
Assignee: |
Kinex Pharmaceuticals, LLC
Buffalo
NY
|
Family ID: |
38704923 |
Appl. No.: |
12/308773 |
Filed: |
June 29, 2007 |
PCT Filed: |
June 29, 2007 |
PCT NO: |
PCT/US2007/015271 |
371 Date: |
October 15, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11480163 |
Jun 29, 2006 |
7838542 |
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12308773 |
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Current U.S.
Class: |
514/235.2 ;
514/254.09; 514/323; 514/363; 514/383; 514/407; 514/419; 514/622;
544/143; 544/373; 546/201; 548/139; 548/246; 548/265.4; 548/364.7;
548/492; 564/172; 564/173 |
Current CPC
Class: |
C07D 401/12 20130101;
A61P 9/00 20180101; C07D 403/06 20130101; A61P 43/00 20180101; C07D
413/06 20130101; A61P 27/02 20180101; A61P 19/10 20180101; C07D
403/12 20130101; A61P 35/00 20180101; C07D 209/42 20130101; C07D
417/12 20130101; A61P 19/02 20180101; C07D 413/12 20130101; A61P
9/10 20180101; A61P 27/16 20180101; A61P 3/04 20180101; A61P 17/06
20180101; A61P 3/10 20180101; C07D 401/06 20130101 |
Class at
Publication: |
514/235.2 ;
548/492; 544/143; 546/201; 548/364.7; 544/373; 548/139; 548/265.4;
548/246; 564/173; 564/172; 514/419; 514/323; 514/407; 514/254.09;
514/363; 514/383; 514/622 |
International
Class: |
A61K 31/404 20060101
A61K031/404; C07D 209/42 20060101 C07D209/42; C07D 413/06 20060101
C07D413/06; C07D 401/06 20060101 C07D401/06; C07D 403/12 20060101
C07D403/12; C07D 403/06 20060101 C07D403/06; C07D 417/12 20060101
C07D417/12; C07D 413/12 20060101 C07D413/12; C07C 233/75 20060101
C07C233/75; A61K 31/5377 20060101 A61K031/5377; A61K 31/454
20060101 A61K031/454; A61K 31/4155 20060101 A61K031/4155; A61K
31/496 20060101 A61K031/496; A61K 31/433 20060101 A61K031/433; A61K
31/4196 20060101 A61K031/4196; A61K 31/166 20060101 A61K031/166;
A61P 19/10 20060101 A61P019/10; A61P 3/04 20060101 A61P003/04; A61P
3/10 20060101 A61P003/10; A61P 27/02 20060101 A61P027/02; A61P 9/00
20060101 A61P009/00; A61P 9/10 20060101 A61P009/10 |
Claims
1. A compound according to Formula V: ##STR00586## or a salt,
solvate, hydrate, or prodrug thereof, wherein R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 are the same or different and are
independently H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a,
C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2,
NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, benzyl,
heteroaryl, biaryl, heterobiaryl, heterocycle, and branched,
unbranched, or cyclic alkyl; R.sub.6, and R.sub.7 are the same or
different and are independently H, branched or unbranched, or
(CH.sub.2).sub.t--Z, wherein Z is aryl, heteroaryl, biaryl, cyclic
alkyl, or heterocycle, or R.sub.6 and R.sub.7 together form a
heterocycle; t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; R.sub.a,
R.sub.b, and R.sub.c are the same or different and are
independently H, aryl, heteroaryl, biaryl, heterobiaryl, or
branched, unbranched, or cyclic alkyl; P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q, further wherein lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is linear or branched
alkyl; K is C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or ##STR00587##
L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or ##STR00588##
M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H;
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or ##STR00589##
Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or ##STR00590##
R.sub.19, R.sub.20 and R.sub.21 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.19 and
R.sub.20 taken together with the attached nitrogen atom form a five
membered ring; wherein any of R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 and R.sub.a, R.sub.b, and R.sub.c is
substituted or unsubstituted; provided that at least one of
R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6 is P.
2. The compound of claim 1, wherein at least one of R.sub.1,
R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is selected from halogen,
boronic acid, hydroxyl, phosphonic acid, sulfamic acid, guanidine,
carboxylic acid, aldehyde, amide, and hydroxymethylphosphonic
acid.
3. The compound according to any one of claims 1-2, wherein at
least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is
halogen.
4. The compound according to any one of claims 1-3, wherein at
least one of R.sub.6 and R.sub.7 is ##STR00591## wherein *R.sub.8
is the point of attachment and is (CH.sub.2).sub.x, wherein x is 0,
1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, CH.sub.2CHOH,
CH(CH.sub.3)(R-isomer), or CH(CH.sub.3)(S-isomer); and each of
R.sub.9, R.sub.10, R.sub.11, R.sub.12, and R.sub.13 is the same or
different and independently are H, C(O)R.sub.a,
C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a,
OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, P', aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle, or branched, cyclic, or
unbranched alkyl, P' is SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2,
OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-K', O--C(O)-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L', NH-lower (C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-M', or
O-aryl-Q', further wherein lower (C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, or C.sub.6) alkyl is linear or branched alkyl; K'
is C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22, NR.sub.22R.sub.23,
SO.sub.2R.sub.24, glycoside, lower C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6 alkoxy, or ##STR00592## L' is aryl, OH,
C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22, NR.sub.22R.sub.23,
SO.sub.2R.sub.24, glycoside, lower C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6 alkoxy, or ##STR00593## M' is aryl, OH,
C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22, NR.sub.22R.sub.23,
SO.sub.2R.sub.24, glycoside, lower C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6 alkoxy, or ##STR00594## Q' is aryl, OH,
C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22, NR.sub.22R.sub.23,
SO.sub.2R.sub.24, glycoside, lower C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6 alkoxy, or ##STR00595## R.sub.22,
R.sub.23 and R.sub.24 are independently C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.22 and R.sub.23 taken
together with the attached nitrogen atom form a five membered ring;
wherein R.sub.a, R.sub.b, and R.sub.c are the same or different and
independently are H, aryl, heteroaryl, biaryl, heterobiaryl,
branched, cyclic, or unbranched alkyl; wherein any of R.sub.9,
R.sub.10, R.sub.11, R.sub.12, and R.sub.13 are substituted or
unsubstituted; and provided that if one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, or R.sub.5 is not P, then at least one of
R.sub.9, R.sub.10, R.sub.11, R.sub.12, and R.sub.13 is P'.
5. A compound of Formula VI: ##STR00596## or a salt, solvate,
hydrate, or prodrug thereof, wherein R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are each the same or
different and independently are H, C(O)R.sub.a,
C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a,
OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle, and branched, cyclic, or
unbranched alkyl; R.sub.a, R.sub.b, and R.sub.c are the same or
different and are independently H, aryl, heteroaryl, biaryl,
heterobiaryl, and branched, cyclic, or unbranched alkyl; P is
SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K,
O--C(O)-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or
C.sub.6) alkyl-L, NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-M, or O-aryl-Q, further wherein lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is
linear or branched alkyl; K is C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00597## L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00598## M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C4, C.sub.5, C.sub.6 alkoxy, or
##STR00599## Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00600## R.sub.19, R.sub.20 and R.sub.21 are independently
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or
R.sub.19 and R.sub.20 taken together with the attached nitrogen
atom form a five membered ring; and x is 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10; provided that at least one of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is P.
6. Use of a compound of Formula V in the manufacture of a
medicament for protection against or treatment of hearing loss in a
subject: ##STR00601## or a salt, solvate, hydrate, or prodrug
thereof, wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5
are the same or different and are independently H, C(O)R.sub.a,
C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a,
OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, benzyl,
heteroaryl, biaryl, heterobiaryl, heterocycle, and branched,
unbranched, or cyclic alkyl; R.sub.6, and R.sub.7 are the same or
different and are independently H, branched or unbranched, or
(CH.sub.2).sub.t--Z, wherein Z is aryl, heteroaryl, biaryl, cyclic
alkyl, or heterocycle, or R.sub.s and R.sub.7 together form a
heterocycle; t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; R.sub.a,
R.sub.b, and R.sub.c are the same or different and are
independently H, aryl, heteroaryl, biaryl, heterobiaryl, or
branched, unbranched, or cyclic alkyl; P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q, further wherein lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is linear or branched
alkyl; K is C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or ##STR00602##
L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or ##STR00603##
M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or ##STR00604##
Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or ##STR00605##
R.sub.19, R.sub.20 and R.sub.21 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.19 and
R.sub.20 taken together with the attached nitrogen atom form a five
membered ring; wherein any of R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 and R.sub.a, R.sub.b, and R.sub.c is
substituted or unsubstituted; provided that at least one of
R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6 is P.
7. Use of a compound of Formula VII in the manufacture of a
medicament for protection against or treatment of a disease
selected from osteoporosis, obesity, diabetes, eye disease, stroke,
atherosclerosis, neuropathic pain, and hepatitis B in a subject:
##STR00606## or a salt, solvate, hydrate, or prodrug thereof,
wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are the
same or different and are independently IL C(O)R.sub.a,
C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a,
OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, benzyl,
heteroaryl, biaryl, heterobiaryl, heterocycle, and branched,
unbranched, or cyclic alkyl; R.sub.6, and R.sub.7 are the same or
different and are independently H, branched or unbranched, or
(CH.sub.2).sub.t--Z, wherein Z is aryl, heteroaryl, biaryl, cyclic
alkyl, or heterocycle, or R.sub.6 and R.sub.7 together form a
heterocycle; t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; R.sub.a,
R.sub.b, and R.sub.c are the same or different and are
independently H, aryl, heteroaryl, biaryl, heterobiaryl, or
branched, unbranched, or cyclic alkyl; P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q, further wherein lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is linear or branched
alkyl; K is C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or ##STR00607##
L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or ##STR00608##
M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or ##STR00609##
Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or ##STR00610##
R.sub.19, R.sub.20 and R.sub.21 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.19 and
R.sub.20 taken together with the attached nitrogen atom form a five
membered ring; wherein any of R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 and R.sub.a, R.sub.b, and R.sub.c is
substituted or unsubstituted.
8. Use of a compound of Formula V in the manufacture of a
medicament for prevention or treatment of a proliferative disorder
in a subject: ##STR00611## or a salt, solvate, hydrate, or prodrug
thereof, wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5
are the same or different and are independently H, C(O)R.sub.a,
C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a,
OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, benzyl,
heteroaryl, biaryl, heterobiaryl, heterocycle, and branched,
unbranched, or cyclic alkyl; R.sub.6, and R.sub.7 are the same or
different and are independently H, branched or unbranched, or
(CH.sub.2).sub.t--Z, wherein Z is aryl, heteroaryl, biaryl, cyclic
alkyl, or heterocycle, or R.sub.6 and R.sub.7 together form a
heterocycle; t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; R.sub.a,
R.sub.b, and R.sub.c are the same or different and are
independently H, aryl, heteroaryl, biaryl, heterobiaryl, or
branched, unbranched, or cyclic alkyl; P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3 C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q, further wherein lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is linear or branched
alkyl; K is C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or ##STR00612##
L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or ##STR00613##
M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or ##STR00614##
Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or ##STR00615##
R.sub.19, R.sub.20 and R.sub.21 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.19 and
R.sub.20 taken together with the attached nitrogen atom form a five
membered ring; wherein any of R.sub.1, R.sub.2, R.sub.3, R.sub.4
R.sub.5, R.sub.6, and R.sub.7 and R.sub.a, R.sub.b, and R.sub.c is
substituted or unsubstituted; provided that at least one of
R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6 is P.
9. Use of a compound of Formula VI in the manufacture of a
medicament for protection against or treatment of hearing loss in a
subject: ##STR00616## or a salt, solvate, hydrate, or prodrug
thereof, wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5,
R.sub.6, and R.sub.7 are each the same or different and
independently are H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b,
C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a,
OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b,
NR.sub.aC(O)NR.sub.bR.sub.c, NR.sub.aC(O)OR.sub.b,
NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle, and branched, cyclic, or
unbranched alkyl; R.sub.a, R.sub.b, and R.sub.c are the same or
different and are independently H, aryl, heteroaryl, biaryl,
heterobiaryl, and branched, cyclic, or unbranched alkyl; P is
SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K,
O--C(O)-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or
C.sub.6) alkyl-L, NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-M, or O-aryl-Q, further wherein lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is
linear or branched alkyl; K is C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00617## L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00618## M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00619## Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00620## R.sub.19, R.sub.20 and R.sub.21 are independently
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or
R.sub.19 and R.sub.20 taken together with the attached nitrogen
atom form a five membered ring; and x is 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10; provided that at least one of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is P.
10. Use of a compound of Formula VIII in the manufacture of a
medicament for protection against or treatment of a disease
selected from osteoporosis, obesity, diabetes, eye disease, stroke,
atherosclerosis, neuropathic pain, and hepatitis B in a subject:
##STR00621## or a salt, solvate, hydrate, or prodrug thereof,
wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 are each the same or different and independently are H,
C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a, OH,
OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle, and branched, cyclic, or
unbranched alkyl; R.sub.a, R.sub.b, and R.sub.c are the same or
different and are independently H, aryl, heteroaryl, biaryl,
heterobiaryl, and branched, cyclic, or unbranched alkyl; P is
SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K,
O--C(O)-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or
C.sub.6) alkyl-L, NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-M, or O-aryl-Q, further wherein lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is
linear or branched alkyl; K is C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00622## L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00623## M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00624## Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00625## R.sub.19, R.sub.20 and R.sub.21 are independently
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or
R.sub.19 and R.sub.20 taken together with the attached nitrogen
atom form a five membered ring; and x is 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10.
11. Use of a compound of Formula VIII in the manufacture of a
medicament for the prevention or treatment of a proliferative
disorder in a subject: ##STR00626## or a salt, solvate, hydrate, or
prodrug thereof, wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 are each the same or different and
independently are H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b,
C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a,
OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b,
NR.sub.aC(O)NR.sub.bR.sub.c, NR.sub.aC(O)OR.sub.b,
NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle, and branched, cyclic, or
unbranched alkyl; R.sub.a, R.sub.b, and R.sub.c are the same or
different and are independently H, aryl, heteroaryl, biaryl,
heterobiaryl, and branched, cyclic, or unbranched alkyl; P is
SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K,
O--C(O)-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or
C.sub.6) alkyl-L, NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-M, or O-aryl-Q, further wherein lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is
linear or branched alkyl; K is C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00627## L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00628## M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00629## Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00630## R.sub.19, R.sub.20 and R.sub.21 are independently
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or
R.sub.19 and R.sub.20 taken together with the attached nitrogen
atom form a five membered ring; and x is 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10.
12.-13. (canceled)
14. A compound selected from: ##STR00631## ##STR00632##
##STR00633## or a salt, solvate, hydrate, or prodrug thereof.
15. Use of a compound according to claim 12 in the manufacture of a
medicament for protection against or treatment of a disease
selected from osteoporosis, obesity, diabetes, eye disease, stroke,
atherosclerosis, neuropathic pain, hepatitis B, and proliferative
disorder in a subject.
16. Use according to any one of claims 6-11 and 14-15, wherein the
administration of the compound is carried out orally, parentally,
subcutaneously, intravenously, intramuscularly, intraperitoneally,
by intranasal instillation, by intracavitary or intravesical
instillation, topically, intraarterially, intralesionally, by
metering pump, or by application to mucous membranes.
17. A pharmaceutical composition comprising a compound according to
any one of claim 1-5 or 14 and a pharmaceutically acceptable
excipient.
Description
BACKGROUND OF THE INVENTION
[0001] Signal transduction is any process by which a cell converts
one kind of signal or stimulus into another. Processes referred to
as signal transduction often involve a sequence of biochemical
reactions inside the cell, which are carried out by enzymes and
linked through second messengers. In many transduction processes,
an increasing number of enzymes and other molecules become engaged
in the events that proceed from the initial stimulus. In such cases
the chain of steps is referred to as a "signaling cascade" or a
"second messenger pathway" and often results in a small stimulus
eliciting a large response. One class of molecules involved in
signal transduction is the kinase family of enzymes. The largest
group of kinases are protein kinases, which act on and modify the
activity of specific proteins. These are used extensively to
transmit signals and control complex processes in cells.
[0002] Protein kinases are a large class of enzymes which catalyze
the transfer of the .gamma.-phosphate from ATP to the hydroxyl
group on the side chain of Ser/Thr or Tyr in proteins and peptides
and are intimately involved in the control of various important
cell functions, perhaps most notably: signal transduction,
differentiation, and proliferation. There are estimated to be about
2,000 distinct protein kinases in the human body, and although each
of these phosphorylate particular protein/peptide substrates, they
all bind the same second substrate, ATP, in a highly conserved
pocket. Protein phosphatases catalyze the transfer of phosphate in
the opposite direction.
[0003] A tyrosine kinase is an enzyme that can transfer a phosphate
group from ATP to a tyrosine residue in a protein. Phosphorylation
of proteins by kinases is an important mechanism in signal
transduction for regulation of enzyme activity. The tyrosine
kinases are divided into two groups; those that are cytoplasmic
proteins and the transmembrane receptor-linked kinases. In humans,
there are 32 cytoplasmic protein tyrosine kinases and 58
receptor-linked protein-tyrosine kinases. The hormones and growth
factors that act on cell surface tyrosine kinase-linked receptors
are generally growth-promoting and function to stimulate cell
division (e.g., insulin, insulin-like growth factor 1, epidermal
growth factor).
[0004] Inhibitors of various known protein kinases or protein
phosphatases have a variety of therapeutic applications. One
promising potential therapeutic use for protein kinase or protein
phosphatase inhibitors is as anti-cancer agents. About 50% of the
known oncogene products are protein tyrosine kinases (PTKs) and
their kinase activity has been shown to lead to cell
transformation.
[0005] The PTKs can be classified into two categories, the membrane
receptor PTKs (e.g. growth factor receptor PTKs) and the
non-receptor PTKs (e.g. the Src family of proto-oncogene products).
There are at least 9 members of the Src family of non-receptor
PTK's with pp60.sup.c-src (hereafter referred to simply as "Src")
being the prototype PTK of the family wherein the approximately 300
amino acid catalytic domains are highly conserved. The
hyperactivation of Src has been reported in a number of human
cancers, including those of the colon, breast, lung, bladder, and
skin, as well as in gastric cancer, hairy cell leukemia, and
neuroblastoma. Overstimulated cell proliferation signals from
transmembrane receptors (e.g. EGFR and p185HER2/Neu) to the cell
interior also appear to pass through Src. Consequently, it has
recently been proposed that Src is a universal target for cancer
therapy, because hyperactivation (without mutation) is involved in
tumor initiation, progression, and metastasis for many important
human tumor types.
[0006] Because kinases are involved in the regulation of a wide
variety of normal cellular signal transduction pathways (e.g., cell
growth, differentiation, survival, adhesion, migration, etc.),
kinases are thought to play a role in a variety of diseases and
disorders. Thus, modulation of kinase signaling cascades may be an
important way to treat or prevent such diseases and disorders.
[0007] An important contribution to the protein kinase field has
been the x-ray structural work with the serine kinase
cAMP-dependent protein kinase ("PKA") bound to the 20-residue
peptide derived from the heat stable inhibitor protein, PKI(5-24),
and Mg.sub.2ATP (Taylor et al., 1993). This structural work is
particularly valuable because PKA is considered to be a prototype
for the entire family of protein kinases since they have evolved
from a single ancestral protein kinase. Sequence alignments of PKA
with other serine and tyrosine kinases have identified a conserved
catalytic core of about 260 residues and 11 highly conserved
residues within this core (Taylor et al., 1993). Two highly
conserved residues of particular note for the work proposed herein
are the general base Asp-166 which is proposed to interact with the
substrate OH and the positively charged residue, Lys-168 for serine
kinases and an Arg for tyrosine kinases (Knighton et al., 1993),
which is proposed to interact with the .gamma.-phosphate of ATP to
help catalyze transfer of this phosphate. Two additional important
PKA crystal structures have been reported (Madhusudan et al.,
1994), one for the ternary PKA:ADP:PKI(5-24) complex wherein the
PKI Ala 21 has been replaced with Ser (thereby becoming a
substrate), and one for the binary PKA:PKI(5-24) complex wherein
the PKI Ala 21 has been replaced with phosphoserine (an end product
inhibitor). The ternary complex shows the serine OH donating a
H-bond to Asp-166 and accepting a H-bond from the side chain of Lys
168. The binary complex shows the phosphate group of phosphoserine
forming a salt bridge with the Lys-168 side chain and within
H-bonding distance of the Asp-166 carboxyl group. These structures
support the earlier proposed roles for Asp-166 and Lys-168 in the
catalytic mechanism.
[0008] The x-ray structures of PKA show that the enzyme consists of
two lobes wherein the smaller lobe binds ATP and the larger lobe
the peptide substrate. Catalysis occurs at the cleft between the
lobes. The crystallographic and solution structural studies with
PKA have indicated that the enzyme undergoes major conformational
changes from an "open" form to the "closed" catalytically active
form as it binds the substrates (Cox et al., 1994). These
conformational changes are presumed to involve the closing of the
cleft between the two lobes as the substrates bind bringing the
.gamma.-phosphate of ATP and the Ser OH in closer proximity for
direct transfer of the phosphate.
[0009] However, many inhibitors of protein kinases and protein
phosphatases still lack the specificity and potency desired for
therapeutic use. Due to the key roles played by protein kinases and
protein phosphatases in a number of different diseases, including
cancer, psoriasis, arthrosclerosis, Type II diabetes, obesity, and
their role in regulating immune system activity, modulators of
protein kinase cascades are needed.
SUMMARY OF THE INVENTION
[0010] One aspect of the invention provides a compound having the
Formula I:
##STR00001##
wherein X is a halogen, and R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, and R.sub.6 are the same or different, and selected from
H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a,
OH, OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2,
NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O)2Rb, NR.sub.aS(O)OR.sub.b, NR.sub.aS(O).sub.2OR.sub.b,
NR.sub.aP(O)OR.sub.bOR.sub.c, NR.sub.aP(O)OR.sub.bR.sub.c,
NR.sub.aP(O)OR.sub.bOR.sub.c, SR.sub.a, S(O)R.sub.a,
S(O).sub.2R.sub.a, S(O)OR.sub.a, S(O).sub.2OR.sub.a,
S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, aryl, heteroaryl,
biaryl, heterobiaryl, heterocyclic compound, and alkyl (branched,
cyclic, or unbranched), having from 1 to 20 carbon atoms,
optionally containing a double or triple bond and optionally
substituted with a heteroatom or other functional groups, such as a
carboxylic acid, carboxylic ester, alcohol, alkoxy, thioether,
amide, thioamide, urea, urethane, sulfoxide, sulfone,phosphonic
acid, phosphonic ester, phosphinic acid, phosphinic ester, boronic
acid, aryl, heteroaryl, biaryl, and heterobiaryl, or R.sub.5 and
R.sub.6 together form a heterocyclic compound. R.sub.a, R.sub.b,
and R.sub.c are the same or different and selected from the group
consisting of H, aryl, heteroaryl, biaryl, heterobiaryl, and alkyl
(branched, cyclic, or unbranched), optionally substituted with a
heteroatom or other functional groups such as a carboxylic acid,
carboxylic ester, alcohol, alkoxy, thioether, amide, thioamide,
urea, urethane, sulfoxide, sulfone, phosphonic acid, phosphonic
ester, phosphinic acid, phosphinic ester, boronic acid, aryl,
heteroaryl, biaryl, and heterobiaryl. It is understood that all
open substitution positions in the above side chains can contain
further substitutions.
[0011] In one embodiment, at least one of R.sub.5 or R.sub.6 is
##STR00002##
wherein R.sub.7* is the point of attachment and is
(CH.sub.2).sub.x, wherein X is selected from 0, 1, 2, 3, 4, 5, 6,
7, 8, 9, and 10, CH.sub.2CHOH, CH(CH.sub.3)(R-isomer), or
CH(CH.sub.3)(S-isomer), and each of R.sub.8, R.sub.9, R.sub.10,
R.sub.11, and R.sub.12 are the same or different and selected from
H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a,
OH, OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2,
NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O)2Rb, NR.sub.aS(O)OR.sub.b, NR.sub.aS(O).sub.2OR.sub.b,
NR.sub.aP(O)OR.sub.bOR.sub.c, NR.sub.aP(O)OR.sub.bR.sub.c,
NR.sub.aP(O)OR.sub.bOR.sub.c, SR.sub.a, S(O)R.sub.a, S(O)2R.sub.a,
S(O)OR.sub.a, S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b,
S(O).sub.2NR.sub.aR.sub.b, P(O)OR.sub.aOR.sub.b, B(OH).sub.2,
halogen, aryl, heteroaryl, biaryl, heterobiaryl, heterocyclic
compound, and alkyl (branched, cyclic, or unbranched), having from
1 to 20 carbon atoms, optionally containing a double or triple bond
and optionally substituted with a heteroatom or other functional
groups, such as a carboxylic acid, carboxylic ester, alcohol,
ether, thioether, amide, thioamide, urea, urethane, sulfoxide,
sulfone, phosphonic acid, phosphonic ester, phosphinic acid,
phosphinic ester, boronic acid, aryl, heteroaryl, biaryl, and
heterobiaryl. R.sub.a, R.sub.b, and R.sub.c can be the same or
different and are selected from the group consisting of H, aryl,
heteroaryl, biaryl, heterobiaryl, and alkyl (branched, cyclic, or
unbranched), optionally substituted with a heteroatom or other
functional groups such as a carboxylic acid, carboxylic ester,
alcohol, ether, thioether, amide, thioamide, urea, urethane,
sulfoxide, sulfone, phosphonic acid, phosphonic ester, phosphinic
acid, phosphinic ester, boronic acid, aryl, heteroaryl, biaryl, and
heterobiaryl. It is understood that any of R.sub.8, R.sub.9,
R.sub.10, R.sub.11, and R.sub.12 are substituted or
unsubstituted.
[0012] In another embodiment, at least one of R.sub.5 or R.sub.6
is
##STR00003##
wherein the asterisk indicates the point of attachment to the
nitrogen.
[0013] Another aspect of the invention is a compound having the
Formula II:
##STR00004##
wherein X is a halogen, e.g., fluorine, and R.sub.1, R.sub.2,
R.sub.3, and R.sub.4 are specificity side chain elements and
defined as described above. In one embodiment, R.sub.1 is H,
R.sub.2 is
##STR00005##
R.sub.3 is H, and R.sub.4 is H. In another embodiment, the compound
is substituted at any other position on the indole ring.
[0014] Another aspect of the invention is a method for identifying
inhibitors of protein kinases. The method involves providing at
least one first module having one or more functional groups each
capable of covalently or non-covalently binding to catalytic
residues of the protein kinase, wherein at least one of the one or
more functional groups is a halogen, combining at least one first
module with at least one second module which provides a non-peptide
scaffold to form one or more combinations of the first and second
modules, screening the one or more combinations of the first and
second modules for protein kinase inhibition, and selecting
combinations of the first and second modules which inhibit protein
kinase activity. As used herein, a module is a single molecular
entity or a collection of functional groups. As used herein, a
non-peptide scaffold is a molecule which may include peptide bonds,
so long as a part of the molecule is not a peptide.
[0015] Another aspect of the invention is a method of inhibiting a
protein kinase. The protein kinase is contacted by a compound
comprising at least one first module having one or more functional
groups each capable of covalently or non-covalently binding to
catalytic residues of the protein kinase, wherein the one or more
functional groups comprise a halogen, and a second module which
provides a non-peptide scaffold. The combination of at least one
first module and second module inhibits the protein kinase
activity.
[0016] Another aspect of the invention is a method of treating a
condition, responsive to a protein kinase inhibitor, in a subject.
Another aspect of the invention is use of a compound of the
invention in the manufacture of a medicament for treating a
condition, responsive to a protein kinase inhibitor, in a subject.
A protein kinase inhibitor is administered to a subject. The
protein kinase inhibitor has at least one first module having one
or more functional groups each capable of covalently or
non-covalently binding to catalytic residues of the protein kinase,
wherein the one or more functional groups comprise a halogen, and a
second module which provides a non-peptide scaffold. The
combination of at least one first module and second module inhibits
protein kinase activity in the subject.
[0017] Another aspect of the invention is a method for identifying
inhibitors of protein phosphatases. The method involves providing
at least one first module having one or more functional groups each
capable of covalently or non-covalently binding to catalytic
residues of the protein phosphatase, combining at least one first
module with at least one second module which provides a non-peptide
scaffold to form one or more combinations of the first and second
modules, screening the one or more combinations of the first and
second modules for phosphatase inhibition, and selecting
combinations of the first and second modules which inhibit protein
phosphatase activity.
[0018] Another aspect of the present invention is a method of
inhibiting a protein phosphatase. Another aspect of the invention
is use of a compound of the invention in the manufacture of a
medicament for inhibiting a protein phosphatase. The protein
phosphatase is contacted by a compound comprising at least one
first module having one or more functional groups each capable of
covalently or non-covalently binding to catalytic residues of the
protein phosphatase, and a second module which provides a
non-peptide scaffold. The combination of at least one first module
and second module inhibits the protein phosphatase activity.
[0019] Another aspect of the invention is a method of treating a
condition, responsive to a protein phosphatase inhibitor, in a
subject. Another aspect of the invention is use of a compound of
the invention in the manufacture of a medicament for treating a
condition, responsive to a protein phosphatase inhibitor, in a
subject. A protein phosphatase inhibitor is administered to a
subject. The protein phosphatase inhibitor has at least one first
module having one or more functional groups each capable of
covalently or non-covalently binding to catalytic residues of the
protein phosphatase, and a second module which provides a non-
peptide scaffold. The combination of at least one first module and
second module inhibits protein phosphatase activity in the
subject.
[0020] Another aspect of the invention is a compound according to
Formula V:
##STR00006##
or a salt, solvate, hydrate, or prodrug thereof. R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.s are the same or different and are
independently H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a,
C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2,
NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, benzyl,
heteroaryl, biaryl, heterobiaryl, heterocycle, and branched,
unbranched, or cyclic alkyl. R.sub.6 and R.sub.7 are the same or
different and are independently H, branched or unbranched, or
(CH.sub.2).sub.t--Z, wherein Z is aryl, heteroaryl, biaryl, cyclic
alkyl, or heterocycle, or R.sub.6 and R.sub.7 together form a
heterocycle. t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. R.sub.a,
R.sub.b, and R.sub.c are the same or different and are
independently H, aryl, heteroaryl, biaryl, heterobiaryl, or
branched, unbranched, or cyclic alkyl. P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q, further wherein lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is linear or branched
alkyl. K is C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00007##
L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00008##
M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00009##
Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00010##
R.sub.19, R.sub.20 and R.sub.21 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.19 and
R.sub.20 taken together with the attached nitrogen atom form a five
membered ring. Any of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5,
R.sub.6, and R.sub.7 and R.sub.a, R.sub.b, and R.sub.c is
substituted or unsubstituted. At least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, and R.sub.6 is P.
[0021] In one embodiment, P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q, further where K is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, glycoside, and heterocycle; further where L is
SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, glycoside, and heterocycle; and
further where M is SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19, NR.sub.19R.sub.20,
glycoside, and heterocycle.
[0022] In one embodiment, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is selected from halogen, boronic
acid, hydroxyl, phosphonic acid, sulfamic acid, guanidine,
carboxylic acid, aldehyde, amide, and hydroxymethylphosphonic acid.
In another embodiment, at least one of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, and R.sub.5 is halogen. In another embodiment, at least
one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is boronic
acid. In another embodiment, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is hydroxyl. In another embodiment,
at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is
amide. For example, amide is vicinal tricarbonyl amide. In one
embodiment, R.sub.3 is halogen. For example, R.sub.3 is fluorine.
In another embodiment, R.sub.3 is hydroxyl.
[0023] In one embodiment, at least one of R.sub.6 and R.sub.7
is
##STR00011##
where *R.sub.8 is the point of attachment. In one embodiment
R.sub.8 is (CH.sub.2).sub.x, where X is 0, 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10. In another embodiment R.sub.8 is CH.sub.2CHOH,
CH(CH.sub.3)(R-isomer), or CH(CH.sub.3)(S-isomer). In another
embodiment, each of R.sub.9, R.sub.10, R.sub.11, R.sub.12, and
R.sub.13 are the same or different and each of R.sub.9, R.sub.10,
R.sub.11, R.sub.12, and R.sub.13 independently are H, C(O)R.sub.a,
C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a,
OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, P', aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle, or branched, cyclic, or
unbranched alkyl, P' is SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2,
OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-K', O--C(O)-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L', NH-lower (C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-M', or
O-aryl-Q', further where lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl is linear or branched alkyl. K' is
C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22, NR.sub.22R.sub.23,
SO.sub.2R.sub.24, glycoside, lower C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00012##
L' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00013##
M' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00014##
Q' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00015##
R.sub.22, R.sub.23 and R.sub.24 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.22 and
R.sub.23 taken together with the attached nitrogen atom form a five
membered ring. R.sub.a, R.sub.b, and R.sub.c are the same or
different and independently are H, aryl, heteroaryl, biaryl,
heterobiaryl, branched, cyclic, or unbranched alkyl; wherein any of
R.sub.9, R.sub.10, R.sub.11, R.sub.12, and R.sub.13 are substituted
or unsubstituted; and provided that if one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, or R.sub.5 is not P, then at least one of
R.sub.9, R.sub.10, R.sub.11, R.sub.12, and R.sub.13 is P'.
[0024] In another embodiment, P' is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K', O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L',
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M', or O-aryl-Q'. K' is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, glycoside, or heterocycle. L' is SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.22, NR.sub.22R.sub.23, glycoside, or heterocycle. M' is
SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.22, NR.sub.22R.sub.23, glycoside, or heterocycle. In
another embodiment, at least one of R.sub.6 and R.sub.7 is
##STR00016##
[0025] Another aspect of the invention includes a compound of
Formula VI:
##STR00017##
or a salt, solvate, hydrate, or prodrug. R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are each the same or
different and independently are H, C(O)R.sub.a,
C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a,
OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle, and branched, cyclic, or
unbranched alkyl. R.sub.a, R.sub.b, and R.sub.c are the same or
different and are independently H, aryl, heteroaryl, biaryl,
heterobiaryl, and branched, cyclic, or unbranched alkyl. P is
SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K,
O--C(O)-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or
C.sub.6) alkyl-L, NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-M, or O-aryl-Q, further where lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is
linear or branched alkyl. K is C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00018##
L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00019##
M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00020##
Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00021##
R.sub.19, R.sub.20 and R.sub.21 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.19 and
R.sub.20 taken together with the attached nitrogen atom form a five
membered ring. X is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. At least one
of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is P.
[0026] In one embodiment, P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q. K is SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19, NR.sub.19R.sub.20,
glycoside, or heterocycle. L is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, glycoside, or heterocycle. M is SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, glycoside, or heterocycle.
[0027] One aspect of the invention includes use of a compound of
Formula V in the manufacture of a medicament to protect against or
treat hearing loss in a subject. Another aspect of the invention
includes a method of protecting against or treating hearing loss in
a subject comprising administering a compound having the Formula
V:
##STR00022##
or a salt, solvate, hydrate, or prodrug thereof. R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 are the same or different and are
independently H, C(O)R.sub.a, C(O)NR.sub.2R.sub.b, C(O)OR.sub.a,
C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2,
NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, benzyl,
heteroaryl, biaryl, heterobiaryl, heterocycle, and branched,
unbranched, or cyclic alkyl. R.sub.6 and R.sub.7 are the same or
different and are independently H, branched or unbranched or
(CH.sub.2).sub.t--Z, wherein Z is aryl, heteroaryl, biaryl, cyclic
alkyl, or heterocycle, or R.sub.6 and R.sub.7 together form a
heterocycle. t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. R.sub.a,
R.sub.b, and R.sub.c are the same or different and are
independently H, aryl, heteroaryl, biaryl, heterobiaryl, or
branched, unbranched, or cyclic alkyl. P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q, further wherein lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is linear or branched
alkyl. K is C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00023##
L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00024##
M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00025##
Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00026##
R.sub.19, R.sub.20 and R.sub.21 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.19 and
R.sub.20 taken together with the attached nitrogen atom form a five
membered ring. Any of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5,
R.sub.6, and R.sub.7 and R.sub.a, R.sub.b, and R.sub.c is
substituted or unsubstituted. At least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is P.
[0028] In one embodiment, P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q. K is SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19, NR.sub.19R.sub.20,
glycoside, or heterocycle. L is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, glycoside, or heterocycle. M is SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, glycoside, or heterocycle.
[0029] In one embodiment, the compound inhibits one or more
components of a protein kinase signaling pathway. In another
embodiment, the compound is an allosteric inhibitor. In another
embodiment, the compound does not inhibit ATP binding to a protein
kinase. In one embodiment, the compound inhibits a Src family
protein kinase. For example, the Src family protein kinase is
pp60.sup.c-src tyrosine kinase.
[0030] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically (e.g., by
administering drops into the ear), intraarterially,
intralesionally, by metering pump, or by application to mucous
membranes. In one embodiment, the compound is administered in
combination with a drug that causes hearing loss e.g., cis platinum
or an aminoglycoside antibiotic. In another embodiment, the
compound is administered in combination with a drug that targets
hairy cells. In one embodiment, the compound is administered with a
pharmaceutically acceptable carrier. In one embodiment, the
compound is administered before initiation of hearing loss. In
another embodiment, the compound is administered after inititiation
of hearing loss.
[0031] In one embodiment, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is selected from halogen, boronic
acid, hydroxyl, phosphonic acid, sulfamic acid, guanidine,
carboxylic acid, aldehyde, amide, and hydroxymethylphosphonic acid.
In another embodiment, at least one of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, and R.sub.5 is a halogen. In another embodiment, at least
one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is boronic
acid. In another embodiment, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is hydroxyl. In another embodiment,
at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is
amide. For example, amide is vicinal tricarbonyl amide. In one
embodiment, R.sub.3 is halogen. For example, R.sub.3 is fluorine.
In one embodiment, R.sub.3 is hydroxyl.
[0032] In one embodiment, at least one of R.sub.6 and R.sub.7
is
##STR00027##
*R.sub.8 is the point of attachment and is (CH.sub.2).sub.x,
wherein X is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, CH.sub.2CHOH,
CH(CH.sub.3)(R-isomer), or CH(CH.sub.3)(S-isomer). Each of R.sub.9,
R.sub.10, R.sub.11, R.sub.12, and R.sub.13 is the same or different
and independently are H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b,
C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a,
OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b,
NR.sub.aC(O)NR.sub.bR.sub.c, NR.sub.aC(O)OR.sub.b, NR.sub.a
C(O)SR.sub.b, NR.sub.aS(O)R.sub.b, NR.sub.aS(O).sub.2R.sub.b,
NR.sub.aS(O)OR.sub.b, NR.sub.aS(O).sub.2OR.sub.b,
NR.sub.aP(O)OR.sub.bOR.sub.c, NR.sub.aP(O)OR.sub.bR.sub.c,
NR.sub.aP(O)OR.sub.bOR.sub.c, SR.sub.a, S(O)R.sub.a,
S(O).sub.2R.sub.a, S(O)OR.sub.a, S(O).sub.2OR.sub.a,
S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, P', aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle, or branched, cyclic, or
unbranched alkyl. P' is SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2,
OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-K', O--C(O)-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L', NH-lower (C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-M', or
O-aryl-Q', further where lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl is linear or branched alkyl. K' is
C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22, NR.sub.22R.sub.23,
SO.sub.2R.sub.24, glycoside, lower C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00028##
L' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00029##
M' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00030##
Q' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00031##
R.sub.22, R.sub.23 and R.sub.24 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.22 and
R.sub.23 taken together with the attached nitrogen atom form a five
membered ring. R.sub.a, R.sub.b, and R.sub.c are the same or
different and independently are H, aryl, heteroaryl, biaryl,
heterobiaryl, branched, cyclic, or unbranched alkyl. Any of
R.sub.9, R.sub.10, R.sub.11, R.sub.12, and R.sub.13 are substituted
or unsubstituted. If one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, or
R.sub.5 is not P, then at least one of R.sub.9, R.sub.10, R.sub.11,
R.sub.12, and R.sub.13 is P'.
[0033] In one embodiment, P' is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K', O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L',
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M', or O-aryl-Q'. K' is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, glycoside, or heterocycle. L' is SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.22, NR.sub.22R.sub.23, glycoside, or heterocycle. M' is
SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.22, NR.sub.22R.sub.23, glycoside, or heterocycle. In
another embodiment, at least one of R.sub.6 or R.sub.7 and
##STR00032##
[0034] One aspect of the invention is use of a compound of Formula
V in the manufacture of a medicament for preventin or treating a
proliferative disease in a subject. Another aspect of the invention
includes a method of preventing or treating a proliferative disease
in a subject comprising administering a compound having the Formula
V:
##STR00033##
or a salt, solvate, hydrate, or prodrug thereof. R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 are the same or different and are
independently H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a,
C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2,
NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, benzyl,
heteroaryl, biaryl, heterobiaryl, heterocycle, and branched,
unbranched, or cyclic alkyl. R.sub.6 and R.sub.7 are the same or
different and are independently H, branched or unbranched, or
(CH.sub.2).sub.t--Z, wherein Z is aryl, heteroaryl, biaryl, cyclic
alkyl, or heterocycle, or R.sub.6 and R.sub.7 together form a
heterocycle. t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. R.sub.a,
R.sub.b, and R.sub.c are the same or different and are
independently H, aryl, heteroaryl, biaryl, heterobiaryl, or
branched, unbranched, or cyclic alkyl. P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q, further wherein lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is linear or branched
alkyl. K is C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00034##
L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00035##
M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00036##
Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00037##
R.sub.19, R.sub.20 and R.sub.21 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.19 and
R.sub.20 taken together with the attached nitrogen atom form a five
membered ring; wherein any of R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 and R.sub.a, R.sub.b, and R.sub.c is
substituted or unsubstituted. At least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, and R.sub.6 is P.
[0035] In one embodiment, P is is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q. K is SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19, NR.sub.19R.sub.20,
glycoside, and heterocycle. L is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, glycoside, and heterocycle. M is SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, glycoside, and heterocycle.
[0036] In one embodiment, the compound inhibits one or more
components of a protein kinase signaling pathway. In another
embodiment, the compound is an allosteric inhibitor. In another
embodiment, the compound is a peptide substrate inhibitor. In
another embodiment, the compound does not inhibit ATP binding to a
protein kinase. In another embodiment, the compound inhibits a Src
family protein kinase. For example, the Src family protein kinase
is pp60.sup.c-scr tyrosine kinase.
[0037] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membrane. In another embodiment, the compound
is administered with a pharmaceutically acceptable carrier. In one
embodiment, the compound is administered before initiation of the
proliferative disease. In another embodiment, the compound is
administered after inititiation of the proliferative disease.
[0038] In one embodiment, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is selected from halogen, boronic
acid, hydroxyl, phosphonic acid, sulfamic acid, guanidine,
carboxylic acid, aldehyde, amide, and hydroxymethylphosphonic acid.
In another embodiment, at least one of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, and R.sub.5 is halogen. In another embodiment, at least
one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is boronic
acid. In another embodiment, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is hydroxyl. In another embodiment,
at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is
amide. For example, the amide is vicinal tricarbonyl amide. In one
embodiment, R.sub.3 is halogen. For example, R.sub.3 is fluorine.
In one embodiment, R.sub.3 is hydroxyl. In one embodiment, at least
one of R.sub.6 and R.sub.7 is
##STR00038##
*R.sub.8 is the point of attachment and is (CH.sub.2).sub.x,
wherein X is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, CH.sub.2CHOH,
CH(CH.sub.3)(R-isomer), or CH(CH.sub.3)(S-isomer). Each of R.sub.9,
R.sub.10, R.sub.11, R.sub.12, and R.sub.13 is the same or different
and independently are H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b,
C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a,
OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b,
NR.sub.aC(O)NR.sub.bR.sub.c, NR.sub.aC(O)OR.sub.b,
NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, P', aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle, or branched, cyclic, or
unbranched alkyl. P' is SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2,
OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-K', O--C(O)-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L', NH-lower (C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-M', or
O-aryl-Q', further wherein lower (C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, or C.sub.6) alkyl is linear or branched alkyl. K'
is C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22, NR.sub.22R.sub.23,
SO.sub.2R.sub.24, glycoside, lower C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00039##
L' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00040##
M' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00041##
Q' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00042##
R.sub.22, R.sub.23 and R.sub.24 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.22 and
R.sub.23 taken together with the attached nitrogen atom form a five
membered ring. R.sub.a, R.sub.b, and R.sub.c are the same or
different and independently are H, aryl, heteroaryl, biaryl,
heterobiaryl, branched, cyclic, or unbranched alkyl. R.sub.9,
R.sub.10, R.sub.11, R.sub.12, and R.sub.13 are substituted or
unsubstituted. If one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, or
R.sub.5 is not P, then at least one of R.sub.9, R.sub.10, R.sub.11,
R.sub.12, and R.sub.13 is P'.
[0039] In one embodiment, P' is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K', O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L',
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M', or O-aryl-Q'. K' is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, glycoside, or heterocycle. L' is SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.22, NR.sub.22R.sub.23, glycoside, or heterocycle. M' is
SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.22, NR.sub.22R.sub.23, glycoside, or heterocycle.
[0040] In one embodiment, at least one of R.sub.6 and R.sub.7
is
##STR00043##
[0041] One aspect of the invention is use of a compound of Formula
VII in the manufacture of a medicament for protecting against or
treating osteoporosis in a subject. Another aspect of the invention
includes a method of protecting against or treating osteoporosis in
a subject comprising administering a compound of Formula VII:
##STR00044##
or a salt, solvate, hydrate, or prodrug thereof. R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 are the same or different and are
independently H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a,
C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2,
NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.aR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, benzyl,
heteroaryl, biaryl, heterobiaryl, heterocycle, and branched,
unbranched, or cyclic alkyl. R.sub.6 and R.sub.7 are the same or
different and are independently'H, branched or unbranched or
(CH.sub.2).sub.t--Z, wherein Z is aryl, heteroaryl, biaryl, cyclic
alkyl, or heterocycle, or R.sub.6 and R.sub.7 together form a
heterocycle. t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. R.sub.a,
R.sub.b, and R.sub.c are the same or different and are
independently H, aryl, heteroaryl, biaryl, heterobiaryl, or
branched, unbranched, or cyclic alkyl. P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q, further wherein lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is linear or branched
alkyl. K is C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NR.sub.19R.sub.20,
SO.sub.2R.sub.21, glycoside, lower C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00045##
L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00046##
M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00047##
Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00048##
R.sub.19, R.sub.20 and R.sub.21 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.19 and
R.sub.20 taken together with the attached nitrogen atom form a five
membered ring; wherein any of R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 and R.sub.a, R.sub.b, and R.sub.c is
substituted or unsubstituted.
[0042] In one embodiment, P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q. K is SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19, NR.sub.19R.sub.20,
glycoside, or heterocycle. L is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, glycoside, or heterocycle. M is SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, glycoside, or heterocycle.
[0043] In one embodiment, the compound inhibits one or more
components of a protein kinase signaling pathway. In another
embodiment, the compound is an allosteric inhibitor. In another
embodiment, the compound is a peptide substrate inhibitor. In
another embodiment, the compound does not inhibit ATP binding to a
protein kinase. In another embodiment, the compound inhibits a Src
family protein kinase. For example, the Src family protein kinase
is pp60.sup.c-src tyrosine kinase.
[0044] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membranes. In one embodiment, the compound is
administered with a pharmaceutically acceptable carrier. In another
embodiment, the compound is administered before initiation of
osteoporosis. In another embodiment, the compound is administered
after inititiation of osteoporosis.
[0045] In another embodiment, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is selected from halogen, boronic
acid, hydroxyl, phosphonic acid, sulfamic acid, guanidine,
carboxylic acid, aldehyde, amide, and hydroxymethylphosphonic
acid.
[0046] In another embodiment, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is a halogen. In one embodiment, at
least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is a
boronic acid. In another embodiment, at least one of R.sub.1,
R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is hydroxyl. In another
embodiment, at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5 is amide. For example, the amide group is vicinal
tricarbonyl amide. In one embodiment, R.sub.3 is halogen. For
example, R.sub.3 is fluorine. In one embodiment, R.sub.3 is
hydroxyl.
[0047] In one embodiment, at least one of R.sub.6 and R.sub.7
is
##STR00049##
*R.sub.8 is the point of attachment and is (CH.sub.2).sub.x,
wherein X is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, CH.sub.2CHOH,
CH(CH.sub.3)(R-isomer), or CH(CH.sub.3)(S-isomer). Each of R.sub.9,
R.sub.10, R.sub.11, R.sub.12, and R.sub.13 is the same or different
and independently are H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b,
C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a,
OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b,
NR.sub.aC(O)NR.sub.bR.sub.c, NR.sub.aC(O)OR.sub.b,
NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, P', aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle and branched, cyclic, or
unbranched alkyl, P' is SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2,
OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-K', O--C(O)-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L', NH-lower (C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-M', or
O-aryl-Q', further wherein lower (C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, or C.sub.6) alkyl is linear or branched alkyl. K'
is C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22, NR.sub.22R.sub.23,
SO.sub.2R.sub.24, glycoside, lower C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00050##
L' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00051##
M' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00052##
Q' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00053##
R.sub.22, R.sub.23 and R.sub.24 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.22 and
R.sub.23 taken together with the attached nitrogen atom form a five
membered ring. R.sub.a, R.sub.b, and R.sub.c are the same or
different and independently are H, aryl, heteroaryl, biaryl,
heterobiaryl, branched, cyclic, or unbranched alkyl; and wherein
any of R.sub.9, R.sub.10, R.sub.11, R.sub.12, and R.sub.13 are
substituted or unsubstituted.
[0048] In one embodiment, P' is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K', O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L',
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M', or O-aryl-Q'. K' is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, glycoside, or heterocycle. L' is SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.22, NR.sub.22R.sub.23, glycoside, or heterocycle; and
further wherein M' is SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22, NR.sub.22R.sub.23,
glycoside, or heterocycle. In one embodiment, at least one of
R.sub.6 and R.sub.7 is
##STR00054##
In another embodiment, the compound is
##STR00055##
[0049] One aspect of the invention is use of a compound of Formula
VI in the manufacture of a medicament for protecting against or
treating hearing loss in a subject. Another aspect of the invention
includes a method of protecting against or treating hearing loss in
a subject comprising administering a compound of Formula VI:
##STR00056##
or a salt, solvate, hydrate, or prodrug thereof. R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are each the same
or different and independently are H, C(O)R.sub.a,
C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a,
OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle, and branched, cyclic, or
unbranched alkyl. R.sub.a, R.sub.b, and R.sub.c are the same or
different and are independently H, aryl, heteroaryl, biaryl,
heterobiaryl, and branched, cyclic, or unbranched alkyl; P is
SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K,
O--C(O)-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or
C.sub.6) alkyl-L, NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-M, or O-aryl-Q, further wherein lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is
linear or branched alkyl. K is C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00057##
L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00058##
M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00059##
Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00060##
R.sub.19, R.sub.20 and R.sub.21 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.19 and
R.sub.20 taken together with the attached nitrogen atom form a five
membered ring. x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. At least one
of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is P.
[0050] In one embodiment, P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q. K is SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NHR.sub.19, NR.sub.19R.sub.20, glycoside, or
heterocycle. L is SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19, NR.sub.19R.sub.20,
glycoside, or heterocycle. M is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, glycoside, or heterocycle.
[0051] In one embodiment, the compound inhibits one or more
components of a protein kinase signaling pathway. In another
embodiment, the compound is an allosteric inhibitor. In one
embodiment, the compound is a peptide substrate inhibitor. In
another embodiment, the compound does not inhibit ATP binding to a
protein kinase. In another embodiment, the compound inhibits a Src
family protein kinase. For example, the Src family protein kinase
is pp60.sup.c-src tyrosine kinase.
[0052] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically (e.g., by
administering drops into the ear), intraarterially,
intralesionally, by metering pump, or by application to mucous
membranes. In one embodiment, the compound is administered in
combination with a drug that causes hearing loss. In another
embodiment, the compound is administered with a pharmaceutically
acceptable carrier. In another embodiment, the compound is
administered before initiation of hearing loss. In another
embodiment, the compound is administered after inititiation of
hearing loss.
[0053] One aspect of the invention is use of a compound of Formula
VIII in the manufacture of a medicament for protecting against or
treating osteoporosis in a subject. Another aspect of the invention
includes a method of protecting against or treating osteoporosis in
a subject comprising administering a compound of Formula VIII:
##STR00061##
or a salt, solvate, hydrate, or prodrug thereof. R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are each the same
or different and independently are H, C(O)R.sub.a,
C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a,
OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle, and branched, cyclic, or
unbranched alkyl. R.sub.a, R.sub.b, and R.sub.c are the same or
different and are independently H, aryl, heteroaryl, biaryl,
heterobiaryl, and branched, cyclic, or unbranched alkyl. P is
SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K,
O--C(O)-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or
C.sub.6) alkyl-L, NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-M, or O-aryl-Q, further wherein lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is
linear or branched alkyl. K is C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00062##
L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00063##
M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00064##
Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00065##
R.sub.19, R.sub.20 and R.sub.21 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.19 and
R.sub.20 taken together with the attached nitrogen atom form a five
membered ring. x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
[0054] In one embodiment, P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q. K is SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19, NR.sub.19R.sub.20,
glycoside, or heterocycle. L is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, glycoside, or heterocycle. M is SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, glycoside, or heterocycle.
[0055] In one embodiment, the compound inhibits one or more
components of a protein kinase signaling pathway. In another
embodiment, the compound is an allosteric inhibitor. In another
embodiment, the compound is a peptide substrate inhibitor. In
another embodiment, the compound does not inhibit ATP binding to a
protein kinase. In another embodiment, the compound inhibits a Src
family protein kinase. For example, the Src family protein kinase
is pp60.sup.c-src tyrosine kinase.
[0056] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membranes. In one embodiment, the compound is
administered with a pharmaceutically acceptable carrier. In one
embodiment, the compound is administered before the onset of
osteoporosis. In another embodiment, the compound is administered
after onset of osteoporosis.
[0057] One aspect of the invention is use of a compound of Formula
VIII in the manufacture of a medicament for preventing or treating
a proliferative disorder in a subject. Another aspect of the
invention includes a method of preventing or treating a
proliferative disorder in a subject comprising administering a
compound of Formula VIII:
##STR00066##
or a salt, solvate, hydrate, or prodrug thereof. R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are each the same
or different and independently are H, C(O)R.sub.a,
C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a,
OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle, and branched, cyclic, or
unbranched alkyl. R.sub.a, R.sub.b, and R.sub.c are the same or
different and are independently H, aryl, heteroaryl, biaryl,
heterobiaryl, and branched, cyclic, or unbranched alkyl. P is
SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2, OPO.sub.3H.sub.2,O-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K,
O--C(O)-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or
C.sub.6) alkyl-L, NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-M, or O-aryl-Q, further wherein lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is
linear or branched alkyl. K is C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00067##
L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1,C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00068##
M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00069##
Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00070##
R.sub.19, R.sub.20 and R.sub.21 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.19 and
R.sub.20 taken together with the attached nitrogen atom form a five
membered ring. x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
[0058] In one embodiment, the compound inhibits one or more
components of a protein kinase signaling pathway. In another
embodiment, the compound is an allosteric inhibitor. In another
embodiment, the compound is a peptide substrate inhibitor. In one
embodiment, the compound does not inhibit ATP binding to a protein
kinase. In another embodiment, the compound inhibits a Src family
protein kinase. For example, the Src family protein kinase is
pp60.sup.c-src tyrosine kinase.
[0059] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membranes. In one embodiment, the compound is
administered with a pharmaceutically acceptable carrier. In another
embodiment, the compound is administered before onset of the
proliferative disease. In another embodiment, the compound is
administered after onset of the proliferative disease.
[0060] One aspect of the invention is use of a compound of the
invention e.g., of Formula VII or VIII in the manufacture of a
medicament for protecting against or treating ophthalmic disease.
Another aspect of the invention includes a method of protecting
against or treating ophthalmic disease (e.g., macular degeneration,
retinopathy, macular edema, etc.) in a subject comprising
administering a compound of Formula VII or VIII. In one embodiment,
the compound inhibits one or more components of a protein kinase
signaling pathway. In another embodiment, the compound is an
allosteric inhibitor. In another embodiment, the compound is a
peptide substrate inhibitor.sub.-- In another embodiment, the
compound does not inhibit ATP binding to a protein kinase. In
another embodiment, the compound inhibits a Src family protein
kinase.
[0061] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically (e.g., by
administration of drops or a cream to the eye), intraarterially,
intralesionally, by metering pump, or by application to mucous
membranes. In one embodiment, the compound is administered with a
pharmaceutical acceptable carrier. In another embodiment, the
compound is administered before initiation of ophthalmic disease.
In another embodiment, the compound is administered after
inititiation of ophthalmic disease.
[0062] One aspect of the invention is use of a compound of the
invention e.g., Formula VII or VIII in the manufacture of a
medicament for protecting against or treating diabetes in a
subject. Another aspect of the invention includes a method of
protecting against or treating diabetes in a subject comprising
administering a compound of Formula VII or VIII. In one embodiment,
the compound inhibits one or more components of a protein kinase
signaling pathway. In another embodiment, the compound is an
allosteric inhibitor. In another embodiment, the compound is a
peptide substrate inhibitor. In another embodiment, the compound
does not inhibit ATP binding to a protein kinase. In another
embodiment, the compound inhibits a Src family protein kinase.
[0063] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membranes. In one embodiment, the compound is
administered with a pharmaceutically acceptable carrier. In another
embodiment, the compound is administered before the onset of
diabetes. In another embodiment, the compound is administered after
the onset of diabetes.
[0064] One aspect of the invention is use of a compound of the
invention e.g, Formula VII or VIII in the manufacture of a
medicament for protecting against or treating obesity in a subject.
Another aspect of the invention includes a method of protecting
against or treating obesity in a subject comprising administering a
compound of Formula VII or VIII. In one embodiment, the compound
inhibits one or more components of a protein kinase signaling
pathway. In another embodiment, the compound is an allosteric
inhibitor. In another embodiment, the compound is a peptide
substrate inhibitor. In another embodiment, the compound does not
inhibit ATP binding to a protein kinase. In another embodiment, the
compound inhibits a Src family protein kinase.
[0065] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membranes. In one embodiment, the compound is
administered with a pharmaceutically acceptable carrier. In another
embodiment, the compound is administered before the onset of
obesity. In another embodiment, the compound is administered after
the onset of obesity.
[0066] One aspect of the invention is use of a compound of the
invention e.g., Formula VII or VIII in the manufacture of a
medicament for protecting against or treating stroke in a subject.
Another aspect of the invention includes a method of protecting
against or treating stroke in a subject comprising administering a
compound of Formula VII or VIII. In one embodiment, the compound
inhibits one or more components of a protein kinase signaling
pathway. In another embodiment, the compound is an allosteric
inhibitor. In another embodiment, the compound is a peptide
substrate inhibitor. In another embodiment, the compound does not
inhibit ATP binding to a protein kinase. In another embodiment, the
compound inhibits a Src family protein kinase.
[0067] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membranes. In one embodiment, the compound is
administered with a pharmaceutically acceptable carrier. In another
embodiment, the compound is administered before a stroke occurs in
a subject. In another embodiment, the compound is administered
after a stroke has occurred in a subject.
[0068] One aspect of the invention is use of a compound of the
invention e.g,. Formula VII or VIII in the manufacture of a
medicament for protecting against or treating athrosclerosis.
Another aspect of the invention includes a method of protecting
against or treating athrosclerosis in a subject comprising
administering .a compound of Formula VII or VIII. In one
embodiment, the compound inhibits one or more components of a
protein kinase signaling pathway. In another embodiment, the
compound is an allosteric inhibitor. In another embodiment, the
compound is a peptide substrate inhibitor. In another embodiment,
the compound does not inhibit ATP binding to a protein kinase. In
another embodiment, the compound inhibits a Src family protein
kinase.
[0069] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membranes. In one embodiment, the compound is
administered with a pharmaceutically acceptable carrier. In another
embodiment, the compound is administered before the onset of
athrosclerosis. In another embodiment, the compound is administered
after the onset of athrosclerosis.
[0070] One aspect of the invention is use of a compound of the
invention e.g., Formula VII or VIII in the manufacture of a
medicament for regulating immune system activity in a subject.
Another aspect of the invention includes a method of regulating
immune system activity in a subject comprising administering a
compound of Formula VII or VIII. In one embodiment, the compound
inhibits one or more components of a protein kinase signaling
pathway. In another embodiment, the compound is an allosteric
inhibitor. In another embodiment, the compound is a peptide
substrate inhibitor. In another embodiment, the compound does not
inhibit ATP binding to a protein kinase. In another embodiment, the
compound inhibits a Src family protein kinase.
[0071] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membranes. In one embodiment, the compound is
administered with a pharmaceutically acceptable carrier.
[0072] One aspect of the invention is use of a compound of the
invention e.g., Formula VII or VIII in the manufacture of a
medicament for protecting against or treating chronic neuropathic
pain in a subject. Another aspect of the invention includes a
method of protecting against or treating chronic neuropathic pain
in a subject comprising administering a compound of Formula VII or
VIII. In one embodiment, the compound inhibits one or more
components of a protein kinase signaling pathway. In another
embodiment, the compound is an allosteric inhibitor. In another
embodiment, the compound is a peptide substrate inhibitor. In
another embodiment, the compound does not inhibit ATP binding to a
protein kinase. In another embodiment, the compound inhibits a Src
family protein kinase.
[0073] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membranes. In one embodiment, the compound is
administered with a pharmaceutically acceptable carrier. In one
embodiment, the compound is administered before the onset of
chronic neuropathic pain. In another embodiment, the compound is
administered after the onset of chronic neuropathic pain.
[0074] One aspect of the invention is use of a compound of the
invention e.g, Formula VII or VIII in the manufacture of a
medicament for protecting against or treating hepatitis B in a
subject. Another aspect of the invention includes a method of
protecting against or treating hepatitis B in a subject comprising
administering a compound of Formula VII or VIII. In one embodiment,
the compound inhibits one or more components of a protein kinase
signaling pathway. In another embodiment, the compound is an
allosteric inhibitor. In another embodiment, the compound is a
peptide substrate inhibitor. In another embodiment, the compound
does not inhibit ATP binding to a protein kinase. In another
embodiment, the compound inhibits a Src family protein kinase.
[0075] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membranes. In one embodiment, the compound is
administered with a pharmaceutically acceptable carrier. In one
embodiment, the compound is administered before the onset of
hepatitis B. In another embodiment, the compound is administered
after the onset of hepatitis B.
BRIEF DESCRIPTION OF THE DRAWINGS
[0076] FIG. 1 depicts the modular strategy for developing
non-peptide protein kinase inhibitors. Step 1 utilizes one or more
first modules ("M.sub.1's") to identify promising non-peptide
scaffolds. Step 2 enhances the potency by adding specificity
elements. During this step the scaffolds are validated. Whether the
inhibitor is non-ATP competitive can also be determined. In step 3,
the potency and selectivity are further enhanced using
combinatorial libraries to optimize M.sub.1 and specificity
elements.
[0077] FIG. 2 provides a depiction of the x-ray structure of
(PKA):Mg.sub.2ATP:pseudosubstrate inhibitor.
[0078] FIG. 3 provides a general module M.sub.1 design features for
binding to the conserved protein kinase catalytic region.
[0079] FIG. 4 shows that the boronic acid "inhibitors" 21 and 22
were shown to be substrates for PICA.
[0080] FIG. 5 demonstrates the binding interactions of Src
substrate Ac-Ile-Tyr-Gly-Glu-Phe-NH.sub.2 (SEQ. ID. No. 1) in model
Src active site.
[0081] FIG. 6 shows the design of naphthalene-based Src inhibitor
scaffolds.
[0082] FIG. 7 shows the design of isoquinoline and indole-based Src
inhibitor scaffolds.
[0083] FIG. 8 provides an example of the chemistry used to prepare
the naphthalene inhibitors, which is described in Marsilje 2000. A
boronic acid functionality can be put in place of a M.sub.1
hydroxyl groups in the Src inhibitors from Table V using the Pd
(0)-catalyzed cross-coupling methodology wherein either an aryl
triflate (Ishiyama et al, 1997) or an aryl halide (Ishiyama, 1995)
can be coupled with the commercially available pinacol ester of
diboron.
[0084] FIG. 9 shows a synthetic scheme that can be followed, in
order to attach hydrophobic S.sub.2 selectivity elements to the
naphthalene scaffold.
[0085] FIG. 10 shows successful model reactions with naphthalene
chemistry, which can be converted to the solid phase in preparation
for synthesizing combinatorial libraries of this scaffold in a
96-well plate format. The chemistry has been carried out on the
less active naphthalene regioisomer represented by 44 because this
compound is readily obtained from commercially available
3,5-dihydroxy-2-naphthoic acid, as described in Marsilje 2000.
[0086] FIG. 11 provides a possible strategy for modifying the
naphthalene scaffold in combinatorial libraries.
[0087] FIG. 12 shows the conversion of the triflate functionality
formed in reaction 2 from intermediate 69 (FIG. 11) to an amine
(Wolfe et al, 1997) and then a series of amides or other amine
derivatives.
[0088] FIG. 13 shows modeling a series of hydroxy-containing
analogs of the boronic acid M.sub.1 group shown in the Src and IRTK
(insulin receptor protein tyrosine kinase) active sites.
[0089] FIG. 14 shows results from testing of the non-peptide Src
inhibitor 43-meta (Table V) in the LA25 and NRK cell lines.
[0090] FIG. 15A shows a comparison of taxol and doxorubicin (they
were more effective than etoposide & cisplatin in this tumor
cell culture) with the three Src inhibitors mentioned above
utilizing ovarian tumor cells from tumor N015. FIG. 15B shows the
results from tests of the src inhibitors for inhibition of normal
human fibroblast cell growth. No inhibition of normal cell growth
(both subconfluent and confluent; some enhanced growth was observed
instead) was found, indicating that these inhibitors are not toxic
to normal cells even at a 10-fold higher concentration. FIG. 15C
provides the structures of the src inhibitors TOM 2-32, TOM 2-47,
and KLM 2-31.
[0091] FIG. 16A shows a comparison of taxol and doxorubicin (they
were more effective than etoposide and cisplatin in this tumor cell
culture) with the three Src inhibitors (45, 43-meta, and 49-meta
from Table V) utilizing ovarian tumor cells from tumor N015. FIG.
16B shows the results from tests of the Src inhibitors for
inhibition of normal human fibroblast cell growth. No inhibition of
normal cell growth (both subconfluent and confluent; some enhanced
growth was observed instead) was found, indicating that these
inhibitors are not toxic to normal cells even at a 10-fold higher
concentration. FIG. 16C shows the results from tests of two of the
Src inhibitors for inhibition of is v-Src stimulated LA25 cell
growth. FIG. 16D shows the results from tests of two of the Src
inhibitors for inhibition of normal rat kidney cell growth. FIG.
16E provides the structures of the Src inhibitors 45, 43-meta, and
49-meta.
[0092] FIG. 16 is a graph showing average threshold shifts (dB) in
chinchilla cochleas after exposure to 0.5 kHz, 1 kHz, 2 kHz, 4 kHz,
and 8 kHz band noise prior to experimental manipulation.
[0093] FIG. 17 is a graph showing average threshold shifts (dB) in
chinchilla cochleas after exposure to 0.5 kHz band noise at day 0,
day 1, day 3, and day 20 after experimental manipulation.
[0094] FIG. 18 is a graph showing average threshold shifts (dB) in
chinchilla cochleas after exposure to 1 kHz band noise at day 0,
day 1, day 3, and day 20 after experimental manipulation.
[0095] FIG. 19 is a graph showing average threshold shifts (dB) in
chinchilla cochleas after exposure to 2 kHz band noise at day 0,
day 1, day 3, and day 20 after experimental manipulation.
[0096] FIG. 20 is a graph showing average threshold shifts (dB) in
chinchilla cochleas after exposure to 4 kHz band noise at day 0,
day 1, day 3, and day 20 after experimental manipulation.
[0097] FIG. 21 is a graph showing average threshold shifts (dB) in
chinchilla cochleas after exposure. to 8 kHz band noise at day 0,
day 1, day 3, and day 20 after experimental manipulation.
[0098] FIG. 22 is a graph showing the average dB threshold shifts
in chinchilla cochleas at day 20 for control and treated ears.
[0099] FIG. 23 is a graph showing average threshold shifts (dB) in
chinchilla cochleas after exposure to 0.5 kHz, 1 kHz, 2 kHz, 4 kHz,
and 8 kHz band noise prior to experimental manipulation.
[0100] FIG. 24 is a graph showing average threshold shifts (dB) in
chinchilla cochleas after exposure to 0.5 kHz, 1 kHz, 2 kHz, 4 kHz,
and 8 kHz band noise on day 1 after experimental manipulation.
[0101] FIG. 25 is a graph showing average threshold shifts (dB) in
chinchilla cochleas after exposure to 0.5 kHz, 1 kHz, 2 kHz, 4 kHz,
and 8 kHz band noise on day 3 after experimental manipulation.
[0102] FIG. 26 is a graph showing average threshold shifts (dB) in
chinchilla cochleas after exposure to 0.5 kHz, 1 kHz, 2 kHz, 4 kHz,
and 8 kHz band noise on day 7 after experimental manipulation.
[0103] FIG. 27 is a graph showing average threshold shifts (dB) in
chinchilla cochleas after exposure to 0.5 kHz, 1 kHz, 2 kHz, 4 kHz,
and 8 kHz band noise on day 20 after experimental manipulation.
[0104] FIG. 28 is a graph showing average threshold shifts (dB) in
chinchilla cochleas after exposure to 8000 Hz on day 1, day 3, day
7, and day 20.
[0105] FIGS. 29A-F are SEM images of chinchilla cochleas. FIG. 29A
shows a split (marked by S) of the reticular lamina after exposure
to an impulse noise. FIG. 29B shows focal adhesion kinase (FAK)
staining in a cochlea exposed to an octave band noise centered at 4
kHz (OBN) at 105 dB (SPL). FIG. 29C shows a small lesion with a few
apoptotic nuclei (marked with arrow) from a cochlea exposed to an
OBN at 110 dB. FIG. 29D shows FAK staining for the lesion shown in
FIG. 29C. FIG. 29E shows a confocal scanning level a few microns
lower than in FIG. 29D, demonstrating that the lesion extends well
below the cuticular plate and into the cell body (marked with
arrow). FIG. 29F shows FAK staining in a cochlea exposed to impulse
noise at 155 dB (SPL). In this figure, many outer hair cells have
lost their cuticular plate integrity. The remaining outer hair
cells show strong FAK fluorescence in the cuticular plates.
[0106] FIGS. 30A-B are confocal images of chinchilla cochleas
exposed to high level noise. In FIG. 30A, the chinchilla cochlea
was pre-treated with CH-65. In FIG. 30B, the chinchilla cochlea was
left untreated.
[0107] FIG. 31A is a graph indicating the effect of AZ28 and
KX2-391 on Src autophosphorylation in c-Src/NIH-3T3 cells; FIG. 31B
is a graph indicating the effect of AZ28 and KX2-391 on Src
autophosphorylation in HT-29 cells.
[0108] FIG. 32A is a graph indicating the effect of AZ28 and
KX2-391 on FAK phosphorylation in c-Src/NIH-3T3 cells; FIG. 32B is
a graph indicating the effect of AZ28 and KX2-391 on FAK
phosphorylation in HT-29 cells.
[0109] FIG. 33A is a graph indicating the effect of AZ28 and
KX2-391 on Shc phosphorylation in c-Src/NIH-3T3 cells; FIG. 33B is
a graph indicating the effect of AZ28 and KX2-391 on Shc
phosphorylation in HT-29 cells.
[0110] FIG. 34 is a graph indicating the effect of AZ28 and KX2-391
on paxillin phosphorylation in c-Src/NIH-3T3 cells.
[0111] FIG. 35A is a graph indicating the effect of AZ28 and
KX2-391 on caspase-3 cleavage in c-Src/NIH-3T3 cells; FIG. 35B is a
graph indicating the effect of AZ28 and KX2-391 on caspase-3
cleavage in HT-29 cells.
[0112] FIG. 36A is a graph indicating the effect of AZ28 and
KX2-391 on total phosphotyrosine levels in c-Src/NIH-3T3 cells;
FIG. 36B is a graph indicating the effect of AZ28 and KX2-391 on
total phosphotyrosine levels in HT-29 cells.
[0113] FIG. 37 is a graph indicating the effect of AZ28 and KX2-391
on autophosphorylation of PDGFR in c-Src/NIH-3T3 cells.
[0114] FIG. 38A is a graph indicating the effect of AZ28 and
KX2-391 on autophosphorylation of FAK in c-Src/NIH-3T3 cells; FIG.
38B is a graph indicating the effect of AZ28 and KX2-391 on
autophosphorylation of FAK in HT-29 cells.
[0115] FIG. 39A is a graph indicating the effect of AZ28 and
KX2-391 on autophosphorylation of EGFR in c-Src/NIH-3T3 cells; FIG.
39B is a graph indicating the effect of AZ28 and KX2-391 on
autophosphorylation of EGFR in HT-29 cells.
[0116] FIG. 40 is a bar chart showing the average threshold shifts
(dB) in chinchilla cochleas after exposure to 0.5 kHz, 1 kHz, 2
kHz, 4 kHz, and 8 kHz band noise on day 1 after experimental
manipulation.
[0117] FIG. 41 is a graph showing the average threshold shifts (dB)
in chinchilla cochleas after exposure to 0.5 kHz, 1 kHz, 2 kHz, 4
kHz, and 8 kHz band noise on day 7 after experimental
manipulation.
[0118] FIG. 42 is a graph showing the average threshold shifts (dB)
in chinchilla cochleas after exposure to 0.5 kHz, 1 kHz, 2 kHz, 4
kHz, and 8 kHz band noise on day 21 after experimental
manipulation.
[0119] FIG. 43 is a line graph showing the threshold shifts (dB) in
guinea pig cochleas after exposure to 2 kHz, 4 kHz, 8 kHz, 12 kHz,
16 kHz and 20 kHz band noise after treatment with cisplatin.
[0120] FIG. 44 is a line graph showing the threshold shifts (dB) in
KX1-004-treated guinea pig cochleas after exposure to 2 kHz, 4 kHz,
8 kHz, 12 kHz, 16 kHz and 20 kHz band noise after treatment with
cisplatin.
[0121] FIG. 45 is a line graph showing the average threshold shifts
(dB) in KX1-004-treated guinea pig cochleas and untreated control
guinea pig cochleas after exposure to 2 kHz, 4 kHz, 8 kHz, 12 kHz,
16 kHz and 20 kHz band noise after treatment with cisplatin.
[0122] FIG. 46 is a series of illustrations depicting the effect of
compounds on osteoclast formation.
[0123] FIG. 47 is a bar chart demonstrating the effect of compounds
on osteoclast formation.
[0124] FIG. 48 is a series of illustrations showing the effect of
compounds on osteoclast survival.
[0125] FIG. 49 is a bar chart depicting the effect of compounds on
osteoclast survival.
[0126] FIG. 50A is a bar chart demonstrating the effect of
compounds on bone resorption in vitro.
[0127] FIG. 50B is a bar chart showing the effect of compounds on
resorption pit formation.
[0128] FIG. 51A is a series of illustrations depicting the effect
of compounds on osteoclast formation on bone slices.
[0129] FIG. 51B is a series of illustrations demonstrating the
effect of compounds on the formation of resorption pits on bone
slices.
[0130] FIG. 52 is a bar chart showing the effect of compounds on
alkaline phosphatase expression by osteoblasts.
[0131] FIG. 53 is a bar chart depicting the effect of compounds on
protein expression by osteoblasts.
DETAILED DESCRIPTION OF THE INVENTION
[0132] Because kinases are involved in the regulation of a wide
variety of normal cellular signal transduction pathways (e.g., cell
growth, differentiation, survival, adhesion, migration, etc.),
kinases are thought to play a role in a variety of diseases and
disorders. Thus, modulation of kinase signaling cascades may be an
important way to treat or prevent such diseases and disorders. Such
diseases and disorders include, for example, cancers, osteoporosis,
cardiovascular disorders, immune system dysfunction, type II
diabetes, obesity, and transplant rejection.
[0133] Compounds of the invention are useful in modulation a
component of the kinase signaling cascade. Compounds of the
invention are useful in the manufacture of a medicament that
modulates a component of the kinase signaling cascade. Some
compounds may be useful in modulation of more than one component of
a kinase signaling cascade. The phrase "modulates one or more
components of a protein kinase signaling cascade" means that one or
more components of the kinase signaling cascade are affected such
that the functioning of a cell changes. Components of a protein
kinase signaling cascade include any proteins involved directly or
indirectly in the kinase signaling pathway including second
messengers and upstream and downstream targets.
[0134] A number of protein kinases and phosphatases are known, and
are targets for the development of therapeutics. See, e.g., Hidaka
and Kobayashi, Annu. Rev. Pharmacol. Toxicol, 1992, 32:377-397;
Davies et al., Biochem. J., 2000, 351:95-105, each of which is
incorporated by reference herein.
[0135] One family of kinases, the protein tyrosine kinases are
divided into two large families: receptor tyrosine kinases, or RTKs
(e.g., insulin receptor kinase (IRK), epidermal growth factor
receptor (EGFR), basic fibroblast growth factor receptor (FGFR),
platelet-derived growth factor receptor (PDGFR), vascular
endothelial growth factor receptor (VEGFR-2 or Flk1/KDR), and nerve
growth factor receptor (NGFR)) and nonreceptor tyrosine kinases, or
NRTKs (e.g., the Src family (Src, Fyn, Yes, Blk, Yrk, Fgr, lick,
Lck, and Lyn), Fak, Jak, Abl and Zap70). See, for example, Parang
and Sun, Expert Opin. Ther. Patents, 2005, 15:1183-1207,
incorporated by reference herein.
[0136] Because of the role of Src kinases in a variety of cancers,
these kinases are the subject of a number of studies relating to
the development of Src inhibitors as cancer therapeutics, including
highly metastatic cancer cell growth. Src inhibitors are sought as
therapeutics for a variety of cancers, including, for example,
colon cancer, precancerous colon lesions, ovarian cancer, breast
cancer, epithelial cancers, esophageal cancer, non-small cell lung
cancer, pancreatic cancer, and others. See, e.g., Frame, Biochim.
Biophys. Acta, 2002, 1602:114-130 and Parang and Sun, Expert Opin.
Ther. Patents, 2005, 15:1183-1207.
[0137] Inhibition of other kinases may be useful in the treatment
and modulation of other types of diseases and disorders. For
example, various eye diseases may be inhibited or prevented by
administration of VEGF receptor tyrosine kinase inhibitors.
Inhibitors of the tyrosine phosphatase PTP-1B and/or glycogen
phosphorylase may provide treatments for Type II diabetes or
obesity. Inhibitors of p56lck may be useful in treating immune
system disorders. Other targets include HIV reverse transcriptase,
thromboxane synthase, EGFRTK, p55 fyn, etc.
[0138] Compounds of the invention may be Src signaling inhibitors
that bind in the Src peptide substrate site. Compounds of the
invention may be useful in the manufacture of a medicament that
comprises a Src signaling inhibitor that binds in the Src peptide
substrate. The activity of various compounds of the invention has
been studied in c-Src (527F, constitutively active and
transforming) transformed NIH3T3 cells and in human colon cancer
cells (HT29). For example, in these cell lines, KX2-391 was shown
to reduce the phosphorylation level of known Src protein substrates
in a dose-dependent fashion and in good correlation with growth
inhibitory effects. Thus, in some embodiments, compounds of the
invention may directly inhibit Src, and may do so by binding in the
peptide binding site (as opposed to binding at an allosteric
site).
[0139] Molecular modeling experiments have been performed which
show that compounds of the invention fit into the model Src
substrate site (See, e.g., U.S. Pat. Nos. 7,005,445 and 7,070,936).
Modeling is also used to retool the Src kinase inhibitor scaffolds
in order to target other kinases, simply by using a different set
of side chains present on the molecules and/or modifying the
scaffold itself.
[0140] Without wishing to be bound by theory, it is believed that
the conformation of some kinases (e.g., Src) outside cells relative
to the conformation inside cells is markedly different, because
inside cells, many kinases are is embedded in multiprotein
signaling complexes. Thus, because the peptide substrate binding
site is not well formed in an isolated kinase (as shown by Src
x-ray structures), it is believed that the activity against
isolated kinase for a peptide substrate binding inhibitor would be
weak. Binding to this site in an isolated kinase assay requires the
inhibitor to capture the very small percentage of total protein in
an isolated enzyme assay that is in the same conformation that
exists inside cells. This requires a large excess of the inhibitor
to drain significant amounts of the enzyme from the catalytic cycle
in the assay in order to be detectable.
[0141] However, for cell-based assays, a large inhibitor excess is
not needed because the peptide binding site is expected to be
formed. In cell-based Src assays, SH2 & SH3 domain binding
proteins have already shifted the Src conformation so that the
peptide substrate binding site is fully formed. Thus, low
concentrations of the inhibitor can remove the enzyme from the
catalytic cycle since all of the enzyme is in the tight binding
conformation.
[0142] The vast majority of known kinase inhibitors are ATP
competitive and show poor selectivity in a panel of isolated kinase
assays. However, many of the compounds of the invention are thought
to be peptide substrate binding inhibitors. Thus, traditional high
throughput screening of compounds against isolated enzymes, such as
Src, would not result in the discovery of compounds of the
invention.
[0143] There is considerable recent literature support for
targeting pp60c-src (Src) as a broadly useful approach to cancer
therapy without resulting in serious toxicity. For example, tumors
that display enhanced EGF receptor PTK signaling, or overexpress
the related Her-2/neu receptor, have constitutively activated Src
and enhanced tumor invasiveness. Inhibition of Src in these cells
induces growth arrest, triggers apoptosis, and reverses the
transformed phenotype (Karni et al. (1999) Oncogene 18(33):
4654-4662). It is known that abnormally elevated Src activity
allows transformed cells to grow in an anchorage-independent
fashion. This is apparently caused by the fact that extracellular
matrix signaling elevates Src activity in the FAK/Src pathway, in a
coordinated fashion with mitogenic signaling, and thereby blocks an
apoptotic mechanism which would normally have been activated.
Consequently FAK/Src inhibition in tumor cells may induce apoptosis
because the apoptotic mechanism which would have normally become
activated upon breaking free from the extracellular matrix would be
induced (Hisano, et al., Proc. Annu. Meet. Am. Assoc. Cancer Res.
38:A1925 (1997)). Additionally, reduced VEGF mRNA expression was
noted upon Src inhibition and tumors derived from these
Src-inhibited cell lines showed reduced angiogenic development
(Ellis et al., Journal of Biological Chemistry 273 (2):1052-1057
(1998)).
[0144] For example, a knock-out of the Src gene in mice led to only
one defect, namely osteoclasts that fail to form ruffled borders
and consequently do not resorb bone. However, the osteoclast bone
resorb function was rescued in these mice by inserting a kinase
defective Src gene (Schwartzberg et al., (1997) Genes &
Development 11: 2835-2844). This suggested that Src kinase activity
can be inhibited in vivo without triggering the only known toxicity
because the presence of the Src protein is apparently sufficient to
recruit and activate other PTKs (which are essential for
maintaining osteoclast function) in an osteoclast essential
signaling complex.
[0145] Src has been proposed to be a "universal" target for cancer
therapy since it has been found to be overactivated in a growing
number of human tumors (Levitzki, Current Opinion in Cell Biology,
8, 239-244 (1996); Levitzki, Anti-Cancer Drug Design, 11, 175-182
(1996)). The potential benefits of Src inhibition for cancer
therapy appear to be four-fold inhibition of uncontrolled cell
growth caused by autocrine growth factor loop effects, inhibition
of metastasis due to triggering apoptosis upon breaking free from
the cell matrix, inhibition of tumor angiogenesis via reduced VEGF
levels, low toxicity.
[0146] Prostate cancer cells have been reported to have both an
over expression of paxillin and p130cas and are hyperphosphorylated
(Tremblay et al., hit. J. Cancer, 68, 164-171, 1996) and may thus
be a prime target for Src inhibitors.
[0147] As described herein, a compound of the invention may be used
to protect against or prevent hearing loss in a subject. A compound
of the invention may be used in the manufacture of a medicament to
protect against or prevent hearing loss in a subject. In order to
protect against hearing loss, the compound may be administered
prior to noise exposure or exposure to a drug which induces hearing
loss. Such drugs may include chemotherapeutic drugs (e.g.,
platinum-based drugs which target hair cells) and aminoglycoside
antibiotics. A compound of the invention may provide a synergistic
effect with certain cancer drugs. For example, promising inhibitors
can be screened in primary human tumor tissue assays, particularly
to look for synergy with other known anti-cancer drugs. In
addition, the protein kinase inhibitors may reduce toxicity of
certain cancer drugs (e.g., platinum-based drugs which are toxic to
the cochlea and kidney), thereby allowing increased dosage.
[0148] Alternatively, a compound of the invention may be used to
treat hearing loss in a subject. In this embodiment, the compound
is administered to the subject subsequent to the initiation of
hearing loss to reduce the level of hearing loss. A compound of the
invention may be involved in modulating a kinase cascade, e.g. a
kinase inhibitor, a non-ATP competitive inhibitor, a tyrosine
kinase inhibitor, a Src inhibitor or a focal adhesion kinase (FAK)
modulator. Although not wishing to be bound by theory, it is
believed that the administration of kinase inhibitors prevents
apoptosis of cochlear hair cells, thereby preventing hearing loss.
In one embodiment, administration of a compound of the invention is
administered to a subject suffering from hearing loss in order to
prevent further hearing loss. In another embodiment, administration
of a compound of the invention is administered to a subject
suffering from hearing loss in order to restore lost hearing. In
particular, following noise exposure, the tight cell junctures
between the cochlear hair cells, as well as the cell-extracellular
matrix interaction, are torn and stressed. The stressing of these
tight cell junctures initiates apoptosis in the cells through a
complex signaling pathway in which tyrosine kinases act as
molecular switches, interacting with focal adhesion kinase to
transduce signals of cell-matrix disruptions to the nucleus. It is
believed that the administration of kinase inhibitors prevents the
initiation of apoptosis in this cascade.
[0149] The identification of apoptosis in the noise-exposed cochlea
has generated a number of new possibilities for the prevention of
noise-induced hearing loss (NIHL) (Hu, et al.; 2000, Acta.
Otolaryngol., 120, 19-24). For example, the ear can be protected
from NIHL by administration of antioxidant drugs to the round
window of the ear (Hight, et al.; 2003, Hear. Res., 179, 21-32; Hu,
et al.; Hear. Res. 113, 198-206). Specifically, NIHL has been
reduced by the administration of FDA-approved antioxidant compounds
(N-L-acetylcysteine (L-NAC) and salicylate) in the chinchilla
(Kopke, et al.; 2000, Hear. Res., 149, 138-146). Moreover, Harris
et al. have recently described prevention of NIHL with Src-PTK
inhibitors (Harris, et al.; 2005, Hear. Res., 208, 14-25). Thus, it
is hypothesized that the administration of a compound of the
instant invention which modulates the activity of kinases, is
useful for treating hearing loss.
[0150] Changes in cell attachment or cell stress can activate a
variety of signals through the activation of integrins and through
the phosphorylation of PTKs, including the Src family of tyrosine
kinases. Src interactions have been linked to signaling pathways
that modify the cytoskeleton and activate a variety of protein
kinase cascades that regulate cell survival and gene transcription
(reviewed in Gianotti and Ruoslahti; 1999, Science, 285,
1028-1032). In fact, recent results have indicated that outer hair
cells (OHC), which had detached at the cell base following an
intense noise exposure, underwent apoptotic cell death.
Specifically, the Src PTK signaling cascade is thought to be
involved in both metabolic- and mechanically-induced initiation of
apoptosis in sensory cells of the cochlea. In a recent study, Src
inhibitors provided protection from a 4 hour, 4 kHz octave band
noise at 106 dB, indicating that Src-PTKs might be activated in
outer hair cells following noise exposure (Harris, et al.; 2005,
Hear. Res., 208, 14-25). Thus, compounds of the instant invention
that modulate the activity of Src, are useful in treating hearing
loss.
[0151] The present invention relates to a method for protecting
against or treating osteoporosis in a subject. This method involves
administering an effective amount of a compound of the invention to
the subject to protect against or to treat osteoporosis. In order
to protect against osteoporosis, the compound may be administered
prior to the development of osteoporosis. Alternatively, the
compound may be used to treat osteoporosis in a subject.. In this
embodiment, the compound is administered to the subject subsequent
to the initiation of osteoporosis to reduce the level of
osteoporosis. A compound of the invention may be used in the
manufacture of a medicament to protect against or treat
osteoporosis in a subject.
[0152] A compound of the invention can be, e.g. a non-ATP
competitive inhibitor. The compound of the invention can modulate a
kinase signaling cascade, depending upon the particular side chains
and scaffold modifications selected. The compound of the invention
can be a kinase inhibitor. For example, the compound can be a
protein tyrosine kinase (PTK) inhibitor. The proline-rich tyrosine
kinase (PYK2; also known as cell adhesion kinase .beta., related
adhesion focal tyrosine kinase, or calcium-dependent tyrosine
kinase) and focal adhesion kinase (FAK) are members of a distinct
family of non receptor protein-tyrosine kinases that are regulated
by a variety of extracellular stimuli (Avraham, et al.; 2000, Cell
Signal., 12, 123-133; Schlaepfer, et al.; 1999, Prog. Biophys. Mol.
Biol., 71, 435-478). The compound of the invention can be a Src
inhibitor. It has been shown that Src deficiency is associated with
osteoporosis in mice, because of loss of osteoclast function
(Soriano, et al.; 1991, Cell, 64, 693-702). Alternatively, the
compound of the invention can modulate the expression of
interleukin-1 receptor associated kinase M (IRAK-M). Mice that lack
IRAK-M develop severe osteoporosis, which is associated with the
accelerated differentiation of osteoclasts, an increase in the
half-life of osteoclasts, and their activation (Hongmei, et al.;
2005, J. Exp. Med., 201, 1169-1177).
[0153] Multinucleated osteoclasts originate from the fusion of
mononuclear phagocytes and play a major role in bone development
and remodeling via the resorption of bone. Osteoclasts are
multinucleated, terminally differentiated cells that degrade
mineralized matrix. In normal bone tissue, there is a balance
between bone formation by osteoblasts and bone resorption by
osteoclasts. When the balance of this dynamic and highly regulated
process is disrupted, bone resorption can exceed bone formation
resulting in quantitative bone loss. Because osteoclasts are
essential for the development and remodeling of bone, increases in
their number and/or activity lead to diseases that are associated
with generalized bone loss (e.g., osteoporosis) and others with
localized bone loss (e.g., rheumatoid arthritis, periodontal
disease).
[0154] Osteoclasts and osteoblasts both command a multitude of
cellular signaling pathways involving protein kinases. Osteoclast
activation is initiated by adhesion to bone, cytoskeletal
rearrangement, formation of the sealing zone, and formation of the
polarized ruffled membrane. It is believed that protein-tyrosine
kinase 2 (PYK2) participates in the transfer of signals from the
cell surface to the cytoskeleton, as it is tyrosine phosphorylated
and activated by adhesion-initiated signaling in osteoclasts
(Duong, et al.; 1998, J. Clin. Invest., 102, 881-892). Recent
evidence has indicated that the reduction of PYK2 protein levels
results in the inhibition of osteoclast formation and bone
resorption in vitro (Duong, et al.; 2001, J. Bio. Chem., 276,
7484-7492). Therefore, the inhibition of PYK2 or other protein
tyrosine kinases might reduce the level of osteoporosis by
decreasing osteoclast formation and bone resorption. Thus, without
wishing to be bound by theory, it is hypothesized that the
administration of a compound of the instant invention will modulate
kinase (e.g. PTK) activity and therefore result in the inhibition
of osteoclast formation and/or bone resporption, thereby treating
osteoporosis.
[0155] Src tyrosine kinase stands out as a promising therapeutic
target for bone disease as validated by Src knockout mouse studies
and in vitro cellular experiments, suggesting a regulatory role for
Src in both osteoclasts (positive) and osteoblasts (negative). In
osteoclasts, Src plays key roles in motility, polarization,
survival, activation (ruffled border formation) and adhesion, by
mediating various signal transduction pathways, especially in
cytokine and integrin signaling (Parang and Sun; 2005, Expert Opin.
Ther. Patents, 15, 1183-1207). Moreover, targeted disruption of the
src gene in mice induces osteopetrosis, a disorder characterized by
decreased bone resorption, without showing any obvious
morphological or functional abnormalities in other tissues or cells
(Soriano, et al.; 1991, Cell, 64, 693-702). The osteopetrotic
phenotype of src.sup.-/- mice is cell-autonomous and results from
defects in mature osteoclasts, which normally express high levels
of Src protein (Home, et al.; 1991, Cell, 119, 1003-1013). By
limiting the effectiveness of Src tyrosine kinase, which triggers
osteoclast activity and inhibits osteoblasts, Src inhibitors are
thought to lessen bone break down and encourage bone formation.
Because osteoclasts normally express high levels of Src, inhibition
of Src kinase activity might be useful in the treatment of
osteoporosis (Missbach, et al.; 1999, Bone, 24, 437-449). Thus, the
PTK inhibitors of the instant invention that modulate the activity
of Src, are useful in treating osteoporosis.
[0156] As described herein, a compound of the invention may be used
to protect against or prevent obesity in a subject. In order to
protect against obesity, the compound may be administered prior to
the development of obesity in a subject. Alternatively, the
compound may be used to treat obesity in a subject. A compound of
the instant invention may be involved in modulating a kinase
signaling cascade, e.g., a kinase inhibitor, a non-ATP competitive
inhibitor, a tyrosine kinase inhibitor, a protein tyrosine
phosphatase inhibitor, or a protein-tyrosine phosphatase 1B
inhibitor. A compound of the invention may be used in the
manufacture of a medicament to protect against or prevent obesity
in a subject
[0157] Obesity is associated with diabetes and increased insulin
resistance in insulin responsive tissues, such as skeletal muscle,
liver, and white adipose tissue (Klaman, et al.; 2000, Mol. Cell.
Biol., 20, 5479-5489). Insulin plays a critical role in the
regulation of glucose homeostasis, lipid metabolism, and energy
balance. Insulin signaling is initiated by binding of insulin to
the insulin receptor (IR), a receptor tyrosine kinase. Insulin
binding evokes a cascade of phosphorylation events, beginning with
the autophosphorylation of the IR on multiple tyrosyl residues.
Autophosphorylation enhances IR kinase activity and triggers
downstream signaling events. The stimulatory effects of protein
tyrosine kinases and the inhibitory effects of protein tyrosine
phosphatases largely define the action of insulin. Appropriate
insulin signaling minimizes large fluctuations in blood glucose
concentrations nd ensures adequate delivery of glucose to cells.
Since insulin stimulation leads to multiple tyrosyl phosphorylation
events, enhanced activity of one or more protein-tyrosine
phosphatases (PTPs) could lead to insulin resistance, which may
lead to obesity. Indeed, increased PTP activity has been reported
in several insulin-resistant states, including obesity (Ahmad, et
al.; 1997, Metabolism, 46, 1140-1145). Thus, without wishing to be
bound by theory, the administration of a compound of the instant
invention modulates kinase (e.g., PTP) activity, thereby treating
obesity in a subject.
[0158] Insulin signaling begins with the activation of the IR via
tyrosine phosphorylation and culminates in the uptake of glucose
into cells by the glucose transporter, GLUT4 (Saltiel and Kahn;
2001, Nature, 414, 799-806). The activated IR must then be
deactivated and returned to a basal state, a process that is
believed to involve protein-tyrosine phosphatase-1B (PTP-1B)
(Ahmad, et al; 1997, J. Biol. Chem., 270, 20503-20508). Disruption
of the gene that codes for PTP-1B in mice results in sensitivity to
insulin and increased resistance to diet-induced obesity (Elchebly,
et al.; 1999, Science, 283, 1544-1548; Klaman, et al.; 2000, Mol.
Biol., 20, 5479-5489). The decreased adiposity in PTP-1B deficient
mice was due to a marked reduction in fat cell mass without a
decrease in adipocyte number (Klaman, et al.; 2000, Mol. Cell.
Biol., 20, 5479-5489). Moreover, leanness in PTP-1B-deficient mice
was accompanied by increased basal metabolic rate and total energy
expenditure, without marked alteration of uncoupling protein mRNA
expression. The disruption of the PTP-1B gene demonstrated that
altering the activity of PTP-1B can modulate insulin signaling and
dietary-induced obesity in vivo. Thus, without wishing to be bound
by theory, the administration of a compound of the instant
invention that modulates insulin signaling (e.g., PTP-1B activity),
is useful in treating obesity in a subject.
[0159] As described herein, a compound of the invention may be used
to protect against or prevent diabetes in a subject. In order to
protect against diabetes, the compound may be administered prior to
the development of diabetes in a subject. Alternatively, the
compound may be used to treat diabetes in a subject. The compound
of the instant invention may be involved in modulating a kinase
signaling cascade, e.g. a kinase inhibitor, a non-ATP competitive
inhibitor, a tyrosine kinase inhibitor, a phosphatase and tension
homologue on chromosome 10 (PTEN) inhibitor, or a sequence homology
2-containing inositol 5'-phosphatase 2 (SHIP2) inhibitor. A
compound of the invention may be used in the manufacture of a
medicament to protect against or prevent diabetes in a subject
[0160] Type 2 diabetes mellitus (T2DM) is a disorder of
dysregulated energy metabolism. Energy metabolism is largely
controlled by the hormone insulin, a potent anabolic agent that
promotes the synthesis and storage of proteins, carbohydrates and
lipids, and inhibits their breakdown and release back into the
circulation. Insulin action is initiated by binding to its tyrosine
kinase receptor, which results in autophosphorylation and increased
catalytic activity of the kinase (Patti, et al.; 1998, J. Basic
Clin. Physiol. Pharmacol. 9, 89-109). Tyrosine phosphorylation
causes insulin receptor substrate (IRS) proteins to interact with
the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K),
leading to the activation of the enzyme and its targeting to a
specific subcellular location, depending on the cell type. The
enzyme generates the lipid product
phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P.sub.3),
which regulates the localization and activity of numerous proteins
(Kido, et al.; 2001, J. Clin. Endocrinol. Metab., 86, 972-979).
PI3K has an essential role in insulin-stimulated glucose uptake and
storage, inhibition of lipolysis and regulation of hepatic gene
expression (Saltiel, et al.; 2001, Nature, 414, 799-806).
Overexpression of dominant-interfering forms of PI3K can block
glucose uptake and translocation of glutamate transporter four,
GLUT4, to the plasma membrane (Quon, et al.; 1995, Mol. Cell.
Biol., 15, 5403-5411). Thus, the administration of a compound of
the instant invention that modulates kinase (e.g. PI3K) activity,
and therefore results in increased glucose uptake, is useful in
treating diabetes.
[0161] PTEN is a major regulator of PI3K signaling in may cell
types, and functions as a tumor suppressor due to antagonism of the
anti-apoptotic, proliferative and hypertrophic activities of the
PI3K pathway (Goberdhan, et al.; 2003, Hum. Mol. Genet., 12,
R239-R248; Leslie, et al.; 2004, J. Biochem., 382, 1-11). Although
not wishing to be bound by theory, it is believed that PTEN
attenuates the PI3K pathway by dephosphorylation of the
Ptdlns(3,4,5)P.sub.3 molecule, degrading this important lipid
second messenger to PtdIns(4,5)P.sub.2. In a recent study,
reduction of endogenous PTEN protein by 50% using small interfering
RNA (siRNA) enhanced insulin-dependent increases in
PtdIns(3,4,5)P.sub.3 levels, and glucose uptake (Tang, et al.;
2005, J. Biol. Chem., 280, 22523-22529). Thus, without wishing to
be bound by theory, it is hypothesized that the administration of a
compound of the instant invention that modulates PTEN activity, and
therefore results in increased glucose uptake, is useful for
treating diabetes.
[0162] PtdIns(3,4,5)P.sub.3 levels are also controlled by the
family of SRC homology 2 (SH2)-containing inositol 5'-phosphatase
(SHIP) proteins, SHIP1 and SHIP2 (Lazar and Saltiel; 2006, Nature
Reviews, 5, 333-342). SHIP2, expressed in skeletal muscle, among
other insulin-sensitive tissues, catalyzes the conversion of
PtdIns(3,4,5)P.sub.3 into PtdIns(3,4)P.sub.2 (Pesesse, et al.;
1997; Biochem Biophys. Res. Commun., 239, 697-700; Backers, et al.;
2003, Adv. Enzyme Regul., 43, 15-28; Chi, et al.; 2004, J. Biol.
Chem., 279, 44987-44995; Sleeman, et al.; 2005, Nature Med., 11,
199-205). Overexpression of SHIP2 markedly reduced
insulin-stimulated PtdIns(3,4,5)P.sub.3 levels, consistent with the
proposed capacity of SHIP2 to attenuate the activation of
downstream effectors of PI3K (Ishihara, et al.; 1999, Biochem.
Biophys. Res. Commun., 260, 265-272). Thus, without wishing to be
bound by theory, it is hypothesized that the administration of a
compound of the instant invention which modulates SHIP2 activity,
and therefore results in increased glucose uptake, is useful for
treating diabetes.
[0163] As described herein, a compound of the invention may be used
to protect against or prevent eye disease in a subject. In order to
protect against eye disease, the compound may be administered prior
to the development of eye disease in a subject. Alternatively, the
compound may be used to treat eye disease in a subject, e.g.
macular degeneration, retinopathy, and macular edema. The compound
of the instant invention may be involved in modulating a kinase
cascade, e.g. a kinase inhibitor, a non-ATP competitive inhibitor,
a tyrosine kinase inhibitor, e.g. a vascular endothelial growth
factor (VEGF) receptor tyrosine kinase inhibitor. A compound of the
invention may be used in the manufacture of a medicament to protect
against or prevent eye disease in a subject
[0164] Vision-threatening neovascularization of the physiologically
avascular cornea can occur. The proliferative retinopathies,
principally diabetic retinopathy and age-related macular
degeneration, are characterized by increased vascular permeability,
leading to retinal edema and subretinal fluid accumulation, and the
proliferation of new vessels that are prone to hemorrhage.
Angiogenesis, the formation of new blood vessels from preexisting
capillaries, is an integral part of both normal development and
numerous pathological processes. VEGF, a central mediator of the
complex cascade of angiogenesis and a potent permeability factor,
is an attractive target for novel therapeutics. VEGF is the ligand
for two membrane-bound tyrosine kinase receptors, VEGFR-1 and
VEGFR-2. Ligand binding triggers VEGFR dimerization and
transphosphorylation with subsequent activation of an intracellular
tyrosine kinase domain. The ensuing intracellular signaling axis
results in vascular endothelial cell proliferation, migration, and
survival. Thus, without wishing to be bound by theory, it is
hypothesized that the administration of a compound of the instant
invention which modulates kinase activity, e.g. tyrosine kinase
activity, and results in the inhibition of angiogenesis and/or
neovascularization, is useful for treating an eye disease, e.g.
macular degeneration, retinopathy and/or macular edema.
[0165] Macular degeneration is characterized by VEGF-mediated
retinal leakage (an increase in vascular permeability) and by the
abnormal growth of small blood vessels in the back of the eye
(angiogenesis). VEGF has been identified in neovascular membranes
in both diabetic retinopathy and age-related macular degeneration,
and intraocular levels of the factor correlate with the severity of
neovascularization in diabetic retinopathy (Kvanta, et al.; 1996,
Invest. Ophthal. Vis. Sci., 37, 1929-1934.; Aiello, et al.; 1994,
N. Engl. J. Med., 331, 1480-1487). Therapeutic antagonism of VEGF
in these models results in significant inhibition of both retinal
and choroidal neovascularization, as well as a reduction in
vascular permeability (Aiello, et al.; 1995, Proc. Natl. Acad. Sci.
USA., 92, 10457-10461; Krzystolik, et al.; 2002, Arch. Ophthal.,
120, 338-346; Qaum, et al.; 2001, Invest. Ophthal. Vis. Sci., 42,
2408-2413). Thus, without wishing to be bound by theory, it is
hypothesized that the administration of a compound of the instant
invention which modulates VEGF activity, and results in the
inhibition of angiogenesis and/or neovascularization, is useful for
treating an eye disease, e.g. macular degeneration, retinopathy
and/or macular edema.
[0166] The compounds of the invention are used in methods of
treating, preventing, ameliorating a stroke in a subject who is at
risk of suffering a stroke, is suffering from a stroke or has
suffered a stroke. The compounds of the invention are useful in
methods of treating patients who are undergoing post-stroke
rehabilitation. A compound of the invention may be used in the
manufacture of a medicament to treat, prevent or ameliorate stroke
in a subject
[0167] A stroke, also known as a cerebrovascular accident (CVA), is
an acute neurological injury whereby the blood supply to a part of
the brain is interrupted due to either blockage of an artery or
rupture of a blood vessel. The part of the brain in which blood
supply is interrupted no longer receives oxygen and/or nutrients
carried by the blood. The brain cells become damaged or necrotic,
thereby impairing function in or from that part of the brain. Brain
tissue ceases to function if deprived of oxygen for more than 60 to
90 seconds and after a few minutes will suffer irreversible injury
possibly leading to a death of the tissue, i.e., infarction.
[0168] Strokes are classified into two major types: ischemic, i.e.,
blockage of a blood vessel supplying the brain, and hemorrhagic,
i.e., bleeding into or around the brain. The majority of all
strokes are ischemic strokes. Ischemic stroke is commonly divided
into thrombotic stroke, embolic stroke, systemic hypoperfusion
(Watershed stroke), or venous thrombosis. In thrombotic stroke, a
thrombus-forming process develops in the affected artery, the
thrombus, i.e., blood clot, gradually narrows the lumen of the
artery, thereby impeding blood flow to distal tissue. These clots
usually form around atherosclerotic plaques. There are two types of
thrombotic strokes, which are categorized based on the type of
vessel on which the thrombus is formed. Large vessel thrombotic
stroke involves the common and internal carotids, vertebral, and
the Circle of Willis. Small vessel thrombotic stroke involves the
intracerebral arteries, branches of the Circle of Willis, middle
cerebral artery stem, and arteries arising from the distal
vertebral and basilar artery.
[0169] A thrombus, even if non-occluding, can lead to an embolic
stroke if the thrombus breaks off, at which point it becomes an
embolus. An embolus refers to a traveling particle or debris in the
arterial bloodstream originating from elsewhere. Embolic stroke
refers to the blockage of arterial access to a part of the brain by
an embolus. An embolus is frequently a blood clot, but it can also
be a plaque that has broken off from an atherosclerotic blood
vessel or a number of other substances including fat, air, and even
cancerous cells. Because an embolus arises from elsewhere, local
therapy only solves the problem temporarily. Thus, the source of
the embolus must be identified. There are four categories of
embolic stroke: those with a known cardiac source; those with a
potential cardiac or aortic source (from trans-thoracic or
trans-esophageal echocardiogram); those with an arterial source;
and those with unknown source.
[0170] Systemic hypoperfusion is the reduction of blood flow to all
parts of the body. It is most commonly due to cardiac pump failure
from cardiac arrest or arrhythmias, or from reduced cardiac output
as a result of myocardial infarction, pulmonary embolism,
pericardial effusion, or bleeding. Hypoxemia (i.e., low blood
oxygen content) may precipitate the hypoperfusion. Because the
reduction in blood flow is global, all parts of the brain may be
affected, especially the "watershed" areas which are border zone
regions supplied by the major cerebral arteries. Blood flow to
these area has not necessary stopped, but instead may have lessened
to the point where brain damage occurs.
[0171] Veins in the brain function to drain the blood back to the
body. When veins are occluded due to thrombosis, the draining of
blood is blocked and the blood backs up, causing cerebral edema.
This cerebral edema can result in both ischemic and hemorrhagic
strokes. This commonly occurs in the rare disease sinus vein
thrombosis.
[0172] Stroke is diagnosed in a subject or patient using one or
more of a variety of techniques known in the art, such as, for
example, neurological examination, blood tests, CT scans (without
contrast enhancements), MRI scans, Doppler ultrasound, and
arteriography (i.e., roentgenography of arteries after injection of
radiopacque material into the blood stream). If a stroke is
confirmed on imaging, various other studies are performed to
determine whether there is a peripheral source of emboli. These
studies include, e.g., an ultrasound/doppler study of the carotid
arteries (to detect carotid stenosis); an electrocardiogram (ECG)
and echocardiogram (to identify arrhythmias and resultant clots in
the heart which may spread to the brain vessels through the
bloodstream); a Holter monitor study to identify intermittent
arrhythmias and an angiogram of the cerebral vasculature (if a
bleed is thought to have originated from an aneurysm or
arteriovenous malformation).
[0173] Compounds useful in these methods of treating, preventing or
ameliorating stroke or a symptom associated with stroke are
compounds that modulate kinase signaling cascade preceding, during
or after a stroke. In some embodiments, the compound is a kinase
inhibitor. For example, the compound is a tyrosine kinase
inhibitor. In an embodiment, the tyrosine kinase inhibitor is an
Src inhibitor. Preferably, the compound used in the methods of
treating, preventing or ameliorating stroke or a symptom associated
with stroke described herein is an allosteric inhibitor of kinase
signaling cascade preceding, during or after a stroke. Preferably,
the compound used in the methods of treating, preventing or
ameliorating stroke or a symptom associated with stroke described
herein is a non-ATP competitive inhibitor of kinase signaling
cascade preceding, during or after a stroke.
[0174] Inhibition of Src activity has been shown to provide
cerebral protection during stroke. (See Paul et al., Nature
Medicine, vol. 7(2):222-227 (2001), which is hereby incorporated by
reference in its entirety). Vascular endothelia growth factor
(VEGF), which is produced in response to the ischemic injury, has
been shown to promote vascular permeability. Studies have shown
that the Src kinase regulates VEGF-mediated VP in the brain
following stroke, and administration of an Src inhibitor before and
after stroke reduced edema, improved cerebral perfusion and
decreased infarct volume after injury occurred. (Paul et al.,
2001). Thus, Src inhibition may be useful in the prevention,
treatment or amelioration of secondary damage following a
stroke.
[0175] The compounds of the invention prevent, treat or ameliorate
stroke or a symptom associated with stroke. Symptoms of a stroke
include sudden numbness or weakness, especially on one side of the
body; sudden confusion or trouble speaking or understanding speech;
sudden trouble seeing in one or both eyes; sudden trouble with
walking, dizziness, or loss of balance or coordination; or sudden
severe headache with no known cause.
[0176] Generally there are three treatment stages for stroke:
prevention, therapy immediately after the stroke, and post-stroke
rehabilitation. Therapies to prevent a first or recurrent stroke
are based on treating the underlying risk factors for stroke, such
as, .e.g., hypertension, high cholesterol, atrial fibrillation, and
diabetes. Acute stroke therapies try to stop a stroke while it is
happening by quickly dissolving the blood clot causing an ischemic
stroke or by stopping the bleeding of a hemorrhagic stroke.
Post-stroke rehabilitation helps individuals overcome disabilities
that result from stroke damage. Medication or drug therapy is the
most common treatment for stroke. The most popular classes of drugs
used to prevent or treat stroke are anti-thrombotics (e.g.,
anti-platelet agents and anticoagulants) and thrombolytics. The
compounds are administered to a patient who is at risk of suffering
a stroke, is suffering from a stroke or has suffered a stroke at a
time before, during, after, or any combination thereof, the
occurrence of a stroke. The compounds of the invention are
administered alone, in pharmaceutical compositions, or in
combination with any of a variety of known treatments, such as, for
example, an anti-platelet medication (e.g., aspirin, clopidogrel,
dipyridamole), an anti-coagulant (e.g., warfarin), or a
thrombolytic medication (e.g., tissue plasminogen activator (t-PA),
reteplase, Urokinase, streptokinase, tenectaplase, lanoteplase, or
anistreplase.
[0177] The compounds of the invention are used in methods of
treating, preventing, ameliorating atherosclerosis or a symptom
thereof in a subject who is at risk for or suffering from
atherosclerosis. The compounds of the invention are used in the
manufacture of a medicament to treat, prevent, or ameliorate
atherosclerosis in a subject.
[0178] Atherosclerosis is a disease affecting the arterial blood
vessel and is commonly referred to as a "hardening" of the
arteries. It is caused by the formation of multiple plaques within
the arteries. Atherosclerotic plaques, though compensated for by
artery enlargement, eventually lead to plaque ruptures and stenosis
(i.e., narrowing) of the artery, which, in turn, leads to an
insufficient blood supply to the organ it feeds. Alternatively, if
the compensating artery enlargement process is excessive, a net
aneurysm results. These complications are chronic, slowly
progressing and cumulative. Most commonly, soft plaque suddenly
ruptures, causing the formation of a blood clot (i.e., thrombus)
that rapidly slows or stops blood flow, which, in turn, leads to
death of the tissues fed by the artery. This catastrophic event is
called an infarction. For example, coronary thrombosis of a
coronary artery causes a myocardial infarction, commonly known as a
heart attack. A myocardial infarction occurs when an
atherosclerotic plaque slowly builds up in the inner lining of a
coronary artery and then suddenly ruptures, totally occluding the
artery and preventing blood flow downstream.
[0179] Atherosclerosis and acute myocardial infarction are
diagnosed in a patient using any of a variety of clinical and/or
laboratory tests such as, physical examination, radiologic or
ultrasound examination and blood analysis. For example, a doctor or
clinical can listen to a subject's arteries to detect an abnormal
whooshing sound, called a bruit. A bruit can be heard with a
stethoscope when placed over the affected artery. Alternatively, or
in addition, the clinician or physician can check pulses, e.g., in
the leg or foot, for abnormalities such as weakness or absence. The
physician or clinical may perform blood work to check for
cholesterol levels or to check the levels of cardiac enzymes, such
as creatine kinase, troponin and lactate dehydrogenase, to detect
abnormalities. For example, troponin sub-units I or T, which are
very specific for the myocardium, rise before permanent injury
develops. A positive troponin in the setting of chest pain may
accurately predict a high likelihood of a myocardial infarction in
the near future. Other tests to diagnose atherosclerosis and/or
myocardial infarction include, for example, EKG (electrocardiogram)
to measure the rate and regularity of a subject's heartbeat; chest
X-ray, measuring ankle/brachial index, which compares the blood
pressure in the ankle with the blood pressure in the arm;
ultrasound analysis of arteries; CT scan of areas of interest;
angiography; an exercise stress test, nuclear heart scanning; and
magnetic resonance imaging (MRI) and positron emission tomography
(PET) scanning of the heart.
[0180] Compounds useful in these methods of treating, preventing or
ameliorating atherosclerosis or a symptom thereof are compounds
that modulate kinase signaling cascade in a patient at risk for or
suffering from atherosclerosis. In some embodiments, the compound
is a kinase inhibitor. For example, the compound is a tyrosine
kinase inhibitor. In an embodiment, the tyrosine kinase inhibitor
is an Src inhibitor. Preferably, the compound used in the methods
of treating, preventing or ameliorating atherosclerosis or a
symptom thereof described herein is an allosteric inhibitor of
kinase signaling cascade involved in atherosclerosis. Preferably,
the compound used in the methods of treating, preventing or
ameliorating atherosclerosis or a symptom associated with
atherosclerosis described herein is a non-ATP competitive inhibitor
of kinase signaling cascade involved in atherosclerosis.
[0181] Cellular signal transduction by Src is believed to play a
key role in increased permeability of vessels, known as vascular
permeability (VP). Vascular endothelia growth factor (VEGF), which
is produced in response to the ischemic injury, including, e.g.,
myocardial infarction, has been shown to promote vascular
permeability. Studies have shown that the inhibition of Src kinase
decreases VEGF-mediated VP. (See Parang and Sun, Expert Opin. Ther.
Patents, vol. 15(9): 1183-1206 (2005), which is hereby incorporated
by reference in its entirety). Mice treated with an Src inhibitor
demonstrated reduced tissue damage associated with trauma or injury
to blood vessels after myocardial infarction, as compared to
untreated mice. (See e.g., U.S. Patent Publication Nos. 20040214836
and 20030130209 by Cheresh et al., the contents of which are hereby
incorporated by reference in their entirety). Thus, Src inhibition
may be useful in the prevention, treatment or amelioration of
secondary damage following injury due to atherosclerosis, such as,
for example, myocardial infarction.
[0182] The compounds of the invention prevent, treat or ameliorate
stroke or a symptom associated with atherosclerosis.
Atherosclerosis generally does not produce symptoms until it
severely narrows the artery and restricts blood flow, or until it
causes a sudden obstruction. Symptoms depend on where the plaques
and narrowing develop, e.g., in the heart, brain, other vital
organs and legs or almost anywhere in the body. The initial
symptoms of atherosclerosis may be pain or cramps when the body
requires more oxygen, for example during exercise, when a person
may feel chest pain (angina) because of lack of oxygen to the heart
or leg cramps because of lack of oxygen to the legs. Narrowing of
the arteries supplying blood to the brain may cause dizziness or
transient ischaemic attacks (TIA's) where the symptoms and signs of
a stroke last less than 24 hours. Typically, these symptoms develop
gradually.
[0183] Symptoms of myocardial infarction are characterized by
varying degrees of chest pain, discomfort, sweating, weakness,
nausea, vomiting, and arrhythmias, sometimes causing loss of
consciousness. Chest pain is the most common symptom of acute
myocardial infarction and is often described as a tightness,
pressure, or squeezing sensation. Pain may radiate to the jaw,
neck, arms, back, and epigastrium, most often to the left arm or
neck. Chest pain is more likely caused by myocardial infarction
when it lasts for more than 30 minutes. Patients suffering from a
myocardial infarction may exhibit shortness of breath (dyspnea)
especially if the decrease in myocardial contractility due to the
infarct is sufficient to cause left ventricular failure with
pulmonary congestion or even pulmonary edema.
[0184] The compounds of the invention are administered alone, in
pharmaceutical compositions, or in combination with any of a
variety of known treatments for atherosclerosis, such as, for
example, cholesterol-lowering drugs (e.g., statins), anti-platelet
medications, or anti-coagulants.
[0185] The compounds of the invention are used in methods of
treating, preventing, ameliorating neuropathic pain, such as
chronic neuropathic pain, or a symptom thereof in a subject who is
at risk of suffering from, is suffering from, or has suffered
neuropathic pain. The compounds of the invention are used in the
manufacture of a medicament to treat, prevent, or ameliorate
neuropathic pain in a subject or a symption thereof in a subject
who is at risk of suffering from, is suffering from, or has
suffered from neuropathic pain.
[0186] Neuropathic pain, also known as neuralgia, is qualitatively
different from ordinary nociceptive pain. Neuropathic pain usually
presents as a steady burning and/or "pins and needles" and/or
"electric shock" sensations. The difference between nociceptive
pain and neuropathic pain is due to the fact that "ordinary",
nociceptive pain stimulates only pain nerves, while a neuropathy
often results in the stimulation of both pain and non-pain sensory
nerves (e.g., nerves that respond to touch, warmth, cool) in the
same area, thereby producing signals that the spinal cord and brain
do not normally expect to receive.
[0187] Neuropathic pain is a complex, chronic pain state that
usually is accompanied by tissue injury. With neuropathic pain, the
nerve fibers themselves may be damaged, dysfunctional or injured.
These damaged nerve fibers send incorrect signals to other pain
centers. The impact of nerve fiber injury includes a change in
nerve function both at the site of injury and areas around the
injury.
[0188] Neuropathic pain is diagnosed in a subject or patient using
one or more of a variety of laboratory and/or clinical techniques
known in the art, such as, for example, physical examination.
[0189] Compounds useful in these methods of treating, preventing or
ameliorating neuropathic pain, such as chronic neuropathic pain, or
a symptom associated with neuropathic pain are compounds that
modulate kinase signaling cascade involved in neuropathic pain. In
some embodiments, the compound is a kinase inhibitor. For example,
the compound is a tyrosine kinase inhibitor. In an embodiment, the
tyrosine kinase inhibitor is an Src inhibitor. Preferably, the
compound used in the methods of treating, preventing or
ameliorating neuropathic pain or a symptom thereof is an allosteric
inhibitor of kinase signaling cascade involved in neuropathic pain.
Preferably, the compound used in the methods of treating,
preventing or ameliorating neuropathic pain or a symptom thereof is
a non-ATP competitive inhibitor of kinase signaling cascade
involved in neuropathic pain.
[0190] c-Src has been shown to regulate the activity of
N-methyl-D-aspartate (NMDA) receptors. (See Yu et al., Proc. Natl.
Acad. Sci. USA, vol. 96:7697-7704 (1999), which is hereby
incorporated by reference in its entirety). Studies have shown that
PP2, a low molecular weight Src kinase inhibitor, decreases
phosphorylation of the NMDA receptor NM2 subunit. (See Guo et al.,
J. Neuro., vol. 22:6208-6217 (2002), which is hereby incorporated
by reference in its entirety). Thus, Src inhibition, which in turn,
inhibits the activity NMDA receptors, may be useful in the
prevention, treatment or amelioration of neuropathic pain, such as
chronic neuropathic pain.
[0191] The compounds of the invention prevent, treat or ameliorate
neuropathic pain, such as chronic neuropathic pain, or a symptom
associated with neuropathic pain. Symptoms of neuropathic pain
include shooting and burning pain, tingling and numbness.
[0192] The compounds of the invention are administered alone, in
pharmaceutical compositions, or in combination with any of a
variety of known treatments, such as, for example, analgesics,
opioids, tricyclic antidepressants, anticonvulsants and serotonin
norepinephrine reuptake inhibitors
[0193] The compounds of the invention are used in methods of
treating, preventing, ameliorating hepatitis B or a symptom thereof
in a subject who is at risk for or suffering from hepatitis B. The
compounds of the invention are used in the manufacture of a
medicament to treat, prevent, or ameliorate hepatitis B or a
symptom thereof in a subject who is at risk for or suffering from
hepatitis B.
[0194] The hepatitis B virus, a member of the Hepadnavirus family,
consists of a proteinaceous core particle containing the viral
genome in the form of double stranded DNA with single-stranded
regions and an outer lipid-based envelope with embedded proteins.
The envelope proteins are involved in viral binding and release
into susceptible cells. The inner capsid relocates the DNA genome
to the cell's nucleus where viral mRNAs are transcribed. Three
subgenomic transcripts encoding the envelope proteins are made,
along with a transcript encoding the X protein. A fourth
pre-genomic RNA is transcribed, which is exported to the cytosol
and translates the viral polymerase and core proteins. Polymerase
and pre-genomic RNA are encapsidated in assembling core particles,
where reverse transcription of the pre-genomic RNA to genomic DNA
occurs by the polymerase protein. The mature core particle then
exits the cell via normal secretory pathways, acquiring an envelope
along the way.
[0195] Hepatitis B is one of a few known non-retroviral viruses
that employ reverse transcription as part of the replication
process. Other viruses which use reverse transcription include,
e.g., HTLV or HIV.
[0196] During HBV infection, the host immune response is
responsible for both hepatocellular damage and viral clearance.
While the innate immune response does not play a significant role
in these processes, the adaptive immune response, particularly
virus-specific cytotoxic T lymphocytes (CTLs), contributes to
nearly all of the liver injury associated with HBV infection. By
killing infected cells and by producing antiviral cytokines capable
of purging HBV from viable hepatocytes, CTLs also eliminate the
virus. Although liver damage is initiated and mediated by the CTLs,
antigen-nonspecific inflammatory cells can worsen CTL-induced
immunopathology and platelets may facilitate the accumulation of
CTLs into the liver.
[0197] Hepatitis B is diagnosed in a patient using any of a variety
of clinical and/or laboratory tests such as, physical examination,
and blood or serum analysis. For example, blood or serum is assayed
for the presence of viral antigens and/or antibodies produced by
the host. In a common test for Hepatitis B, detection of hepatitis
B surface antigen (HBsAg) is used to screen for the presence of
infection. It is the first detectable viral antigen to appear
during infection with this virus; however, early in an infection,
this antigen may not be present and it may be undetectable later in
the infection as it is being cleared by the host. During this
`window` in which the host remains infected but is successfully
clearing the virus, IgM antibodies to the hepatitis B core antigen
(anti-HBc IGM) may be the only serologic evidence of disease.
[0198] Shortly after the appearance of the HBsAg, another antigen
named as the hepatitis B e antigen (HBeAg) will appear.
Traditionally, the presence of HBeAg in a host's serum is
associated with much higher rates of viral replication; however,
some variants of the hepatitis B virus do not produce the "e"
antigen at all. During the natural course of an infection, the
HBeAg may be cleared, and antibodies to the "e" antigen (anti-HBe)
will arise immediately afterward. This conversion is usually
associated with a dramatic decline in viral replication. If the
host is able to clear the infection, eventually the HBsAg will
become undetectable and will be followed by antibodies to the
hepatitis B surface antigen (anti-HBs). A person negative for HBsAg
but positive for anti-HBs has either cleared an infection or has
been vaccinated previously. A number of people who are positive for
HBsAg may have very little viral multiplication, and hence may be
at little risk of long-term complications or of transmitting
infection to others.
[0199] Compounds useful in these methods of treating, preventing or
ameliorating hepatitis B or a symptom thereof are compounds that
modulate kinase signaling cascade in a patient at risk for or
suffering from hepatitis B. In some embodiments, the compound is a
kinase inhibitor. For example, the compound is a tyrosine kinase
inhibitor. In an embodiment, the tyrosine kinase inhibitor is an
Src inhibitor. Preferably, the compound used in the methods of
treating, preventing or ameliorating hepatitis B or a symptom
thereof described herein is an allosteric inhibitor of kinase
signaling cascade involved in hepatitis B. Preferably, the compound
used in the methods of treating, preventing or ameliorating
hepatitis B or a symptom associated with hepatitis B described
herein is a non-ATP competitive inhibitor of kinase signaling
cascade involved in hepatitis B.
[0200] Src plays a role in the replication of the hepatitis B
virus. The virally encoded transcription factor HBx activates Src
in a step that is required from propagation of the HBV virus. (See
e.g., Klein et al., EMBO J., vol. 18:5019-5027 (1999); Klein et
al., Mol. Cell. Biol., vol. 17:6427-6436 (1997), each of which is
hereby incorporated by reference in its entirety). Thus, Src
inhibition, which in turn, inhibits Src-mediated propagation of the
HBV virus, may be useful in the prevention, treatment or
amelioration of hepatitis B or a symptom thereof.
[0201] The compounds of the invention prevent, treat or ameliorate
hepatitis B or a symptom associated with hepatitis B. Symptoms of
hepatitis B typically develop within 30-180 days of exposure to the
virus. However, up to half of all people infected with the
hepatitis B virus have no symptoms. The symptoms of hepatitis B are
often compared to flu, and include, e.g., appetite loss; fatigue;
nausea and vomiting, itching all over the body; pain over the liver
(e.g., on the right side of the abdomen, under the lower rib cage),
jaundice, and changes in excretory functions.
[0202] The compounds of the invention are administered alone, in
pharmaceutical compositions, or in combination with any of a
variety of known treatments for hepatitis B, such as, for example,
interferon alpha, lamivudine (Epivir-HBV) and baraclude
(entecavir).
[0203] As described herein, the compounds of the invention may be
used to regulate immune system activity in a subject, thereby
protecting against or preventing autoimmune disease, e.g.,
rheumatoid arthritis, multiple sclerosis, sepsis and lupus as well
as transplant rejection and allergic diseases. Alternatively, the
compound may be used to treat autoimmune disease in a subject. For
example, the compound may result in reduction in the severity of
symptoms or halt impending progression of the autoimmune disease in
a subject. The compound of the invention may be involved in
modulating a kinase signaling cascade, e.g., a kinase inhibitor, a
non-ATP competitive inhibitor, a tyrosine kinase inhibitor, e.g., a
Src inhibitor, a p59fyn (Fyn) inhibitor or a p561ck (Lck)
inhibitor. The compounds of the invention are used in the
manufacture of a medicament to regulate immune system activity in a
subject
[0204] Autoimmune diseases are diseases caused by a breakdown of
self-tolerance such that the adaptive immune system responds to
self antigens and mediates cell and tissue damage. Autoimmune
diseases can be organ specific (e.g., thyroiditis or diabetes) or
systemic (e.g., systemic lupus erythematosus). T cells modulate the
cell-mediated immune response in the adaptive immune system. Under
normal conditions, T cells express antigen receptors (T cell
receptors) that recognize peptide fragments of foreign proteins
bound to self major histocompatibility complex molecules. Among the
earliest recognizable events after T cell receptor (TCR)
stimulation are the activation of Lek and Fyn, resulting in TCR
phosphorylation on tyrosine residues within immunoreceptor
tyrosine-based activation motifs (Zamoyska, et al.; 2003, Immunol.
Rev., 191, 107-118). Tyrosine kinases, such as Lck (which is a
member of the Src family of protein tyrosine kinases) play an
essential role in the regulation of cell signaling and cell
proliferation by phosphorylating tyrosine residues of peptides and
proteins (Levitzki; 2001, Top. Curr. Chem., 211, 1-15; Longati, et
al.; 2001, Curr. Drug Targets, 2, 41-55; Qian, and Weiss; 1997,
Curr. Opin. Cell Biol., 9, 205-211). Thus, although not wishing to
be bound by theory, it is hypothesized that the administration of a
compound of the instant invention which modulates tyrosine kinase
(e.g., Src) activity is useful in the treatment of autoimmune
disease.
[0205] The tyrosine kinases Ick and fyn are both activated in the
TCR pathway; thus, inhibitors of Ick and/or fyn have potential
utility as autoimmune agents (Palacios and Weiss; 2004, Oncogene,
23, 7990-8000). Lck and Fyn are predominantly expressed by T cells
through most of their lifespan. The roles of Lck and Fyn in T cell
development, homeostasis and activation have been demonstrated by
animal and cell line studies (Parang and Sun; 2005, Expert Opin.
The. Patents, 15, 1183-1207). Lck activation is involved in
autoimmune diseases and transplant rejection (Kamens, et al.; 2001,
Curr. Opin. Investig. Drugs, 2, 1213-1219). Results have shown that
the lck (-) Jurkat cell lines are unable to proliferate, produce
cytokines, and generate increases in intracellular calcium,
inositol phosphate, and tyrosine phosphorylation in response to T
cell receptor stimulation (Straus and Weiss; 1992, Cell., 70,
585-593; Yamasaki, et al.; 1996, Mol. Cell. Biol., 16, 7151-7160).
Therefore, an agent inhibiting lck would effectively block T cell
function, act as an immunosuppressive agent, and have potential
utility in autoimmune diseases, such as rheumatoid arthritis,
multiple sclerosis, and lupus, as well as in the area of transplant
rejection and allergic diseases (Ranke and Pollok; 1995,
Inflammation Res., 44, 357-371). Thus, although not wishing to be
bound by theory, it is hypothesized that the administration of a
compound of the instant invention which modulates one or more
members of the Src family of protein tyrosine kinases (e.g., lck
and/or fyn) is useful in the treatment of autoimmune disease.
[0206] The present invention provides inhibitors of protein kinases
and/or protein phosphatases. In one embodiment, the protein kinase
and/or protein phosphatase inhibitor is a non-peptide inhibitor
having the following Formula I:
##STR00071##
wherein X is a halogen, and R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, and R.sub.6 are the same or different, and selected from
H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a,
OH, OR., OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
OSO.sub.3H, OPO.sub.3H.sub.2, P(O)OR.sub.aOR.sub.b, B(OH).sub.2,
halogen, aryl, O-aryl-Q, heteroaryl, biaryl, heterobiaryl,
heterocyclic compound, and alkyl (branched, cyclic, or unbranched),
having from 1 to 20 carbon atoms, optionally containing a double or
triple bond and optionally substituted with a heteroatom or other
functional groups, such as a carboxylic acid, carboxylic ester,
alcohol, alkoxy, thioether, amide, C(O)NH.sub.2, thioamide, urea,
urethane, sulfoxide, sulfone, SO.sub.3H, OSO.sub.3H, phosphonic
acid, phosphonic ester, phosphinic acid, phosphinic ester,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, boronic acid, aryl, heteroaryl,
biaryl, heterocycle, NH.sub.2, alkyl and dialkyl amine, glucoside,
and heterobiaryl, or R.sub.5 and R.sub.6 together form a
heterocyclic compound. R.sub.a, R.sub.b, and R.sub.c are the same
or different and selected from H, aryl, heteroaryl, biaryl,
heterobiaryl, and alkyl (branched, cyclic, or unbranched),
optionally substituted with a heteroatom or other functional groups
such as a carboxylic acid, carboxylic ester, alcohol, alkoxy,
thioether, amide, C(O)NH.sub.2, thioamide, urea, urethane,
sulfoxide, sulfone, SO.sub.3H, OSO.sub.3H, phosphonic acid,
phosphonic ester, phosphinic acid, phosphinic ester,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, boronic acid, aryl, heteroaryl,
biaryl, heterocycle, NH2, alkyl and dialkyl amine, glucoside, and
heterobiaryl. Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glucoside, alkoxy,
or
##STR00072##
It is understood that all open substitution positions in the above
side chains can contain further substitutions. Examples of suitable
R groups are provided in Table VI, below.
[0207] In one embodiment, at least one of R.sub.5 or R.sub.6 is
##STR00073##
wherein R.sub.7* is the point of attachment and is
(CH.sub.2).sub.x, wherein X is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10, CH.sub.2CHOH, CH(CH.sub.3)(R-isomer), or
CH(CH.sub.3)(S-isomer), and each of R.sub.8, R.sub.9, R.sub.10,
R.sub.11, and R.sub.12 are the same or different and selected from
H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a,
OH, OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2,
NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.b,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
OSO.sub.3H, OPO.sub.3H.sub.2, P(O)OR.sub.aOR.sub.b, B(OH).sub.2,
halogen, aryl, O-aryl-Q, heteroaryl, biaryl, heterobiaryl,
heterocyclic compound, and alkyl (branched, cyclic, or unbranched),
preferably having from 1 to 20 carbon atoms, optionally containing
a double or triple bond and optionally substituted with a
heteroatom or other functional groups, such as a carboxylic acid,
carboxylic ester, alcohol, alkoxy, thioether, amide, C(O)NH.sub.2,
thioamide, urea, urethane, sulfoxide, sulfone, SO.sub.3H,
OSO.sub.3H, phosphonic acid, phosphonic ester, phosphinic acid,
phosphinic ester, boronic acid, aryl, heteroaryl, biaryl,
heterocycle, NH.sub.2, alkyl and dialkyl amine, glucoside, and
heterobiaryl. R.sub.a, R.sub.b, and R.sub.c can be the same or
different and are selected from the group consisting of H, aryl,
heteroaryl, biaryl, heterobiaryl, and alkyl (branched, cyclic, or
unbranched), optionally substituted with a heteroatom or other
functional groups such as a carboxylic acid, carboxylic ester,
alcohol, alkoxy, thioether, amide, C(O)NH.sub.2, thioamide, urea,
urethane, sulfoxide, sulfone, SO.sub.3H, OSO.sub.3H, phosphonic
acid, phosphonic ester, phosphinic acid, phosphinic ester,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, boronic acid, aryl, heteroaryl,
biaryl, heterocycle, NH.sub.2, alkyl and dialkyl amine, glucoside,
and heterobiaryl. Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.311.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glucoside, alkoxy,
or
##STR00074##
It is understood that all open substitution positions in the above
side chains can contain further substitutions. In one embodiment,
each of R.sub.8, R.sub.9, R.sub.10, R.sub.11, and R.sub.12 is
selected from OCH.sub.3, OCH.sub.2CH.sub.3, H, CH.sub.3, OH,
CH.sub.2OH, CF.sub.3, OCF.sub.3, CFO, C.sub.6H.sub.5,
OC.sub.6H.sub.5, OCH.sub.2C.sub.6H.sub.5,
OCH.sub.2CH.sub.2CH.sub.3, CHO, CO.sub.2H, CO.sub.2CH.sub.3,
CH.sub.2CO.sub.2H, CH.sub.2CO.sub.2CH.sub.3, NO.sub.2, and
halogen.
[0208] In another embodiment, at least one of R.sub.5 or R.sub.6
is
##STR00075##
wherein the asterisk indicates the point of attachment to the
nitrogen.
[0209] In a preferred embodiment, the non-peptide inhibitor
inhibits the activity of pp60.sup.c-src tyrosine kinase,
pp56.sup.lck tyrosine kinase, or pp55.sup.fyn tyrosine kinase.
[0210] In another preferred embodiment, the non-peptide inhibitor
inhibits the activity of protein tyrosine phosphatase 1B
(PTP-1B).
[0211] Another compound of the present invention has the following
Formula II:
##STR00076##
wherein X is a halogen, e.g., fluorine, and R.sub.1, R.sub.2,
R.sub.3, and R.sub.4 are specificity elements. As used herein,
specificity elements or specificity side chains are side chains
which will bind in unique binding pockets for individual proteins.
Thus, the side chains used will depend on the particular protein to
be inhibited. To identify suitable side chains, known peptide
binding side chains may be used to identify analogues which are
then used in combinatorial chemistry techniques to expand the
library of possible side chains.
[0212] In one embodiment, R.sub.1 is H, R.sub.2 is
##STR00077##
R.sub.3 is H, and R.sub.4 is H. In another embodiment, the compound
is substituted at any other position on the indole ring.
[0213] The compounds of the present invention provide activity
against tyrosine kinases, such as pp60.sup.c-src, and are expected
to improve the ability of the compound to inhibit tyrosine kinases
in vivo, since one easily metabolized OH group has been removed. In
particular, an OH group at the 5-position on the indole ring has
been substituted with a halogen. The halogen is a hydrogen bond
acceptor, useful with catalytic residues which are hydrogen bond
donors. In addition, the halogen is not metabolized in phase H
metabolism and is electronegative, leading to in vivo benefits
(see, e.g., Park et al., 2001). Some members of this class are also
inhibitors of the opposing enzymes, i.e., phosphotyrosine
phosphatases. These compounds are inhibitors of pp60.sup.c-src, of
highly metastatic prostate cancer cell growth, and are non-toxic in
mice upon high dose acute i.p. administration, as described in
Example 1, below. Some of these compounds may be found to have
other biological activities upon broader testing (e.g., inhibit
glycogen phosphorylase for Type II diabetes, HIV reverse
transcriptase, or thromboxane synthase). Thus, these compounds may
be used as tyrosine kinase inhibitors for therapeutic applications,
such as cancer. Tyrosine kinase inhibitors have other potential
therapeutic applications as well (e.g., immunosuppressants in the
case of p56lck) and inhibitors of the tyrosine phosphatase PTP-1B
may provide drugs for treating Type II diabetes or obesity.
[0214] The present invention also provides a method for identifying
inhibitors of protein kinases. The general modular strategy for the
development of non-peptide PTK inhibitors is outlined in FIG. 1.
Basically, at least one first module having a one or more
functional groups for binding to catalytic residues of the protein
kinase (in a preferred embodiment, at least one of the functional
groups is a halogen) is combined with at least one second module
which provides a non-peptide scaffold. The functional group(s) of
the at least one first module are each capable of covalently or
non-covalently binding with catalytic residues of the protein
kinase. Thus, each functional group of each first module is capable
of reversible or irreversible bond formation, either covalently or
non-covalently, to catalytic residues of the protein kinase when
the protein kinase the first module are combined under conditions
effective for such binding. Combinations of the first and second
modules which inhibit protein kinase activity are then selected.
Step 1 begins with protein kinase inhibitor information which was
already generated, i.e. pentapeptide scaffolds which bind in the
substrate specificity sites of PICA or Src have already been used
to position various rationally designed functional groups (i.e.
module "M.sub.1" or "first module") to interact with the conserved
catalytic residues, MgATP or MgADP. A selection of preferred
functional groups have now been identified in this fashion to serve
as the initial M.sub.I module for Step 1. These M.sub.1 functional
groups have been utilized to identify promising non-peptide
scaffolds for Src inhibitors in Step 1. It was anticipated that
these bare non-peptide scaffolds, with only an M.sub.1 appendage,
would have low binding affinity and be relatively non-selective
among the protein tyrosine kinases (PTKs). A lack of selectivity at
the level of Step 1 is viewed as an advantage for the development
of a general strategy which can be reapplied to additional PTKs.
Therefore, the suite of non-peptide scaffolds identified in Step 1
can be recycled for use against additional PTKs by re-screening
them and carrying the better ones through Steps 2 and 3, all using
the new PTK target. The potency of these bare scaffolds from Step 1
may be increased enough by the attachment of one or two initial
specificity elements (S.sub.e) to allow for the validation of the
scaffold as non-ATP competitive and amenable to further potency
enhancements using combinatorial chemistry in a rationally guided
fashion. Promising Src non-peptide M.sub.2 (second module)
scaffolds identified in Step 1 have undergone Step 2 and displayed
a one to two order-of-magnitude increase in potency against Src as
well as non-competitive binding relative to ATP.
[0215] Validation of the scaffolds at the level of Step 2 before
undertaking the resource intensive combinatorial library synthesis
and testing of Step 3 is important for three reasons: 1) to develop
the chemistry for appending the specificity element (S.sub.n) side
chains; 2) to determine that these inhibitors are not
ATP-competitive; and 3) to determine that the potency is responding
to the side chain S.sub.n properties and attachment points as would
be expected based upon the working model for the Src:inhibitor
complex (this provides some confidence that rationally guided
choices can be made for the ranges of individual selectivity
elements S.sub.n to include in the focused libraries of Step
3).
[0216] It is in Step 3 that high potency and specificity for a
particular PTK is anticipated because numerous combinations of
M.sub.1 functional groups (and close analogs M.sub.1') with
selectivity elements (S.sub.n) will be evaluated experimentally via
combinatorial chemistry and high-throughput screening. Potency and
selectivity may be further increased if necessary by appending
additional specificity elements (see optional S.sub.n's in FIG.
1).
[0217] In each of the Steps 1-3, molecular modeling studies with
the IRTK:peptide:AMP-PNP crystal structure, the model of the
Src:peptide complex and the models for the Src complex with the
individual families of inhibitors based upon a particular scaffold
will be used as qualitative guides. These modeling studies have
been remarkably helpful thus far in guiding the inhibitor design
efforts as detailed later. Combining structure-based design and
combinatorial chemistry technologies in this fashion provides a
synergy wherein the major individual deficiencies of these
technologies used in isolation are addressed by the strengths of
the other. The major deficiency of structure-based design is the
difficulty in quantitatively predicting ligand binding affinities,
which is particularly challenging due to the complex effects of
solvation and entropy (Ajay & Murcko, 1995). The major strength
of structure-based design is its capability to predict what types
of molecules are likely to be good ligands. Structure-based design
can determine the rough boundaries (proteins have some flexibility
which need to be taken into account) for molecular size and shape
as well as indicate where hydrophobic, H-bonding, and ionic
interactions are likely to occur. On the other hand, the major
deficiency of combinatorial chemistry is that "molecular space" for
drug-sized molecules (i.e. MW ca. 500 or less) is so large that one
could not hope to sample all of this molecular space with a high
density of coverage in a reasonable sized combinatorial library. A
recent estimate (Bohacek et al., 1996) of the number of possible
compounds containing up to 30 atoms chosen only from carbon,
nitrogen, oxygen and sulfur (in addition to H's) is 10.sup.60
compounds. This is in the molecular weight range of typical drug
molecules and still does not include additional diversity provided
by other atoms, e.g. halogens. Consequently, additional constraints
need to be used to identify regions of molecular space wherein
particular drug candidates are likely to be located.
Structure-based design can drastically reduce the volume of
molecular space to be explored by identifying the types of
molecules which have a higher probability of being good ligands.
The inability to quantitatively predict which of these "focused"
combinatorial library members will in fact be the tightest binding
ligands (i.e. the quantitation problem) is then resolved by
employing an efficient combinatorial synthesis and high-throughput
testing of the library.
[0218] In the earlier peptide based serine and tyrosine kinase
inhibitor design efforts, PKA was used as a convenient qualitative
model for designing the protein kinase inhibitor module M.sub.1 for
interaction with the conserved catalytic residues. There is much
more structural and kinetic information available for PKA than any
other protein kinase.
[0219] The crystal structure of PKA complexed with Mg.sub.2ATP and
a pseudosubstrate (i.e. OH replaced with H) peptide inhibitor (PKI
5-24 amide) has been solved (Zheng et al., 1993) and the active
site interactions near the P 0 Ala of this inhibitor are shown in
FIG. 2.
[0220] This crystal structure shows Mg.sub.2ATP bound to the small
lobe of PKA and a 20-residue pseudosubstrate peptide inhibitor
bound to the large lobe with the overall conformation of the enzyme
in the closed (i.e. the two lobes are touching) and activated
state. The distances between the P 0 Ala side chain carbon and the
nearby heavy atoms in the complex are shown in .ANG. in FIG. 2.
These distances show that the Ala side chain is within van der
Waals contact distance of the surrounding atoms and indicates that
there is little space for appending bulky M.sub.1 functional groups
to the Ala side chain. However, PKA is a flexible enzyme with open,
closed and intermediate conformations (Cox et al., 1994) and these
more open conformations would result in a retraction back of the
ATP .gamma.-phosphate from the inhibitor Ala thereby creating a
binding cavity for appended M.sub.1 functional groups. Furthermore,
PKA binds MgADP with the same affinity as MgATP (Whitehouse et al.,
1983) and the ratio of ATP/ADP in cells is typically 10/1 (Alberts,
et al. 1994). Therefore, at equilibrium, ca. 10% of the cellular
protein kinase is in the MgADP bound state and this form of the
enzyme can also be targeted with an inhibitor to drain all of the
enzyme from the catalytic cycle into a PKA:MgADP:inhibitor inactive
complex.
[0221] Since the PICA catalytic residues Asp-166 and Lys-168 are
completely conserved in all serine kinases, and the tyrosine
kinases only differ by the substitution of Arg for Lys-168 (Taylor
et al., 1993), this region of the active site was chosen, along
with the adjoining MgATP or MgADP, to target a selection of
inhibitor functional groups which could serve as M.sub.1 and be
broadly useful for developing inhibitors for the entire protein
kinase family. By targeting M.sub.1 to the region of the active
site adjacent to the nucleotide, an orientation point is provided
for the non-peptide inhibitors which can extend into the peptide
binding specificity sites without always competing with ATP/ADP
binding.
[0222] A selection of functional groups which could be utilized as
M.sub.1 was identified first because, although this region of the
active site is very highly conserved, it was expected that each
particular protein kinase will still display some differing
preferences across this selection due to small variations in the
active site conformations and adjoining residues. Furthermore, the
rank order preference among this selection of M.sub.1's may change
somewhat as the M.sub.1 module is appended to different non-peptide
scaffolds. This expectation is based upon the potential for each
non-peptide scaffold to bind in somewhat different orientations
with each individual protein kinase and with each particular set of
selectivity element (S.sub.n) side chains. Pentapeptide scaffolds
were chosen for the initial screening of functional groups for
M.sub.1 because the binding orientation of these larger peptide
scaffolds is likely to be very consistent and predictable (i.e.
closely resembling that observed by x-ray) throughout the series
and could be more confidently assumed to position each tested
M.sub.1 functionality adjacent to the conserved catalytic residues
as intended. Consequently, the goal of this earlier peptide-based
work was to identify a collection of M.sub.1 functional groups
which can be used, not only for the initial screening of
non-peptide scaffolds (Step 1), but also as an initial set of
M.sub.1 side chains which can be further expanded via close analogs
and thereby optimized simultaneously with the other side chains in
the final non-peptide combinatorial libraries (Step 3).
[0223] In order to model the candidate M.sub.1 functional groups in
this conserved catalytic region of the PKA active site, they were
built onto the P 0 Ala position in the PKA ternary structure using
the SYBYL molecular modeling package (Tripos) on a Silicone
Graphics workstation as indicated in FIG. 3.
[0224] A crystal structure of PICA with MgATP and an inhibitor
bound in a more "open" conformation was not available, so initial
modeling studies were carried out on the MgADP bound form of PKA
derived from the ternary complex illustrated in FIG. 2 by simply
deleting the ATP .gamma.-phosphate. Initial modeling studies were
used to provide qualitative guidance for identifying interesting
potential M.sub.1 functional groups for the protein kinase family
before synthesis and testing. The most advanced computational
algorithms for quantitatively predicting the free energy of
binding, such as Free Energy Perturbation methods, are
computationally intensive methods which are not practical at this
point in time for routine use by the non-specialist. Even the most
advanced methods can be inaccurate due to difficulties in sampling,
inadequacies in the molecular mechanics force fields/parameters,
and an incomplete understanding of electrostatics in water (Ajay
& Murcko, 1995). Less rigorous (and easier to use)
computational methods tend to be unreliable in making quantitative
predictions of binding affinities, especially when dealing with
multiple polar and ionic interactions such as those involved in
M.sub.1 binding.
[0225] In order to allow molecular mechanics calculations to be
done with the Silicone Graphics workstation in a reasonable amount
of time, two layers of residues were carved out from the PKA
ternary structure which are surrounding the PKA active site, along
with the peptide inhibitor and Mg.sub.2ADP. The M.sub.1 functional
groups were then appended to the P 0 Ala side chain and the entire
PKA active site:Mg.sub.2ADP:modified peptide inhibitor complex was
then subjected to 300 iterations of molecular mechanics
minimization using the Tripos force field with a distance dependent
dielectric constant after assigning appropriate formal charges and
calculating Gasteiger Marsili point charges using SYBYL. Setting
the maximum number of iterations at 300 was sufficient to remove
any serious strain in the complexes and yet not allow the overall
structure to "drift" significantly from the starting x-ray
structure if convergence is not reached. These minimized complexes
were then visually evaluated to determine if the appended
individual M.sub.1 functional groups were able to engage in
favorable interactions with the conserved catalytic residues and/or
Mg.sub.2ADP. This visual evaluation involved, among other standard
interaction evaluations, measuring atom-atom distances to determine
if hydrogen bonds and ionic interactions were being favorably
formed.
[0226] Favorable intermolecular interactions between an individual
M.sub.1 functionality and the conserved catalytic residues or
Mg.sub.2ADP does not necessarily mean enhanced binding affinity
will be observed for the new inhibitor. Unfavorable desolvation of
both the polar M.sub.1 functionality and the polar PKA active site
residues (as well as complex entropy effects) are not included in
this analysis and may reduce the net binding affinity to the point
that the modified inhibitor may even be less potent that the
corresponding P 0 Ala inhibitor, even though the appended M.sub.1
functionality is interacting with the conserved catalytic residues
and/or MgADP (or MgATP) as intended. Even in cases where this
desolvation penalty results in no net increase in binding affinity,
these M.sub.1 functional groups are still useful as an orienting
groups for correctly positioning the non-peptide inhibitor analogs
in the protein kinase active site. Positioning these polar
functional groups elsewhere within the active site (assuming they
are tethered so as not to be able to extend into bulk solvent while
the scaffold is favorably bound in the active site) is likely to
result in a reduced binding affinity because they were specifically
designed and selected based upon their demonstrated ability (while
appropriately tethered to pentapeptide scaffolds) to be accepted
adjacent to the conserved catalytic residues and MgADP/MgATP. If a
particular M.sub.1 functionality does not correctly position a
non-peptide scaffold in Step 1 then attempts to improve the potency
by rationally appending initial specificity elements in Step 2
would likely fail.
[0227] None of the literature protein kinase assay procedures
contain added ADP. A typical PKA literature assay procedure (Glass
et al., 1989) was modified by adding 10% as much ADP as the ATP
concentration used to reflect the natural 1/10 ratio in the cell.
This protein kinase assay is hereinafter referred to as the
"Literature Mimetic" assay. It has been used for PKA as well as the
Src. An examination of the literature, and commercially available
protein kinase assays, showed that there is poor consistency from
lab to lab and company to company and that all of these assays use
physical chemical conditions which differ considerably from those
known to exist inside cells. Since inhibition of intracellular
protein kinases is the ultimate goal for drug discovery, new
protein kinase assays have been developed which come much closer to
mimicking the overall cytosolic physical chemical conditions known
to exist inside cells. The development of these "Cellular Mimetic"
protein kinase assays, is described herein, along with a novel
method for determining which form of a protein kinase a given
inhibitor binds best to (the STAIRe method). Data was collected
correlating the activity of the new non-peptide Src inhibitors in
the Cellular Mimetic assay with that obtained in the LA25 Src
transformed cell line (see below).
[0228] When these two assay conditions were applied to some of the
pentapeptide-based PKA inhibitors, which were designed as
illustrated in FIG. 3, the results shown in Table I were obtained.
The same assay conditions were also applied to the analogously
designed pentapeptide-based Src inhibitors and obtained the results
shown in Table II.
TABLE-US-00001 TABLE I INITIAL M.sub.1 SCREENING RESULTS WHILE
APPENDED TO THE PKA PENTAPEPTIDE SCAFFOLD ##STR00078## = Attachment
Point K.sub.i (uM), (Conditions*) ##STR00079## ##STR00080##
##STR00081## 76 (L) NT (C) ##STR00082## 18 (L)-Diastereomer A 72
(L)-Diastereomer B NT (C) ##STR00083## ##STR00084## ##STR00085##
##STR00086## ##STR00087## ##STR00088## ##STR00089## ##STR00090##
##STR00091## ##STR00092## ##STR00093## ##STR00094## ##STR00095##
##STR00096## 45 (L) NT (C) *L = Literature Mimetic C = Cellular
Mimetic
The structure identified in Table I as Ac-Arg-Arg-Gly-Ala bonded to
M.sub.1-Ile-NH.sub.2 is SEQ. ID. No. 2.
TABLE-US-00002 TABLE II INITIAL M.sub.1 SCREENING RESULTS WHILE
APPENDED TO THE SRC PENTAPEPTIDE SCAFFOLD ##STR00097## % Inhibition
of 2 mM RR-src phosphorylation by src Assay Conditions Inhibitor (1
mM) Literature Mimetic Cellular Mimetic ##STR00098## 36 0
##STR00099## 51 0 ##STR00100## 83 88 ##STR00101## 68 59
##STR00102## 60 8 ##STR00103## 20 28 ##STR00104## 64 5 ##STR00105##
24 0
[0229] The structure identified in Table II as Ac-Ile-Tyr bonded to
M.sub.1-Gly-Glu-Phe-NH.sub.2 is SEQ. ID. No. 3.
[0230] The standard pentapeptide sequence chosen for the majority
of PKA inhibitors in Table I was derived from the pseudosubstrate
sequence of the peptide inhibitor which was bound to PKA, when the
crystal structure illustrated in FIG. 1 was solved. The standard
pentapeptide sequence used for Src in Table II,
Ac-Ile-Xaa-Gly-Glu-Phe-NH.sub.2 (SEQ. ID. No. 3), was described in
Nair, Kim et al., 1995. Some of the chemistry used to prepare the
PKA inhibitors is described in Nair, Lee & Hangauer 1995. The
synthetic methodology used to develop a number of the Src
inhibitors is described in Lai et al., 1998.
[0231] The collective results in Tables I and II show that both the
serine kinase PKA and the PTK Src can accommodate a variety of
large polar M.sub.1 functional groups at the P 0 phosphorylation
position. Furthermore, using the STAIRe methodology (see Choi et
al. 1996), the sulfamic acid inhibitor 8, and related inhibitors,
were shown to actually bind best when MgATP (not MgADP or no
nucleotide) is also bound. This was a somewhat surprising result
since these inhibitors are analogs of the "end product inhibitors"
1 and 12 which must bind simultaneously with MgADP just following
phosphate transfer in the generally accepted reaction mechanism for
protein kinases.
[0232] These results also demonstrate that both PKA and Src can
show a large difference in binding affinity for structurally very
similar inhibitors. For example, the sulfamic acid PKA inhibitor 8
(Table I) has a K.sub.i of 0.16 .mu.M under Literature Mimetic
assay conditions (L) whereas the isosteric sulfonamide 7 is
1,875.times. less potent (K.sub.i=300 .mu.M). The sulfamic acid
inhibitor 8 is also isosteric with the end product phosphate
inhibitor 1 yet it binds much more tightly under both Literature
Mimetic assay conditions (31.times.) and Cellular Mimetic (C) assay
conditions (108 X). The beneficial effect of an oxygen atom
positioned analogously to that in the substrate Ser is illustrated
by comparison of phosphonate 2 to phosphate 1 and also ether 6 to
phosphate 1. This oxygen atom can also be positioned as a
serine-like OH side chain and enhance binding (compare 2 to 3A and
4A) wherein the closer serine mimic 4A is the more active. The
difference in activity of the diasteromeric inhibitors 3A or B and
4A or B suggests a specific interaction with the active site
catalytic residue Asp-166 may in fact be occurring as intended in
the M.sub.1 design (FIG. 3).
[0233] The Src inhibition results (Table II) show that the end
product inhibitor 12 drops in activity upon going from Literature
Mimetic assay conditions to the higher ionic strength Cellular
Mimetic assay conditions, analogous to the PKA end product
inhibitor 1. However, whereas all of the PKA inhibitors with polar
M.sub.1 functional groups were less active under Cellular Mimetic
assay conditions, three of the Src inhibitors 14, 15, and 17 held
their activity under these higher ionic strength assay conditions.
Also, the hydroxyphosphonate Src inhibitor 13 (a mixture of the R
and S diastereomers) is analogous to the PKA inhibitor 3A and both
are roughly in the same activity range as their corresponding end
product inhibitors, 12 and 1 respectively, under Literature Mimetic
assay conditions. Shortening the side chain length in the
phosphonate Src inhibitor 13 by one carbon atom (and necessarily
removing the attached OH at the same time) to give 14 improved the
activity (analogous to the PKA inhibitor comparison 3 to 4) and,
more importantly, resulted in equivalent activity under Cellular
Mimetic assay conditions. The Src results with 16-19 (particularly
17, see later for an analogous .alpha.-tricarbonyl acid M.sub.1
analog appended to non-peptide Src inhibitors) also suggests that
similar amides may be useful M.sub.1 functional groups to explore
with non-peptide Src inhibitors.
[0234] Non-peptide Src inhibitors are preferred to peptide scaffold
based compounds, partly because some of these inhibitors have a
dual effect on Src. For example, phosphonate inhibitor 14 not only
inhibits Src by competitively binding in the active site but it
also activates Src by binding to the SH.sub.2 site thereby
releasing the intramolecular autoinhibition mechanism (Xu et al.,
1997). This opposing effect gives an unusual IC.sub.50 curve for
14, wherein at low inhibitor concentrations Src is stimulated (to a
maximum of 70%) in a smooth dose-response fashion (due to initial
tighter SH2 binding) followed by a typical IC.sub.50 inhibition
curve at higher inhibitor concentrations (due to lower affinity
blockade of the active site). This opposing activation effect of
the pentapeptide inhibitors makes them appear to be less potent
active site inhibitors than they in fact are, and makes it
difficult to accurately rank M.sub.1 groups while appended to this
pentapeptide scaffold. However, the better M.sub.1 groups
identified with the Src pentapeptide scaffold must still be
accommodated in the catalytic region of the active site and hence
are useful orienting groups for the ongoing non-peptide Src
inhibitor studies as intended. Since PKA does not have an SH2
domain, this complication is not a factor in interpreting the PKA
pentapeptide inhibitor M.sub.1 testing data.
[0235] The results in Tables I and II also show how much effect the
assay conditions can have on both inhibitor potencies and the rank
order of activity. For example, as shown in Table I, switching from
the Literature Mimetic (L) assay conditions to the Cellular Mimetic
(C) assay conditions can change the potency from as little as
3-fold (inhibitor 10) to as much as 108-fold (inhibitor 1). Also,
whereas inhibitor 10 is less potent than 1 under Literature Mimetic
conditions, it is more potent under Cellular Mimetic conditions.
The Src inhibitor data presented in Table II show that many of the
inhibitors lose their potency upon going from Literature Mimetic
assay conditions to Cellular Mimetic assay conditions. The rank
order of potency against Src is also sensitive to the assay
conditions. Whereas inhibitor 18 is more potent than inhibitor 17
under Literature Mimetic conditions, the opposite is true under
Cellular Mimetic conditions. Since activity within cells is the
goal, the Cellular Mimetic Src assay was selected as the standard
assay for testing potential non-peptide Src inhibitors. Activity
within the Cellular Mimetic assay is a necessary, but not
sufficient, condition for activity within cells. As will be
described later, the Cellular Mimetic Src assay will be followed up
with cell culture assays wherein cell penetration, metabolism, and
binding to other cellular components are also factors in the
measured potency.
[0236] The next class of M.sub.1 functionality which was explored
was the boronic acid group. This functional group is an intriguing
candidate for M.sub.I for a number of reasons: 1) It can exist in a
non-ionic state so that it should not prevent passive absorption of
non-peptide inhibitors across cell membranes. 2) The planar,
trigonal, boron acids might form reversible tetrahedral covalent
borate complexes (a well known property of boronic acids, see
Loomis & Durst, 1992) through their vacant 2p orbitals with
anions present in the protein kinase active site, such as the
catalytic Asp carboxyl group, or the ATP/ADP terminal phosphate
oxygens. This ability to form borate complexes with active site
nucleophiles has been extensively utilized to develop slow binding
inhibitors of serine proteases (e.g. see Kettner & Shenvi,
1984), wherein the nucleophilic serine OH forms a covalent bond
with the vacant 2p orbital in the boronic acid resulting in a
tetrahedral borate complex (e.g. see Skordalakes et al., 1997).
Also, an intramolecular complex of a boronic acid with a urea
NH.sub.2 was used to prepare transition state analogs inhibitors of
dihydroorotase (Kinder et al., 1990). 3) Boronic acids act as Lewis
acids and are converted to tetrahedral hydrates in water by forming
borate complexes with water or hydroxide ions. Therefore, it is
also possible that these boronic acid hydrates may function as
phosphate mimics and M.sub.1 modules as proposed in FIG. 2. This
hydration property was utilized by Baggio et al. (1997) wherein a
hydrated boronic acid functioned as a transition state analog
inhibitor functionality for arginase. These researchers evaluated
the inhibited complex by x-ray and showed that the hydrated boronic
acid functionality formed two hydrogen bonds with the active site
catalytic Glu-277 carboxyl side chain and one of the other hydrated
boronic acid OH's interacted with two catalytic Mn.sup.2+'s in the
active site. These binding interactions are closely analogous to
those proposed in protein kinase active sites, i.e. H-bonds to the
catalytic Asp side chain carboxyl group and interactions with the
active site Mg.sup.2+'s (see FIGS. 2 and 4). The use of boronic
acids for protein kinase inhibitors has not been explored
previously.
[0237] In the area of pentapeptide-based PICA inhibitors, the
boronic acid functionality has been prepared and tested as a
potential M.sub.1 module utilizing the four inhibitors 21-24 shown
in Table III (see Hsiao & Hangauer, 1998, for some of the
chemistry used to prepare these compounds).
TABLE-US-00003 TABLE III PKA INHIBITION RESULTS WITH BORONIC
ACID-CONTAINING PEPTIDE INHIBITORS IC.sub.50 .mu.M (cond. IC.sub.50
.mu.M (Cond. IC.sub.50 .mu.M (cond. IC.sub.50 .mu.M (cond.
Ac-RRGXI- L, 0 h L, 4 h C, 0 h C, 4 H NH.sub.2, X = preincubation)
preincubation) preincubation) preincubation) 20 Ala 278 (K.sub.i =
9 .mu.M) 417 41 (K.sub.i = 25 .mu.M) 50 ##STR00106## 249 *500 .mu.M
34% inh 764 *2000 .mu.M 19% sti ##STR00107## 81 *65 *1753 *2000
.mu.M 71% sti ##STR00108## 398 133 2000 .mu.M 16% inh *2000 .mu.M
5% inh ##STR00109## 1000 .mu.M 33% inh 1000 .mu.M 44% inh 2000
.mu.M 6% sti 1734 .mu.M *Very distorted IC.sub.50 curve: Suggests
Inhibitor is also a substrate. L = Literature Mimetic Assay
Conditions. C = Cellular Mimetic Assay Conditions. Inh =
Inhibition. Sti = Stimulation.
The structure identified in Table III as Ac-RRGXI-NH.sub.2 is SEQ.
ID. No. 4.
[0238] While testing these boronic acid-containing PKA inhibitors,
the corresponding pentapeptide pseudosubstrate inhibitor 20 was
included as an internal control while investigating time-dependent
inhibition as shown in Table HI. Under Literature Mimetic assay
conditions, and no preincubation, the initial results suggested
that the shortest chain L-amino acid 21 was binding with the same
affinity as the pseudosubstrate inhibitor 20 (i.e. K.sub.i ca. 9
.mu.M). As this side chain was increased in length (to 23 and then
24) binding affinity appeared to decrease. When the stereochemistry
of the unnatural amino acid was inverted from L in 21 to D in 22
binding affinity appeared to increase 3-fold. This improvement in
binding may occur as a result that the boronic acid OH in 21 is
positioned at the same chain length as L-homoserine whereas the
natural substrate, L-serine, has a one carbon shorter side chain.
Modeling results with the PKA ternary structure indicated that the
boronic acid OH can be retracted back somewhat by inverting the
a-carbon stereochemistry from L in 21 to D in 22 and then
repositioning the side chain to more closely mimic the positioning
of the natural substrate L-serine OH adjacent to the catalytic
residues (Asp-166 and Arg-168). The modeling results were
subsequently supported by the finding that, upon incubation of PKA
with these inhibitors for up to four hours without adding the
competing peptide substrate (Kemptamide: LRRASLG-NH.sub.2 (SEQ. ID.
No. 5)), both 21 and 22 function as substrates with the
D-diastereomer 22 being phosphorylated faster.
[0239] The fact that these boronic acid inhibitors are also
substrates, became much more obvious by the greatly distorted
IC.sub.50 curves obtained under the Cellular Mimetic conditions,
both with and without preincubation (both PKA and Src are more
active enzymes under the Cellular Mimetic conditions than under
Literature Mimetic conditions). In the assay used to obtain these
results, the P.sup.32 phosphorylated Kemptamide product (25
generated from .gamma.-P.sup.32 ATP) was isolated at the end of the
substrate incubation period by binding to phosphocellulose filter
paper via the three cationic groups (two Arg's and the N-terminus)
and the level of phosphorylated product isolated on the paper is
then measured by liquid scintillation counting (cpm's). The boronic
acid inhibitors 21-24 have two Arg's in their sequence also and
therefore will bind to the phosphocellulose paper in addition to
Kemptamide (although not as consistently or completely due to one
less positive charge). Consequently, when analyzed as inhibitors,
the amount of phosphorylated Kemptamide produced was not only
counted, but also the amount of phosphorylated inhibitor
simultaneously produced (e.g. see 26 below). The net result is that
distorted IC.sub.50 curves are obtained which show net
"stimulation" at higher inhibitor concentrations in some cases. The
D diastereomer 22 gives the greatest apparent "stimulation" (71%)
when preincubated with PKA for four hours under Cellular Mimetic
conditions followed by the L diastereomer 21 (19%) and then the one
carbon homolog 23 (5%), indicating all three are substrates for PKA
(Table III). The underlying substrate behavior of these
"inhibitors" makes an accurate measurement of their inhibition
potency impossible with the current assay. However, it does appear
from the data that homologating the boronic acid functionality out
with only CH.sub.2 groups (homologations with boronic acid
non-peptide Src inhibitors may also be carried out) decreases the
binding affinity and ability to function as a substrate.
##STR00110##
[0240] Phosphorylated Kemptamide is SEQ. ID. No. 6. Phosphorylated
22 is SEQ. ID. No. 4. The boronic acid "inhibitors" 21 and 22 were
shown to be substrates for PKA by running the same assay, but
without adding Kemptamide, and stopping the reaction at various
time points as shown in FIG. 4. The graphs show their respective
rates and levels of phosphorylation with the typical loss of
initial velocity kinetics with time (due to substrate depletion and
end product inhibition), analogous to a standard L-Ser substrate
such as Kemptamide. The comparison of 21 to 22 shown was done in
the same assay run, at identical boronic acid substrate
concentrations, and with identical Cellular Mimetic assay solutions
so that the cpm's could be directly compared. The graphs show that
initial velocity conditions were lost within one hour for D isomer
22 whereas the linearity appears to have been lost somewhat slower
with the L isomer 21 suggesting a slower consumption of starting
material. That the boronic acid moiety would be phosphorylated by
PKA was surprising, but it is even more surprising that the
phosphonic-boronic acid mixed anhydride produced (e.g. 26) was
stable enough to survive the pH 7.2/37.degree. C. assay incubation
and then be isolated by binding to phosphocellulose paper after
acid quenching of the reaction with 10% TCA and washing the
phosphocellulose paper with 25 mM phosphoric acid (3.times.). An
STN substructure search was run on mixed anhydrides of phosphoric
and boronic acids and found only three references to experiments
and theoretical calculations for the analogous putative (but not
proven) anhydride formed from boric acid and phosphoric acid as a
solid surface impregnated catalyst for the partial oxidation of
ethane to acetaldehyde at 823.degree. K (Zhanpeisov & Otsuka,
1992, Otsuka et al., 1992, Murakami et al., 1990). However, this
highly unusual anhydride has never before been synthesized free of
a solid surface, isolated, or characterized. Thus, this is a novel
enzymatic reaction and chemical entity with interesting
possibilities for protein kinase inhibitor designs.
[0241] The next class of M.sub.1 functionality which was explored
was the halogen group. This functional group is an intriguing
candidate for M.sub.1 for a number of reasons: 1) it is a good
hydrogen bond acceptor, and 2) it reduces the rate of metabolism,
leading to in vivo benefits.
[0242] The halogen functionality has been prepared and tested as a
potential M.sub.1 module utilizing the inhibitor shown below (see
Example 1, for the chemistry used to prepare this compound):
##STR00111##
[0243] This inhibitor was tested for Src inhibition using the assay
procedure set forth in Example 1. The results obtained are shown in
Table VII, which indicates an IC.sub.50 of 40 .mu.M for the above
inhibitor (1a in Table VII). This inhibitor includes a non-peptide
scaffold (indole) which was chosen based on the screening method
described below.
[0244] The Src and PKA pentapeptide scaffold tethered M.sub.1
evaluations described above have resulted in identifying a variety
of orienting M.sub.1 groups which could be used for screening
potential non-peptide scaffolds as indicated in Step 1 (FIG. 1).
The boronic acid (from 22), the phosphonate (from 14), and the
sulfamic acid (from 8) were chosen from the menu of potential
M.sub.1's for the Src non-peptide scaffold screening. Among these
choices, the boronic acid M.sub.1 group has proven effective for
Step 1 screening of non-peptide scaffolds.
[0245] The most useful crystal structures available for the design
of non-peptide Src inhibitors, which do not compete with ATP, are
the native Src structure and the IRTK:peptide:AMP-PNP ternary
structure. For all of the modeling studies discussed below, the
SYBYL molecular modeling software package is used on a Silicone
Graphics Workstation.
[0246] Since the Src and IRTK structures are only used as
qualitative guides in designing the non-peptide scaffolds and
combinatorial libraries, the active sites along with two layers of
surrounding residues were carved out from the native Src and IRTK
ternary structures, analogous to the previous PKA modeling studies.
The IRTK:peptide:AMP-PNP ternary. structure active site region was
used as the template structure to guide the building of the Src
residue sequence 424-418 back onto the Src structure using the
comparative homology modeling technique (see Hutchins & Greer,
1991). These residues were disordered in the native Src crystal
structure and therefore not visible by x-ray. They were
reintroduced because they help form the P+1 to P+3 binding sites
for peptide substrates which are important for some of the modeling
studies. The analogous residues in the IRTK ternary structure are
seen by x-ray and directly interact with the bound peptide
substrate. In fact, it is probably the presence of the bound
peptide substrate which induces order in the positioning of this
sequence so that it is visible by x-ray. The Src pentapeptide
substrate Ac-Ile-Tyr-Gly-Glu-Phe-NH.sub.2 (SEQ. ID. No. 1) (Nair et
al., 1995) was then docked into the Src active site again using the
IRTK ternary structure as a template. Small adjustments were then
manually made to partially clean up this complex, all of the
hydrogen atoms were added, appropriate formal and partial charges
(calculated via the Gasteiger Marsili method) were added, and then
the entire complex was subjected to 300 iterations of molecular
mechanics minimization using the Tripos force field, analogous to
the previous PKA modeling procedure. A schematic representation of
this modeled complex is given in FIG. 5. Any inaccuracies in this
Src:peptide and the Src:inhibitor models are accommodated by
experimentally evaluating a range of side chains, the number and
diversity of which is scaled roughly to the level of uncertainty
for the structure of their particular binding region in the Src
model active site (see later), in a combinatorial fashion.
[0247] As shown in FIG. 5 the residues 424-418 built back into the
Src interact with the P+1 to P+3 substrate residues,
Gly-Glu-Phe-NH.sub.2 respectively, through beta sheet type hydrogen
bonding interactions with the substrate main chain (analogous to
the IRTK peptide substrate). Lys 423 engages in two important
interactions: 1) the .gamma. and .gamma. CH.sub.2's fold over the
top of the P O Tyr phenyl ring engaging in a hydrophobic binding
interaction and then 2) the remaining
CH.sub.2--CH.sub.2--NH.sub.3.sup.+ of this side chain extends away
to form a salt bridge with the P+2 Glu side chain as indicated. The
rest of the P 0 Tyr hydrophobic binding pocket is formed by Pro 425
under the phenyl ring and part of the Cys 277 side chain above the
phenyl ring. Using a large combinatorial peptide Src substrate
library, Songyang et al. (1995) found that the most commonly chosen
side chain for the P+1 position was Gly followed by Glu. The
present model indicates that a P+1 Glu side chain may form a salt
bridge with nearby Arg 469 as indicated in FIG. 5. Previously,
researchers found that only Glu was chosen for the P+2 position and
the present model indicates that this side chain forms a salt
bridge with the Lys 423 side chain. At the P+3 position Phe was
very strongly preferred and the model indicates that this side
chain forms a stacking interaction with the Phe 424 side chain. At
the P-1 position Songyang et al. found that Ile was the most
preferred residue followed by Val and then Leu. The model shows a
hydrophobic pocket for binding the P-1 side chain formed mainly by
Trp 428, Ala 390 and Leu 347. One might expect that the P 0 Tyr
side main chain will strongly interact (though hydrogen bonding)
with the active site in a catalytically competent complex because
enzymes often form more critical interactions in this region close
to where the reaction will be occurring. The IRTK ternary complex
does not show a good hydrogen bond to either the P 0 Tyr NH or
carbonyl. The nearest candidate residue for this interaction in the
IRTK structure is Asn 1215 wherein the side chain NH.sub.2 is 3.71
.ANG. from the Tyr carbonyl oxygen. When the IRTK ternary structure
is overlayed onto the Src native structure, using the four residues
mentioned in the Background and Significance section, Asn 468 from
the Src structure was found to be positioned very close to the
analogous IRTK Asn 1215. This suggests that this conserved residue
is performing an important role and might move a little closer
(i.e. about 1 .ANG.) to the substrate P 0 NH and carbonyl in a
catalytically active complex and form the hydrogen bonding
interactions indicated in FIG. 5. Finally, the catalytic Arg 388
and Asp 386 are correctly positioned in the Src model to catalyze
the transfer of the y-phosphate from ATP to the Tyr OH.
[0248] The Src:peptide substrate complex can now be used to model
potential non-peptide scaffolds and determine preferred
substitution positions for the specificity elements, all with an
appropriately attached M.sub.1 functionality, before choosing new
scaffolds to experimentally evaluate. The IRTK:peptide:AMP-PNP
ternary structure can also be used to model these potential
scaffolds and preferred substitution positions. These scaffolds
have broad utility for the development of selective PTK inhibitors
by further developing them with appropriate specificity elements
following the strategy outlined in FIG. 1.
[0249] The first non-peptide scaffold evaluated with this
Src:peptide substrate model was the naphthalene scaffold. This is
the first use of bicyclic aromatic scaffolds for non-peptide PTK
inhibitors, which do not compete with ATP. The naphthalene
scaffold's utility for this purpose was demonstrated by developing
a non-peptide inhibitor of the 1RTK and EGF receptor PTK
(Saperstein et al., 1989). The 1RTK ternary complexes were
subsequently used to adapt this scaffold for Src inhibition (see
Marsilje et al., 2000). The naphthalene scaffold was docked into
the Src active site by first carrying out a least squares fitting
of atoms a-d onto the peptide substrate as indicated in FIG. 6. In
this way the naphthalene scaffold is related to the peptide
substrate by the cyclization shown by the arrow in FIG. 6 and an
appended OH as a substitute for the substrate Tyr NH. This is
essentially the same process used to dock this scaffold into the
IRTK structure as described in Marsilje 2000. The peptide substrate
was then deleted from the active site, various M.sub.1 functional
groups and specificity elements S.sub.2 and S.sub.3 were then added
to the scaffold as indicated and the complexes were then
individually minimized for 300 iterations. This same process was
also used to design the isoquinoline and indole scaffolds whose
binding modes are indicated in FIG. 7.
[0250] In all of these modeled complexes, selectivity element
S.sub.2 consists of various hydrophobic side chains which can bind
in the same pocket as the substrate P-1 Ile side chain and
selectivity element S.sub.3 consists of various molecular fragments
which can bind in the P.sub.+1 to P+3 region of the peptide
substrate binding sites (FIG. 5). Since the active site region
where M.sub.1 binds is highly conserved among all of the protein
kinases, the small menu of M.sub.1 functional groups previously
identified using peptide scaffolds served as the initial M.sub.1
groups for attachment to the scaffolds at the indicated positions.
Of the two selectivity elements binding sites, the structure of the
hydrophobic binding cavity for S.sub.2 is known with greater
confidence in the Src model than is the P+1 to P+3 binding region
for S.sub.3. This is because the S.sub.3 binding site was
constructed partially by comparative homology modeling whereas the
S.sub.2 site is largely unchanged from the structure determined by
x-ray for native Src. In view of these varied levels of confidence
in the modeled binding sites for M.sub.1, S.sub.2 and S.sub.3, the
combinatorial library diversity is scaled such that the greatest
variety and number of side chains in the combinatorial libraries
are at the S.sub.3 site followed by the S.sub.2 site and then
M.sub.1.
[0251] The Src results using M.sub.1 functional groups to
experimentally identify promising non-peptide scaffolds are listed
in Table IV.
TABLE-US-00004 TABLE IV INITIAL STEP 1 RESULTS % SRC INHIBITION IN
CELLULAR MIMETIC ASSAY % Inhibition of 2 mM RR-src at Inhibitor
Inhibitor Concentration () ##STR00112## ##STR00113## M.sub.1 27
##STR00114## 59 (1 mM) 13 (100 .mu.M) IC.sub.50 = 950 .mu.M K.sub.i
= 554 .mu.M NON-ATP COMPETITIVE 28 ##STR00115## 31 (1 mM) IC.sub.50
= 1.6 mM K.sub.i = 963 .mu.M NON-ATP COMPETITIVE 29 ##STR00116## 0
(1 mM) 30 ##STR00117## 14 (1 mM) 31 ##STR00118## 0 (100 .mu.M)
##STR00119## M.sub.1 32 ##STR00120## 0 (100 .mu.M) 33 ##STR00121##
1 (1 mM) 34 ##STR00122## 0 (100 .mu.M) ##STR00123## 35 X = O 10
(100 .mu.M) 36 X = S 12 (100 .mu.M) 37 ##STR00124## 13 (500 .mu.M)
##STR00125## M.sub.1 38 ##STR00126## 62 (500 .mu.M) NON-ATP
COMPETITIVE 39 ##STR00127## 11 (500 .mu.M) 40 ##STR00128## 13 (100
.mu.M) 41 ##STR00129## 14 (100 .mu.M)
[0252] The data in Table IV allows a number of conclusions to be
drawn: 1) Low, but measurable, inhibition potency can be obtained
with an appropriate M.sub.1 group attached to a scaffold (e.g. 27
and 38). 2) 1 mM inhibitor concentrations for this type of
screening is higher than desirable but 100 .mu.M is too low.
Screening of scaffolds bearing an M.sub.1 group would optimally be
conducted at 500 .mu.M. 3) The boronic acid, sulfamic acid, and
phosphonic acid M.sub.1 functional groups, which had been
identified using the PKA pentapeptide scaffold (22, Table III and
8, Table I) or the Src pentapeptide scaffold (14, Table II),
respectively, give measurable activity when placed at the 2
position of the naphthalene ring (27, 28 and 30 respectively), the
preferred position for M.sub.1 identified in the model naphthalene
inhibitor:Src complex (FIG. 6). Moving the boronic acid or
phosphonic acid M.sub.1 groups to the 1 position (32 or 33) reduced
activity. 4) The related M.sub.1 sulfonamide functionality, which
was poor on the PKA pentapeptide scaffold (7 & 9, Table 1) is
also poor when appended to the 2 (31) or 1 (34) position of the
naphthalene scaffold. The sulfonic acid analog at the naphthalene 2
position (29) is completely inactive, even at 1 mM. 5) The
naphthalene scaffold can be replaced with a benzofuran (35) or a
benzothiophene (26) scaffold without a noticeable reduction in
activity when the boronic acid M.sub.1 group is positioned
analogous to the 2 position on a naphthalene. 6) The boronic acid
M.sub.1 group also provides active compounds when appended to the
isoquinoline (37) or indole (38) scaffolds at the positions
indicated by modeling results (FIG. 7). However, the indole
scaffold is clearly favored over the isoquinoline scaffold
suggesting that a hydrogen bond donating ability to Asn 468 (see
FIG. 7) is important for higher activity (this would require the
protonated isoquinoline which is disfavored by the adjacent
electron withdrawing ester group). This conclusion is also
supported by considering that a peptide substrate may position a
hydrogen bond donating peptide bond NH at a similar position (FIG.
6) and by finding that an equivalently positioned phenolic OH (FIG.
6) improves potency (phenolic OH's are much better H-bond donors
than acceptors). 8) When directly compared to other M.sub.1 groups,
the boronic acid group is superior (e.g. 27 vs. 28-31, 38 vs. 32).
9) A biphenyl scaffold modeled into the Src and IRTK active sites
and found promising binding modes for this scaffold. Combinatorial
libraries were developed with the biphenyl scaffold (see Pavia et
al., 1996), and the modeling results were encouraging. Therefore,
the para (40) and meta (41) isomers were evaluated with the boronic
acid M.sub.1 group. Both biphenyl compounds showed potency
equivalent to the best naphthalene boronic acid (27) and therefore
provide another scaffold geometry (the two phenyl rings are not
planar) for further evaluation and development.
[0253] Since the bare scaffolds, with only an M.sub.1 group
appended, often have low binding affinity, the IC.sub.50's and
K.sub.i's for the 2-naphthalene boronic acid and sulfamic acid
inhibitors were determined to ensure that a typical dose/response
IC.sub.50 curve is obtained. This analysis provided the typical
shape dose/response curves seen with more potent inhibitors. The
IC.sub.50's and K.sub.i's of these simple inhibitors also confirmed
that the boronic acid inhibitor 27 is more potent than the sulfamic
acid analog 28 and has a K.sub.i of about 554 .mu.M.
[0254] The next issue addressed with these simple inhibitors before
proceeding to elaborate them further was their mode of inhibition,
specifically whether they are ATP-competitive inhibitors. In the
case of the naphthalene inhibitors 27 and 28, their IC.sub.50's
were monitored as the ATP concentration was increased in three
steps up to 1 mM. As a comparison, the IC.sub.50 of the
pentapeptide phosphonic acid Src inhibitor 14 (Table II) was also
monitored. If any of these inhibitors were competing with ATP, then
their IC.sub.50's should have increased proportionally with the ATP
concentration (i.e. the dashed line). As shown, the IC.sub.50's for
all three inhibitors remained essentially constant as the ATP
concentration was increased demonstrating that they are not
ATP-competitive inhibitors. A very similar, but much less costly
(commercial Src is expensive), analysis was conducted with the
indole boronic acid inhibitor 38. In this case, the % inhibition
was monitored with 38 at a constant 500 .mu.M inhibitor
concentration but with increasing ATP concentrations of 200, 500
and 1,000 .mu.M. Once again the inhibitor potency was not reduced
by the increasing ATP concentration demonstrating that 38 is also
non-ATP competitive.
[0255] The initial results obtained in Step 1 suggests that it is
possible to identify promising scaffolds for further elaboration
with this procedure. The biggest uncertainty with Step 1 is that
some of the scaffolds identified in this way might not be binding
in the fashion suggested by the prior modeling evaluations. This is
essentially a "false positive" problem. These "false positives"
will likely fail in Step 2, when they are evaluated for improved
binding using the modeled complexes as a guide. Some false positive
results can be accepted in Step 1 because the bare scaffolds with
only the M.sub.1 group attached are easily obtained. For further
inhibitor development, one may return to Step 1 each time new
scaffolds are needed to carry through Steps 2 and 3. The best
M.sub.1 generated can be used each time Step 1 is repeated.
Currently, the boronic acid M.sub.1 group has been used since it
has a proven ability to give measurable activity with bare
scaffolds. Also the boronic acid M.sub.1 group offers multiple
interesting possibilities for covalent and non-covalent
interactions with the conserved catalytic residues since it can: 1)
hydrate, 2) form borate complexes with electron rich active site
atoms, and/or 3) be phosphorylated and then react with active site
nucleophiles or engage in additional non-covalent interactions.
From the data in Table IV, the naphthalene and indole scaffolds
were chosen as M.sub.2 for the first efforts in Step 2 (the
biphenyl scaffold is also a preferred scaffold). It is also worth
mentioning that naphthylalanine and analogs can be successfully
substituted for the P 0 tyrosine in Src peptide substrates (e.g.
see Alfaro-Lopez et al., 1998) further supporting the notion that
naphthalene and related scaffolds can bind at the P 0 site.
[0256] In comparing the naphthalene vs. indole scaffold results
with a boronic acid M.sub.1 group (i.e. 27 vs. 38, Table IV) the
indole hydrogen bond donating NH and/or the adjacent ester group
appeared to be the reason for the enhanced potency. Consequently,
for Step 2 one of the first attempts was to add a hydroxyl group
and an amide (with S.sub.2) to the naphthalene scaffold at the
adjacent positions suggested by the modeling results (FIG. 6). For
the indole scaffold one priority was to prepare some amide analogs
to see if potency can be increased with the S.sub.2 specificity
element (FIG. 7). In order to facilitate the synthesis of these
initial analogs, an OH was temporarily substituted for the boronic
acid M.sub.1 group. The OH group is also known to interact with the
catalytic residues, as required for an M.sub.1 group, because it is
the natural substrate M.sub.1 whose phosphorylation rate is
accelerated by interactions with the catalytic residues. The
results obtained for some of the initial analogs are given in Table
V along with a side by side comparison, in the Cellular Mimetic Src
assay, to two literature Src inhibitors 50 and 51 which are
reported be non-ATP competitive. Some of these results and
additional analogs are described in Marsilje 2000.
TABLE-US-00005 TABLE V INITIAL STEP 2 RESULTS % SRC INHIBITION IN
CELLULAR MIMETIC ASSAY % Inhibition of 2 mM RR-src Inhibitor at
Inhibitor Concentration () 42 ##STR00130## 47 (100 .mu.M) 43
##STR00131## Ortho: 39 (100 .mu.M) Meta: 89 (100 .mu.M) IC.sub.50 =
18 .mu.M, K.sub.i = 10 .mu.M Para: 23 (100 .mu.M) NON-ATP
COMPETITIVE 44 ##STR00132## 45 (100 .mu.M) 45 ##STR00133## 51 (100
.mu.M) IC.sub.50 = 170 .mu.M NON-ATP COMPETITIVE 46 ##STR00134##
Ortho: 42 (100 .mu.M) Meta: In progress Para: 42 (100 .mu.M) 47
##STR00135## 40 (500 .mu.M) 48 ##STR00136## Ortho: 43 (100 .mu.M)
Meta: 30 (100 .mu.M) Para: 45 (100 .mu.M) 49 ##STR00137## Ortho: 24
(100 .mu.M) Meta: 74 (100 .mu.M) Para: 54 (100 .mu.M) 50
##STR00138## 30 (100 .mu.M) Lit. IC.sub.50 = 118 nM 51 ##STR00139##
37 (100 .mu.M) Lit. IC.sub.50 = 18 .mu.M 52 ##STR00140## 41 (100
.mu.M) Lit. IC.sub.50 = 66 .mu.M for p56.sup.lck
[0257] Inhibitor 50, and analogs (Huang et al., 1995), were of
particular interest because the iminochromene scaffold is closely
related to the naphthalene scaffold and it's binding mode would be
expected to be very similar based upon the model (FIG. 6). Partly
because of this close analogy, the amides of hydroxyanilines with
the naphthalene and indole scaffolds were examined as shown in
Table V. Also, the modeling studies with these hydroxyaniline amide
derivatives in the Src active site indicated that the hydroxyl
group may be able to engage in hydrogen bonding interactions with
the Src Phe 424-Ala 422 backbone peptide bonds analogous to peptide
substrates (see FIG. 5). These modeling studies also indicated that
the homologous hydroxybenzylamides should be active and, more
importantly, provide a substitution position (i.e. the benzylic
carbon) for appending side chains to bind in the P-1 side chain
pocket (e.g. to Arg 469, FIG. 5).
[0258] The data in Table V allow the following conclusions to be
drawn: 1) Adding an amide extension onto both the naphthalene and
indole scaffolds can increase potency as predicted by the models
for these scaffolds bound in the Src active site (ca. 5-fold in the
cases of 42 vs. 43-meta & 47 vs 48). 2) Adding a hydroxyl group
to the naphthalene scaffold adjacent to the amide increases potency
(about 5-fold, 43-meta vs. 44) as predicted by the Src model, and
also suggests Mn 468 does hydrogen bond with this OH. 3) Moving the
M.sub.1 OH group from the position predicted to be best in the Src
model to the adjacent position reduces potency by one order of
magnitude (43-meta to 45). 4) The indole scaffold is less
responsive than the naphthalene scaffold to regiochemistry of the
hydroxyaniline extension (48 vs. 43). 5) The naphthalene and the
indole scaffolds accept the one carbon homologation provided by
using hydroxybenzylamides (46 vs. 43 & 49 vs. 48). 6) The two
M.sub.1 hydroxy regioisomers of the naphthalene scaffold are both
non-ATP competitive (see Marsilje 2000). 7) All of the methyl
hydroxyaniline and hydroxybenzylamide inhibitors were found to be
less active suggesting that the hydroxyl group in the amide
extension is functioning as a hydrogen bond donor. In this regard
it is worth mentioning that in another Src peptide substrate
combinatorial library study, Ser and Thr were identified as two of
the most preferred residues at the P+2 position (Alfaro-Lopez et
al., 1998), suggesting that there are other binding opportunities
for an amide extension OH other than to the Phe 424-Ala 422 peptide
bonds suggested by the modeling studies. 8) The most potent non-ATP
competitive, non-peptide, Src inhibitor previously disclosed in the
literature (50) is not nearly as potent as reported when tested
under the Cellular Mimetic assay conditions (IC.sub.50=118 nM
reported by Huang et al., 1995 vs only 30% inhibition at 100 .mu.M)
and is less potent than a number of the current inhibitors
(especially 43-meta) including the most analogous inhibitor (52 vs.
45). The structure-activity-relationship (SAR) reported for hydroxy
regioisomers of 50 in their assay (Huang et al., 1995) also does
not correspond with the SAR which was obtained for the related
naphthalene inhibitors. For example, their iminochromene analog of
the most potent naphthalene inhibitor 43-meta is 230-fold less
potent than 50 in their Src assay. An important advantage of the
naphthalene scaffold over the iminochromene scaffold is that it
allows a highly desirable S.sub.2 specificity element to be added
for accessing the P-1 hydrophobic site (see FIG. 6) whereas the
analogous position can not be substituted on the iminochromene
scaffold because it is occupied by the ring oxygen atom.
[0259] The inhibitor potencies in the Src Cellular Mimetic assay
can be further calibrated against other literature non-ATP,
non-peptide Src inhibitors. Two additional examples are 51 (ST 638,
Shiraishi et al., 1989) which is a member of the "tyrphostin"
family of erbstatin analogs (see Lawrence & Niu, 1998) and the
natural product PTK inhibitor piceatannol 52 (Thakkar et al.,
1993). In the Cellular Mimetic assay all of these known inhibitors
are less potent than had been reported suggesting that the assay is
particularly demanding in terms of achieving high potency. The
initial testing of Src inhibitors is carried out using a single
concentration (in triplicate) because commercial Src is too
expensive to do full IC.sub.50 curves on every inhibitor. It should
be mentioned, however, that an IC.sub.50 dose response curve is not
linear and the difference between ca. 50% inhibition at 100 .mu.M
and a ca. 90% inhibition at 100 .mu.M is actually a factor of 10
and not a factor of 2 (e.g. 45 vs. 43-meta). Consequently, the
literature Src inhibitors 50-52 are greater than an
order-of-magnitude less active than the currently most potent
inhibitor 43-meta.
[0260] The discrepancies found within the literature reporting the
potency of these inhibitors, the sensitivity to assay conditions
described earlier with the PKA inhibitors, and the lack of
consistency among numerous labs and commercial protein kinase assay
kits highlights this overlooked, but crucial, problem in the field.
Although the inhibitors produced by the present invention may be
more potent under other assay conditions, the Cellular Mimetic
assay should be used, which mimics the intracellular physical
chemical conditions as closely as possible, as the primary potency
and rank order guide for evaluating the inhibitors before choosing
compounds to proceed to whole cell or tissue assays. As will be
discussed in more detail later, the most potent naphthalene-based
inhibitor thus far from the Cellular Mimetic assay (i.e. 43-meta,
IC.sub.50=18 .mu.M and K.sub.i=10 .mu.M) is also effective in
specifically blocking pp60.sup.v-arc stimulated cell proliferation
with a similar IC.sub.50 of ca. 25 .mu.M. This suggests that not
only is the Cellular Mimetic Src assay predictive, but also that
this class of naphthalene-based inhibitors can readily pass through
cell membranes and inhibit intracellular Src.
[0261] Analogs of a number of the naphthalene and indole inhibitors
above can be prepared with the boronic acid or halogen M.sub.1
group in place of the M.sub.1 OH and/or with a S.sub.2 hydrophobic
specificity element attached for binding in the Src P-1 site as
illustrated in FIGS. 6 and 7. The naphthalene and indole scaffolds
can then be taken through to Step 3 as described below. Each time
Step 2 is repeated with new scaffolds from Step 1, the best
selectivity elements S.sub.2 and/or S.sub.3 which have discovered
with previous scaffolds will be used in the combinatorial libraries
of Step 3. Even though the optimal combination of M.sub.1, S.sub.2,
and S.sub.3 is likely to be different for each scaffold, those
found optimal with the previous related scaffold (e.g. going from
the naphthalene to the indole scaffold) should be suitable for
utilization as better initial specificity elements in Step 2 with
the new scaffold. The same process will be repeated each time there
is a need to try another scaffold until sufficient potency,
selectivity, and suitable pharmaceutical properties are achieved
for the Src inhibitors or, subsequently, for inhibitors of
additional therapeutically important PTK's.
[0262] Some of the chemistry used to prepare the naphthalene
inhibitors is described in Marsilje 2000. For attaching a boronic
acid functionality in place of a M.sub.1 hydroxyl group in the Src
inhibitors from Table V, the Pd (0)-catalyzed cross-coupling
methodology was used wherein either an aryl triflate (Ishiyama et
al., 1997) or an aryl halide (Ishiyama, 1995) can be coupled with
the commercially available pinacol ester of diboron. An
illustrative example recently completed is given in FIG. 8.
[0263] The example shown in FIG. 8 demonstrates that it is possible
to selectively triflate the less hindered OH at the M.sub.1
position and this has been proven by its removal to 56 with
subsequent .sup.1H NMR verification of the substitution pattern.
The monotriflate 53 was then taken on to the desired boronic acid
55 as indicated. The same reaction sequence also works well for the
regioisomer of 42 which corresponds to inhibitor 45 from Table V.
The synthetic scheme shown in FIG. 9 can be followed, in order to
attach hydrophobic S.sub.2 selectivity elements to the naphthalene
scaffold.
[0264] The naphthalene chemistry can be converted to the solid
phase in preparation for synthesizing combinatorial libraries of
this scaffold in a 96-well plate format. Thus far, model chemistry
has been carried out on the less active naphthalene regioisomer
represented by 44 because this compound is readily obtained from
commercially available 3,5-dihydroxy-2-naphthoic acid as described
in Marsilje 2000. The successful model reactions to date are shown
in FIG. 10.
[0265] These model reactions demonstrate that it is possible to
couple the naphthalene scaffold to the Wang resin (63) and then
carry out chemistry on the triflate [in this case the Pd
(0)-catalyzed cross-coupling to the boronic ester 64] followed by
cleavage under mild conditions (65). The ester in 63 can also be
saponified for subsequent coupling reactions to form amides
containing the S.sub.3 selectivity elements.
[0266] The naphthalene scaffold currently provides three diversity
sites to be explored in the combinatorial libraries, M.sub.1,
S.sub.2, and S.sub.3. Solid phase combinatorial chemistry with
96-well plate reactors similar to that used in previous studies may
be used (Pavia et al., 1996). The greatest number and diversity of
side chains will be used for S.sub.3 followed by S.sub.2 and then
M.sub.1 for the reasons discussed earlier. One possible overall
synthetic strategy, based upon the synthetic model studies above,
for preparing these libraries is shown in FIG. 11.
[0267] Of course if problems arise with this route there are many
other possibilities. For example, if the Mitsunobu coupling to give
67 proceeds in too low a yield (due to the increased steric
congestion of the added adjacent allyl group-but perhaps not a
problem given the 92% loading obtained in FIG. 10), then the
scaffold could be tethered to a resin through the carboxyl group,
rather than the OH, using the acylsulfonamide "safety catch" linker
(Backes et al., 1996) and form the amides last (the excess amines
can be removed after cleavage by filtering through an acidic
resin). Likewise, other linkers and/or resins can be used if the
reduction of the alkene in the presence of benzylic ethers (67 to
68) is desired but problematic. The first use of the chemistry
proposed in FIG. 11 will be to simply prepare a library of 96
amides, containing the boronic acid M.sub.1 group, without having
the allyl side chain in place so that these two potential
complications will not be a problem initially and the most
promising S.sub.3 elements can be quickly identified.
[0268] At least 14 S.sub.2 hydrophobic side chains (includes
linear, branched and cyclic) are identified for further study (28
if the corresponding alkenes are also explored) based upon the
modeling of candidate side chains into the P-1 site of the Src
model (FIG. 6) and on the commercial availability of the needed
halides to prepare the corresponding Wittig reagents. At least 96
commercially available amines are available which will provide
potential S.sub.3 specificity elements including: 1) hydrocarbons
(4), 2) alkyl groups containing hydrogen bond acceptors (4), 3)
alkyl groups containing both hydrogen bond acceptors and donors
(19), 4) alkyl/aryl groups containing hydrogen bond acceptors and
donors (25), 5) aryl hydrogen bond acceptors and donors (10), 6)
heterocyclic hydrogen bond acceptors and donors (20), 7) side
chains containing cationic groups (4), 8) side chains containing
anionic groups (9), and the 3-amino phenol side chain from
inhibitor 43-meta as an internal control for Src activity. A broad
range of amines were included for S.sub.3, in order not to overly
bias the library here due to the higher level of uncertainty for
this binding site in the Src model.
[0269] The indole scaffold can be developed into a combinatorial
library in much the same fashion. In this case, the indole NH would
be used as the tether point for attachment to the Wang (or other)
resin since the analogous Mitsunobu reaction is known (Bhagwat
& Gude, 1994). A large amount of synthetic methodology has been
developed for the synthesis of substituted indoles and have
designed a route to include the S.sub.2 hydrophobic side chain (see
FIG. 7) (Ezquerra et al., 1996).
[0270] The triflate functionality formed in reaction 2 from
intermediate 69 (FIG. 11) can be converted to an amine (Wolfe et
al., 1997) and then a series of amides or other amine derivatives
following the reaction sequence shown in FIG. 12. In fact, the
triflate is a versatile synthetic handle and could be converted
into other functional groups as well.
[0271] When the amine 72 is available, the known M.sub.1's (e.g.
the sulfamic acid from Src inhibitor 28 Table V and amide-acid 17
Table III) can be evaluated with this more developed scaffold and
evaluate some new amine derivatives as potential M.sub.1's. For
example the hydrated tricarbonyl amide M.sub.1 group shown in
structure 73 (and it's non-hydrated precursor) is accessible via
the synthetic methodology (see Lai et al., 1996) and could form a
variety of interesting interactions with the conserved catalytic
residues.
[0272] Following the modeling procedure described above, a series
of hydroxy-containing analogs of the boronic acid M.sub.1 group
shown in FIG. 13 were modeled in the Src and IRTK active sites and
the illustrated interactions/binding modes were found as some of
the interesting possibilities. By phosphorylating the boronic acid,
additional interesting possibilities are available (e.g. suicide
type inhibition via reaction of the resulting mixed anhydride with
an active site nucleophile). The presence of additional hydroxyl
groups on the Tyr-mimetic phenyl ring is necessary and common among
many PTK inhibitors (e.g. Piceatannol 52, Table V) and was shown to
be beneficial on the side chain with the PKA phosphonate inhibitors
(e.g. 2 vs. 3 and 4, Table I). Consequently, adding one or more
OH's to the boronic acid inhibitor M.sub.1 design as illustrated in
FIG. 13 may considerably enhance potency. These OH groups would
also extend the boronic acid side chain past the catalytic Asp and
Arg residues without suffering a penalty for covering them with
hydrocarbon as was probably the case with the PKA boronic acid
homologs (23 and 24, Table III). One possible route to the
hydroxyboronic acids 76 and 77 utilizes the chiral boronic ester
homologation methodology of Matteson (e.g. see Matteson et al.,
1987, 1988 & 1990).
[0273] Thus, in a preferred embodiment of the invention, the first
module is produced by attaching the first module to a peptide
scaffold. One or more functional groups are identified which
preferentially bind to catalytic residues of the protein kinase,
wherein at least one of the one or more functional groups is a
halogen. Further, the first module is combined with the second
module so that the second module substitutes for the peptide
scaffold.
[0274] Preferred first modules have a two or more functional
groups, including a halogen and one or more additional functional
groups such as a boronic acid group, a hydroxyl group, phosphonic
acid, sulfamic acid, a guanidino group, carboxylic acid, an
aldehyde, an amide, and hydroxymethylphosphonic acid. More
preferred additional functional groups are boronic acid groups, a
hydroxyl group, or an amide group. An even more preferred amide
group is a vicinal tricarbonyl amide.
[0275] Preferred second modules include indole, naphthalene,
biphenyl, isoquinoline, benzofuran, and benzothiophene. More
preferred second modules are an indole or naphthalene. In some
embodiments of the invention more than one first module may be
bound to the second module. In addition, the first module may have
a linear chain comprising between one and three carbon atoms which
links the first module to the second module. In alternative
embodiments, one of the carbon atoms in the linear chain is
substituted with a nitrogen, oxygen or sulfur atom.
[0276] The methods and compounds of the invention are broadly
applicable to any protein kinase. Preferred protein kinases are
protein tyrosine kinases and protein serine kinases (a.k.a.
serine-threonine kinases). Preferred protein tyrosine kinases are
pp60.sup.c-arc, p56.sup.lck, p55.sup.fyn, ZAP kinase, platelet
derived growth factor receptor tyrosine kinase, Bcr-Abl, VEGF
(vascular endothelial growth factor) receptor tyrosine kinase,
epidermal growth factor receptor tyrosine kinase, and epidermal
growth factor receptor-like tyrosine kinases. A more preferred
protein tyrosine kinase is pp60.sup.c-src. Preferred serine protein
kinases include MAP (mitogen activated protein) kinase, protein
kinase C, and CDK (cyclin dependent protein kinase).
[0277] The method of the present invention may further consist of
adding one or more specificity side chain elements to the
combination of the first and second modules, as described above.
Specificity side chains can increase potency and specificity of the
inhibitor. Suitable specificity side chains are described above (R
groups for above structures) and in the Examples, which follow.
[0278] Once a promising second module is identified it is not
necessary to repeat all the steps of the method. Rather, the first
module, specificity side chains, or a combination the two may be
modified to improve the original inhibitor, i.e an inhibitor which
has an increased ability to inhibit protein kinase activity when
compared to the unmodified first inhibitor.
[0279] The present method is designed to preferentially provide
protein kinase inhibitors which do not act by inhibiting ATP
binding to the protein kinase. Inhibitors of protein kinases which
act by inhibiting ATP binding may be potent but often lack
specificity and are therefore often not good drug candidates.
Therefore, protein kinase inhibitors which inhibit protein kinase
activity but do not inhibit or only weakly inhibit ATP binding to
the protein kinase are preferred.
[0280] In another embodiment, the present invention provides a
method of inhibiting a protein kinase. The protein kinase is
contacted with a compound having at least one first module which
has one or more functional groups capable of covalently or
non-covalently binding to catalytic residues of the protein kinase,
wherein the one or more functional groups comprise a halogen, and a
second module which provides a non-peptide scaffold. The
combination of the at least one first module and second module
inhibits the protein kinase activity.
[0281] The present invention further provides a method of treating
a condition, responsive to a protein kinase inhibitor, in a
subject. An effective dose of a protein kinase inhibitor is
administered to a subject. The protein kinase inhibitor has at
least one first module having one or more functional groups each
capable of covalently or non-covalently binding to catalytic
residues of the protein kinase, wherein the one or more functional
groups comprise a halogen, and a second module which provides a
non-peptide scaffold, where the combination of the at least one
first module and second module inhibits protein kinase
activity.
[0282] Another aspect of the present invention is a method for
identifying inhibitors of protein phosphatases. The method involves
providing at least one first module having one or more functional
groups each capable of covalently or non-covalently binding to
catalytic residues of the protein phosphatase, combining at least
one first module with at least one second module which provides a
non-peptide scaffold to form one or more combinations of the first
and second modules, screening the one or more combinations of the
first and second modules for protein phosphatase inhibition, and
selecting combinations of the first and second modules which
inhibit protein phosphatase activity.
[0283] Suitable first and second modules and functional groups are
described above. In a preferred embodiment, the at least one first
module comprises a halogen, most preferably, fluorine. Examples of
suitable non-peptide protein phosphatase inhibitors are shown in
Table VIII, below.
[0284] Suitable protein phosphatases include, but are not limited
to, PTP-1B. Other suitable protein phosphatases are described, for
example, in Zhang, 2002; McCluskey et al., 2002a; Zhang 2001;
McCluskey et al., 2001; Pestell et al., 2000; Moller et al., 2000;
Ripka, 2000; Kennedy, 1999; Johnson et al., 2002; McCluskey
2002b.
[0285] As described above, this method is designed to
preferentially provide phosphatase inhibitors which bind to the
substrate peptide binding site.
[0286] The present invention also relates to a method of inhibiting
a protein phosphatase. The protein phosphatase is contacted by a
compound comprising at least one first module having one or more
functional groups each capable of covalently or non-covalently
binding to catalytic residues of the protein phosphatase, and a
second module which provides a non-peptide scaffold. The
combination of at least one first module and second module inhibits
the protein phosphatase activity.
[0287] In one embodiment, the compound has the following Formula
III:
##STR00141##
wherein R.sub.1 through R.sub.7 may be the same or different, and
are selected from the group consisting of H, C(O)R.sub.a,
C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a,
OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, aryl, heteroaryl,
biaryl, heterobiaryl, heterocyclic compound, and alkyl (branched,
cyclic, or unbranched), preferably having from 1 to 20 carbon
atoms, optionally containing a double or triple bond and optionally
substituted with a heteroatom or other functional groups, such as a
carboxylic acid, carboxylic ester, alcohol, ether, thioether,
amide, thioamide, urea, urethane, sulfoxide, sulfone, phosphonic
acid, phosphonic ester, phosphinic acid, phosphinic ester, boronic
acid, aryl, heteroaryl, biaryl, and heterobiaryl, or R.sub.5 and
R.sub.6 together form a heterocyclic compound. R.sub.a, R.sub.b,
and R.sub.c can be the same or different and are selected from the
group consisting of H, aryl, heteroaryl, biaryl, heterobiaryl, and
alkyl (branched, cyclic, or unbranched), optionally substituted
with a heteroatom or other functional groups such as a carboxylic
acid, carboxylic ester, alcohol, ether, thioether, amide,
thioamide, urea, urethane, sulfoxide, sulfone, phosphonic acid,
phosphonic ester, phosphinic acid, phosphinic ester, boronic acid,
aryl, heteroaryl, biaryl, and heterobiaryl. It is understood that
any of R.sub.1 through R.sub.7 and R.sub.a through R.sub.c may be
substituted or unsubstituted. In a preferred embodiment, R.sub.3 is
a halogen, most preferably, fluorine.
[0288] In another embodiment, at least one of R.sub.6 or R.sub.7
is
##STR00142##
wherein R.sub.8* is the point of attachment and is
(CH.sub.2).sub.x, wherein X is from 0 to 10, CH.sub.2CHOH,
CH(CH.sub.3)(R-isomer), or CH(CH.sub.3)(S-isomer), and each of
R.sub.9 through R.sub.13 may be the same or different and are
selected from the group consisting of H, C(O)R.sub.a,
C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a,
OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.2P(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, aryl, heteroaryl,
biaryl, heterobiaryl, heterocyclic compound, and alkyl (branched,
cyclic, or unbranched), preferably having from 1 to 20 carbon
atoms, optionally containing a double or triple bond and optionally
substituted with a heteroatom or other functional groups, such as a
carboxylic acid, carboxylic ester, alcohol, ether, thioether,
amide, thioamide, urea, urethane, sulfoxide, sulfone, phosphonic
acid, phosphonic ester, phosphinic acid, phosphinic ester, boronic
acid, aryl, heteroaryl, biaryl, and heterobiaryl. R.sub.a, R.sub.b,
and R.sub.c can be the same or different and are selected from the
group consisting of H, aryl, heteroaryl, biaryl, heterobiaryl, and
alkyl (branched, cyclic, or unbranched), optionally substituted
with a heteroatom or other functional groups such as a carboxylic
acid, carboxylic ester, alcohol, ether, thioether, amide,
thioamide, urea, urethane, sulfoxide, sulfone, phosphonic acid,
phosphonic ester, phosphinic acid, phosphinic ester, boronic acid,
aryl, heteroaryl, biaryl, and heterobiaryl. It is understood that
any of R.sub.9 through R.sub.13 and R.sub.a through R.sub.c may be
substituted or unsubstituted. In a preferred embodiment, each of
R.sub.9 through R.sub.13 may be selected from the group consisting
of OCH.sub.3, OCH.sub.2CH.sub.3, H, CH.sub.3, OH, CH.sub.2OH,
CF.sub.3, OCF.sub.3, CFO, C.sub.6H.sub.5, OC.sub.6H.sub.5,
OCH.sub.2C.sub.6H.sub.5, OCH.sub.2CH.sub.2CH.sub.3, CHO, CO.sub.2H,
CO.sub.2CH.sub.3, CH.sub.2CO.sub.2H, CH.sub.2CO.sub.2CH.sub.3,
NO.sub.2, and halogen.
[0289] In a further embodiment, at least one of R.sub.6 or R.sub.7
is
##STR00143##
wherein the asterisk indicates the point of attachment to the
nitrogen.
[0290] In yet a further embodiment, the compound has the Formula
IV:
##STR00144##
wherein R.sub.1 through R.sub.7 are each the same or different and
are selected from the group consisting of H, C(O)R.sub.a,
C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a,
OC(O)R.sub.a, OC(O)R.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, aryl, heteroaryl,
biaryl, heterobiaryl, heterocyclic compound, and alkyl (branched,
cyclic, or unbranched), preferably having from 1 to 20 carbon
atoms, optionally containing a double or triple bond and optionally
substituted with a heteroatom or other functional groups, such as a
carboxylic acid, carboxylic ester, alcohol, ether, thioether,
amide, thioamide, urea, urethane, sulfoxide, sulfone, phosphonic
acid, phosphonic ester, phosphinic acid, phosphinic ester, boronic
acid, aryl, heteroaryl, biaryl, and heterobiaryl. R.sub.a, R.sub.b,
and R.sub.c can be the same or different and are selected from the
group consisting of H, aryl, heteroaryl, biaryl, heterobiaryl, and
alkyl (branched, cyclic, or unbranched), optionally substituted
with a heteroatom or other functional groups such as a carboxylic
acid, carboxylic ester, alcohol, ether, thioether, amide,
thioamide, urea, urethane, sulfoxide, sulfone, phosphonic acid,
phosphonic ester, phosphinic acid, phosphinic ester, boronic acid,
aryl, heteroaryl, biaryl, and heterobiaryl. It is understood that
all open substitution positions in the above side chains can
contain further substitutions.
[0291] Another aspect of the present invention relates to a method
of treating a condition, responsive to a protein phosphatase
inhibitor, in a subject. A protein phosphatase inhibitor is
administered to a subject. The protein phosphatase inhibitor has at
least one first module having one or more functional groups each
capable of covalently or non-covalently binding to catalytic
residues of the protein phosphatase, and a second module which
provides a non-peptide scaffold. The combination of at least one
first module and second module inhibits protein phosphatase
activity in the subject.
[0292] Protein phosphatase inhibitors may be used in various
therapeutic techniques, including, but not limited to, treatment of
Type II diabetes, obesity, and cancer (Zhang, 2002; McCluskey et
al., 2002a; Zhang 2001; McCluskey et al., 2001; Pestell et al.,
2000; Moller et al., 2000; Ripka, 2000; Kennedy, 1999; Johnson et
al., 2002; McCluskey 2002b).
[0293] Examples of other suitable compounds for the above-described
methods include:
##STR00145##
wherein any of the individual R's can be a halogen-containing
M.sub.1, and the remaining R groups can be H, C(O)R.sub.a,
C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a,
OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, aryl, heteroaryl,
biaryl, and alkyl group (branched, cyclic or unbranched) optionally
containing a double or triple bond and/or a heteroatom or other
functional groups, such as a carboxylic acid, carboxylic ester,
alcohol, ether, thioether, amide, thioamide, urea, urethane,
sulfoxide, sulfone, phosphonic acid, phosphonic ester, phosphinic
acid, phosphinic ester, boronic acid, aryl, heteroaryl, biaryl, and
heterobiaryl. R.sub.a, R.sub.b, and R.sub.c can be the same or
different and are selected from the group consisting of H, aryl,
heteroaryl, biaryl, heterobiaryl, and alkyl (branched, cyclic or
unbranched) optionally substituted with a heteroatom or other
functional groups such as a carboxylic acid, carboxylic ester,
alcohol, ether, thioether, amide, thioamide, urea, urethane,
sulfoxide, sulfone, phosphonic acid, phosphonic ester, phosphinic
acid, phosphinic ester, boronic acid, aryl, heteroaryl, biaryl, and
heterobiaryl. It is understood that all open substitution positions
in the above side chains can contain further substitutions;
##STR00146##
wherein any of the individual R's can be M.sub.1, and the remaining
R groups can be H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a,
C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2,
NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, aryl, heteroaryl,
biaryl, and alkyl group (branched, cyclic or unbranched) optionally
containing a double or triple bond and/or a heteroatom or other
functional groups, such as a carboxylic acid, carboxylic ester,
alcohol, ether, thioether, amide, thioamide, urea, urethane,
sulfoxide, sulfone, phosphonic acid, phosphonic ester, phosphinic
acid, phosphinic ester, boronic acid, aryl, heteroaryl, biaryl, and
heterobiaryl. R.sub.a, R.sub.b, and R.sub.c can be the same or
different and are selected from the group consisting of H, aryl,
heteroaryl, biaryl, heterobiaryl, and alkyl (branched, cyclic or
unbranched) optionally substituted with a heteroatom or other
functional groups such as a carboxylic acid, carboxylic ester,
alcohol, ether, thioether, amide, thioamide, urea, urethane,
sulfoxide, sulfone, phosphonic acid, phosphonic ester, phosphinic
acid, phosphinic ester, boronic acid, aryl, heteroaryl, biaryl, and
heterobiaryl. It is understood that all open substitution positions
in the above side chains can contain further substitutions;
##STR00147##
wherein any of the individual R's can be M.sub.1, and the remaining
R groups can be H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a,
C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2,
NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, aryl, heteroaryl,
biaryl, and alkyl group (branched, cyclic or unbranched) optionally
containing a double or triple bond and/or a heteroatom or other
functional groups, such as a carboxylic acid, carboxylic ester,
alcohol, ether, thioether, amide, thioamide, urea, urethane,
sulfoxide, sulfone, phosphonic acid, phosphonic ester, phosphinic
acid, phosphinic ester, boronic acid, aryl, heteroaryl, biaryl, and
heterobiaryl. R.sub.a, R.sub.b, and R.sub.c can be the same or
different and are selected from the group consisting of H, aryl,
heteroaryl, biaryl, heterobiaryl, and alkyl (branched, cyclic or
unbranched) optionally substituted with a heteroatom or other
functional groups such as a carboxylic acid, carboxylic ester,
alcohol, ether, thioether, amide, thioamide, urea, urethane,
sulfoxide, sulfone, phosphonic acid, phosphonic ester, phosphinic
acid, phosphinic ester, boronic acid, aryl, heteroaryl, biaryl, and
heterobiaryl. It is understood that all open substitution positions
in the above side chains can contain further substitutions;
##STR00148##
wherein any of the individual R's can be M.sub.1, and the remaining
R groups can be H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a,
C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2,
NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aR.sub.b, B(OH).sub.2, halogen, aryl, heteroaryl,
biaryl, and alkyl group (branched, cyclic or unbranched) optionally
containing a double or triple bond and/or a heteroatom or other
functional groups, such as a carboxylic acid, carboxylic ester,
alcohol, ether, thioether, amide, thioamide, urea, urethane,
sulfoxide, sulfone, phosphonic acid, phosphonic ester, phosphinic
acid, phosphinic ester, boronic acid, aryl, heteroaryl, biaryl, and
heterobiaryl. R.sub.a, R.sub.b, and R.sub.c can be the same or
different and are selected from the group consisting of H, aryl,
heteroaryl, biaryl, heterobiaryl, and alkyl (branched, cyclic or
unbranched) optionally substituted with a heteroatom or other
functional groups such as a carboxylic acid, carboxylic ester,
alcohol, ether, thioether, amide, thioamide, urea, urethane,
sulfoxide, sulfone, phosphonic acid, phosphonic ester, phosphinic
acid, phosphinic ester, boronic acid, aryl, heteroaryl, biaryl, and
heterobiaryl. It is understood that all open substitution positions
in the above side chains can contain further substitutions;
##STR00149##
wherein any of the individual R's can be M.sub.1, and the remaining
R groups can be H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a,
C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2,
NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, aryl, heteroaryl,
biaryl, and alkyl group (branched, cyclic or unbranched) optionally
containing a double or triple bond and/or a heteroatom or other
functional groups, such as a carboxylic acid, carboxylic ester,
alcohol, ether, thioether, amide, thioamide, urea, urethane,
sulfoxide, sulfone, phosphonic acid, phosphonic ester, phosphinic
acid, phosphinic ester, boronic acid, aryl, heteroaryl, biaryl, and
heterobiaryl. R.sub.a, R.sub.b, and R.sub.c can be the same or
different and are selected from the group consisting of H, aryl,
heteroaryl, biaryl, heterobiaryl, and alkyl (branched, cyclic or
unbranched) optionally substituted with a heteroatom or other
functional groups such as a carboxylic acid, carboxylic ester,
alcohol, ether, thioether, amide, thioamide, urea, urethane,
sulfoxide, sulfone, phosphonic acid, phosphonic ester, phosphinic
acid, phosphinic ester, boronic acid, aryl, heteroaryl, biaryl, and
heterobiaryl. It is understood that all open substitution positions
in the above side chains can contain further substitutions;
##STR00150##
wherein any of the individual R's can be M.sub.1, and the remaining
R groups can be H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a,
C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2,
NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, aryl, heteroaryl,
biaryl, and alkyl group (branched, cyclic or unbranched) optionally
containing a double or triple bond and/or a heteroatom or other
functional groups, such as a carboxylic acid, carboxylic ester,
alcohol, ether, thioether, amide, thioamide, urea, urethane,
sulfoxide, sulfone, phosphonic acid, phosphonic ester, phosphinic
acid, phosphinic ester, boronic acid, aryl, heteroaryl, biaryl, and
heterobiaryl. R.sub.a, R.sub.b, and R.sub.c can be the same or
different and are selected from the group consisting of H, aryl,
heteroaryl, biaryl, heterobiaryl, and alkyl (branched, cyclic or
unbranched) optionally substituted with a heteroatom or other
functional groups such as a carboxylic acid, carboxylic ester,
alcohol, ether, thioether, amide, thioamide, urea, urethane,
sulfoxide, sulfone, phosphonic acid, phosphonic ester, phosphinic
acid, phosphinic ester, boronic acid, aryl, heteroaryl, biaryl, and
heterobiaryl. It is understood that all open substitution positions
in the above side chains can contain further substitutions;
##STR00151##
wherein any of the individual R's can be M.sub.1, and the remaining
R groups can be H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a,
C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2,
NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, aryl, heteroaryl,
biaryl, and alkyl group (branched, cyclic or unbranched) optionally
containing a double or triple bond and/or a heteroatom or other
functional groups, such as a carboxylic acid, carboxylic ester,
alcohol, ether, thioether, amide, thioamide, urea, urethane,
sulfoxide, sulfone, phosphonic acid, phosphonic ester, phosphinic
acid, phosphinic ester, boronic acid, aryl, heteroaryl, biaryl, and
heterobiaryl. R.sub.a, R.sub.b, and R.sub.c can be the same or
different and are selected from the group consisting of H, aryl,
heteroaryl, biaryl, heterobiaryl, and alkyl (branched, cyclic or
unbranched) optionally substituted with a heteroatom or other
functional groups such as a carboxylic acid, carboxylic ester,
alcohol, ether, thioether, amide, thioamide, urea, urethane,
sulfoxide, sulfone, phosphonic acid, phosphonic ester, phosphinic
acid, phosphinic ester, boronic acid, aryl, heteroaryl, biaryl, and
heterobiaryl. It is understood that all open substitution positions
in the above side chains can contain further substitutions;
##STR00152##
wherein any of the individual R's can be M.sub.1, and the remaining
R groups can be H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a,
C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.4, NH.sub.2,
NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.2P(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, aryl, heteroaryl,
biaryl, and alkyl group (branched, cyclic or unbranched) optionally
containing a double or triple bond and/or a heteroatom or other
functional groups, such as a carboxylic acid, carboxylic ester,
alcohol, ether, thioether, amide, thioamide, urea, urethane,
sulfoxide, sulfone, phosphonic acid, phosphonic ester, phosphinic
acid, phosphinic ester, boronic acid, aryl, heteroaryl, biaryl, and
heterobiaryl. R.sub.a, R.sub.b, and R.sub.c can be the same or
different and are selected from the group consisting of H, aryl,
heteroaryl, biaryl, heterobiaryl, and alkyl (branched, cyclic or
unbranched) optionally substituted with a heteroatom or other
functional groups such as a carboxylic acid, carboxylic ester,
alcohol, ether, thioether, amide, thioamide, urea, urethane,
sulfoxide, sulfone, phosphonic acid, phosphonic ester, phosphinic
acid, phosphinic ester, boronic acid, aryl, heteroaryl, biaryl, and
heterobiaryl. It is understood that all open substitution positions
in the above side chains can contain further substitutions;
##STR00153##
wherein any of the individual R's can be M.sub.1, and the remaining
R groups can be H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a,
C(O)SR.sub.4, OH, OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2,
NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, aryl, heteroaryl,
biaryl, and alkyl group (branched, cyclic or unbranched) optionally
containing a double or triple bond and/or a heteroatom or other
functional groups, such as a carboxylic acid, carboxylic ester,
alcohol, ether, thioether, amide, thioamide, urea, urethane,
sulfoxide, sulfone, phosphonic acid, phosphonic ester, phosphinic
acid, phosphinic ester, boronic acid, aryl, heteroaryl, biaryl, and
heterobiaryl. R.sub.a, R.sub.b, and R.sub.c can be the same or
different and are selected from the group consisting of H, aryl,
heteroaryl, biaryl, heterobiaryl, and alkyl (branched, cyclic or
unbranched) optionally substituted with a heteroatom or other
functional groups such as a carboxylic acid, carboxylic ester,
alcohol, ether, thioether, amide, thioamide, urea, urethane,
sulfoxide, sulfone, phosphonic acid, phosphonic ester, phosphinic
acid, phosphinic ester, boronic acid, aryl, heteroaryl, biaryl, and
heterobiaryl. It is understood that all open substitution positions
in the above side chains can contain further substitutions.
[0294] Another aspect of the invention is a compound according to
Formula V:
##STR00154##
or a salt, solvate, hydrate, or prodrug thereof. R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 are the same or different and are
independently H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a,
C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2,
NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, benzyl,
heteroaryl, biaryl, heterobiaryl, heterocycle, and branched,
unbranched, or cyclic alkyl. R.sub.6 and R.sub.7 are the same or
different and are independently H, branched or unbranched or
(CH.sub.2).sub.t--Z, wherein Z is aryl, heteroaryl, biaryl, cyclic
alkyl, or heterocycle, or R.sub.6 and R.sub.7 together form a
heterocycle. t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. R.sub.a,
R.sub.b, and R.sub.c are the same or different and are
independently H, aryl, heteroaryl, biaryl, heterobiaryl, or
branched, unbranched, or cyclic alkyl. P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q, further wherein lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is linear or branched
alkyl. K is C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00155##
L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00156##
M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00157##
Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00158##
R.sub.19, R.sub.20 and R.sub.21 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.19 and
R.sub.20 taken together with the attached nitrogen atom form a five
membered ring. Any of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5,
R.sub.6, and R.sub.7 and R.sub.a, R.sub.b, and R.sub.c is
substituted or unsubstituted. At least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, and R.sub.6 is P.
[0295] In one embodiment, P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q, further where K is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, glycoside, and heterocycle; further where L is
SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, glycoside, and heterocycle; and
further where M is SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19, NR.sub.19R.sub.20,
glycoside, and heterocycle.
[0296] In one embodiment, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is selected from halogen, boronic
acid, hydroxyl, phosphonic acid, sulfamic acid, guanidine,
carboxylic acid, aldehyde, amide, and hydroxymethylphosphonic acid.
In another embodiment, at least one of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, and R.sub.5 is halogen. In another embodiment, at least
one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is boronic
acid. In another embodiment, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is hydroxyl. In another embodiment,
at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is
amide. For example, amide is vicinal tricarbonyl amide. In one
embodiment, R.sub.3 is halogen. For example, R.sub.3 is fluorine.
In another embodiment, R.sub.3 is hydroxyl.
[0297] In one embodiment, at least one of R.sub.6 and R.sub.7 is
(CH.sub.2).sub.t--Z. In another embodiment, Z is aryl. In one
embodiment, aryl is unsubstituted. In one embodiment aryl is
monosubstituted. In another embodiment, aryl is disubsituted. In
another embodiment, aryl is trisubstituted. In one embodiment aryl
is substituted with hydroxyl, halogen, phenoxy(--OC.sub.6H.sub.5),
alkoxy, CF.sub.3, alkyl, hydroxymethyl, aryl, OCF.sub.3,
benzyloxy(--OCH.sub.2C.sub.6H.sub.5), nitro, aldehyde, or
alkoxycarboxy (also referred to as ester e.g., --C(O)OEt). In one
embodiment, (CH.sub.2) is substituted, wherein one or more of the
hydrogen atoms of the methylene group are replaced with one or more
substituents. Methylene substitutents are selected from
hydroxmethyl, alkyl (e.g., methyl), hydroxyl, and aryl. In another
embodiment, at least one of R.sub.6 and R.sub.7 is branched or
unbranched alkyl. In one embodiment, branched or unbranched alkyl
is unsubstituted. In another embodiment, branched or unbranched
alkyl is substituted with hydroxyl. In another embodiment, Z is
cyclic alkyl. In one embodiment, cyclic alkyl is substituted. In
another embodiment, cyclic alkyl is substituted with hydroxyl. In
another embodiment, cyclic alkyl is unsubstituted. In one
embodiment, t is 0. In another embodiment, t is 1. In another
embodiment, t is 2.
[0298] In one embodiment, R.sub.6 and R.sub.7 form a ring, where
the ring is selected from pyrrolidine, piperidine, and
morpholine.
[0299] In one embodiment, at least one of R.sub.6 and R.sub.7
is
##STR00159##
where *R.sub.8 is the point of attachment. In one embodiment
R.sub.8 is (CH.sub.2).sub.x, where X is 0, 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10. In another embodiment R.sub.8 is CH.sub.2CHOH,
CH(CH.sub.3)(R-isomer), or CH(CH.sub.3)(S-isomer). In another
embodiment, each of R.sub.9, R.sub.10, R.sub.11, R.sub.12, and
R.sub.13 are the same or different and each of R.sub.9, R.sub.10,
R.sub.11, R.sub.12, and R.sub.13 independently are H, C(O)R.sub.a,
C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a,
OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, P', aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle, or branched, cyclic, or
unbranched alkyl, P' is SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2,
OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-K', O--C(O)-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L', NH-lower (C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-M', or
O-aryl-Q', further where lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl is linear or branched alkyl. K' is
C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22, NR.sub.22R.sub.23,
SO.sub.2R.sub.24, glycoside, lower C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00160##
L' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.2,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00161##
M' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00162##
Q' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00163##
R.sub.22, R.sub.23 and R.sub.24 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.22 and
R.sub.23 taken together with the attached nitrogen atom form a five
membered ring. R.sub.a, R.sub.b, and R.sub.c are the same or
different and independently are H, aryl, heteroaryl, biaryl,
heterobiaryl, branched, cyclic, or unbranched alkyl; wherein any of
R.sub.9, R.sub.10, R.sub.11, R.sub.12, and R.sub.13 are substituted
or unsubstituted; and provided that if one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, or R.sub.5 is not P, then at least one of
R.sub.9, R.sub.10, R.sub.11, R.sub.12, and R.sub.13 is P'.
[0300] In another embodiment, P' is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K', O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L',
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M', or O-aryl-q. K' is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, glycoside, or heterocycle. L' is SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.22, NR.sub.22R.sub.23, glycoside, or heterocycle. M' is
SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.22, NR.sub.22R.sub.23, glycoside, or heterocycle.
In another embodiment, at least one of R.sub.6 and R.sub.7 is
##STR00164##
[0301] Another aspect of the invention includes a compound of
Formula VI:
##STR00165##
or a salt, solvate, hydrate, or prodrug. R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are each the same or
different and independently are H, C(O)R.sub.a,
C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a,
OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle, and branched, cyclic, or
unbranched alkyl. R.sub.a, R.sub.b, and R.sub.c are the same or
different and are independently H, aryl, heteroaryl, biaryl,
heterobiaryl, and branched, cyclic, or unbranched alkyl. P is
SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K,
O--C(O)-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or
C.sub.6) alkyl-L, NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-M, or O-aryl-Q, further where lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is
linear or branched alkyl. K is C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00166##
L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00167##
M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00168##
Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00169##
R.sub.19, R.sub.20 and R.sub.21 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.19 and
R.sub.20 taken together with the attached nitrogen atom form a five
membered ring. X is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. At least one
of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is P.
[0302] In one embodiment, P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q. K is SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19, NR.sub.19R.sub.20,
glycoside, or heterocycle. L is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, glycoside, or heterocycle. M is SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, glycoside, or heterocycle.
[0303] Another aspect of the invention includes a method of
protecting against or treating hearing loss in a subject comprising
administering a compound having the Formula V:
##STR00170##
or a salt, solvate, hydrate, or prodrug thereof. R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 are the same or different and are
independently H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a,
C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2,
NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, benzyl,
heteroaryl, biaryl, heterobiaryl, heterocycle, and branched,
unbranched, or cyclic alkyl. R.sub.6 and R.sub.7 are the same or
different and are independently H, branched or unbranched, or
(CH.sub.2).sub.t--Z, wherein Z is aryl, heteroaryl, biaryl, cyclic
alkyl, or heterocycle, or R.sub.6 and R.sub.7 together form a
heterocycle. t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. R.sub.a,
R.sub.b, and R.sub.c are the same or different and are
independently H, aryl, heteroaryl, biaryl, heterobiaryl, or
branched, unbranched, or cyclic alkyl. P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q, further wherein lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is linear or branched
alkyl. K is C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00171##
L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00172##
M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00173##
Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00174##
R.sub.19, R.sub.20 and R.sub.21 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.19 and
R.sub.20 taken together with the attached nitrogen atom form a five
membered ring. Any of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5,
R.sub.6, and R.sub.7 and R.sub.a, R.sub.b, and R.sub.c is
substituted or unsubstituted. At least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 is P.
[0304] In one embodiment, P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q. K is SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19, NR.sub.19R.sub.20,
glycoside, or heterocycle. L is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, glycoside, or heterocycle. M is SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, glycoside, or heterocycle.
[0305] In one embodiment, the compound inhibits one or more
components of a protein kinase signaling pathway. In another
embodiment, the compound is an allosteric inhibitor. In another
embodiment, the compound does not inhibit ATP binding to a protein
kinase. In one embodiment, the compound binds to a peptide binding
site. In one embodiment, the compound inhibits a Src family protein
kinase. For example, the Src family protein kinase is
pp60.sup.c-src tyrosine kinase. In another embodiment, the Src
family protein kinase is PYK2.
[0306] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically (e.g., by
administering drops into the ear), intraarterially,
intralesionally, by metering pump, or by application to mucous
membranes. In one embodiment, the compound is administered in
combination with a drug that causes hearing loss e.g., cis platinum
or an aminoglycoside antibiotic. In another embodiment, the
compound is administered in combination with a drug that targets
hairy cells. In one embodiment, the compound is administered with a
pharmaceutically acceptable carrier. In one embodiment, the
compound is administered before initiation of hearing loss. In
another embodiment, the compound is administered after inititiation
of hearing loss. In one embodiment, at least one of R.sub.1,
R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is selected from halogen,
boronic acid, hydroxyl, phosphonic acid, sulfamic acid, guanidine,
carboxylic acid, aldehyde, amide, and hydroxymethylphosphonic acid.
In another embodiment, at least one of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, and R.sub.5 is a halogen. In another embodiment, at least
one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is boronic
acid. In another embodiment, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is hydroxyl. In another embodiment,
at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is
amide. For example, amide is vicinal tricarbonyl amide. In one
embodiment, R.sub.3 is halogen. For example, R.sub.3 is fluorine.
In one embodiment, R.sub.3 is hydroxyl.
[0307] In one embodiment, at least one of R.sub.6 and R.sub.7 is
(CH.sub.2).sub.t--Z. In another embodiment, Z is aryl. In one
embodiment, aryl is unsubstituted. In one embodiment aryl is
monosubstituted. In another embodiment, aryl is disubsituted. In
another embodiment, aryl is trisubstituted. In one embodiment aryl
is substituted with hydroxyl, halogen, phenoxy (--OC.sub.6H.sub.5),
alkoxy, CF.sub.3, alkyl, hydroxymethyl, aryl, OCF.sub.3, benzyloxy
(--OCH.sub.2C.sub.6H.sub.5), nitro, aldehyde, or alkoxycarboxy
(also referred to as ester e.g., --C(O)OEt. In one embodiment,
(CH.sub.2) is substituted, wherein one or more of the hydrogen
atoms of the methylene group are replaced with one or more
substituents. Methylene substitutents are selected from
hydroxmethyl, alkyl (e.g., methyl), hydroxyl, and aryl. In another
embodiment, at least one of R.sub.6 and R.sub.7 is branched or
unbranched alkyl. In one embodiment, branched or unbranched alkyl
is unsubstituted. In another embodiment, branched or unbranched
alkyl is substituted with hydroxyl. In another embodiment, Z is
cyclic alkyl. In one embodiment, cyclic alkyl is substituted. In
another embodiment, cyclic alkyl is substituted with hydroxyl. In
another embodiment, cyclic alkyl is unsubstituted. In one
embodiment, t is 0. In another embodiment, t is 1. In another
embodiment, t is 2.
[0308] In one embodiment, R.sub.6 and R.sub.7 form a ring, where
the ring is selected from pyrrolidine, piperidine, and
morpholine.
[0309] In one embodiment, at least one of R.sub.6 and R.sub.7
is
##STR00175##
*R.sub.8 is the point of attachment and is (CH.sub.2).sub.x,
wherein X is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, CH.sub.2CHOH,
CH(CH.sub.3)(R-isomer), or CH(CH.sub.3)(S-isomer). Each of R.sub.9,
R.sub.10, R.sub.11, R.sub.12, and R.sub.13 is the same or different
and independently are H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b,
C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a,
OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b,
NR.sub.aC(O)NR.sub.bR.sub.c, NR.sub.aC(O)OR.sub.b,
NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bR.sub.c, SR.sub.a,
S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a, S(O).sub.2OR.sub.a,
S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, P', aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle, or branched, cyclic, or
unbranched alkyl. P' is SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2,
OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-K', O--C(O)-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L', NH-lower (C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-M', or
O-aryl-Q', further where lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl is linear or branched alkyl. K' is
C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22, NR.sub.22R.sub.23,
SO.sub.2R.sub.24, glycoside, lower C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00176##
L' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00177##
M' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00178##
Q' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00179##
R.sub.22, R.sub.23 and R.sub.24 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.22 and
R.sub.23 taken together with the attached nitrogen atom form a five
membered ring. R.sub.a, R.sub.b, and R.sub.c are the same or
different and independently are H, aryl, heteroaryl, biaryl,
heterobiaryl, branched, cyclic, or unbranched alkyl. Any of
R.sub.9, R.sub.10, R.sub.11, R.sub.12, and R.sub.13 are substituted
or unsubstituted. If one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, or
R.sub.5 is not P, then at least one of R.sub.9, R.sub.10, R.sub.11,
R.sub.12, and R.sub.13 is P'.
[0310] In one embodiment, P' is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K', O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L',
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M', or O-aryl-Q'. K' is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, glycoside, or heterocycle. L'is SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.22, NR.sub.22R.sub.23, glycoside, or heterocycle. M' is
SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.22, NR.sub.22R.sub.23, glycoside, or heterocycle. In
another embodiment, at least one of R.sub.6 or R.sub.7 and
##STR00180##
[0311] Another aspect of the invention includes a method of
preventing or treating a proliferative disease in a subject
comprising administering a compound having the Formula V:
##STR00181##
or a salt, solvate, hydrate, or prodrug thereof. R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 are the same or different and are
independently H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a,
C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2,
NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, benzyl,
heteroaryl, biaryl, heterobiaryl, heterocycle, and branched,
unbranched, or cyclic alkyl. R.sub.6 and R.sub.7 are the same or
different and are independently H, branched or unbranched, or
(CH.sub.2).sub.t--Z, wherein Z is aryl, heteroaryl, biaryl, cyclic
alkyl, or heterocycle, or R.sub.6 and R.sub.7 together form a
heterocycle. t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. R.sub.a,
R.sub.b, and R.sub.c are the same or different and are
independently H, aryl, heteroaryl, biaryl, heterobiaryl, or
branched, unbranched, or cyclic alkyl. P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q, further wherein lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is linear or branched
alkyl. K is C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00182##
L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00183##
M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00184##
Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00185##
R.sub.19, R.sub.20 and R.sub.21 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.19 and
R.sub.20 taken together with the attached nitrogen atom form a five
membered ring; wherein any of R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 and R.sub.a, R.sub.b, and R.sub.c is
substituted or unsubstituted. At least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, and R.sub.6 is P.
[0312] In one embodiment, P is is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q. K is SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19, NR.sub.19R.sub.20,
glycoside, and heterocycle. L is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, glycoside, and heterocycle. M is SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, glycoside, and heterocycle.
[0313] In one embodiment, the compound inhibits one or more
components of a protein kinase signaling pathway. In another
embodiment, the compound is an allosteric inhibitor. In another
embodiment, the compound is a peptide substrate inhibitor. In
another embodiment, the compound does not inhibit ATP binding to a
protein kinase. In another embodiment, the compound binds to a
peptide binding site. In another embodiment, the compound inhibits
a Src family protein kinase. For example, the Src family protein
kinase is pp60.sup.c-arc tyrosine kinase. In another embodiment,
the Src family protein kinase is PYK2.
[0314] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membrane. In another embodiment, the compound
is administered with a pharmaceutically acceptable carrier. In one
embodiment, the compound is administered before initiation of the
proliferative disease. In another embodiment, the compound is
administered after inititiation of the proliferative disease.
[0315] In one embodiment, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is selected from halogen, boronic
acid, hydroxyl, phosphonic acid, sulfamic acid, guanidine,
carboxylic acid, aldehyde, amide, and hydroxymethylphosphonic acid.
In another embodiment, at least one of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, and R.sub.5 is halogen. In another embodiment, at least
one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is boronic
acid. In another embodiment, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is hydroxyl. In another embodiment,
at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is
amide. For example, the amide is vicinal tricarbonyl amide. In one
embodiment, R.sub.3 is halogen. For example, R.sub.3 is fluorine.
In one embodiment, R.sub.3 is hydroxyl.
[0316] In one embodiment, at least one of R.sub.6 and R.sub.7 is
(CH.sub.2).sub.t--Z. In another embodiment, Z is aryl. In one
embodiment, aryl is unsubstituted. In one embodiment aryl is
monosubstituted. In another embodiment, aryl is disubsituted. In
another embodiment, aryl is trisubstituted. In one embodiment aryl
is substituted with hydroxyl, halogen, phenoxy(--OC.sub.6H.sub.5),
alkoxy, CF.sub.3, alkyl, hydroxymethyl, aryl, OCF.sub.3,
benzyloxy(--OCH.sub.2C.sub.6H.sub.5), nitro, aldehyde, or
alkoxycarboxy (also referred to as ester e.g., --C(O)OEt). In one
embodiment, (CH.sub.2) is substituted, wherein one or more of the
hydrogen atoms of the methylene group are replaced with one or more
substituents. Methylene substitutents are selected from
hydroxmethyl, alkyl (e.g., methyl), hydroxyl, and aryl. In another
embodiment, at least one of R.sub.6 and R.sub.7 is branched or
unbranched alkyl. In one embodiment, branched or unbranched alkyl
is unsubstituted. In another embodiment, branched or unbranched
alkyl is substituted with hydroxyl. In another embodiment, Z is
cyclic alkyl. In one embodiment, cyclic alkyl is substituted. In
another embodiment, cyclic alkyl is substituted with hydroxyl. In
another embodiment, cyclic alkyl is unsubstituted. In one
embodiment, t is 0. In another embodiment, t is 1. In another
embodiment, t is 2.
[0317] In one embodiment, R.sub.6 and R.sub.7 form a ring, where
the ring is selected from pyrrolidine, piperidine, and
morpholine.
In one embodiment, at least one of R.sub.6 and R.sub.7 is
##STR00186##
*R.sub.8 is the point of attachment and is (CH.sub.2).sub.x,
wherein X is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, CH.sub.2CHOH,
CH(CH.sub.3)(R-isomer), or CH(CH.sub.3)(S-isomer). Each of R.sub.9,
R.sub.10, R.sub.11, R.sub.12, and R.sub.13 is the same or different
and independently are H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b,
C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.3,
OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b,
NR.sub.aC(O)NR.sub.bR.sub.c, NR.sub.aC(O)OR.sub.b,
NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.2, S(O).sub.2R.sub.a, S(O)OR.sub.3,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, P', aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle, or branched, cyclic, or
unbranched alkyl. P' is SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2,
OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-K', O--C(O)-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L', NH-lower (C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-M', or
O-aryl-Q', further wherein lower (C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, or C.sub.6) alkyl is linear or branched alkyl. K'
is C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22, NR.sub.22R.sub.23,
SO.sub.2R.sub.24, glycoside, lower C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00187##
L' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00188##
M' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00189##
Q' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00190##
R.sub.22, R.sub.23 and R.sub.24 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.22 and
R.sub.23 taken together with the attached nitrogen atom form a five
membered ring. R.sub.a, R.sub.b, and R.sub.c are the same or
different and independently are H, aryl, heteroaryl, biaryl,
heterobiaryl, branched, cyclic, or unbranched alkyl. R.sub.9,
R.sub.10, R.sub.11, R.sub.12, and R.sub.13 are substituted or
unsubstituted. If one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, or
R.sub.5 is not P, then at least one of R.sub.9, R.sub.10, R.sub.11,
R.sub.12, and R.sub.13 is P'.
[0318] In one embodiment, P' is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K', O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L',
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M', or O-aryl-Q'. K' is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, glycoside, or heterocycle. L' is SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.22, NR.sub.22R.sub.23, glycoside, or heterocycle. M' is
SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.22, NR.sub.22R.sub.23, glycoside, or heterocycle.
[0319] In one embodiment, at least one of R.sub.6 and R.sub.7
is
##STR00191##
[0320] Another aspect of the invention includes a method of
protecting against or treating osteoporosis in a subject comprising
administering a compound of Formula VII:
##STR00192##
or a salt, solvate, hydrate, or prodrug thereof. R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 are the same or different and are
independently H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b, C(O)OR.sub.a,
C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2,
NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.a,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, benzyl,
heteroaryl, biaryl, heterobiaryl, heterocycle, and branched,
unbranched, or cyclic alkyl. R.sub.6 and R.sub.7 are the same or
different and are independently H, branched or unbranched, or
(CH.sub.2).sub.t--Z, wherein Z is aryl, heteroaryl, biaryl, cyclic
alkyl, or heterocycle, or R.sub.6 and R.sub.7 together form a
heterocycle. t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. R.sub.a,
R.sub.b, and R.sub.c are the same or different and are
independently H, aryl, heteroaryl, biaryl, heterobiaryl, or
branched, unbranched, or cyclic alkyl. P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q, further wherein lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is linear or branched
alkyl. K is C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00193##
L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00194##
M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00195##
Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00196##
R.sub.19, R.sub.20 and R.sub.21 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.19 and
R.sub.20 taken together with the attached nitrogen atom form a five
membered ring; wherein any of R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 and R.sub.a, R.sub.b, and R.sub.c is
substituted or unsubstituted.
[0321] In one embodiment, P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q. K is SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19, NR.sub.19R.sub.20,
glycoside, or heterocycle. L is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, glycoside, or heterocycle. M is SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, glycoside, or heterocycle.
[0322] In one embodiment, the compound inhibits one or more
components of a protein kinase signaling pathway. In another
embodiment, the compound is an allosteric inhibitor. In another
embodiment, the compound is a peptide substrate inhibitor. In
another embodiment, the compound does not inhibit ATP binding to a
protein kinase. In one embodiment, the compound binds to a peptide
binding site. In another embodiment, the compound inhibits a Src
family protein kinase. For example, the Src family protein kinase
is pp60.sup.c-src tyrosine kinase. In another embodiment, the Src
family protein kinase is PYK2.
[0323] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membranes. In one embodiment, the compound is
administered with a pharmaceutically acceptable carrier. In another
embodiment, the compound is administered before initiation of
osteoporosis. In another embodiment, the compound is administered
after inititiation of osteoporosis.
[0324] In another embodiment, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is selected from halogen, boronic
acid, hydroxyl, phosphonic acid, sulfamic acid, guanidine,
carboxylic acid, aldehyde, amide, and hydroxymethylphosphonic acid.
In another embodiment, at least one of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, and R.sub.5 is a halogen. In one embodiment, at least one
of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is a boronic
acid. In another embodiment, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 is hydroxyl. In another embodiment,
at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 is
amide. For example, the amide group is vicinal tricarbonyl amide.
In one embodiment, R.sub.3 is halogen. For example, R.sub.3 is
fluorine. In one embodiment, R.sub.3 is hydroxyl.
[0325] In one embodiment, at least one of R.sub.6 and R.sub.7 is
(CH.sub.2).sub.t--Z. In another embodiment, Z is aryl. In one
embodiment, aryl is unsubstituted. In one embodiment aryl is
monosubstituted. In another embodiment, aryl is disubsituted. In
another embodiment, aryl is trisubstituted. In one embodiment aryl
is substituted with hydroxyl, halogen, phenoxy (--OC.sub.6H.sub.5),
alkoxy, CF.sub.3, alkyl, hydroxymethyl, aryl, OCF.sub.3, benzyloxy
(--OCH.sub.2C.sub.6H.sub.5), nitro, aldehyde, or alkoxycarboxy
(also referred to as ester e.g., --C(O)OEt). In one embodiment,
(CH.sub.2) is substituted, wherein one or more of the hydrogen
atoms of the methylene group are replaced with one or more
substituents. Methylene substitutents are selected from
hydroxmethyl, alkyl (e.g., methyl), hydroxyl, and aryl. In another
embodiment, at least one of R.sub.6 and R.sub.7 is branched or
unbranched alkyl. In one embodiment, branched or unbranched alkyl
is unsubstituted. In another embodiment, branched or unbranched
alkyl is substituted with hydroxyl. In another embodiment, Z is
cyclic alkyl. In one embodiment, cyclic alkyl is substituted. In
another embodiment, cyclic alkyl is substituted with hydroxyl. In
another embodiment, cyclic alkyl is unsubstituted. In one
embodiment, t is 0. In another embodiment, t is 1. In another
embodiment, t is 2.
[0326] In one embodiment, R.sub.6 and R.sub.7 form a ring, where
the ring is selected from pyrrolidine, piperidine, and
morpholine.
[0327] In one embodiment, at least one of R.sub.6 and R.sub.7
is
##STR00197##
*R.sub.8 is the point of attachment and is (CH.sub.2).sub.x,
wherein X is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, CH.sub.2CHOH,
CH(CH.sub.3)(R-isomer), or CH(CH.sub.3)(S-isomer). Each of R.sub.9,
R.sub.10, R.sub.11, R.sub.12, and R.sub.13 is the same or different
and independently are H, C(O)R.sub.a, C(O)NR.sub.aR.sub.b,
C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a, OC(O)R.sub.a,
OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b, NR.sub.aC(O)R.sub.b,
NR.sub.aC(O)NR.sub.bR.sub.c, NR.sub.aC(O)OR.sub.b,
NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, halogen, P', aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle and branched, cyclic, or
unbranched alkyl, P' is SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2,
OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-K', O--C(O)-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L', NH-lower (C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.s, or C.sub.6) alkyl-M', or
O-aryl-Q', further wherein lower (C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, or C.sub.6) alkyl is linear or branched alkyl. K'
is C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22, NR.sub.22R.sub.23,
SO.sub.2R.sub.24, glycoside, lower C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00198##
L' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00199##
M' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00200##
Q' is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, SO.sub.2R.sub.24, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00201##
R.sub.22, R.sub.23 and R.sub.24 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.22 and
R.sub.23 taken together with the attached nitrogen atom form a five
membered ring. R.sub.a, R.sub.b, and R.sub.c are the same or
different and independently are H, aryl, heteroaryl, biaryl,
heterobiaryl, branched, cyclic, or unbranched alkyl; and wherein
any of R.sub.9, R.sub.10, R.sub.11, R.sub.12, and R.sub.13 are
substituted or unsubstituted.
[0328] In one embodiment, P' is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K', O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L',
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M', or O-aryl-Q'. K' is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22,
NR.sub.22R.sub.23, glycoside, or heterocycle. L' is SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.22, NR.sub.22R.sub.23, glycoside, or heterocycle; and
further wherein M' is SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.22, NR.sub.22R.sub.23,
glycoside, or heterocycle. In one embodiment, at least one of
R.sub.6 and R.sub.7 is
##STR00202##
In another embodiment, the compound is
##STR00203##
[0329] Another aspect of the invention includes a method of
protecting against or treating hearing loss in a subject comprising
administering a compound of Formula VI:
##STR00204##
or a salt, solvate, hydrate, or prodrug thereof. R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are each the same
or different and independently are H, C(O)R.sub.a,
C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.4, OH, OR.sub.a,
OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle, and branched, cyclic, or
unbranched alkyl. R.sub.a, R.sub.b, and R.sub.c are the same or
different and are independently H, aryl, heteroaryl, biaryl,
heterobiaryl, and branched, cyclic, or unbranched alkyl;
[0330] P is SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2,
OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L, NH-lower (C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-M, or
O-aryl-Q, further wherein lower (C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, or C.sub.6) alkyl is linear or branched alkyl. K
is C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19, NR.sub.19R.sub.20,
SO.sub.2R.sub.21, glycoside, lower C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00205##
L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00206##
M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00207##
Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00208##
R.sub.19, R.sub.20 and R.sub.21 are independeritly C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.19
and R.sub.20 taken together with the attached nitrogen atom form a
five membered ring. x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. At least
one of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and
R.sub.7 is P.
[0331] In one embodiment, P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q. K is SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19, NR.sub.19R.sub.20,
glycoside, or heterocycle. L is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, glycoside, or heterocycle. M is SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, glycoside, or heterocycle.
[0332] In one embodiment, the compound inhibits one or more
components of a protein kinase signaling pathway. In another
embodiment, the compound is an allosteric inhibitor. In one
embodiment, the compound is a peptide substrate inhibitor. In
another embodiment, the compound does not inhibit ATP binding to a
protein kinase. In one embodiment, the compound binds to a peptide
binding site. In another embodiment, the compound inhibits a Src
family protein kinase. For example, the Src family protein kinase
is pp60.sup.c-src tyrosine kinase. In another embodiment, the Src
family protein kinase is PYK2.
[0333] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically (e.g., by
administering drops into the ear), intraarterially,
intralesionally, by metering pump, or by application to mucous
membranes. In one embodiment, the compound is administered in
combination with a drug that causes hearing loss. In another
embodiment, the compound is administered with a pharmaceutically
acceptable carrier. In another embodiment, the compound is
administered before initiation of hearing loss. In another
embodiment, the compound is administered after inititiation of
hearing loss.
[0334] Another aspect of the invention includes a method of
protecting against or treating osteoporosis in a subject comprising
administering a compound of Formula VIII:
##STR00209##
or a salt, solvate, hydrate, or prodrug thereof. R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are each the same
or different and independently are H, C(O)R.sub.a,
C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a,
OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.8S(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle, and branched, cyclic, or
unbranched alkyl. R.sub.a, R.sub.b, and R.sub.c are the same or
different and are independently H, aryl, heteroaryl, biaryl,
heterobiaryl, and branched, cyclic, or unbranched alkyl. P is
SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K,
O--C(O)-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or
C.sub.6) alkyl-L, NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-M, or O-aryl-Q, further wherein lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is
linear or branched alkyl. K is C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00210##
L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00211##
M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00212##
Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00213##
R.sub.19, R.sub.20 and R.sub.21 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.19 and
R.sub.20 taken together with the attached nitrogen atom form a five
membered ring. x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
[0335] In one embodiment, P is SO.sub.3H, OSO.sub.3H,
OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower (C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K, O--C(O)-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-L,
NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6)
alkyl-M, or O-aryl-Q. K is SO.sub.3H, OSO.sub.3H, PO.sub.3H.sub.2,
OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19, NR.sub.19R.sub.20,
glycoside, or heterocycle. L is SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, glycoside, or heterocycle. M is SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, glycoside, or heterocycle.
[0336] In one embodiment, the compound inhibits one or more
components of a protein kinase signaling pathway. In another
embodiment, the compound is an allosteric inhibitor. In another
embodiment, the compound is a peptide substrate inhibitor. In
another embodiment, the compound does not inhibit ATP binding to a
protein kinase. In one embodiment, the compound binds to a peptide
binding site. In another embodiment, the compound inhibits a Src
family protein kinase. For example, the Src family protein kinase
is pp60.sup.c-src tyrosine kinase. In another embodiment, the Src
family protein kinase is PYK2.
[0337] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membranes. In one embodiment, the compound is
administered with a pharmaceutically acceptable carrier. In one
embodiment, the compound is administered before the onset of
osteoporosis. In another embodiment, the compound is administered
after onset of osteoporosis.
[0338] A method of preventing or treating a proliferative disorder
in a subject comprising administering a compound of Formula
VIII:
##STR00214##
or a salt, solvate, hydrate, or prodrug thereof. R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 are each the same
or different and independently are H, C(O)R.sub.a,
C(O)NR.sub.aR.sub.b, C(O)OR.sub.a, C(O)SR.sub.a, OH, OR.sub.a,
OC(O)R.sub.a, OC(O)OR.sub.a, NH.sub.2, NR.sub.aR.sub.b,
NR.sub.aC(O)R.sub.b, NR.sub.aC(O)NR.sub.bR.sub.c,
NR.sub.aC(O)OR.sub.b, NR.sub.aC(O)SR.sub.b, NR.sub.aS(O)R.sub.b,
NR.sub.aS(O).sub.2R.sub.b, NR.sub.aS(O)OR.sub.b,
NR.sub.aS(O).sub.2OR.sub.b, NR.sub.aP(O)OR.sub.bOR.sub.c,
NR.sub.aP(O)OR.sub.bR.sub.c, NR.sub.aP(O)OR.sub.bOR.sub.c,
SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, S(O)OR.sub.a,
S(O).sub.2OR.sub.a, S(O)NR.sub.aR.sub.b, S(O).sub.2NR.sub.aR.sub.b,
P(O)OR.sub.aOR.sub.b, B(OH).sub.2, P, halogen, aryl, heteroaryl,
biaryl, heterobiaryl, heterocycle, and branched, cyclic, or
unbranched alkyl. R.sub.a, R.sub.b, and R.sub.c are the same or
different and are independently H, aryl, heteroaryl, biaryl,
heterobiaryl, and branched, cyclic, or unbranched alkyl. P is
SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2, OPO.sub.3H.sub.2, O-lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl-K,
O--C(O)-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or
C.sub.6) alkyl-L, NH-lower (C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, or C.sub.6) alkyl-M, or O-aryl-Q, further wherein lower
(C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, or C.sub.6) alkyl is
linear or branched alkyl. K is C(O)NH.sub.2, COOH, SO.sub.3H,
OSO.sub.3H, PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2,
NHR.sub.19, NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00215##
L is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00216##
M is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00217##
Q is aryl, OH, C(O)NH.sub.2, COOH, SO.sub.3H, OSO.sub.3H,
PO.sub.3H.sub.2, OPO.sub.3H.sub.2, NH.sub.2, NHR.sub.19,
NR.sub.19R.sub.20, SO.sub.2R.sub.21, glycoside, lower C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6 alkoxy, or
##STR00218##
R.sub.19, R.sub.20 and R.sub.21 are independently C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, or C.sub.6 alkyl or R.sub.19 and
R.sub.20 taken together with the attached nitrogen atom form a five
membered ring. x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
[0339] In one embodiment, the compound inhibits one or more
components of a protein kinase signaling pathway. In another
embodiment, the compound is an allosteric inhibitor. In another
embodiment, the compound is a peptide substrate inhibitor. In one
embodiment, the compound does not inhibit ATP binding to a protein
kinase. In one embodiment, the compound binds to a peptide binding
site. In another embodiment, the compound inhibits a Src family
protein kinase. For example, the Src family protein kinase is
pp60.sup.c-src tyrosine kinase. In another embodiment, the Src
family protein kinase is PYK2.
[0340] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membranes. In one embodiment, the compound is
administered with a pharmaceutically acceptable carrier. In another
embodiment, the compound is administered before onset of the
proliferative disease. In another embodiment, the compound is
administered after onset of the proliferative disease.
[0341] Another aspect of the invention includes a method of
protecting against or treating ophthalmic disease (e.g., macular
degeneration, retinopathy, macular edema, etc.) in a subject
comprising administering a compound of Formula VII or VIII. In one
embodiment, the compound inhibits one or more components of a
protein kinase signaling pathway. In another embodiment, the
compound is an allosteric inhibitor. In another embodiment, the
compound is a peptide substrate inhibitor. In another embodiment,
the compound does not inhibit ATP binding to a protein kinase. In
one embodiment, the compound binds to a peptide binding site. In
another embodiment, the compound inhibits a Src family protein
kinase.
[0342] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically (e.g., by
administration of drops or a cream to the eye), intraarterially,
intralesionally, by metering pump, or by application to mucous
membranes. In one embodiment, the compound is administered with a
pharmaceutical acceptable carrier. In another embodiment, the
compound is administered before initiation of ophthalmic disease.
In another embodiment, the compound is administered after
inititiation of ophthalmic disease.
[0343] Another aspect of the invention includes a method of
protecting against or treating diabetes in a subject comprising
administering a compound of Formula VII or VIII. In one embodiment,
the compound inhibits one or more components of a protein kinase
signaling pathway. In another embodiment, the compound is an
allosteric inhibitor. In another embodiment, the compound is a
peptide substrate inhibitor. In another embodiment, the compound
does not inhibit ATP binding to a protein kinase. In one
embodiment, the compound binds to a peptide binding site. In
another embodiment, the compound inhibits a Src family protein
kinase.
[0344] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membranes. In one embodiment, the compound is
administered with a pharmaceutically acceptable carrier. In another
embodiment, the compound is administered before the onset of
diabetes. In another embodiment, the compound is administered after
the onset of diabetes.
[0345] Another aspect of the invention includes a method of
protecting against or treating obesity in a subject comprising
administering a compound of Formula VII or VIII. In one embodiment,
the compound inhibits one or more components of a protein kinase
signaling pathway. In another embodiment, the compound is an
allosteric inhibitor. In another embodiment, the compound is a
peptide substrate inhibitor. In another embodiment, the compound
does not inhibit ATP binding to a protein kinase. In one
embodiment, the compound binds to a peptide binding site. In
another embodiment, the compound inhibits a Src family protein
kinase.
[0346] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membranes. In one embodiment, the compound is
administered with a pharmaceutically acceptable carrier. In another
embodiment, the compound is administered before the onset of
obesity. In another embodiment, the compound is administered after
the onset of obesity.
[0347] Another aspect of the invention includes a method of
protecting against or treating stroke in a subject comprising
administering a compound of Formula VII or VIII. In one embodiment,
the compound inhibits one or more components of a protein kinase
signaling pathway. In another embodiment, the compound is an
allosteric inhibitor. In another embodiment, the compound is a
peptide substrate inhibitor. In another embodiment, the compound
does not inhibit ATP binding to a protein kinase. In one
embodiment, the compound binds to a peptide binding site. In
another embodiment, the compound inhibits a Src family protein
kinase.
[0348] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membranes. In one embodiment, the compound is
administered with a pharmaceutically acceptable carrier. In another
embodiment, the compound is administered before a stroke occurs in
a subject. In another embodiment, the compound is administered
after a stroke has occurred in a subject.
[0349] Another aspect of the invention includes a method of
protecting against or treating athrosclerosis in a subject
comprising administering a compound of Formula VII or VIII. In one
embodiment, the compound inhibits one or more components of a
protein kinase signaling pathway. In another embodiment, the
compound is an allosteric inhibitor. In another embodiment, the
compound is a peptide substrate inhibitor. In another embodiment,
the compound does not inhibit ATP binding to a protein kinase. In
one embodiment, the compound binds to a peptide binding site. In
another embodiment, the compound inhibits a Src family protein
kinase.
[0350] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membranes. In one embodiment, the compound is
administered with a pharmaceutically acceptable carrier. In another
embodiment, the compound is administered before the onset of
athrosclerosis. In another embodiment, the compound is administered
after the onset of athrosclerosis.
[0351] Another aspect of the invention includes a method of
regulating immune system activity in a subject comprising
administering a compound of Formula VII or VIII. In one embodiment,
the compound inhibits one or more components of a protein kinase
signaling pathway. In another embodiment, the compound is an
allosteric inhibitor. In another embodiment, the compound is a
peptide substrate inhibitor. In another embodiment, the compound
does not inhibit ATP binding to a protein kinase. In one
embodiment, the compound binds to a peptide binding site. In
another embodiment, the compound inhibits a Src family protein
kinase.
[0352] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membranes. In one embodiment, the compound is
administered with a pharmaceutically acceptable carrier.
[0353] Another aspect of the invention includes a method of
protecting against or treating chronic neuropathic pain in a
subject comprising administering a compound of Formula VII or VIII.
In one embodiment, the compound inhibits one or more components of
a protein kinase signaling pathway. In another embodiment, the
compound is an allosteric inhibitor. In another embodiment, the
compound is a peptide substrate inhibitor. In another embodiment,
the compound does not inhibit ATP binding to a protein kinase. In
one embodiment, the compound binds to a peptide binding site. In
another embodiment, the compound inhibits a Src family protein
kinase.
[0354] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membranes. In one embodiment, the compound is
administered with a pharmaceutically acceptable carrier. In one
embodiment, the compound is administered before the onset of
chronic neuropathic pain. In another embodiment, the compound is
administered after the onset of chronic neuropathic pain.
[0355] Another aspect of the invention includes a method of
protecting against or treating hepatitis B in a subject comprising
administering a compound of Formula VII or VIII. In one embodiment,
the compound inhibits one or more components of a protein kinase
signaling pathway. In another embodiment, the compound is an
allosteric inhibitor. In another embodiment, the compound is a
peptide substrate inhibitor. In another embodiment, the compound
does not inhibit ATP binding to a protein kinase. In one
embodiment, the compound binds to a peptide binding site. In
another embodiment, the compound inhibits a Src family protein
kinase.
[0356] In one embodiment, the administration of the compound is
carried out orally, parentally, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
intracavitary or intravesical instillation, topically,
intraarterially, intralesionally, by metering pump, or by
application to mucous membranes. In one embodiment, the compound is
administered with a pharmaceutically acceptable carrier. In one
embodiment, the compound is administered before the onset of
hepatitis B. In another embodiment, the compound is administered
after the onset of hepatitis B.
[0357] The present invention also provides a method for testing
compounds for an ability to inhibit protein kinase or protein
phosphatase activity. Compounds are produced as described above.
The activity of the protein kinase or protein phosphatase is
measured in the presence of the inhibitor at the same temperature,
pH, ionic strength, osmolarity, and free magnesium concentration as
found in a cell which expresses the protein kinase or protein
phosphatase. The level of protein kinase or protein phosphatase
activity is compared to the level of activity from the protein
kinase or protein phosphatase without the presence of the
inhibitor. Such an assay system which mimics physiological
conditions provides the most relevant inhibition data. The assay
may be conducted in an automated assay system. Furthermore, the
assay may be combined with a combinatorial chemistry method to
rapidly screen numerous candidates.
[0358] The Pierce 96-well plate non-radioactive ELISA PTK assay
method may be adapted to the Cellular Mimetic assay conditions for
initial Src screening of the 96-well plate combinatorial libraries.
This high throughput assay utilizes the same RR-SRC peptide
substrate, except that it is biotinylated so that it can be
attached to the NeutrAvidin-coated wells in their commercial
96-well plates. This high throughput inhibition assay can be run by
incubating Src with the RR-SRC substrate prebound to the wells
followed by adding their anti-phosphotyrosine antibody
(PY20)-horseradish peroxidase (HRP) conjugate and their HRP
substrate to quantitate the level of phospho-RR-SRC produced via
measuring the level of HRP product with a 96-well plate UV reader.
Standard low throughput P.sup.32-ATP radioactive assays have been
used, but a 96-well plate format is preferred, especially with a
non-radioactive assay if possible. As very potent Src inhibitors
are developed, a panel of protein kinase assays could be set up
with commercially available protein kinases, using the Cellular
Mimetic protein kinase assay conditions, and test these inhibitors
across the panel to obtain an initial assessment of specificity. A
more complete specificity assessment, involving the full ca. 2,000
protein kinases, will need to be conducted in cell culture and in
vivo.
[0359] Active Src inhibitors can be studied in a set of
side-by-side cell-based assays using normal rat kidney (NRK) cells
and a temperature-sensitive pp60.sup.v-Src transformant of this
cell line (LA25). The LA25 transformant engages in anchorage- and
serum-independent growth at the "permissive" temperature of
33.degree. C. due to activation of pp60.sup.v-src but not at the
"non-permissive" temperature of 40.degree. C. at which
pp60.sup.v-src is not activated (Li et al., 1996). The use of this
pair of closely related cell lines for testing the Src inhibitors
at both the permissive and non-permissive temperatures allows one
to determine if a given Src inhibitor is blocking cell growth due
to specific blockade of the Src signaling pathway, by a different
mechanism or by a general cytotoxic effect. Results from initial
testing of the non-peptide Src inhibitor 43-meta (Table V) in this
pair of cell lines are shown in FIG. 14.
[0360] As shown in this graph the growth of the LA25 cells at the
permissive temperature of 33.degree. C. is inhibited by ca. 50% at
a 25 .mu.M concentration of 43-meta relative to the LA25 cell
growth at the non-permissive 40.degree. C. as a control. The lack
of cell toxicity of 43-meta is evidenced by the fact that as its
concentration is increased up to 400 .mu.M, the basal growth of the
NRK non-transformed cells, the LA25 cells at the non-permissive
40.degree. C., and the LA25 cells at the permissive temperature of
33.degree. C. (but with pp60.sup.v-src fully inhibited by 43-meta)
not only does not decrease but actually increases somewhat
(presumably due to a non-Src related activity of this compound).
Since the 43-meta solutions were prepared with a low concentration
of DMSO for solubilization, a DMSO control was also run at the same
concentration.
[0361] Moreover, promising Src inhibitors can be screened in
primary human tumor tissue assays, particularly to look for synergy
with other known anti-cancer drugs.
DEFINITIONS
[0362] For convenience, certain terms used in the specification,
examples and appended claims are collected here.
[0363] Protein kinases are a large class of enzymes which catalyze
the transfer of the .gamma.-phosphate from ATP to the hydroxyl
group on the side chain of Ser/Thr or Tyr in proteins and peptides
and are intimately involved in the control of various important
cell functions, perhaps most notably: signal transduction,
differentiation, and proliferation. There are estimated to be about
2,000 distinct protein kinases in the human body, and although each
of these phosphorylate particular protein/peptide substrates, they
all bind the same second substrate ATP in a highly conserved
pocket. About 50% of the known oncogene products are protein
tyrosine kinases (PTKs), and their kinase activity has been shown
to lead to cell transformation.
[0364] The PTKs can be classified into two categories, the membrane
receptor PTKs (e.g. growth factor receptor PTKs) and the
non-receptor PTKs (e.g. the Src family of proto-oncogene products
and focal adhesion kinase (FAK)). The hyperactivation of Src has
been reported in a number of human cancers, including those of the
colon, breast, lung, bladder, and skin, as well as in gastric
cancer, hairy cell leukemia, and neuroblastoma.
[0365] "inhibits one or more components of a protein kinase
signaling cascade" means that one or more components of the kinase
signaling cascade are effected such that the functioning of the
cell changes. Components of a protein kinase signaling cascade
include any proteins involved directly or indirectly in the kinase
signaling pathway including second messengers and upstream and
downstream targets.
[0366] "Treating", includes any effect, e.g., lessening, reducing,
modulating, or eliminating, that results in the improvement of the
condition, disease, disorder, etc. "Treating" or "treatment" of a
disease state includes: (1) preventing the disease state, i.e.
causing the clinical symptoms of the disease state not to develop
in a subject that may be exposed to or predisposed to the disease
state, but does not yet experience or display symptoms of the
disease state; (2) inhibiting the disease state, i.e., arresting
the development of the disease state or its clinical symptoms; or
(3) relieving the disease state, i.e., causing temporary or
permanent regression of the disease state or its clinical
symptoms.
[0367] "Disease state" means any disease, disorder, condition,
symptom, or indication.
[0368] As used herein, the term "cell proliferative disorder"
refers to conditions in which the unregulated and/or abnormal
growth of cells can lead to the development of an unwanted
condition or disease, which can be cancerous or non-cancerous, for
example a psoriatic condition. As used herein, the terms "psoriatic
condition" or "psoriasis" refers to disorders involving
keratinocyte hyperproliferation, inflammatory cell infiltration,
and cytokine alteration.
[0369] In one embodiment, the cell proliferation disorder is
cancer. As used herein, the term "cancer" includes solid tumors,
such as lung, breast, colon, ovarian, brain, liver, pancreas,
prostate, malignant melanoma, non-melanoma skin cancers, as well as
hematologic tumors and/or malignancies, such as childhood leukemia
and lymphomas, multiple myeloma, Hodgkin's disease, lymphomas of
lymphocytic and cutaneous origin, acute and chronic leukemia such
as acute lymphoblastic, acute myelocytic or chronic myelocytic
leukemia, plasma cell neoplasm, lymphoid neoplasm and cancers
associated with AIDS.
[0370] In addition to psoriatic conditions, the types of
proliferative diseases which may be treated using the compositions
of the present invention are epidermic and dermoid cysts, lipomas,
adenomas, capillary and cutaneous hemangiomas, lymphangiomas, nevi
lesions, teratomas, nephromas, myofibromatosis, osteoplastic
tumors, and other dysplastic masses and the like. The proliferative
diseases can include dysplasias and disorders of the like.
[0371] An "effective amount" of a compound of the disclosed
invention is the quantity which, when administered to a subject
having a disease or disorder, results in regression of the disease
or disorder in the subject. Thus, an effective amount of a compound
of the disclosed invention is the quantity which, when administered
to a subject having a cell proliferation disorder, results in
regression of cell growth in the subject. The amount of the
disclosed compound to be administered to a subject will depend on
the particular disorder, the mode of administration,
co-administered compounds, if any, and the characteristics of the
subject, such as general health, other diseases, age, sex,
genotype, body weight and tolerance to drugs. The skilled artisan
will be able to determine appropriate dosages depending on these
and other factors.
[0372] As used herein, the term "effective amount" refers to an
amount of a compound, or a combination of compounds, of the present
invention effective when administered alone or in combination as an
anti-proliferative agent. For example, an effective amount refers
to an amount of the compound present in a formulation or on a
medical device given to a recipient patient or subject sufficient
to elicit biological activity, for example, anti-proliferative
activity, such as e.g., anti-cancer activity or anti-neoplastic
activity. The combination of compounds optionally is a synergistic
combination. Synergy, as described, for example, by Chou and
Talalay, Adv. Enzyme Regul. vol. 22, pp. 27-55 (1984), occurs when
the effect of the compounds when administered in combination is
greater than the additive effect of the compounds when administered
alone as a single agent. In general, a synergistic effect is most
clearly demonstrated at sub-optimal concentrations of the
compounds. Synergy can be in terms of lower cytotoxicity, or
increased anti-proliferative effect, or some other beneficial
effect of the combination compared with the individual
components.
[0373] "A therapeutically effective amount" means the amount of a
compound that, when administered to a mammal for treating a
disease, is sufficient to effect such treatment for the disease.
The "therapeutically effective amount" will vary depending on the
compound, the disease and its severity and the age, weight, etc.,
of the mammal to be treated.
[0374] A therapeutically effective amount of one or more of the
compounds can be formulated with a pharmaceutically acceptable
carrier for administration to a human or an animal. Accordingly,
the compounds or the formulations can be administered, for example,
via oral, parenteral, or topical routes, to provide an effective
amount of the compound. In alternative embodiments, the compounds
prepared in accordance with the present invention can be used to
coat or impregnate a medical device, e.g., a stent.
[0375] The term "prophylactically effective amount" means an
effective amount of a compound or compounds, of the present
invention that is administered to prevent or reduce the risk of
unwanted cellular proliferation.
[0376] "Pharmacological effect" as used herein encompasses effects
produced in the subject that achieve the intended purpose of a
therapy. In one embodiment, a pharmacological effect means that
primary indications of the subject being treated are prevented,
alleviated, or reduced. For example, a pharmacological effect would
be one that results in the prevention, alleviation or reduction of
primary indications in a treated subject. In another embodiment, a
pharmacological effect means that disorders or symptoms of the
primary indications of the subject being treated are prevented,
alleviated, or reduced. For example, a pharmacological effect would
be one that results in the prevention or reduction of primary
indications in a treated subject.
[0377] With respect to the chemical compounds useful in the present
invention, the following terms can be applicable:
[0378] The term "substituted," as used herein, means that any one
or more hydrogens on the designated atom is replaced with a
selection from the indicated group, provided that the designated
atom's normal valency is not exceeded, and that the substitution
results in a stable compound. When a substituent is keto (i.e.,
.dbd.O), then 2 hydrogens on the atom are replaced. Keto
substituents are not present on aromatic moieties. Ring double
bonds, as used herein, are double bonds that are formed between two
adjacent ring atoms (e.g., C.dbd.C, C.dbd.N, or N.dbd.N).
[0379] The present invention is intended to include all isotopes of
atoms occurring in the present compounds. Isotopes include those
atoms having the same atomic number but different mass numbers. By
way of general example and without limitation, isotopes of hydrogen
include tritium and deuterium, and isotopes of carbon include C-13
and C-14.
[0380] The compounds described herein may have asymmetric centers.
Compounds of the present invention containing an asymmetrically
substituted atom may be isolated in optically active or racemic
forms. It is well known in the art how to prepare optically active
forms, such as by resolution of racemic forms or by synthesis from
optically active starting materials. 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. All chiral, diastereomeric, racemic, and geometric isomeric
forms of a structure are intended, unless the specific
stereochemistry or isomeric form is specifically indicated. All
tautomers of shown or described compounds are also considered to be
part of the present invention.
[0381] When any variable (e.g., R.sub.1) occurs more than one time
in any constituent or formula for a compound, its definition at
each occurrence is independent of its definition at every other
occurrence. Thus, for example, if a group is shown to be
substituted with 0-2 R.sub.1 moieties, then the group may
optionally be substituted with up to two R.sub.1 moieties and
R.sup.1 at each occurrence is selected independently from the
defmition of R.sub.1. Also, combinations of substituents and/or
variables are permissible, but only if such combinations result in
stable compounds.
[0382] When a bond to a substituent is shown to cross a bond
connecting two atoms in a ring, then such substituent may be bonded
to any atom in the ring. When a substituent is listed without
indicating the atom via which such substituent is bonded to the
rest of the compound of a given formula, then such substituent may
be bonded via any atom in such substituent. Combinations of
substituents and/or variables are permissible, but only if such
combinations result in stable compounds.
[0383] Compounds of the present invention that contain nitrogens
can be converted to N-oxides by treatment with an oxidizing agent
(e.g., 3-chloroperoxybenzoic acid (m-CPBA) and/or hydrogen
peroxides) to afford other compounds of the present invention.
Thus, all shown and claimed nitrogen-containing compounds are
considered, when allowed by valency and structure, to include both
the compound as shown and its N-oxide derivative (which can be
designated as N.fwdarw.O or N.sup.+--O.sup.-). Furthermore, in
other instances, the nitrogens in the compounds of the present
invention can be converted to N-hydroxy or N-alkoxy compounds. For
example, N-hydroxy compounds can be prepared by oxidation of the
parent amine by an oxidizing agent such as m-CPBA. All shown and
claimed nitrogen-containing compounds are also considered, when
allowed by valency and structure, to cover both the compound as
shown and its N-hydroxy (i.e., N--OH) and N-alkoxy (i.e., N--OR,
wherein R is substituted or unsubstituted C.sub.1-6 alkyl,
C.sub.1-6 alkenyl, C.sub.1-6 alkynyl, C.sub.3-14 carbocycle, or
3-14-membered heterocycle) derivatives.
[0384] Compounds of the invention include water solubilizing groups
Wermuth, The Practice of Medicinal Chemistry 2003, p. 617. e.g.,
SO.sub.3H, OSO.sub.3H, OPO.sub.3H.sub.2, OPO.sub.3H.sub.2,
amines,
##STR00219##
tetrazole, etc.
[0385] When an atom or chemical moiety is followed by a subscripted
numeric range (e.g., C.sub.1-6), the invention is meant to
encompass each number within the range as well as all intermediate
ranges. For example, "C.sub.1-6 alkyl" is meant to include alkyl
groups with 1, 2, 3, 4, 5, 6, 1-6, 1-5, 1-4, 1-3, 1-2, 2-6, 2-5,
2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, and 5-6 carbons.
[0386] As used herein, "alkyl" is intended to include both branched
and straight-chain saturated aliphatic hydrocarbon groups having
the specified number of carbon atoms. For example, C.sub.1-6 alkyl
is intended to include C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5,
and C.sub.6 alkyl groups. Examples of alkyl include, but are not
limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl,
t-butyl, n-pentyl, s-pentyl, and n-hexyl. "Alkyl" further includes
alkyl groups that have oxygen, nitrogen, sulfur or phosphorous
atoms replacing one or more hydrocarbon backbone carbon atoms. In
certain embodiments, a straight chain or branched chain alkyl has
six or fewer carbon atoms in its backbone (e.g., C.sub.1-C.sub.6
for straight chain, C.sub.3-C.sub.6 for branched chain), and in
another embodiment, a straight chain or branched chain alkyl has
four or fewer carbon atoms. Likewise, cycloalkyls have from three
to eight carbon atoms in their ring structure, and in another
embodiment, cycloalkyls have five or six carbons in the ring
structure.
[0387] Unless the number of carbons is otherwise specified, "lower
alkyl" includes an alkyl group, as defined above, but having from
one to ten, or in another embodiment from one to six, carbon atoms
in its backbone structure. "Lower alkenyl" and "lower alkynyl" have
chain lengths of, for example, 2-5 carbon atoms.
[0388] The term "substituted alkyl" refers to alkyl moieties having
substituents replacing a hydrogen on one or more carbons of the
hydrocarbon backbone. Such substituents can include, for example,
alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkylamino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, allcylaryl, or an
aromatic or heteroaromatic moiety. Cycloalkyls can be further
substituted, e.g., with the substituents described above. An
"alkylaryl" or an "aralkyl" moiety is an alkyl substituted with an
aryl (e.g., phenylmethyl(benzyl)).
[0389] "Alkenyl" includes unsaturated aliphatic groups analogous in
length and possible substitution to the alkyls described above, but
that contain at least one double bond. For example, the term
"alkenyl" includes straight-chain alkenyl groups (e.g., ethenyl,
propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl,
decenyl), branched-chain alkenyl groups, cycloalkenyl (e.g.,
alicyclic) groups (e.g., cyclopropenyl, cyclopentenyl,
cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl
substituted cycloalkenyl groups, and cycloalkyl or cycloalkenyl
substituted alkenyl groups. The term "alkenyl" further includes
alkenyl groups, which include oxygen, nitrogen, sulfur or
phosphorous atoms replacing one or more hydrocarbon backbone
carbons. In certain embodiments, a straight chain or branched chain
alkenyl group has six or fewer carbon atoms in its backbone (e.g.,
C.sub.2-C.sub.6 for straight chain, C.sub.3-C.sub.6 for branched
chain). Likewise, cycloalkenyl groups may have from three to eight
carbon atoms in their ring structure, and in one embodiment,
cycloalkenyl groups have five or six carbons in the ring structure.
The term "C.sub.2-C.sub.6" includes alkenyl groups containing two
to six carbon atoms. The term "C.sub.3-C.sub.6" includes alkenyl
groups containing three to six carbon atoms.
[0390] The term "substituted alkenyl" refers to alkenyl moieties
having substituents replacing a hydrogen on one or more hydrocarbon
backbone carbon atoms. Such substituents can include, for example,
alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkylamino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety.
[0391] "Alkynyl" includes unsaturated aliphatic groups analogous in
length and possible substitution to the alkyls described above, but
which contain at least one triple bond. For example, "alkynyl"
includes straight-chain alkynyl groups (e.g., ethynyl, propynyl,
butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl),
branched-chain alkynyl groups, and cycloalkyl or cycloalkenyl
substituted alkynyl groups. The term "alkynyl" further includes
alkynyl groups having oxygen, nitrogen, sulfur or phosphorous atoms
replacing one or more hydrocarbon backbone carbons. In certain
embodiments, a straight chain or branched chain alkynyl group has
six or fewer carbon atoms in its backbone (e.g., C.sub.2-C.sub.6
for straight chain, C.sub.3-C.sub.6 for branched chain). The term
"C.sub.2-C.sub.6" includes alkynyl groups containing two to six
carbon atoms. The term "C.sub.3-C.sub.6" includes alkynyl groups
containing three to six carbon atoms.
[0392] The term "substituted alkynyl" refers to alkynyl moieties
having substituents replacing a hydrogen on one or more hydrocarbon
backbone carbon atoms. Such substituents can include, for example,
alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkylamino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety.
[0393] "Aryl" includes groups with aromaticity, including 5- and
6-membered "unconjugated", or single-ring, aromatic groups that may
include from zero to four heteroatoms, as well as "conjugated", or
multicyclic, systems with at least one aromatic ring. Examples of
aryl groups include benzene, phenyl, pyrrole, furan, thiophene,
thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole,
oxazole, isooxazole, pyridine, pyrazine, pyridazine, and
pyrimidine, and the like. Furthermore, the term "aryl" includes
multicyclic aryl groups, e.g., tricyclic, bicyclic, e.g.,
naphthalene, benzoxazole, benzodioxazole, benzothiazole,
benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline,
isoquinoline, napthridine, indole, benzofuran, purine, benzofuran,
deazapurine, or indolizine. Those aryl groups having heteroatoms in
the ring structure may also be referred to as "aryl heterocycles",
"heterocycles," "heteroaryls" or "heteroaromatics" or the entire
compound may be referred to as "heterocyclic compound". The
aromatic ring can be substituted at one or more ring positions with
such substituents as described above, as for example, halogen,
hydroxyl, aldehyde, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, O-benzyl, carboxylate,
alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl,
alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl,
alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylhydroxy,
alkylthiocarbonyl, phenoxy (O--C.sub.6H.sub.5), phosphate,
phosphonato, phosphinato, cyano, amino (including alkylamino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, --OCF.sub.3, cyano,
azido, heterocyclyl, alkylaryl, or an aromatic (e.g., phenyl) or
heteroaromatic moiety. Aryl groups can also be fused or bridged
with alicyclic or heterocyclic rings, which are not aromatic so as
to form a multicyclic system (e.g., tetralin,
methylenedioxyphenyl).
[0394] As used herein, "halo" or "halogen" refers to fluoro,
chloro, bromo, and iodo. The term "perhalogenated" generally refers
to a moiety wherein all hydrogens are replaced by halogen
atoms.
[0395] "Counterion" is used to represent a small, negatively
charged species such as chloride, bromide, hydroxide, acetate, and
sulfate.
[0396] The term "non-hydrogen substituent" refers to substituents
other than hydrogen. Non-limiting examples include alkyl groups,
alkoxy groups, halogen groups, hydroxyl groups, aryl groups,
etc.
[0397] As used herein, "carbocycle" or "carbocyclic ring" is
intended to mean any stable monocyclic, bicyclic, or tricyclic ring
having the specified number of carbons, any of which may be
saturated, unsaturated, or aromatic. For example a C.sub.3-14
carbocycle is intended to mean a mono-, bi-, or tricyclic ring
having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms.
Examples of carbocycles include, but are not limited to,
cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl,
cyclohexyl, cycloheptenyl, cycloheptyl, cycloheptenyl, adamantyl,
cyclooctyl, cyclooctenyl, cyclooctadienyl, fluorenyl, phenyl,
naphthyl, indanyl, adamantyl, and tetrahydronaphthyl. Bridged rings
are also included in the definition of carbocycle, including, for
example, [3.3.0]bicyclooctane, [4.3.0]bicyclononane,
[4.4.0]bicyclodecane, and [2.2.2]bicyclooctane. A bridged ring
occurs when one or more carbon atoms link two non-adjacent carbon
atoms. In one embodiment, bridge rings are one or two carbon atoms.
It is noted that a bridge always converts a monocyclic ring into a
tricyclic ring. When a ring is bridged, the substituents recited
for the ring may also be present on the bridge. Fused (e.g.,
naphthyl and tetrahydronaphthyl) and spiro rings are also
included.
[0398] As used herein, the term "glycoside" means any molecule in
which a sugar group is bonded through its anomeric carbon to
another group. Examples of glycosides include, for example methyl
.alpha.-D-glucopyranoside,
##STR00220##
and methyl .beta.-D-glucopyranoside,
##STR00221##
Because a glycoside is bonded through its anomeric carbon to
another group, it is also known as a non-reducing sugar (i.e., it
is not subject to attack by reagents that attack carbonyl
groups).
[0399] As used herein, the term "heterocycle" or "heterocyclic" is
intended to mean any stable monocyclic, bicyclic, or tricyclic ring
which is saturated, unsaturated, or aromatic and comprises carbon
atoms and one or more ring heteroatoms, e.g., 1 or 1-2 or 1-3 or
1-4 or 1-5 or 1-6 heteroatoms, independently selected from the
group consisting of nitrogen, oxygen, and sulfur. A bicyclic or
tricyclic heterocycle may have one or more heteroatoms located in
one ring, or the heteroatoms may be located in more than one ring.
The nitrogen and sulfur heteroatoms may optionally be oxidized
(i.e., N.fwdarw.O and S(O).sub.p, where p=1 or 2). When a nitrogen
atom is included in the ring it is either N or NH, depending on
whether or not it is attached to a double bond in the ring (i.e., a
hydrogen is present if needed to maintain the tri-valency of the
nitrogen atom). The nitrogen atom may be substituted or
unsubstituted (i.e., N or NR wherein R is H or another substituent,
as defined). The heterocyclic ring may be attached to its pendant
group at any heteroatom or carbon atom that results in a stable
structure. The heterocyclic rings described herein may be
substituted on carbon or on a nitrogen atom if the resulting
compound is stable. A nitrogen in the heterocycle may optionally be
quatemized. In one embodiment, when the total number of S and O
atoms in the heterocycle exceeds 1, then these heteroatoms are not
adjacent to one another. Bridged rings are also included in the
definition of heterocycle. A bridged ring occurs when one or more
atoms (i.e., C, O, N, or S) link two non-adjacent carbon or
nitrogen atoms. Bridges include, but are not limited to, one carbon
atom, two carbon atoms, one nitrogen atom, two nitrogen atoms, and
a carbon-nitrogen group. It is noted that a bridge always converts
a monocyclic ring into a tricyclic ring. When a ring is bridged,
the substituents recited for the ring may also be present on the
bridge. Spiro and fused rings are also included.
[0400] As used herein, the term "aromatic heterocycle" or
"heteroaryl" is intended to mean a stable 5, 6, or 7-membered
monocyclic or bicyclic aromatic heterocyclic ring or 7, 8, 9, 10,
11, or 12-membered bicyclic aromatic heterocyclic ring which
consists of carbon atoms and one or more heteroatoms, e.g., 1 or
1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, independently selected
from the group consisting of nitrogen, oxygen, and sulfur. In the
case of bicyclic heterocyclic aromatic rings, only one of the two
rings needs to be aromatic (e.g., 2,3-dihydroindole), though both
may be (e.g., quinoline). The second ring can also be fused or
bridged as defined above for heterocycles. The nitrogen atom may be
substituted or unsubstituted (ie., N or NR wherein R is H or
another substituent, as defined). The nitrogen and sulfur
heteroatoms may optionally be oxidized (i.e., N.fwdarw.O and
S(O).sub.p, where p=1 or 2). It is to be noted that total number of
S and O atoms in the aromatic heterocycle is not more than 1.
[0401] Examples of heterocycles include, but are not limited to,
acridinyl, azocinyl, benzimidazolyl, benzofuranyl,
benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl,
benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,
benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl,
carbolinyl, chromenyl, chromenyl, cinnolinyl, decahydroquinolinyl,
2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran,
furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl,
1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,
3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl,
isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,
methylenedioxyphenyl, morpholinyl, naphthyridinyl,
octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,
1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,
1,2,4-oxadiazol5(4H)-one, oxazolidinyl, oxazolyl, oxindolyl,
pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl,
phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,
piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,
pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl,
pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole,
pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,
pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,
quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,
tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,
tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,
1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,
thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,
thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl,
1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, and
xanthenyl.
[0402] "Acyl" includes compounds and moieties that contain the acyl
radical (CH.sub.3CO--) or a carbonyl group. "Substituted acyl"
includes acyl groups where one or more of the hydrogen atoms are
replaced by for example, alkyl groups, alkynyl groups, halogens,
hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, allcylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkylamino,
diarylamino, arylamino, diarylamino, and alkylarylamino), acylamino
(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and
ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,
thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,
sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,
alkylaryl, or an aromatic or heteroaromatic moiety.
[0403] "Acylamino" includes moieties wherein an acyl moiety is
bonded to an amino group. For example, the term includes
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido
groups.
[0404] "Aroyl" includes compounds and moieties with an aryl or
heteroaromatic moiety bound to a carbonyl group. Examples of aroyl
groups include phenylcarboxy, naphthyl carboxy, etc.
[0405] "Alkoxyalkyl", "alkylaminoalkyl" and "thioalkoxyallcyl"
include alkyl groups, as described above, which further include
oxygen, nitrogen or sulfur atoms replacing one or more hydrocarbon
backbone carbon atoms, e.g., oxygen, nitrogen or sulfur atoms.
[0406] The term "alkoxy" or "alkoxyl" includes substituted and
unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked
to an oxygen atom. Examples of alkoxy groups (or alkoxyl radicals)
include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy
groups. Examples of substituted alkoxy groups include halogenated
alkoxy groups. The alkoxy groups can be substituted with groups
such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkylamino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moieties. Examples of halogen
substituted alkoxy groups include, but are not limited to,
fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy,
dichloromethoxy, and trichloromethoxy.
[0407] The term "thiocarbonyl" or "thiocarboxy" includes compounds
and moieties which contain a carbon connected with a double bond to
a sulfur atom.
[0408] The term "ether" or "alkoxy" includes compounds or moieties
which contain an oxygen bonded to two different carbon atoms or
heteroatoms. For example, the term includes "alkoxyalkyl" which
refers to an alkyl, alkenyl, or alkynyl group covalently bonded to
an oxygen atom which is covalently bonded to another alkyl
group.
[0409] The term "ester" includes compounds and moieties which
contain a carbon or a heteroatom bound to an oxygen atom which is
bonded to the carbon of a carbonyl group. The term "ester" includes
alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl,
propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc. The alkyl,
alkenyl, or alkynyl groups are as defined above.
[0410] The term "thioether" includes compounds and moieties which
contain a sulfur atom bonded to two different carbon or
heteroatoms. Examples of thioethers include, but are not limited to
alkthioalkyls, alkthioalkenyls, and alkthioalkynyls. The term
"alkthioalkyls" include compounds with an alkyl, alkenyl, or
alkynyl group bonded to a sulfur atom which is bonded to an alkyl
group. Similarly, the term "alkthioalkenyls" and alkthioalkynyls"
refer to compounds or moieties wherein an alkyl, alkenyl, or
alkynyl group is bonded to a sulfur atom which is covalently bonded
to an alkynyl group.
[0411] The term "hydroxy" or "hydroxyl" includes groups with an
--OH or --O.sup.-.
[0412] "Polycyclyl" or "polycyclic radical" refers to two or more
cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls,
aryls and/or heterocyclyls) in which two or more carbons are common
to two adjoining rings. Rings that are joined through non-adjacent
atoms are termed "bridged" rings. Each of the rings of the
polycycle can be substituted with such substituents as described
above, as for example, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, awloxycarbonyloxy, carboxylate,
alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl,
aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl,
arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, aminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkylamino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or
an aromatic or heteroaromatic moiety.
[0413] An "anionic group," as used herein, refers to a group that
is negatively charged at physiological pH. Anionic groups include
carboxylate, sulfate, sulfonate, sulfinate, sulfamate, tetrazolyl,
phosphate, phosphonate, phosphinate, or phosphorothioate or
functional equivalents thereof. "Functional equivalents" of anionic
groups are intended to include bioisosteres, e.g., bioisosteres of
a carboxylate group. Bioisosteres encompass both classical
bioisosteric equivalents and non-classical bioisosteric
equivalents. Classical and non-classical bioisosteres are known in
the art (see, e.g., Silverman, R. B. The Organic Chemistry of Drug
Design and Drug Action, Academic Press, Inc.: San Diego, Calif.,
1992, pp. 19-23). In one embodiment, an anionic group is a
carboxylate.
[0414] In the present specification, the structural formula of the
compound represents a certain isomer for convenience in some cases,
but the present invention includes all isomers such as geometrical
isomer, optical isomer based on an asymmetrical carbon,
stereoisomer, tautomer and the like which occur structurally and an
isomer mixture and is not limited to the description of the formula
for convenience, and may be any one of isomer or a mixture.
Therefore, an asymmetrical carbon atom may be present in the
molecule and an optically active compound and a racemic compound
may be present in the present compound, but the present invention
is not limited to them and includes any one. In addition, a crystal
polymorphism may be present but is not limiting, but any crystal
form may be single or a crystal form mixture, or an anhydride or
hydrate. Further, so-called metabolite which is produced by
degradation of the present compound in vivo is included in the
scope of the present invention.
[0415] "Isomerism" means compounds that have identical molecular
formulae but that differ in the nature or the sequence of bonding
of their atoms or in the arrangement of their atoms in space.
Isomers that differ in the arrangement of their atoms in space are
termed "stereoisomers". Stereoisomers that are not mirror images of
one another are termed "diastereoisomers", and stereoisomers that
are non-superimposable mirror images are termed "enantiomers", or
sometimes optical isomers. A carbon atom bonded to four
nonidentical substituents is termed a "chiral center".
[0416] "Chiral isomer" means a compound with at least one chiral
center. It has two enantiomeric forms of opposite chirality and may
exist either as an individual enantiomer or as a mixture of
enantiomers. A mixture containing equal amounts of individual
enantiomeric forms of opposite chirality is termed a "racemic
mixture". A compound that has more than one chiral center has
2.sup.n-1enantiomeric pairs, where n is the number of chiral
centers. Compounds with more than one chiral center may exist as
either an individual diastereomer or as a mixture of diastereomers,
termed a "diastereomeric mixture". When one chiral center is
present, a stereoisomer may be characterized by the absolute
configuration (R or S) of that chiral center. Absolute
configuration refers to the arrangement in space of the
substituents attached to the chiral center. The substituents
attached to the chiral center under consideration are ranked in
accordance with the Sequence Rule of Cahn, Ingold and Prelog. (Cahn
et al, Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Calm et
al., Angew. Chem. 1966, 78, 413; Calm and Ingold, J. Chem. Soc.
1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Calm,
J., Chem. Educ. 1964, 41, 116).
[0417] "Geometric Isomers" means the diastereomers that owe their
existence to hindered rotation about double bonds. These
configurations are differentiated in their names by the prefixes
cis and trans, or Z and E, which indicate that the groups are on
the same or opposite side of the double bond in the molecule
according to the Cahn-Ingold-Prelog rules.
[0418] Further, the structures and other compounds discussed in
this application include all atropic isomers thereof. "Atropic
isomers" are a type of stereoisomer in which the atoms of two
isomers are arranged differently in space. Atropic isomers owe
their existence to a restricted rotation caused by hindrance of
rotation of large groups about a central bond. Such atropic isomers
typically exist as a mixture, however as a result of recent
advances in chromatography techniques, it has been possible to
separate mixtures of two atropic isomers in select cases.
[0419] The terms "crystal polymorphs" or "polymorphs" or "crystal
forms" means crystal structures in which a compound (or salt or
solvate thereof) can crystallize in different crystal packing
arrangements, all of which have the same elemental composition.
Different crystal forms usually have different X-ray diffraction
patterns, infrared spectral, melting points, density hardness,
crystal shape, optical and electrical properties, stability and
solubility. Recrystallization solvent, rate of crystallization,
storage temperature, and other factors may cause one crystal form
to dominate. Crystal polymorphs of the compounds can be prepared by
crystallization under different conditions.
[0420] Additionally, the compounds of the present invention, for
example, the salts of the compounds, can exist in either hydrated
or unhydrated (the anhydrous) form or as solvates with other
solvent molecules. Nonlimiting examples of hydrates include
monohydrates, dihydrates, etc. Nonlimiting examples of solvates
include ethanol solvates, acetone solvates, etc.
[0421] "Solvates" means solvent addition forms that contain either
stoichiometric or non stoichiometric amounts of solvent. Some
compounds have a tendency to trap a fixed molar ratio of solvent
molecules in the crystalline solid state, thus forming a solvate.
If the solvent is water the solvate formed is a hydrate, when the
solvent is alcohol, the solvate formed is an alcoholate. Hydrates
are formed by the combination of one or more molecules of water
with one of the substances in which the water retains its molecular
state as H.sub.2O, such combination being able to form one or more
hydrate.
[0422] "Tautomers" refers to compounds whose structures differ
markedly in arrangement of atoms, but which exist in easy and rapid
equilibrium. It is to be understood that the compounds of the
invention may be depicted as different tautomers. It should also be
understood that when compounds have tautomeric forms, all
tautomeric forms are intended to be within the scope of the
invention, and the naming of the compounds does not exclude any
tautomer form.
[0423] Some compounds of the present invention can exist in a
tautomeric form which are also intended to be encompassed within
the scope of the present invention.
[0424] The compounds, salts and prodrugs of the present invention
can exist in several tautomeric forms, including the enol and imine
form, and the keto and enamine form and geometric isomers and
mixtures thereof. All such tautomeric forms are included within the
scope of the present invention. Tautomers exist as mixtures of a
tautomeric set in solution. In solid form, usually one tautomer
predominates. Even though one tautomer may be described, the
present invention includes all tautomers of the present
compounds
[0425] A tautomer is one of two or more structural isomers that
exist in equilibrium and are readily converted from one isomeric
form to another. This reaction results in the formal migration of a
hydrogen atom accompanied by a switch of adjacent conjugated double
bonds. In solutions where tautomerization is possible, a chemical
equilibrium of the tautomers will be reached. The exact ratio of
the tautomers depends on several factors, including temperature,
solvent, and pH. The concept of tautomers that are interconvertable
by tautomerizations is called tautomerism.
[0426] Of the various types of tautomerism that are possible, two
are commonly observed. In keto-enol tautomerism a simultaneous
shift of electrons and a hydrogen atom occurs. Ring-chain
tautomerism, is exhibited by glucose. It arises as a result of the
aldehyde group (--CHO) in a sugar chain molecule reacting with one
of the hydroxy groups (--OH) in the same molecule to give it a
cyclic (ring-shaped) form.
[0427] Tautomerizations are catalyzed by: Base: 1. deprotonation;
2. formation of a delocalized anion (e.g. an enolate); 3.
protonation at a different position of the anion; Acid: 1.
protonation; 2. formation of a delocalized cation; 3. deprotonation
at a different position adjacent to the cation.
[0428] Common tautomeric pairs are: ketone-enol, amide-nitrile,
lactam-lactim, amide-imidic acid tautomerism in heterocyclic rings
(e.g. in the nucleobases guanine, thymine, and cytosine),
amine-enamine and enamine-enamine. Examples include:
##STR00222##
[0429] It will be noted that the structure of some of the compounds
of the invention include asymmetric carbon atoms. It is to be
understood accordingly that the isomers arising from such asymmetry
(e.g., all enantiomers and diastereomers) are included within the
scope of the invention, unless indicated otherwise. Such isomers
can be obtained in substantially pure form by classical separation
techniques and by stereochemically controlled synthesis.
Furthermore, the structures and other compounds and moieties
discussed in this application also include all tautomers thereof.
Alkenes can include either the E- or Z-geometry, where appropriate.
The compounds of this invention may exist in stereoisomeric form,
therefore can be produced as individual stereoisomers or as
mixtures.
[0430] As used herein, the term "analog" refers to a chemical
compound that is structurally similar to another but differs
slightly in composition (as in the replacement of one atom by an
atom of a different element or in the presence of a particular
functional group, or the replacement of one functional group by
another functional group). Thus, an analog is a compound that is
similar or comparable in function and appearance, but not in
structure or origin to the reference compound.
[0431] As defined herein, the term "derivative", refers to
compounds that have a common core structure, and are substituted
with various groups as described herein. For example, all of the
compounds represented by formula I are indole derivatives, and have
formula I as a common core.
[0432] The term "bioisostere" refers to a compound resulting from
the exchange of an atom or of a group of atoms with another,
broadly similar, atom or group of atoms. The objective of a
bioisosteric replacement is to create a new compound with similar
biological properties to the parent compound. The bioisosteric
replacement may be physicochemically or topologically based.
Examples of carboxylic acid bioisosteres include acyl sulfonimides,
tetrazoles, sulfonates, and phosphonates. See, e.g., Patani and
LaVoie, Chem. Rev. 96, 3147-3176 (1996).
[0433] A "pharmaceutical composition" is a formulation containing
the disclosed compounds in a form suitable for administration to a
subject. In one embodiment, the pharmaceutical composition is in
bulk or in unit dosage form. The unit dosage form is any of a
variety of forms, including, for example, a capsule, an IV bag, a
tablet, a single pump on an aerosol inhaler, or a vial. The
quantity of active ingredient (e.g., a formulation of the disclosed
compound or salt, hydrate, solvate, or isomer thereof) in a unit
dose of composition is an effective amount and is varied according
to the particular treatment involved. One skilled in the art will
appreciate that it is sometimes necessary to make routine
variations to the dosage depending on the age and condition of the
patient. The dosage will also depend on the route of
administration. A variety of routes are contemplated, including
oral, pulmonary, rectal, parenteral, transdermal, subcutaneous,
intravenous, intramuscular, intraperitoneal, inhalational, buccal,
sublingual, intrapleural, intrathecal, intranasal, and the like.
Dosage forms for the topical or transdermal administration of a
compound of this invention include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches and inhalants. In
one embodiment, the active compound is mixed under sterile
conditions with a pharmaceutically acceptable carrier, and with any
preservatives, buffers, or propellants that are required.
[0434] The term "flash dose" refers to compound formulations that
are rapidly dispersing dosage forms.
[0435] The term "immediate release" is defined as a release of
compound from a dosage form in a relatively brief period of time,
generally up to about 60 minutes. The term "modified release" is
defined to include delayed release, extended release, and pulsed
release. The term "pulsed release" is defined as a series of
releases of drug from a dosage form. The term "sustained release"
or "extended release" is defined as continuous release of a
compound from a dosage form over a prolonged period.
[0436] A "subject" includes mammals, e.g., humans, companion
animals (e.g., dogs, cats, birds, and the like), farm animals
(e.g., cows, sheep, pigs, horses, fowl, and the like) and
laboratory animals (e.g., rats, mice, guinea pigs, birds, and the
like). In one embodiment, the subject is human.
[0437] As used herein, the phrase "pharmaceutically acceptable"
refers to those compounds, materials, compositions, carriers,
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.
[0438] "Pharmaceutically acceptable excipient" means an excipient
that is useful in preparing a pharmaceutical composition that is
generally safe, non-toxic and neither biologically nor otherwise
undesirable, and includes excipient that is acceptable for
veterinary use as well as human pharmaceutical use. A
"pharmaceutically acceptable excipient" as used in the
specification and claims includes both one and more than one such
excipient.
[0439] The compounds of the invention are capable of further
forming salts. All of these forms are also contemplated within the
scope of the claimed invention.
[0440] "Pharmaceutically acceptable salt" of a compound means a
salt that is pharmaceutically acceptable and that possesses the
desired pharmacological activity of the parent compound.
[0441] As used herein, "pharmaceutically acceptable salts" refer to
derivatives of the disclosed compounds wherein the parent compound
is modified by making acid or base salts thereof. 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 include
the conventional non-toxic salts or the quaternary ammonium salts
of the parent compound formed, for example, from non-toxic
inorganic or organic acids. For example, such conventional
non-toxic salts include, but are not limited to, those derived from
inorganic and organic acids selected from 2-acetoxybenzoic,
2-hydroxyethane sulfonic, acetic, ascorbic, benzene sulfonic,
benzoic, bicarbonic, carbonic, citric, edetic, ethane disulfonic,
1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic, glutamic,
glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic,
hydrobromic, hydrochloric, hydroiodic, hydroxymaleic,
hydroxynaphthoic, isethionic, lactic, lactobionic, lauryl sulfonic,
maleic, malic, mandelic, methane sulfonic, napsylic, nitric,
oxalic, pamoic, pantothenic, phenylacetic, phosphoric,
polygalacturonic, propionic, salicyclic, stearic, subacetic,
succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, toluene
sulfonic, and the commonly occurring amine acids, e.g., glycine,
alanine, phenylalanine, arginine, etc.
[0442] Other examples include hexanoic acid, cyclopentane propionic
acid, pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid,
cinnamic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic
acid, 4-toluenesulfonic acid, camphorsulfonic acid,
4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylic acid,
3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic
acid, muconic acid, and the like. The invention also encompasses
salts formed when an acidic proton present in the parent compound
either is replaced by a metal ion, e.g., an alkali metal ion, an
alkaline earth ion, or an aluminum ion; or coordinates with an
organic base such as ethanolamine, diethanolamine, triethanolamine,
tromethamine, N-methylglucamine, and the like.
[0443] It should be understood that all references to
pharmaceutically acceptable salts include solvent addition forms
(solvates) or crystal forms (polymorphs) as defined herein, of the
same salt.
[0444] The pharmaceutically acceptable salts of the present
invention can be synthesized from a parent compound that 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; non-aqueous media like ether, ethyl acetate, ethanol,
isopropanol, or acetonitrile can be used. Lists of suitable salts
are found in Remington's Pharmaceutical Sciences, 18th ed. (Mack
Publishing Company, 1990). For example, salts can include, but are
not limited to, the hydrochloride and acetate salts of the
aliphatic amine-containing, hydroxyl amine-containing, and
imine-containing compounds of the present invention.
[0445] The compounds of the present invention can also be prepared
as esters, for example pharmaceutically acceptable esters. For
example a carboxylic acid function group in a compound can be
converted to its corresponding ester, e.g., a methyl, ethyl, or
other ester. Also, an alcohol group in a compound can be converted
to its corresponding ester, e.g., an acetate, propionate, or other
ester.
[0446] The compounds of the present invention can also be prepared
as prodrugs, for example pharmaceutically acceptable prodrugs. The
terms "pro-drug" and "prodrug" are used interchangeably herein and
refer to any compound which releases an active parent drug in vivo.
Since prodrugs are known to enhance numerous desirable qualities of
pharmaceuticals (e.g., solubility, bioavailability, manufacturing,
etc.) the compounds of the present invention can be delivered in
prodrug form. Thus, the present invention is intended to cover
prodrugs of the presently claimed compounds, methods of delivering
the same and compositions containing the same. "Prodrugs" are
intended to include any covalently bonded carriers that release an
active parent drug of the present invention in vivo when such
prodrug is administered to a subject. Prodrugs the present
invention are prepared by modifying functional groups present in
the compound in such a way that the modifications are cleaved,
either in routine manipulation or in vivo, to the parent compound.
Prodrugs include compounds of the present invention wherein a
hydroxy, amino, sulfhydryl, carboxy, or carbonyl group is bonded to
any group that, may be cleaved in vivo to form a free hydroxyl,
free amino, free sulfhydryl, free carboxy or free carbonyl group,
respectively.
[0447] Examples of prodrugs include, but are not limited to, esters
(e.g., acetate, dialkylaminoacetates, formates, phosphates,
sulfates, and benzoate derivatives) and carbamates (e.g.,
N,N-dimethylaminocarbonyl) of hydroxy functional groups, esters
groups (e.g. ethyl esters, morpholinoethanol esters) of carboxyl
functional groups, N-acyl derivatives (e.g. N-acetyl) N-Mannich
bases, Schiff bases and enaminones of amino functional groups,
oximes, acetals, ketals and enol esters of ketone and aldehyde
functional groups in compounds of Formula I, and the like, See
Bundegaard, H. "Design of Prodrugs" p 1-92, Elesevier, New
York-Oxford (1985).
[0448] "Protecting group" refers to a grouping of atoms that when
attached to a reactive group in a molecule masks, reduces or
prevents that reactivity. Examples of protecting groups can be
found in Green and Wuts, Protective Groups in Organic Chemistry,
(Wiley, 2.sup.nd ed. 1991); Harrison and Harrison et al.,
Compendium of Synthetic Organic Methods, Vols. 1-8 (John Wiley and
Sons, 1971-1996); and Kocienski, Protecting Groups, (Verlag,
3.sup.rd ed. 2003).
[0449] The term "amine protecting group" is intended to mean a
functional group that converts an amine, amide, or other
nitrogen-containing moiety into a different chemical group that is
substantially inert to the conditions of a particular chemical
reaction. Amine protecting groups are preferably removed easily and
selectively in good yield under conditions that do not affect other
functional groups of the molecule. Examples of amine protecting
groups include, but are not limited to, formyl, acetyl, benzyl,
t-butyldimethylsilyl, t-butdyldiphenylsilyl, t-butyloxycarbonyl
(Boc), p-methoxybenzyl, methoxymethyl, tosyl, trifluoroacetyl,
trimethylsilyl (TMS), fluorenyl-methyloxycarbonyl,
2-trimethylsilyl-ethyoxycarbonyl,
1-methyl-1-(4-biphenylyl)ethoxycarbonyl, allyloxycarbonyl,
benzyloxycarbonyl (CBZ), 2-trimethylsilyl-ethanesulfonyl (SES),
trityl and substituted trityl groups, 9-fluorenylmethyloxycarbonyl
(FMOC), nitro-veratryloxycarbonyl (NVOC), and the like. Other
suitable amine protecting groups are straightforwardly identified
by those of skill in the art.
[0450] Representative hydroxy protecting groups include those where
the hydroxy group is either acylated or alkylated such as benzyl,
and trityl ethers as well as alkyl ethers, tetrahydropyranyl
ethers, trialkylsilyl ethers and allyl ethers.
[0451] "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.
[0452] In the specification, the singular forms also include the
plural, unless the context clearly dictates otherwise. Unless
defined otherwise, all technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. In the case of
conflict, the present specification will control.
[0453] All percentages and ratios used herein, unless otherwise
indicated, are by weight.
[0454] "Combination therapy" (or "co-therapy") includes the
administration of a compound of the invention and at least a second
agent as part of a specific treatment regimen intended to provide
the beneficial effect from the co-action of these therapeutic
agents. The beneficial effect of the combination includes, but is
not limited to, pharmacokinetic or pharmacodynamic co-action
resulting from the combination of therapeutic agents.
Administration of these therapeutic agents in combination typically
is carried out over a defined time period (usually minutes, hours,
days or weeks depending upon the combination selected).
"Combination therapy" may, but generally is not, intended to
encompass the administration of two or more of these therapeutic
agents as part of separate monotherapy regimens that incidentally
and arbitrarily result in the combinations of the present
invention.
[0455] "Combination therapy" is intended to embrace administration
of these therapeutic agents in a sequential manner, that is,
wherein each therapeutic agent is administered at a different time,
as well as administration of these therapeutic agents, or at least
two of the therapeutic agents, in a substantially simultaneous
manner. Substantially simultaneous administration can be
accomplished, for example, by administering to the subject a single
capsule having a fixed ratio of each therapeutic agent or in
multiple, single capsules for each of the therapeutic agents.
Sequential or substantially simultaneous administration of each
therapeutic agent can be effected by any appropriate route
including, but not limited to, oral routes, intravenous routes,
intramuscular routes, and direct absorption through mucous membrane
tissues. The therapeutic agents can be administered by the same
route or by different routes. For example, a first therapeutic
agent of the combination selected may be administered by
intravenous injection while the other therapeutic agents of the
combination may be administered orally. Alternatively, for example,
all therapeutic agents may be administered orally or all
therapeutic agents may be administered by intravenous injection.
The sequence in which the therapeutic agents are administered is
not narrowly critical.
[0456] "Combination therapy" also embraces the administration of
the therapeutic agents as described above in further combination
with other biologically active ingredients and non-drug therapies
(e.g., surgery or radiation treatment) . Where the combination
therapy further comprises a non-drug treatment, the non-drug
treatment may be conducted at any suitable time so long as a
beneficial effect from the co-action of the combination of the
therapeutic agents and non-drug treatment is achieved. For example,
in appropriate cases, the beneficial effect is still achieved when
the non-drug treatment is temporally removed from the
administration of the therapeutic agents, perhaps by days or even
weeks.
[0457] Throughout the description, where compositions are described
as having, including, or comprising specific components, it is
contemplated that compositions also consist essentially of, or
consist of, the recited components. Similarly, where processes are
described as having, including, or comprising specific process
steps, the processes also consist essentially of, or consist of,
the recited processing steps. Further, it should be understood that
the order of steps or order for performing certain actions are
immaterial so long as the invention remains operable. Moreover, two
or more steps or actions may be conducted simultaneously.
[0458] The compounds, or pharmaceutically acceptable salts thereof,
is administered orally, nasally, transdermally, pulmonary,
inhalationally, buccally, sublingually, intraperintoneally,
subcutaneously, intramuscularly, intravenously, rectally,
intrapleurally, intrathecally and parenterally. In one embodiment,
the compound is administered orally. One skilled in the art will
recognize the advantages of certain routes of administration.
[0459] The dosage regimen utilizing the compounds is selected in
accordance with a variety of factors including type, species, age,
weight, sex and medical condition of the patient; the severity of
the condition to be treated; the route of administration; the renal
and hepatic function of the patient; and the particular compound or
salt thereof employed. An ordinarily skilled physician or
veterinarian can readily determine and prescribe the effective
amount of the drug required to prevent, counter or arrest the
progress of the condition.
[0460] Techniques for formulation and administration of the
disclosed compounds of the invention can be found in Remington: the
Science and Practice of Pharmacy, 19.sup.th edition, Mack
Publishing Co., Easton, Pa. (1995). In an embodiment, the compounds
described herein, and the pharmaceutically acceptable salts
thereof, are used in pharmaceutical preparations in combination
with a pharmaceutically acceptable carrier or diluent. Suitable
pharmaceutically acceptable carriers include inert solid fillers or
diluents and sterile aqueous or organic solutions. The compounds
will be present in such pharmaceutical compositions in amounts
sufficient to provide the desired dosage amount in the range
described herein.
[0461] In one embodiment, the compound is prepared for oral
administration, wherein the disclosed compounds or salts thereof
are combined with a suitable solid or liquid carrier or diluent to
form capsules, tablets, pills, powders, syrups, solutions,
suspensions and the like.
[0462] The tablets, pills, capsules, and the like contain from
about 1 to about 99 weight percent of the active ingredient and a
binder such as gum tragacanth, acacias, corn starch or gelatin;
excipients such as dicalcium phosphate; a disintegrating agent such
as corn starch, potato starch or alginic acid; a lubricant such as
magnesium stearate; and/or a sweetening agent such as sucrose,
lactose, saccharin, xylitol, and the like. When a dosage unit form
is a capsule, it often contains, in addition to materials of the
above type, a liquid carrier such as a fatty oil.
[0463] In some embodiments, various other materials are present as
coatings or to modify the physical form of the dosage unit. For
instance, in some embodiments, tablets are coated with shellac,
sugar or both. In some embodiments, a syrup or elixir contains, in
addition to the active ingredient, sucrose as a sweetening agent,
methyl and propylparabens as preservatives, a dye and a flavoring
such as cherry or orange flavor, and the like.
[0464] For some embodiments relating to parental administration,
the disclosed compounds, or salts, solvates, tautomers or
polymorphs thereof, can be combined with sterile aqueous or organic
media to form injectable solutions or suspensions. In one
embodiment, injectable compositions are aqueous isotonic solutions
or suspensions. The compositions may be sterilized and/or contain
adjuvants, such as preserving, stabilizing, wetting or emulsifying
agents, solution promoters, salts for regulating the osmotic
pressure and/or buffers. In addition, they may also contain other
therapeutically valuable substances. The compositions are prepared
according to conventional mixing, granulating or coating methods,
respectively, and contain about 0.1 to 75%, in another embodiment,
the compositions contain about 1 to 50%, of the active
ingredient.
[0465] For example, injectable solutions are produced using
solvents such as sesame or peanut oil or aqueous propylene glycol,
as well as aqueous solutions of water-soluble
pharmaceutically-acceptable salts of the compounds. In some
embodiments, dispersions are prepared in glycerol, liquid
polyethylene glycols and mixtures thereof in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms. The terms
"parenteral administration" and "administered parenterally" as used
herein means modes of administration other than enteral and topical
administration, usually by injection, and includes, without
limitation, intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal and
intrasternal injection and infusion.
[0466] For rectal administration, suitable pharmaceutical
compositions are, for example, topical preparations, suppositories
or enemas. Suppositories are advantageously prepared from fatty
emulsions or suspensions. The compositions may be sterilized and/or
contain adjuvants, such as preserving, stabilizing, wetting or
emulsifying agents, solution promoters, salts for regulating the
osmotic pressure and/or buffers. In addition, they may also contain
other therapeutically valuable substances. The compositions are
prepared according to conventional mixing, granulating or coating
methods, respectively, and contain about 0.1 to 75%, in another
embodiment, compositions contain about 1 to 50%, of the active
ingredient.
[0467] In some embodiments, the compounds are formulated to deliver
the active agent by pulmonary administration, e.g., administration
of an aerosol formulation containing the active agent from, for
example, a manual pump spray, nebulizer or pressurized metered-dose
inhaler. In some embodiments, suitable formulations of this type
also include other agents, such as antistatic agents, to maintain
the disclosed compounds as effective aerosols.
[0468] A drug delivery device for delivering aerosols comprises a
suitable aerosol canister with a metering valve containing a
pharmaceutical aerosol formulation as described and an actuator
housing adapted to hold the canister and allow for drug delivery.
The canister in the drug delivery device has a headspace
representing greater than about 15% of the total volume of the
canister. Often, the polymer intended for pulmonary administration
is dissolved, suspended or emulsified in a mixture of a solvent,
surfactant and propellant. The mixture is maintained under pressure
in a canister that has been sealed with a metering valve.
[0469] For nasal administration, either a solid or a liquid carrier
can be used. The solid carrier includes a coarse powder having
particle size in the range of, for example, from about 20 to about
500 microns and such formulation is administered by rapid
inhalation through the nasal passages. In some embodiments where
the liquid carrier is used, the formulation is administered as a
nasal spray or drops and includes oil or aqueous solutions of the
active ingredients.
[0470] Also contemplated are formulations that are rapidly
dispersing dosage forms, also known as "flash dose" forms. In
particular, some embodiments of the present invention are
formulated as compositions that release their active ingredients
within a short period of time, e.g., typically less than about five
minutes, in another embodiment, less than about ninety seconds, in
another embodiment, less than about thirty seconds and in another
embodiment, in less than about ten or fifteen seconds. Such
formulations are suitable for administration to a subject via a
variety of routes, for example by insertion into a body cavity or
application to a moist body surface or open wound.
[0471] Typically, a "flash dosage" is a solid dosage form that is
administered orally, which rapidly disperses in the mouth, and
hence does not require great effort in swallowing and allows the
compound to be rapidly ingested or absorbed through the oral
mucosal membranes. In some embodiments, suitable rapidly dispersing
dosage forms are also used in other applications, including the
treatment of wounds and other bodily insults and diseased states in
which release of the medicament by externally supplied moisture is
not possible.
[0472] "Flash dose" forms are known in the art; see for example,
effervescent dosage forms and quick release coatings of insoluble
microparticles in U.S. Pat. Nos. 5,578,322 and 5,607,697; freeze
dried foams and liquids in U.S. Pat. Nos. 4,642,903 and 5,631,023;
melt spinning of dosage forms in U.S. Pat. Nos. 4,855,326,
5,380,473 and 5,518,730; solid, free-form fabrication in U.S. Pat.
No. 6,471,992; saccharide-based carrier matrix and a liquid binder
in U.S. Pat. Nos. 5,587,172, 5,616,344, 6,277,406, and 5,622,719;
and other forms known to the art.
[0473] The compounds of the invention are also formulated as
"pulsed release" formulations, in which the compound is released
from the pharmaceutical compositions in a series of releases (i.e.,
pulses). The compounds are also formulated as "sustained release"
formulations in which the compound is continuously released from
the pharmaceutical composition over a prolonged period.
[0474] Also contemplated are formulations, e.g., liquid
formulations, including cyclic or acyclic encapsulating or
solvating agents, e.g., cyclodextrins, polyethers, or
polysaccharides (e.g., methylcellulose), or in another embodiment,
polyanionic .beta.-cyclodextrin derivatives with a sodium sulfonate
salt group separate from the lipophilic cavity by an alkyl ether
spacer group or polysaccharides. In one embodiment, the agent is
methylcellulose. In another embodiment, the agent is a polyanionic
.beta.-cyclodextrin derivative with a sodium sulfonate salt
separated from the lipophilic cavity by a butyl ether spacer group,
e.g., CAPTISOL.RTM. (CyDex, Overland, Kans.). One skilled in the
art can evaluate suitable agent/disclosed compound formulation
ratios by preparing a solution of the agent in water, e.g., a 40%
by weight solution; preparing serial dilutions, e.g. to make
solutions of 20%, 10, 5%, 2.5%, 0% (control), and the like; adding
an excess (compared to the amount that can be solubilized by the
agent) of the disclosed compound; mixing under appropriate
conditions, e.g., heating, agitation, sonication, and the like;
centrifuging or filtering the resulting mixtures to obtain clear
solutions; and analyzing the solutions for concentration of the
disclosed compound.
[0475] All publications and patent documents cited herein are
incorporated herein by reference as if each such publication or
document was specifically and individually indicated to be
incorporated herein by reference. Citation of publications and
patent documents is not intended as an admission that any is
pertinent prior art, nor does it constitute any admission as to the
contents or date of the same. The invention having now been
described by way of written description, those of skill in the art
will recognize that the invention can be practiced in a variety of
embodiments and that the foregoing description and examples below
are for purposes of illustration and not limitation of the claims
that follow.
EXAMPLES
Example 1
Synthesis and Activity of Indole Derivative Protein Kinase and/or
Protein Phosphatase Inhibitors
[0476] The following results show the solution phase synthesis of
5-fluoroindole-2-carbaxamide libraries and testing of indole
derived protein kinase and/or protein phosphatase inhibitors. These
final products are examples of indole-based inhibitors wherein
synthesis with a 5-fluoro group is illustrated.
A. Synthesis of Intermediates and Sample Reagents:
5-fluoro-3-phenylindole-2-carboxylic acid
##STR00223##
[0477] (a) Preparation of Methyl Ester
##STR00224##
[0479] A mixture of 5-fluoroindole-2-carboxylic acid (6 g, 33.5
mmol) and a freshly prepared methanolic HCl (100 mL) was stirred
overnight at room temperature. The precipitated ester was collected
by filtration, washed with NaHCO.sub.3 saturated solution, water,
and MeOH. The filtrate was treated with saturated NaHCO.sub.3 and
extracted with EtOAc. The organic layer was washed with brine,
dried (MgSO.sub.4), and evaporated in vacuo. The product ester (6
g) was an off white solid and it was used for the next step without
further purification: MP 200-201.degree. C.; .sup.1H NMR
(CDCl.sub.3, 500 MHz) .delta. 8.94 (br, 1H), 7.33 (dd, 1H, J=9.2
and 4.3 Hz), 7.30 (dd, 1H, J=2.2 and 9.2), 7.15 (d, 1H, J=2.1 Hz),
7.07 (ddd, 1H, J=2.5, 8.9 and 9.1 Hz), 3.92 (s, 3H)
(b) Preparation of the 3-iodo Derivative
##STR00225##
[0480] 4.22 g (21.8 mmol) of the methyl ester was dissolved in DMF
(25 mL). In another flask, a solution of iodine (6.09 g, 24 mmol)
and KOH (4.65 g, 82.9 mmol) in DMF (25 mL) was stirred for 30
minutes and added dropwise to the ester solution over 5 minutes.
After stirring for 10 minutes at room temperature, the reaction was
quenched by pouring into a solution of NaHSO.sub.3 (2.2 g),
NH.sub.4OH (25% solution in H.sub.2O) in 300 mL water. The mixture
was stirred for 30 minutes then the precipitated solid product was
collected by filtration and washed with H.sub.2O: .sup.1H NMR
(CDCl.sub.3, 500 MHz) .delta. 9.17 (br, 1H), 7.33 (dd, 1H, J=9.0
and 4.2 Hz) 7.21 (dd, 1H J=9.0 and 2.0 Hz), 7.12 (dt, 1H, J=9.0 and
2.0 Hz), 3.81 (s, 3H).
(c) Suzuki Coupling
##STR00226##
[0482] The iodo derivative was mixed with benzeneboronic acid (2.76
g, 22 mmol), PdCl.sub.2(PPh.sub.3).sub.2 (0.7 g, 1 mmol), and 50 mL
of 2M Na.sub.2CO.sub.3 in dioxane (200 mL). The mixture was stirred
at 90.degree. C. overnight. The solvent was evaporated under
vacuum. The product was extracted with EtOAc (3.times.200 mL). The
combined extract was washed with brine, dried with MgSO.sub.4, and
purified by crystallization (CH.sub.2Cl.sub.2-hexane) and silica
gel chromatography (Hexane-EtOAc 4:1): MP 189.degree. C.; .sup.1H
NMR (CDCl.sub.3, 500 MHz) .delta. 8.94 (br, 1H), 7.51 (dd, 2H,
J=1.5 and 7.9 Hz), 7.45 (ddd, 2H, J=1.8, 7.3 and 7.8), 7.39-7.34
(complex, 2H), 7.25 (dd, 1H, J=2.5 and 8.7 Hz), 7.10 (ddd, 1H,
J=2.5, 8.9 and 9.1 Hz), 3.80 (s, 3H).
(d) Saponification of Methyl Ester
[0483] The ester described above (2.5 g, 9.28 mmol) was dissolved
in THF (30 mL). A solution of LiOH (2.4 g, 100 mmol) in water (20
mL) was added and the mixture was heated at reflux for 1 hour. The
mixture was cooled to room temperature and THF was removed by
vacuum evaporation. The mixture was treated with 2M HCl until it
became acidic. The product was extracted with EtOAc. The organic
layer was washed, dried (brine, Na.sub.2SO.sub.4), and concentrated
under vacuum. The crude solid product was redissolved in
NaHCO.sub.3 (saturated solution) and washed several times with
CH.sub.2Cl.sub.2. The aqueous layer was acidified with ice and 2M
HCl and extracted with EtOAc. After washing, drying, and
rotavaping, the product was collected as white solid (yield 2.3 g,
97%): MP 195-196.degree. C.; .sup.1H NMR (CDCl.sub.3, 500 MHz)
.delta. 8.94 (br, 1H), 7.52 (dd, 2H, J=1.8 and 7.9 Hz), 7.46 (ddd,
2H, J=1.8, 7.3 and 7.6), 7.40 (ddd, 1H, J=1.8, 7.4 and 7.8 Hz),
7.37 (dd, 1H, J=9.0 and 4.2 Hz), 7.25 (dd, 1H, J=2.5 and 8.7 Hz),
7.12 (ddd, 1H, J=2.4, 8.8 and 8.9. Hz).
3-benzyloxy-5-hydroitybenzonitrile
##STR00227##
[0485] To a mixture of 3,5-dihydroxybenzonitrile (1.08 g, 8 mmol)
and K.sub.2CO.sub.3 (1.104 g, 8 mmol) in CH.sub.3CN (50 mL), benzyl
bromide (1.438 g, 8 mmol) was added. The mixture was heated to
reflux for 2 hours. Solvent was evaporated under vacuum. The
residue was treated with EtOAc (200 mL) and 1M HCl (200 mL). The
organic layer was washed, dried, and evaporated in vacuo. The
residue was chromatographed (gradient,
Hexanes-CH.sub.2Cl.sub.2-MeOH) to give
3-benzyloxy-5-hydroxybenzonitrile (529 mg, 29%),
3,5-dibenzyloxybenzonitryl (784 mg, 31%) and 256 mg (23.7 mg, 24%)
of the starting material. The product
3-benzyloxy-5-hydroxybenzonitrile had: MP 144-145.degree. C.;
.sup.1H NMR .delta. 9.15 (s, 1H, OH), 7.47 (d, 2H, J=7.0 Hz, 2' and
6'), 7.40 (ddd, 2H, J=7.0, 7.0, 2.0 Hz, 3' and 5'), 7.34 (dd, 1H,
J=7.7 and 2.1 Hz, 4'), 6.89 (dd, 1H, J=1.5 Hz, 4), 6.78 (d, 2H,
J=1.8 Hz), 5.16 (s, 2H).
3,5-dibenzyloxybenzonitryl
##STR00228##
[0487] This compound had MP 106.degree. C.; .sup.1H NMR
(CDCl.sub.3, 500 MHz) .delta. 7.38 (complex, 10H), 6.83 (d, 2H,
J=2.1 Hz), 6.79 (d, 1H, J=2.1 Hz).
4-benzyloxy-3-hydroxybenzonitrile
##STR00229##
[0489] A mixture of 3,4-dihydroxybenzonitrile (540 mg, 4 mmol),
K.sub.2CO.sub.3 (552 mg, 4 mmol) and benzyl bromide (476 mg, 4
mmol) in acetone (20 mL) was stirred at room temperature for 3
days. The mixture was evaporated under vacuum and subjected to
flash column chromatography (2% MeOH in toluene-hexane, 2:1) to
give the desired product (224 mg, 25%): MP 101.degree. C.; .sup.1H
NMR (Acetone-d.sub.6, 500 MHz) .delta. 8.55 (s, 1H), 7.50 (d, 2H,
J=7.3 Hz), 7.39 (dd, 2H, J=7.0 and 7.3), 7.34 (dd, 1H, J=7.0 and
7.3), 7.2 (m, complex, 3H), 5.25 (s, 2H).
3-hydroxy-4-propyloxybenzonitrile
##STR00230##
[0491] This compound was prepared following a similar procedure
used to prepare 4-benzyIoxy-3-hydroxybenzonitrile in 27% yield: MP
99.degree. C.; .sup.1H NMR (Acetone-d.sub.6, 500 MHz) .delta. 8.33
(s, 1H), 7.21 (dd, 1H, J=8.2 and 1.8 Hz), 7.13 (d, 1H, J=1.8 Hz),
7.08 (d, 1H, J=8.3 Hz), 4.07 (t, 2H, J=6.4 Hz), 1.80 (m, 2H), 1.01
(t, 3H, J=7.3 Hz).
3-benzyloxy-5-hydroxybenzylamine
##STR00231##
[0493] 3-benzyloxy-5-hydroxybenzonitrile (225 mg, 1 mmol) was
dissolved in 2 mL THF. 2 mL of BH.sub.3-THF (1.5 M in THF and
ether) was added dropwise, then the mixture was heated at reflux
temperature for 3 hours. After cooling, the mixture was carefully
poured to 3M HCl (ice cooled) and allowed to stir for 20 hours at
room temperature. The mixture was neutralized with solid
NaHCO.sub.3, thus the product precipitated as a white solid. The
product was collected by filtration, washed with water, and dried
(140 mg, 61%): MP 164-166.degree. C. (dec); .sup.1H NMR
(DMSO-d.sub.6, 400 MHz) .delta. 9.28 (br, 1H), 7.41 (d, 2H, J=6.9
Hz), 7.36 (dd, 2H, J=7.0 and 7.6 Hz), 7.30 (dd, 1H, J=7.0 and 6.6
Hz), 6.43 (s, 1H), 6.32 (s, 1H), 6.21 (dd, 1H, J=2.2 and 2.0 Hz),
4.99 (S, 2H), 3.57 (S, 2H).
3,5-dibenzytoxybenzylamine
##STR00232##
[0495] This compound was prepared according to the procedure used
in preparation of 3-benzyloxy-5-hydroxybenzylamine. The reaction
was quenched via addition of MeOH and the mixture was left to stir
overnight. The solvent was removed and the product was obtained by
flash column chromatography (CH.sub.2Cl.sub.2-Hexanes containing 5%
MeOH) as clear thick oil (90%): .sup.1H NMR (Acetone-4, 500 MHz)
.delta. 7.46 (d, 4H, J=7.6), 7.37 (dd, 4H, J=7.3 and 7.6), 7.31
(dd, 2H, J=7.3 and 7.0), 6.65 (d, 1H, J=2.1 Hz), 6.64 (d, 1H, J=2.0
Hz), 6.52 (dd, 1H, J=2.0 and 2.2 Hz), 5.07 (s, 4H), 4.35 (s, 2H),
1.97 (br, 1H), 1.85 (br, 1H).
4-benzyloxy-3-hydroxybenzylamine
##STR00233##
[0497] This compound was prepared according to procedure used in
preparation of 3,5-dibenzyloxybenzylamine, starting from
4-benzyloxy-3-hydroxybenzonitrile. Yield was 33%. MP
122-125.degree. C.; .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta. 8.9
(br, 1H), 7.44 (d, 2H, J=7.4 Hz), 7.35 (dd, 2H, J=7.0 and 7.7 Hz),
7.28 (dd, 1H, J=7.0 and 7.3), 6.85 (d, 1H, J=7.6 Hz), 6.77 (d, 1H,
J=2.1 Hz), 6.61 (dd, 1H, J=7.4 and 2.2 Hz), 5.05 (s, 2H), 3.55 (s,
1H), 2.50 (br, 2H).
3-hydroxy-4-propyloxybenzylamine
##STR00234##
[0499] This compound was prepared by reduction of
3-hydroxy-4-propyloxybenzonitrile according to procedure described
in preparation of 3,5-dibenzyloxybenzylamine. Yield was 48%: MP
110-113.degree. C. (dec.); .sup.1H NMR (CDCl.sub.3, 400 MHz)
.delta. 6.86 (s, 1H), 6.77 (d, 1H, J=8.4 Hz), 6.74 (d, 1H, J=8.1
Hz), 3.95 (t, 1H, J=6.6 Hz), 3.74 (s, 2H), 2.01 (br, 2H), 1.82 (m,
2H), 1.02 (t, 3H, J=7.4 Hz).
4-hydroxymethylbenzylamine
##STR00235##
[0501] This compound was prepared by reduction of
4-cyanobenzaldehyde according to the procedure described in
preparation of 3,5-dibenzyloxybenzylamine. Yield was 46%: MP
102-123.degree. C.; .sup.1H NMR (Acetone-d.sub.6, 500 MHz) 7.27 (m,
complex 4H), 4.58 (s, 2H), 3.72 (s, 2H), 3.69 (s, 1H) 2.77 (br,
1H), 2.45 (br, 1H).
3-hydroxymethylbenzylamine
##STR00236##
[0503] This compound was prepared by reduction of
3-cyanobenzaldehyde according to the procedure described in
preparation of 3,5-dibenzyloxybenzylamine. Yield was 66%; .sup.1H
NMR (Acetone-d.sub.6, 500 MHz) .delta. 7.32 (s, 1H), 7.23 (dd, 1H,
J=7.6 and 7.0), 7.19, complex, 2H), 4.59 (s, 2H), 4.40 (s, 2H),
4.10 (br, 1H), 1.96 (br, 1H), 1.88 (br, 1H).
2-methoxy-5-nitrobenzaldehyde methyl hemiacetal
##STR00237##
[0505] 2-hydroxy-5-nitrobenzaldehyde (3.34 g, 20 mmol) was
dissolved in acetone (70 mL); k.sub.2CO.sub.3 (5.53 g, 40 mmol) and
iodomethane (14.19 g, 100 mmol) was added and the solution heated
to reflux overnight. Solvent was removed in vacuo and residue was
dissolved in EtOAc. The resulting product was washed with 2M NaOH,
water, and brine and dried. Removal of solvent resulted in a solid
product (2.5 g, 69%) of 2-methoxy-5-nitrobenzaldehyde methyl
hemiacetal: MP 147-148.degree. C. (89.degree. C. reported for the
aldehyde); .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 8.43 (d, 1H,
J=2.9 Hz), 8.24 (dd, 1H, J=2.6 and 9.1 Hz), 7.78 (d, 1H, 16.5 Hz),
6.99 (d, 1H, J=9.1 Hz), 6.83 (d, 1H, J=16.4 Hz). NOTE: This NMR was
taken after about 10 months and the hemiacetal was still existing
and pure.
5-nitro-2-propyloxybenzaldehyde
##STR00238##
[0507] This compound was prepared by the reaction of
2-hydroxy-5-nitrobenzaldehyde and 1-iodopropane using a similar
procedure as described in the preparation of
2-methoxy-5-nitrobenzaldehyde methyl hemiacetal. Yield was 72%: MP
(51-52.degree. C.); .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 10.46
(s, 1H), 8.68 (d, 1H, J=2.9 Hz), 8.39 (dd, 1H, J=2.7 and 9.1 Hz),
7.08 (d, 1H, J=9.1 Hz), 4.16 (t, 2H, J=6.2), 1.93 (m, 2H), 1.98 (t,
3H, J=7.37).
2-hydroxymethyl-4-nitrophenol
##STR00239##
[0509] A solution of 2-hydroxy-5-nitrobenzaldehyde (5.01 g, 30
mmol) in a mixture of 60 mL 1M NaOH and 30 mL MeOH was cooled to
0.degree. C. NaBH.sub.4 (1.13 g, 30 mmol) solution in 15 mL 1M NaOH
and 5 mL MeOH was added slowly. The reaction mixture was stirred
for 24 hours at room temperature. The mixture was poured into ice
cooled 2M HCl and extracted with EtOAc. The organic layer was
washed, dried, and evaporated in vacuo to give the alcohol as a
yellow solid (5.1 g, 100%): MP (112-114.degree. C.); NMR
(DMSO-d.sub.6, 400 MHz) .delta. 11.08 (s, 1H), 8.18 (d, 1H, J=2.5
Hz), 8.00 (dd, 1H, J=2.5 and 8.7 Hz), 6.92 (d, 1H, J=8.8 Hz), 5.20
(br, 1H), 4.49 (s, 2H).
2-methoxy-5-nitrobenzylalcohol
##STR00240##
[0511] This compound was prepared by reduction of
2-methoxy-5-nitrobenzaldehyde methyl hemiacetal using a method
similar to that described. for preparing
2-hydroxymethyl-4-nitrophenol in 76% yield: MP 121-122.degree. C.;
NMR (DMSO-d.sub.6, 500 MHz) .delta. 8.22 (d, 1H, J=1.22 Hz), 8.16
(dd, 1H, J=2.7 and 9.1), 7.16 (d, 1H, J=8.9 Hz), 4.50 (s, 2H), 3.90
(s, 3H).
5-nitro-2-propyloxy-benzylalcohol
##STR00241##
[0513] This compound was prepared by reduction of
5-nitro-2-propyloxybenzaldehyde using a method similar to that
described for preparing 2-hydroxymethyl-4-nitrophenol in 93% yield:
MP (No Sample left for MP); NMR (400 MHz, DMSO-d.sub.6) 8.22 (d,
1H, J=2.6 Hz), 8.13 (dd, 1H, J=2.9 and 9.2 Hz), 7.14 (d, 1H, J=9.2
Hz), 5.41 (t, 1H, J=5.5 Hz), 4.52 (d, 2H, J=5.8 Hz), 4.08 (t, 2H,
J=6.2), 1.75 (m, 2H), 0.98 (t, 3H, J=7.6 Hz).
2-benzyloxy-5-nitrobenzylalcohol
##STR00242##
[0515] This intermediate was prepared by alkylation of
2-hydroxymethyl-4-nitrophenol with benzyl bromide following the
method described for preparation of 2-methoxy-5-nitrobenzaldehyde
methyl hemiacetal in a yield of 84%: MP 81-83.degree. C.; .sup.1H
NMR .delta. (DMSO-d.sub.6, 500 MHz) 8.26 (d, 1H, J=2.9 Hz H-6),
8.15 (dd, 1H, J=2.9 and 9.1 Hz, H-4), 7.46 (d, 2H, J=7.0, 2',6'-Hs)
7.41 (dd, 2H, J=7.0 and 7.7, 3',5'-Hs), 7.34 (d, 1H, J=7 Hz, 4'-H),
7.25 (d, 1H, J=9.1 Hz, 3-H), 5.4 (br, 1H, OH), 5.29 (s, 2H,
CH.sub.2), 4.57 (s, 2H, CH.sub.2).
3-hydroxymethyl-4-methoxyaniline
##STR00243##
[0517] A mixture of 2-methoxy-5-nitrobenzylalcohol (1.02 g, 6.03
mmol) and SnCl.sub.2.H.sub.2O (6.8 g, 30.15 mmol) in EtOH (20 mL)
was heated at 70.degree. C. for 1 hour. After cooling, the mixture
was treated with 2M NaOH and extracted with ether. The organic
layer was washed with water, dried, and evaporated under vacuum to
provide 2.18 g (84%) of the aniline
3-hydroxymethyl-4-methoxyaniline: .sup.1H NMR (DMSO-d.sub.6, 500
MHz) .delta. 6.66 (d, 1H, J=2.2 Hz), 6.61 (d, 1H, J=8.6 Hz), 6.38
(dd, 1H, J=2.4 and 8.2 Hz), 4.81 (t, 1H, J=5.5 Hz), 4.54 (br, 2H),
4.37 (d, 2H, J=5.8 Hz), 3.61 (s, 3H).
3-hydroxymethyl-4-propyloxyaniline
##STR00244##
[0519] This compound was prepared by reduction of
5-nitro-2-propyloxy-benzylalcohol using the method described for
the preparation of 3-hydroxymethyl-4-methoxyaniline in 37% yield:
.sup.1H NMR (DMSO-d.sub.6, 500 MHz) .delta. 6.66 (d, 1H, J=2.5 Hz),
6.60 (d, 1H, J=8.6 Hz), 6.35 (dd, 1H, J=2.7 and 8.5 Hz), 4.79 (t,
1H, J=5.8 Hz), 4.54 (br, 2H), 4.37 (d, 2H, J=6.1 Hz), 3.74 (t, 2H,
J=6.4 Hz), 1.65 (m, 2H), 0.94 (t, 3H, J=7.4 Hz).
4-benzyloxy-3-hydroxymethylaniline
##STR00245##
[0521] This compound was prepared by reduction of
2-benzyloxy-5-nitrobenzylalcohol using the method described for
preparation of 3-hydroxymethyl-4-methoxyaniline in 86% yield:
.sup.1H NMR (DMSO-d.sub.6, 500 MHz) .delta. 7.40 (d, 2H, J=7.3 Hz),
7.36 (dd, 2H, J=7.3 and 7.6 Hz), 7.28 (dd, 1H, J=7.0 and 7.4), 6.70
(d, 1H, J=8.5 Hz), 6.68 (d, 1H, J=2.4 Hz), 6.35 (dd, 1H, J=2.8 and
8.3 Hz), 4.92 (s, 2H), 4.84 (t, 1H, J=5.8 Hz), 4.59 (br, 2H), 4.44
(d, 2H, J=6.4 Hz).
B. Formation of Libraries
General Structure
[0522] ##STR00246## [0523] R.dbd.H, Ph [0524] R.sup.1,
R.sup.2.dbd.H, alkyl, aryl, aralkyl, heterocyclic
1. General Procedures for Amide Coupling
[0525] a. Method A
[0526] To a cold mixture (at 0.degree. C.) of an amine (see Table
VI below for amines) (0.15 mL of 1M solution in CH.sub.2Cl.sub.2,
0.15 mmol), an acid (5-fluoroindole-2-carboxylic acid or
5-fluoro-3-phenylindole-2-carboxylic acid) (0.15 mmol as 0.15 mL of
1M solution in THF) in CH.sub.2Cl.sub.2 (0.5 mL) was added and
cooled to 0.degree. C. Subsequently, a mixture of
1-(3-dimethylaminopropyl)-3-ethylearbodiimide (EDCI) (0.15 mmol)
and Et.sub.3N (0.06 mmol) in CH.sub.2Cl.sub.2 (0.5 mL) was added
and the reaction was shaken in a Bohdan orbital shaker
(Mettler-Toledo Bohdan, Vernon Hills, Ill.) at 0.degree. C. for 30
minutes then at room temperature for 18 hours. After adding 0.5 mL
of CH.sub.2Cl.sub.2 and 0.5 mL MeOH, the mixture was passed through
a cartridge charged with a cationic exchange resin (Dowex
50wX4-200, Aldrich Chemical Co., Milwaukee, Wis., pre-washed with
1M HCl, H.sub.2O, H.sub.2O-MeOH, MeOH, MeOH--CH.sub.2Cl.sub.2). The
eluent was directly passed through a chromatography cartridge
containing silica gel mixed with 10% Na.sub.2CO.sub.3. The product
was eluted with 2 mL CH.sub.2Cl.sub.2-MeOH (2 mL), CH.sub.2Cl.sub.2
(2 mL), and CH.sub.2Cl.sub.2-MeOH (2 mL). The fraction(s)
containing pure product was identified by TLC (EtOAc-Hexane, 1:1).
The compounds were characterized and their relative purity was
estimated using .sup.1H NMR.
[0527] b. Method B
[0528] A mixture of an amine (see Table VI below for amines) (0.1
mmol), an acid (5-fluoroindole-2-carboxylic acid or
5-fluoro-3-phenylindole-2-carboxylic acid) (0.1 mmol as 0.1 mL of
1M solution in DMF), and diisopropylethylamine (DIEA) (0.05 mL, 0.3
mmol) was cooled to 0.degree. C.
(benzotriazol-1-yloxy)tripyrrolidino-phosphonium-hexafluorophosphate
(PyBOP) (0.1 mmol as 0.1 mL of 1M solution in DMF) was added. The
reaction mixture was shaken using an orbital shaker at 0.degree. C.
for 30 minutes then at room temperature for 18 hours. EtOAc was
added to the mixture and the organic solution was washed with 1M
HCl (2.times.1 mL), brine (1 mL) NaHCO.sub.3 (2.times.1 mL), and
brine (1 mL). The organic layer was passed through a silica gel
cartridge containing a top layer of anhydrous MgSO.sub.4 and
moistened with hexane. The product amide was eluted with hexane
(1.times.1 mL), hexane-EtOAc 2:1 (3.times.1 mL), hexane-EtOAc 1:1
(2.times.2 mL), and hexane-EtOAc 1:2 (1.times.2 mL). The
fraction(s) containing pure product was identified by TLC
(EtOAc-hexane 1:1 and EtOAc-hexane 1:2 in the case of
5-fluoro-3-phenylindole-2-carboxylic acid amide derivatives). The
compounds were characterized and their relative purity was
estimated using .sup.1H NMR.
[0529] c. Method C
[0530] A mixture of an amine (see Table VI below for amines) (0.1
mmol), an acid (5-fluoroindole-2-carboxylic acid or
5-fluoro-3-phenylindole-2-carboxylic acid) (0.1 mmol as 0.1 mL of
1M solution in THF), and DIEA (0.05 mL, 0.3 mmol) in 0.4 mL of
CH.sub.2Cl.sub.2-THF (3:1) was cooled to 0.degree. C. PyBrOP (0.1
mmol) was added. The reaction mixture was shaken using an orbital
shaker at 0.degree. C. for 30 minutes then at room temperature for
48 hours (0.1 mL THF and 0.2 mL CH.sub.2Cl.sub.2 were added after
24 hours). EtOAc was added to the mixture and the organic solution
was washed with 1M HCl (2.times.1 mL), brine (1 mL) NaHCO.sub.3
(2.times.1 mL), and brine (1 mL). The organic layer was passed
through a silica gel cartridge containing a top layer of anhydrous
MgSO.sub.4 and moistened with hexane. The product amide was eluted
with hexane (1.times.1 mL), hexane-EtOAc 2:1 (3.times.1 mL),
hexane-EtOAc 1:1 (2.times.2 mL), and hexane-EtOAc 1:2 (1.times.2
mL). The fraction(s) containing pure product was identified by TLC
(EtOAc-hexane 1:1 and EtOAc-hexane 1:2 in the case of
5-fluoro-3-phenylindole-2-carboxylic acid amide derivatives). The
compounds were characterized by .sup.1H NMR.
[0531] d. Method D
Preparation of 5-fluoroindole-2-carboxylic acid chloride
[0532] 5-fluoroindole-2-carboxylic acid (537 mg, 3 mmol) was
dissolved in DME (8 mL). 0.6 mL triethylamine was added and the
mixture cooled to 0.degree. C. Thionyl chloride (0.44 mL, 6 mmol)
mixed with 4 mL DME was added cautiously using addition funnel over
10 minutes while stirring. The mixture was left to stir for 30
minutes. The formed precipitate was filtered off, and the solvent
was evaporated under reduced pressure to give yellow solid of the
acid chloride.
Reaction of amines with 5-fluoroindole-2-carboxylic acid
chloride
[0533] A mixture of an amine (see Table VI below for amines) (1
mmol) and pyridine (0.18 mL) in 1 mL DCM was cooled to 0.degree. C.
5-fluoroindole-2-carboxylic acid chloride (19.8 mg, 1 mmol) was
added, then reaction was stirred at room temperature for 1 hour.
The resulting amide (in DCM) was washed with 1M HCl, then with
Brine. The crude product was purified by silica gel
chromatography.
[0534] e. Representative Examples of Amide Coupling Methods
Synthesis of Compound 1z
##STR00247##
[0535] To a mixture of 3-fluorobenzylamine (2.03 g, 20 mmol) and
5-fluoroindole-2-carboxylic acid (3.58 g, 20 mmol) in DMF (50 mL),
was added a solution of DIEA (6.98 mL, 40 mmol) in 15 mL
CH.sub.2Cl.sub.2. The mixture was cooled to 0.degree. C. and PyBOP
(10.41 g, 20 mmol) was added portion wise. The reaction mixture was
stirred at 0.degree. C. for 30 minutes, then at room temperature
for 4 hours. EtOAc (400 mL) was added to the mixture and the
organic solution was washed with 2M HCl (4.times.200 mL), brine
(200 mL), NaHCO.sub.3 (4.times.200 mL), and brine (2.times.200 mL).
The organic layer was dried (MgSO.sub.4) and concentrated in vacuo
to furnish the crude product as off-white solid. Recrystallization
from MeOH and CH.sub.2Cl.sub.2 provided 5.36 g (93%) of 1z as white
crystals: MP 239-241.degree. C.; .sup.1H NMR (DMSO-d.sub.6, 500
MHz) .delta. 11.73 (s, 1H), 9.12 (t, 1H, J=6.1 Hz), 7.39 (complex,
3H), 7.17 (complex, 2H), 7.07 (dd, 1H, J=2.2 and 9.5 Hz), 7.07 (dd,
1H, J=2.2 and 9.0 Hz), 7.03 (ddd, 1H, J=2.4, 9.1 and 9.2 Hz), 4.52
(d, 2H, J=6.1 Hz); Anal. (C.sub.16H.sub.12F.sub.2N.sub.2O) C,
67.13; H, 4.23; N, 9.79. Found; C, 66.91; H, 4.31; N, 9.81.
Synthesis of Compound 1a
##STR00248##
[0536] (a) Preparation of Methoxy Intermediate
##STR00249##
[0538] Following same procedure mentioned above for the synthesis
of 1z, this compound was prepared starting from 20 mmol of amine
and acid. Purification with flash column chromatography afforded
5.43 g (91%) of the methoxy intermediate as off-white crystalline
solid: MP 192.degree. C.
(b) Demethylation
[0539] A mixture of the methoxy intermediate (5 g, 16.7 mmol) and
CH.sub.2Cl.sub.2 (80 mL) was placed in a multi neck flask equipped
with a dropping funnel and a thermometer. The flask was cooled to
0.degree. C. in an ice/salt bath. A solution of BBr.sub.3 in
CH.sub.2Cl.sub.2 (80 mL) was added dropwise while keeping the
temperature less than 5.degree. C. The mixture was stirred at room
temperature for 3 hours. After addition of ice and 3M HCl (200 mL),
the mixture was left to stir overnight. The precipitated solid
product was collected by filtration, washed with water, and dried.
Crystallization from CH.sub.2Cl.sub.2 and MeOH furnished 4.2 g
(88%) of compound 1a: MP 213.degree. C.; .sup.1H NMR (DMSO-d.sub.6,
400 MHz) .delta. 11.68 (br, 1H), 9.31 (br, 1H), 9.01 (t, 1H, J=6.0
Hz), 7.39 (complex, 2H), 7.14 (s, 1H), 7.09 (dd, 1H, J=8.0 and 7.7
Hz), 7.02 (ddd, 1H, J=9.2, 8.9 and 2.5 Hz), 6.72 (d, 2H, J=7.3 Hz),
6.61 (d, 1H, J=8.4 Hz), 4.41 (d, 2H, J=5.9 Hz), HRMS (EI): Required
M.sup.+ for C.sub.16H.sub.13FN.sub.2O.sub.2, 284.0956; Found,
284.0960; Anal. (C.sub.16H.sub.13FN.sub.2O.sub.2) C, 67.60; H,
4.61; F, N, 9.85. Found C, 67.50;H, 4.65; F, N, 9.76.
[0540] f. Other Representative Compounds Obtained and Relative
Purity Data
[0541] The following are examples of compounds obtained using the
above methods and their relative purity data. Table VI, below,
lists all compounds obtained.
Compound 1bb
##STR00250##
[0543] .sup.1H NMR (acetone-d.sub.6, 400 MHz) .delta. 10.85 (br,
1H), 8.29 (br, 1H), 7.53 (dd, 1H, J=9.9 and 4.6 Hz), 7.36 (d, 1H,
J=7.4), 7.30 (complex, 3H), 7.22 (dd, 1H, J=7.3 and 7.0 Hz), 7.12
(s, 1H) 7.02 (ddd, 1H, J=2.6 and 9.2 and 9.1), 4.60 (d, 2H, J=6.3
Hz).
Compound 1cc
##STR00251##
[0545] .sup.1HNMR (Acetone-d.sub.6, 500 MHz) .delta. 10.74 (br, 2H)
8.01 (d, br, 1H, J=9.0 Hz), 7.45 (dd, 1H, J=9.0 and 4.6 Hz), 7.37
(d, 2H, J=7.4 Hz), 7.34 (d, 2H, J=7.4 Hz), 7.28 (dd, 1H, J=9.5 and
2.5Hz), 7.24 to 7.10 (complex. m, 7H), 6.99 (ddd, 1H, J=9.3, 9.2
and 2.6 Hz), 5.40 (dd, 1H, J=9.0 and 6.4 Hz), 5.20 (dd, 1H, J=6.2
and 4.6 Hz), 4.71 (d, 1H, J=4.6 Hz); LRMS (EI), m/z 356.1
(M.sup.+-H.sub.2O).
Compound 1dd
##STR00252##
[0547] .sup.1H NMR (Acetone-d.sub.6, 500 MHz) .delta. 10.80 (br,
2H) 8.04 (d, br, 1H, J=8.7 Hz), 7.43 (dd, 1H, J=9.0 and 4.8 Hz),
7.38 (dd, 2H, J=8.0 and 1.4 Hz), 7.34 (dd, 2H, J=8.0 and 1.4 Hz),
7.28 (dd, 1H, J=9.6 and 2.4 Hz), 7.25 to 7.11 (complex. m, 7H),
6.98 (ddd, 1H, J=9.2, 9.1 and 2.5 Hz), 5.41 (dd, 1H, J=8.8d 6.6
Hz), 5.21 (dd, 1H, J=6.2 and 4.8 Hz), 4.71 (d, 1H, J=4.6 Hz).
Compound 1bbb
##STR00253##
[0549] .sup.1H NMR (acetone-d.sub.6, 500 MHz) .delta. 10.89 (br,
1H), 8.31 (br, 1H), 7.54 (dd, 1H, J=9.1 and 4.6 Hz), 7.45 (dd, 1H,
J=7.7 and 7.7 Hz), 7.29 (complex, 2H), 7.17-7.08 (complex, 3H),
7.03 (ddd, 1H, J=9.2, 9.1 and 2.6 Hz), 4.66 (d, 2H, J=5.8 Hz).
Compound 1yyy
##STR00254##
[0551] .sup.1H NMR (Acetone-d.sub.6, 500 MHz) .delta. 10.99 (br,
1H), 9.57 (br, 1H), 7.80 (s, 1H), 7.76 (d, 1H, J=83 MHz), 7.57 (dd,
1H, J=9.0 and 4.4 Hz), 7.35 (dd, 1H, 9.2 and 2.4 Hz), 3.34 (s, 1H),
7.29 (dd, 1H, J=7.7 and 7.8 Hz), 7.09 (d, 1H, J=8.8 Hz), 7.06 (ddd,
1H, J=9.2, 8.9 and 2.4 Hz), 4.63 (d, 2H, J=5.8 Hz), 4.24 (t, 1H,
J=5.8 Hz); LRMS (EI) m/z 284.1 (74%, M.sup.+).
Compound 1ccc
##STR00255##
[0553] .sup.1H NMR (acetone-d.sub.6, 500 MHz) .delta. 10.96 (br,
1H), 9.49 (br, 1H), 7.79 (complex, 2H), 7.56 (dd, 1H, J=8.9 and 4.5
Hz), 7.34 (complex, 2H), 7.05 (ddd, 1H, J=9.2, 9.1 and 2.4 Hz),
6.92 (d, 1H, J=8.6 Hz), 4.65 (d, 1H, J=7.0 Hz), 4.06 (t, 1H,
J=5.8), 3.81 (s, 1H); LRMS (EI) m/z 314.12 (51%, M.sup.+).
Compound 1oooo
##STR00256##
[0554] .sup.1H NMR (acetone-d.sub.6, 500 MHz) .delta. 10.94 (br,
1H), 8.31 (br, 1H), 7.52 (dd, 1H, J=8.9 and 4.6), 7.33 (d, 2H,
J=8.9), 7.30 (d, 2H, J=8.5), 7.29 (dd, 1H, J=9.6 and 2.6Hz), 7.13
(s, 1H), 7.02 (ddd, 1H, J=9.3, 9.1 and 2.4 Hz), 4.60 (d, 4H, J=5.8
Hz), 4.15 (t, 1H, J=5.8 Hz); LRMS (EI) m/z 298.12 (100%,
M.sup.+).
Compound 2f
##STR00257##
[0556] .sup.1H NMR (acetone-d.sub.6, 500 MHz) .delta. 11.12 (br,
1H), 8.07 (br, 1H), 7.66-77.60 (m, complex, 5H), 7.52 (m, 1H), 7.36
(d, 1H, J=2.2 Hz), 7.30 (dd, 1H, J=8.7 and 2.6), 7.15-7.09 (m,
complex, 2H), 6.84 (d, 1H, J=8.8 Hz), 4.56 (d, 2H, J=6.1 Hz), 4.04
(t, 1h, J=5.8 Hz), 3.77 (s, 3H).
Compound 2g
##STR00258##
[0558] .sup.1H NMR (Acetone-d.sub.6, 500 MHz) .delta. 11.02 (br,
1H), 7.59 (dd, 1H, J=9.7 and 4.6 Hz), 7.53 (d, 1H, J=9.8 Hz), 7.47
(dd, 2H, J=7.9 and 7.4 Hz), 7.40 (m, 1H), 7.31 (dd, 1H. J=7.0 and
6.5 Hz), 7.08 (complex, 2H), 7.03 (d, 1H, J=7.7 Hz), 6.99 (complex,
2H), 6.91 (br, 1H), 4.48 (d, 2H, J=5.8 Hz); LRMS (EI) m/z 362.14
(85%, M.sup.+).
Compound 2s
##STR00259##
[0560] .sup.1H NMR (Acetone-d.sub.6, 500 MHz) .delta. 11.03 (br,
1H), 7.57-7.45 (complex, 4H), 7.544 (m, 1H), 7.31-7.24 (complex,
2H), 7.13-7.055 (complex, 4H), 6.76 (br, 1H), 4.50 (d, 2H, J=5.2
Hz); LRMS (EI) m/z 362.1 (95%, M.sup.+).
Compound 3q
##STR00260##
[0562] .sup.1H NMR (Acetone-d.sub.6, 500 MHz) .delta. 10.99 (br,
1H), 8.26 (s, 1H), 7.58 (dd, 1H, J=9.5 and 4.6 Hz), 7.51 (dd, 2H,
J=8.2 and 1.4 Hz), 7.46 (dd, 2H, J=7.7 and 7.3 Hz), 7.38 (m, 1H),
7.10-7.05 (complex, 3H), 6.72 (br, 1H), 6.67 (complex, 2H), 6.62
(d, 1H, J=7.6 Hz), 4.38 (d, 2H, J=6.2Hz); LRMS (EI) m/z 360.12
(100%, M.sup.+).
2. General Procedure for Oxidation of Benzyl Alcohol Amide
Derivatives to Benzaldehyde: Preparation of Compounds 3b, 3d, 3e,
3f, 3g, and 3h
[0563] The starting benzyl alcohol amide derivative was dissolved
in a 1:1 mixture of CH.sub.2Cl.sub.2 and THF (5 milmmol),
pyridinium chlorochromate (2 molar equivalent) was added, and the
mixture was stirred at room temperature for 3.5 hours. EtOAc and
water were added. The brown solid was removed by filtration. The
organic phase was washed several times with NaHCO.sub.3, brine,
dried, and concentrated. Product aldehyde was purified by
crystallization and confirmed by .sup.1H NMR (disappearance of the
methylene of benzyl alcohol and appearance of aldehyde peak).
[0564] The following table sets forth the structures made by the
above methods:
TABLE-US-00006 TABLE VI LIBRARY OF 5-FLUOROINDOLE-2-CARBAXAMIDE
COMPOUNDS AND METHOD OF PREPARATION Cmpd Code Structure M. Wt.
Method of Preparation 1a ##STR00261## 284.29 See Example 1 1b
##STR00262## 264.3 Amide Coupling Method A 1c ##STR00263## 264.3
Amide Coupling Method A 1d ##STR00264## 236.24 Amide Coupling
Method A 1e ##STR00265## 252.24 Amide Coupling Method A 1f
##STR00266## 192.19 Amide Coupling Method A 1g ##STR00267## 262.32
Amide Coupling Method A 1h ##STR00268## 234.27 Amide Coupling
Method A 1i ##STR00269## 290.38 Amide Coupling Method A 1j
##STR00270## 272.25 Amide Coupling Method A 1k ##STR00271## 346.35
Amide Coupling Method A 1l ##STR00272## 284.29 Amide Coupling
Method A 1m ##STR00273## 270.26 Amide Coupling Method A 1n
##STR00274## 270.26 Amide Coupling Method A 1o ##STR00275## 314.31
Amide Coupling Method A 1p ##STR00276## 254.26 Amide Coupling
Method A 1q ##STR00277## 304.32 Amide Coupling Method A 1r
##STR00278## 286.26 Amide Coupling Method A 1s ##STR00279## 322.26
Amide Coupling Method A 1t ##STR00280## 282.31 Amide Coupling
Method A 1u ##STR00281## 321.35 Amide Coupling Method A 1v
##STR00282## 232.25 Amide Coupling Method A 1w ##STR00283## 248.25
Amide Coupling Method A 1x ##STR00284## 246.28 Amide Coupling
Method A 1y ##STR00285## 312.34 Amide Coupling Method A 1z
##STR00286## 286.28 Amide Coupling Method A 1aa ##STR00287## 312.34
Amide Coupling Method A 1bb ##STR00288## 268.29 Amide Coupling
Method A 1cc ##STR00289## 374.41 Amide Coupling Method A 1dd
##STR00290## 374.41 Amide Coupling Method A 1ee ##STR00291## 298.31
Amide Coupling Method A 1ff ##STR00292## 394.18 Amide Coupling
Method A 1gg ##STR00293## 282.31 Amide Coupling Method A 1hh
##STR00294## 328.34 Amide Coupling Method A 1ii ##STR00295## 298.31
Amide Coupling Method A 1jj ##STR00296## 336.28 Amide Coupling
Method A 1kk ##STR00297## 336.28 Amide Coupling Method A 1ll
##STR00298## 336.28 Amide Coupling Method A 1mm ##STR00299## 298.31
Amide Coupling Method A 1nn ##STR00300## 274.33 Amide Coupling
Method A 1oo ##STR00301## 284.29 Amide Coupling Method A 1pp
##STR00302## 298.31 Amide Coupling Method A 1qq ##STR00303## 337.18
Amide Coupling Method A 1rr ##STR00304## 358.36 Amide Coupling
Method A 1ss ##STR00305## 337.18 Amide Coupling Method A 1tt
##STR00306## 304.27 Amide Coupling Method A 1uu ##STR00307## 296.34
Amide Coupling Method A 1vv ##STR00308## 347.18 Amide Coupling
Method A 1ww ##STR00309## 320.72 Amide Coupling Method A 1xx
##STR00310## 316.76 Amide Coupling Method A 1yy ##STR00311## 302.73
Amide Coupling Method A 1zz ##STR00312## 312.34 Amide Coupling
Method A 1aaa ##STR00313## 354.27 Amide Coupling Method A 1bbb
##STR00314## 286.28 Amide Coupling Method A 1ccc ##STR00315##
358.36 Amide Coupling Method A 1ddd ##STR00316## 337.18 Amide
Coupling Method A 1eee ##STR00317## 304.27 Amide Coupling Method A
1fff ##STR00318## 404.28 Amide Coupling Method A 1ggg ##STR00319##
337.18 Amide Coupling Method A 1hhh ##STR00320## 304.27 Amide
Coupling Method A 1iii ##STR00321## 352.28 Amide Coupling Method A
1jjj ##STR00322## 347.18 Amide Coupling Method A 1kkk ##STR00323##
320.72 Amide Coupling Method A 1lll ##STR00324## 302.73 Amide
Coupling Method A 1mmm ##STR00325## 354.27 Amide Coupling Method A
1nnn ##STR00326## 282.31 Amide Coupling Method A 1ooo ##STR00327##
344.38 Amide Coupling Method A 1ppp ##STR00328## 352.28 Amide
Coupling Method A 1qqq ##STR00329## 404.43 Amide Coupling Method A
1rrr ##STR00330## 302.73 Amide Coupling Method A 1sss ##STR00331##
354.27 Amide Coupling Method A 1ttt ##STR00332## 354.27 Amide
Coupling Method A 1uuu ##STR00333## 286.28 Amide Coupling Method A
1vvv ##STR00334## 282.31 Amide Coupling Method A 1www ##STR00335##
365.17 Amide Coupling Method A 1xxx ##STR00336## 284.29 Amide
Coupling Method C 1yyy ##STR00337## 284.29 Amide Coupling Method B
1zzz ##STR00338## 270.26 Amide Coupling Method C 1aaaa ##STR00339##
342.36 Amide Coupling Method C 1bbbb ##STR00340## 390.41 Amide
Coupling Method C 1cccc ##STR00341## 314.31 Amide Coupling Method C
1dddd ##STR00342## 390.41 Amide Coupling Method B 1eeee
##STR00343## 328.34 Amide Coupling Method B 1ffff ##STR00344##
282.31 Amide Coupling Method B 1gggg ##STR00345## 300.28 Amide
Coupling Method B 1hhhh ##STR00346## 328.34 Amide Coupling Method B
1iiii ##STR00347## 480.53 Amide Coupling Method B 1jjjj
##STR00348## 390.41 Amide Coupling Method B 1kkkk ##STR00349##
347.18 Amide Coupling Method B 1llll ##STR00350## 314.31 Amide
Coupling Method B 1mmmm ##STR00351## 313.28 Amide Coupling Method B
1nnnn ##STR00352## 342.36 Amide Coupling Method B 1oooo
##STR00353## 298.31 Amide Coupling Method B 1pppp ##STR00354##
298.31 Amide Coupling Method B 1qqqq ##STR00355## 278.32 Amide
Coupling Method B 2a ##STR00356## 354.42 Amide Coupling Method B 2b
##STR00357## 366.43 Amide Coupling Method B 2c ##STR00358## 360.38
Amide Coupling Method B 2d ##STR00359## 418.46 Amide Coupling
Method B 2e ##STR00360## 466.5 Amide Coupling Method B 2f
##STR00361## 390.41 Amide Coupling Method B 2g ##STR00362## 362.37
Amide Coupling Method B 2h ##STR00363## 470.28 Amide Coupling
Method B 2i ##STR00364## 412.38 Amide Coupling Method B 2j
##STR00365## 412.38 Amide Coupling Method B 2k ##STR00366## 466.5
Amide Coupling Method B 2l ##STR00367## 413.27 Amide Coupling
Method B 2m ##STR00368## 434.46 Amide Coupling Method B 2n
##STR00369## 413.27 Amide Coupling Method B 2o ##STR00370## 396.82
Amide Coupling Method B 2p ##STR00371## 392.85 Amide Coupling
Method B 2q ##STR00372## 378.83 Amide Coupling Method B 2r
##STR00373## 430.37 Amide Coupling Method B 2s ##STR00374## 362.37
Amide Coupling Method B 2t ##STR00375## 434.46 Amide Coupling
Method B 2u ##STR00376## 413.27 Amide Coupling Method B 2v
##STR00377## 380.36 Amide Coupling Method B 2w ##STR00378## 480.38
Amide Coupling Method B 2x ##STR00379## 413.27 Amide Coupling
Method B 2y ##STR00380## 380.36 Amide Coupling Method B 2z
##STR00381## 404.43 Amide Coupling Method B 2aa ##STR00382## 428.38
Amide Coupling Method B
2bb ##STR00383## 378.83 Amide Coupling Method B 2cc ##STR00384##
430.37 Amide Coupling Method B 2dd ##STR00385## 556.63 Amide
Coupling Method B 2ee ##STR00386## 466.5 Amide Coupling Method B
2ff ##STR00387## 428.38 Amide Coupling Method B 2gg ##STR00388##
378.83 Amide Coupling Method B 2hh ##STR00389## 430.37 Amide
Coupling Method B 2ii ##STR00390## 431.37 Amide Coupling Method B
2jj ##STR00391## 362.37 Amide Coupling Method B 2kk ##STR00392##
389.38 Amide Coupling Method B 2ll ##STR00393## 441.27 Amide
Coupling Method B 2mm ##STR00394## 418.46 Amide Coupling Method B
2nn ##STR00395## 374.41 Amide Coupling Method B 2oo ##STR00396##
375.41 Amide Coupling Method B 3a ##STR00397## 300.28 Demethylation
of compound 1hh using BBr.sub.3 method in Example 1. 3b
##STR00398## 282.27 Oxidation of 1yyy 3c ##STR00399## 269.29 Amide
Coupling Method C 3d ##STR00400## 340.35 Oxidation of 1aaaa 3e
##STR00401## 388.39 Oxidation of 1bbbb 3f ##STR00402## 312.3
Oxidation of 1cccc 3g ##STR00403## 296.3 Oxidation of 1pppp 3h
##STR00404## 388.39 Oxidation of 2f 3i ##STR00405## 390.41 Amide
Coupling Method B 3j ##STR00406## 291.31 Amide Coupling Method C 3k
##STR00407## 316.28 Amide Coupling Method C 3l ##STR00408## 323.35
Amide Coupling Method C 3m ##STR00409## 262.26 Amide Coupling
Method C 3n ##STR00410## 245.21 Amide Coupling Method C 3o
##STR00411## 450.5 Amide Coupling Method B 3p ##STR00412## 374.41
Amide Coupling Method B 3q ##STR00413## 360.38 See synthesis of 1a
in Example 1 3r ##STR00414## 263.3 Amide Coupling Method D 3s
##STR00415## 259.24 Amide Coupling Method D 3t ##STR00416## 255.25
Amide Coupling Method D 3u ##STR00417## 3v ##STR00418## 3w
##STR00419## 3x ##STR00420## 3y ##STR00421## 3z ##STR00422## 3aa
##STR00423## 3bb ##STR00424## 3cc ##STR00425## 3dd ##STR00426##
C. Inhibition of Human Cancer Cell Line H460 and Isolated Src
[0565] Subsequent to synthesis, several of the above compounds were
tested for the inhibition of the growth of human lung cancer cell
line H460 and the inhibition of isolated Src. To test for
inhibition of H460, the cells were seeded at 600 cells/well in 96
well plates in complete medium-RPMI-1640 containing 5% FCS, 5%
NuSerum IV, 2 mM L-glutamine, and 10 mM HEPES. Following an
overnight incubation, compounds which were solubilized in DMSO and
diluted in RPMI-1640, were added to cells plates. After 72 hours,
cells were fixed, stained, and total protein/well was determined.
Compound concentration which inhibited growth by 50% (IC.sub.50)
was determined and is reported below. To test for inhibition of
isolated Src, the compounds were tested using the assay procedure
described in Lai et al., 1998, with the following assay components,
final concentrations, and conditions: 50.0 mM MOPS, 4.02 mM
MgCl.sub.2, 6.00 mM K.sub.3 citrate (used as a Mg.sup.2+ buffer to
stabilize the free Mg.sup.2+ at 0.5 mM), 99.0 mM KCl, 10.0 mM
2-mercaptoethanol, 198 .mu.M ADP, 10 U full length human purified
recombinant pp60.sup.c-src (Upstate Biotechnology Inc., Lake
Placid, N.Y.), 2.00 mM RR-SRC, 4.0% DMSO, pH 7.2, 37.degree. C.
These overall assay conditions have been shown to reproduce the
intracellular conditions of pH, temperature, free M.sup.2+ (0.5
mM), ionic strength, osmolality, ATP/ADP, and reduction potential.
The results are in Table VII, below.
TABLE-US-00007 TABLE VII INHIBITION OF THE GROWTH OF HUMAN LUNG
CANCER CELL LINE H460 AND THE INHIBITION OF ISOLATED SRC Compound
H460.sup.a IC.sub.50 (.mu.M).sup.b Src IC.sub.50 (.mu.M) 1a 35 .+-.
0.59 IC.sub.50 = 40 1z 15 .+-. 1.6 NT.sup.c 1bb 82 .+-. 3.5 NT 1dd
33 .+-. 0.78 NT 1yyy 104 .+-. 10 NT 1cc 30 .+-. 0.66 NT 1cccc
>100 NT 1oooo 74 .+-. 2.7 NT 2f 13 .+-. 0.46 NT 2s 26 .+-. 0.34
NT 1bbb >100 NT 2g 13 .+-. 0.56 NT 3q 30 .+-. 0.34 NT .sup.aH460
- NSCLC cells. .sup.bAll compounds were solubilized in DMSO and
further diluted in RPMI 1640 containing 5% FCS, 5% NuSerum IV, 2 mM
L-glutamine, and 20 mM HEPES. .sup.cNT = not tested.
[0566] These results show that the use of a phenyl group attached
to the 3 position of the indole ring can significantly improve the
activity of the inhibitor.
D. Inhibition of Epidermal Growth Factor Receptor Tyrosine Kinase
(EGFRTK), p56 lck, p55 fyn, and PTP-1B
[0567] The compounds listed in Table VIII below were tested for
inhibition of EGFRTK, a transmembrane receptor tyrosine kinase, p56
lck, a member of the Src family of non-receptor tyrosine kinases,
p55 fyn, another member of the Src family of non-receptor tyrosine
kinases, and PTP-1B, a phosphotyrosine phosphatase, the opposite of
a kinase and a target for type II diabetes and/or obesity. The data
in the table are the % inhibition of the indicated enzyme by the
compound at a concentration of 10 micromolar. Blanks for a
particular enzyme indicate that inhibition was not found.
TABLE-US-00008 TABLE VIII INHIBITION OF EGFRPTK, p56 lck, p55 fyn,
and PTP-1B Compound PTP-1B @ EGFRTK @ p56 Lck @ p55 fyn @ Structure
MW Code 10 .mu.m 10 .mu.m 10 .mu.m 10 .mu.m ##STR00427## 264.3 1b
##STR00428## 264.3 1c ##STR00429## 236.2 1d ##STR00430## 252.2 1e
##STR00431## 192.2 1f ##STR00432## 262.3 1g 19 ##STR00433## 234.3
1h ##STR00434## 290.4 1i ##STR00435## 272.3 1j ##STR00436## 346.4
1k 13 ##STR00437## 284.3 1l ##STR00438## 270.3 1m ##STR00439##
270.3 1n 12 ##STR00440## 314.3 1o ##STR00441## 254.3 1p
##STR00442## 304.3 1q ##STR00443## 286.3 1r 11 ##STR00444## 322.3
1s ##STR00445## 282.3 1t 11 ##STR00446## 321.4 1u 10 ##STR00447##
232.3 1v ##STR00448## 248.3 1w 10 ##STR00449## 246.3 1x
##STR00450## 312.3 1y ##STR00451## 286.3 1z 26 ##STR00452## 312.3
1aa 12 10 ##STR00453## 268.3 1bb 19 ##STR00454## 374.4 1cc 19
##STR00455## 374.4 1dd 41 ##STR00456## 298.3 1ee 16 ##STR00457##
394.2 1ff 24 ##STR00458## 282.3 1gg ##STR00459## 328.3 1hh
##STR00460## 298.3 1ii ##STR00461## 336.3 1jj 18 ##STR00462## 336.3
1kk ##STR00463## 336.3 1ll ##STR00464## 298.3 1mm ##STR00465##
274.3 1nn ##STR00466## 284.3 1oo 17 ##STR00467## 298.3 1pp
##STR00468## 337.2 1qq ##STR00469## 358.4 1rr 12 ##STR00470## 337.2
1ss 12 ##STR00471## 304.3 1tt ##STR00472## 296.3 1uu ##STR00473##
347.2 1vv ##STR00474## 320.7 1ww ##STR00475## 316.8 1xx 14
##STR00476## 302.7 1yy 10 12 ##STR00477## 312.3 1zz ##STR00478##
354.3 1aaa 12 ##STR00479## 286.3 1bbb ##STR00480## 358.4 1ccc
##STR00481## 337.2 1ddd ##STR00482## 304.3 1eee ##STR00483## 404.3
1fff ##STR00484## 337.2 1ggg ##STR00485## 304.3 1hhh ##STR00486##
352.3 1iii ##STR00487## 347.2 1jjj ##STR00488## 320.7 1kkk
##STR00489## 302.7 1lll ##STR00490## 354.3 1mmm 14 ##STR00491##
282.3 1nnn ##STR00492## 344.4 1ooo ##STR00493## 352.3 1ppp
##STR00494## 404.4 1qqq 11 ##STR00495## 302.7 1rrr 15 ##STR00496##
354.3 1sss ##STR00497## 354.3 1ttt ##STR00498## 286.3 1uuu 13 16
##STR00499## 282.3 1vvv ##STR00500## 365.2 1www ##STR00501## 284.3
1xxx 12 ##STR00502## 284.3 1yyy 11 ##STR00503## 270.3 1zzz
##STR00504## 342.4 1aaaa ##STR00505## 390.4 1bbbb ##STR00506##
314.3 1cccc 20 19 ##STR00507## 390.4 1dddd 16 ##STR00508## 328.3
1eeee ##STR00509## 282.3 1ffff 12 ##STR00510## 300.3 1gggg 25
##STR00511## 328.3 1hhhh 17 ##STR00512## 480.5 1iiii 12
##STR00513## 390.4 1jjjj 15 ##STR00514## 347.2 1kkkk 30
##STR00515## 314.3 1llll ##STR00516## 313.3 1mmmm 29 ##STR00517##
342.4 1nnnn 17 11 ##STR00518## 298.3 1oooo 33 10 ##STR00519## 298.3
1pppp ##STR00520## 278.3 1qqqq 18 ##STR00521## 354.4 2a
##STR00522## 368.4 2b 19 13 ##STR00523## 360.4 2c ##STR00524##
418.5 2d 23 ##STR00525## 466.5 2e 10 ##STR00526## 390.4 2f 18 11
##STR00527## 362.4 2g ##STR00528## 470.3 2h ##STR00529## 412.4 2i
##STR00530## 412.4 2j 20 ##STR00531## 466.5 2k ##STR00532## 413.3
2l ##STR00533## 434.5 2m ##STR00534## 413.3 2n ##STR00535## 396.8
2o 12 ##STR00536## 392.9 2p ##STR00537## 378.8 2q ##STR00538##
430.4 2r ##STR00539## 362.4 2s 33 10 11 ##STR00540## 434.5 2t
##STR00541## 413.3 2u ##STR00542## 380.4 2v ##STR00543## 480.4 2w
12 ##STR00544## 413.3 2x ##STR00545## 380.4 2y 20 ##STR00546##
404.4 2z ##STR00547## 428.4 2aa ##STR00548## 378.8 2bb
##STR00549## 430.4 2cc ##STR00550## 556.6 2dd ##STR00551## 466.5
2ee ##STR00552## 428.4 2ff ##STR00553## 378.8 2gg 12 ##STR00554##
430.4 2hh 12 ##STR00555## 430.4 2ii ##STR00556## 362.4 2jj
##STR00557## 389.4 2kk 24 ##STR00558## 441.3 2ll 10 ##STR00559##
418.5 2mm ##STR00560## 374.4 2nn ##STR00561## 374.4 2oo
E. Mice Toxicity Study
[0568] FIG. 15 shows the results of a maximum tolerated dose (MTD)
study with two indole inhibitors:
##STR00562##
These compounds were administered to SCID mice by intraperitoneal
administration in tween80:EtOH. The results in FIG. 17 show that
compound la is less toxic in mice than compound 2k from Example 4,
since the mice exhibited less weight loss when compound 1a was
administered.
Example 2
Synthesis and Activity of 7-Substituted Indole Derivative Protein
Kinase Inhibitors
[0569] 7-substituted indole derivative protein kinase inhibitors
were synthesized as set forth in Scheme 1, below:
##STR00563##
(2-bromo-4-fluoro-phenyl)-hydrazine (2)
##STR00564##
[0571] Commercially available 2-bromo-4-fluoroaniline 1 (2.36 ml,
20.75 mmol) was added to a stirring solution of concentrated
hydrochloric acid (40 ml) that was cooled to -5.degree. C. This
solution was allowed to age while stirring for 10 minutes. An
aqueous solution of NaNO.sub.2 was added over 15 minutes.
SnCl.sub.2/HCl (10.40 g, 46.1 mmol, 10 ml HCl) was added over 15
minutes and aged for an additional 30 minutes to 1 hour. The
mixture was filtered and washed with dichloromethane. The resulting
solid was dissolved in 1.0M HCl and extracted 3 times with
dichloromethane. The organic layer was vacuum dried overnight to
give 3.53 g (83% yield). .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
7.169 (dd, J=8 Hz, J=2.8 Hz 1H), .delta. 7.076 (dd, J=5.2 Hz, J=9.2
Hz, 1H), .delta. 6.982 (td, J=8.4 Hz, J=2 Hz 1H), .delta. 5.540
(bs, 1H), .delta. 3.590 (bs, 1H).
2-[(2-bromo-4-fluoro-phenyl)-hydrazono]-propionic acid ethyl ester
(3)
##STR00565##
[0573] Commercially available p-toluensulfonic acid (38.37 mg,
0.217 mmol) was added to 60 ml of toluene in a round bottom flask
and magnetic stir bean. The flask was then fitted with a Dean Stark
trap and reflux condenser. The solution was then allowed to stir
for 2 hours. After 2 hours, the solution was cooled and
2-(bromo-4-phenyl)hydrazine (4.135 g, 20.17 mmol) was added. The
solution was then refluxed for an additional 1.5 hours using the
same apparatus. After 1.5 hours, the solution was placed on the
rotary evaporator to remove the toluene. A dark brown tar-like
substance was left in the flask. An appropriate amount of hexanes
were added to the flask and refluxed to dissolve the pure
hydrazine. The hexanes took on a yellow color and were then
decanted hot into another flask leaving the tar-like side product
behind. This was repeated. The flask containing the hexane solution
was refluxed so as to dissolve the precipitating hydrazine and
placed in the freezer to form crystals. 3.6 g (11.9 mmol, 87%
yield) of 3. .sup.1H NMR (Acetone-d.sub.6): .delta. 12.369 (bs,
1H), .delta. 7.646 (dd, J=9.2 Hz, J=5.6 Hz 1H), .delta. 7.449 (dd,
J=8.2 Hz, J=2.8 Hz 1H), .delta. 7.22 (td, J=.about.8.6 Hz, J=2.8
Hz, 1H), .delta. 4.37 (q, J=7.2 Hz, 2H), .delta. 2.203 (s, 3H),
.delta. 1.402 (t, J=7.2 Hz, 3H).
7-bromo-5-fluoro-1H-indole-2-carboxylic acid ethyl ester (4)
##STR00566##
[0575] Commercially available p-toluensulfonic acid dihydrate (2.26
g, 11.9 mmol) was added to 120 ml of toluene and dried under reflux
using a Dean Stark apparatus for 2 hours.
2-[(2-bromo-4-fluoro-phenyl)-hydrazono]-propionic acid ethyl ester
(3.6 g, 11.9 mmol), was added to the cooled solution, and refluxed
for an additional 1.5 hours. After 1.5 hours the solution was
cooled. The toluene was removed under reduced pressure. Then the
solid was refluxed with hexane to isolate the indole ester. The
resulting hexane solution was refluxed to dissolve the
precipitating indole, and placed in the freezer for
crystallization. After removal of supernatant and drying of
crystals gave 3.30 g, 11.543 mmol of 4 (97% yield). .sup.1H NMR
(400 MHz, Acetone-d.sub.6): .delta. 10.85 (bs, 1H), .delta. 7.45
(dd, J=9.2 Hz, J=2.4 Hz, 1H), .delta. 7.39 (dd, J=9.2 Hz, J=2.0 Hz,
1H), .delta. 7.28 (d, J=2.0 Hz, 1H), .delta. 4.36 (q, J=6.8 Hz,
2H), .delta. 1.345 (t, J=6.8 Hz, 1H).
7-bromo-5-fluoro-1H-indole-2-carboxylic acid (5)
##STR00567##
[0577] Tetrahydrofuran (35.2 ml), water (23.5 ml), lithium
hydroxide (2.61 g, 10.9 mmol), and
7-bromo-5-fluoro-1H-indole-2-carboxylic acid ethyl ester (3.11 g,
10.9 mmol) were added to a round bottom flask and mixed with a
magnetic stirrer. This mixture was refluxed for 1 hour. The THF was
removed via rotary evaporator, and the aqueous solution was
acidified with 1M HCL, and extracted with ethyl acetate. .sup.1H
NMR (DMSO-d.sub.6): .delta. 13.206 (bs, 1H), .delta. 11.876 (s,
1H), .delta. 7.498-7.445 (m, 2H), .delta. 7.19 (d, J=2.0 Hz,
1.0H).
7-bromo-5-fluoro-1H-indole-2-carboxylic acid 3-methoxy-benzylamide
(6)
##STR00568##
[0579] To a round bottom flask that has been fire dried, flushed
with a continuous stream of argon, and equipped with a stir bean,
DMF (4.8 ml) was added. To this stirring solution 5 (600 mg, 2.33
mmol), was combined with methoxybenzylamine (328 .mu.L, 2.56 mmol),
and PyBOP (1.33 g, 2.56 mmol). This solution was then cooled to a
temperature of 0 degrees C. After 2 minutes diisopropylamine (1.7
ml, 9.67 mmol) was added and the entire solution was allowed to
stir at room temperature overnight. The reaction was then diluted
with roughly 60 ml of ethyl acetate and extracted 3.times. with
saturated sodium bicarbonate, and 3.times. with 1M HCl in
appropriate volumes to remove any unreacted starting materials. The
ethyl acetate layer was isolated and dried over sodium sulfate. The
ethyl acetate was removed using a rotary evaporator to yield a
brownish film on the sides of the flask. Hexanes were added to the
flask and refluxed. A solid then formed on the sides of the flask,
and the hexanes were removed via rotary evaporator to give 709.0 mg
of 6 (81% yield). .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
11.537 (bs, 1H), .delta. 9.092 (t, J=5.6 Hz, 1H), .delta. 7.49 (dd,
J=9.4 Hz, J=2.4 Hz 1H), .delta. (dd, J=8.8 Hz, J=2 Hz 1H), .delta.
7.268-7.228 (m, 2H), .delta. 6.91 (d, J=6.8 Hz, 2H), .delta. 6.82
(d, J=8.2 Hz, 1H), .delta. 4.48 (d, J=6 Hz, 2H), .delta. 3.729 (s,
3H).
7-bromo-5-fluoro-1H-indole-2-carboxylic acid 3-hydroxy-benzylamide
(7)
##STR00569##
[0581] A stirring solution of methylene chloride (1 ml) was cooled
to -78 degrees in a dry ice acetone bath and flushed with a stream
of argon. To this cold stirring solution 6 (50 mg, 0.133 mmol) was
added. 7 equivalents of BBr.sub.3 was added and allowed to stir at
-78 degrees for 1 hour, and then the solution was allowed to stir
at room temperature overnight. The reaction was then quenched with
excess water, then neutralized with saturated sodium bicarbonate,
and extracted with methylene chloride. The methylene chloride layer
was dried over sodium sulfate and removed under reduced pressure to
yield 35.0 mg of 2 (70% yield). .sup.1H NMR (400 MHz,
Acetone-d.sub.6) .delta. 10.633 (bs, 1H), .delta. 8.42 (d, J=15.6,
2H), 7.48 (dd, J=9.2 Hz, J=2.4 Hz, 1H), .delta. 7.42 (dd, J=9 Hz,
J=2.4 Hz, 1H), .delta. 7.373 (d, J=2.4 Hz, 1H), .delta. 7.217 (t,
J=7.6 Hz, 1H), .delta. 6.932-6.898 (m, 2H), .delta. 6.80 (dd, J=2
Hz, J=8 Hz, 1H), .delta. 4.634 (d, J=5.6 Hz, 2H). Disappearance of
the characteristic methoxy peak at 3.7 ppm indicates a successful
deprotection.
Example 3
Design, Synthesis and Activity of Non-ATP Competitive
Hydroxynaphthalene Derivative Inhibitors of pp60.sup.c-Src Tyrosine
Kinase
[0582] The crystal structure of the autoinhibited human IRTK
catalytic domain (Hubbard et al., 1994) was used to carry out
qualitative molecular modeling studies (SYBYL.TM., 6.4, Tripos
Inc., St. Louis) wherein a naphthalene ring was superimposed upon
the IRTK Tyr 1,162. The IRTK region containing Tyr 1,162 folds back
into the active site, with Tyr 1,162 positioned analogous to a
phosphorylatable Tyr in a peptide substrate, thereby autoinhibiting
the tyrosine kinase. This superimposition indicated that an amide
carbonyl should be placed at the 2-position (Scheme 1) of the
##STR00570##
naphthalene ring to mimic the Tyr 1,162 carbonyl and a hydroxyl
group should be positioned at the 6-position to mimic the Tyr 1,162
hydroxyl group. These modeling studies also indicated that a
hydroxyl group at the 3-position could mimic the Tyr 1,162 NH.
[0583] In order to test these design concepts experimentally, the
2-position carbonyl group was appended as either a methyl ester or
as a series of amides (Table IX). The hydroxy N-phenyl (X=0) and
N-benzyl (X=1) amides were chosen based upon the increase in
pp60.sup.c-src inhibitor potency observed with iminochromene
analogs containing appended hydroxy N-phenyl amide side-chains
(Huang et al., 1995). Analogs wherein the 6-hydroxyl group was
either deleted or moved were also prepared to determine if a drop
in potency occurs as predicted from the modeling studies.
[0584] The series of 2-carbonyl-3,5-dihydroxy naphthalene
inhibitors (1a, 2a-2d, 2i-2l, 2o-2p) and 2-carbonyl-3,7-dihydroxy
naphthalene inhibitors (1c, 2t-2u) were synthesized from
commercially available (Aldrich) 3,5-dihydroxy-2-naphthoic acid and
3,7-dihydroxy-2-naphthoic acid, respectively. The methyl esters 1a
and 1c were obtained by refluxing the respective acid starting
materials for 48 h in methanol pre-saturated with HCl gas. The
amides (2a-2d, 2i-2l, 2o-2p, 2t-2u) were synthesized by coupling
the respective carboxylic acid with commercially available (Aldrich
or Lancaster) amines using one of two methods. The first method
utilized the NBS/PPh.sub.3 methodology as described by Froyen
(Froyen, 1997). The second method utilized IIDQ (Aldrich) as the
coupling reagent. The carboxylic acid was first reacted with 1.0
eq. IIDQ in anhydrous DMF at room temperature for 24 hours. The
respective amine (2.0 eq.) was then added neat and the reaction was
heated to 80.degree. C. for 2-6 hours. After aqueous workup,
purification was achieved by silica gel chromatography and
precipitation from CH.sub.2Cl.sub.2/hexane, followed by preparative
C-18 RP-HPLC (CH.sub.3CN/H.sub.2O), if necessary. The benzyl amines
were commercially available only as their corresponding hydroxyl
protected methyl ethers. Consequently, after amide formation, the
hydroxyl groups were deprotected by treatment with 6 eq. BBr.sub.3
in DCM for 1 minute at -78.degree. C. followed by 1 hour at room
temperature.
TABLE-US-00009 TABLE IX pp60.sup.c-src INHIBITORY ACTIVITY OF
HYDROXYNAPHTHALENE DERIVATIVES AND SELECT PUBLISHED
INHIBITORS..sup.a,b,c (1a-1d) ##STR00571## (2a-2v) ##STR00572## %
Inhibition at 100 .mu.M (std. Cmpd R.sub.1 R.sub.2 R.sub.3 R.sub.4
R.sub.5 R.sub.6 R.sub.7 R.sub.8 dev.) IC.sub.50 (.mu.M) 1a OH OH H
H N/A N/A N/A N/A 5 (+/- 2) n.t. 1b OH H OH H N/A N/A N/A N/A 47
(+/- 3) n.t. 1c OH H H OH N/A N/A N/A N/A 19 (+/- 6) n.t. 1d
NH.sub.2 H H H N/A N/A N/A N/A Inactive n.t. 2a OH OH H H OH H H 0
12 (+/- 4) n.t. 2b OH OH H H H OH H 0 51 (+/- 1) 150 2c OH OH H H H
H OH 0 60 (+/- 7) n.t. 2d OH OH H H OH H OH 0 14 (+/- 2) n.t. 2e OH
H OH H OH H H 0 39 (+/- 5) n.t. 2f OH H OH H H OH H 0 89 (+/- 1) 16
2g OH H OH H H H OH 0 23 (+/- 5) n.t. 2h OH H OH H OH H OH 0 56
(+/- 1) n.t. 2i OH OH H H H OMe H 0 33 (+/- 5) n.t. 2j OH OH H H H
H OMe 0 35 (+/- 8) n.t. 2k OH OH H H OMe H H 1 13 (+/- 3) n.t. 2l
OH OH H H H H OMe 1 14 (+/- 2) n.t. 2m OH H OH H OMe H H 1 inactive
n.t. 2n OH H OH H H H OMe 1 4 (+/- 7) n.t. 2o OH OH H H OH H H 1 41
(+/- 2) n.t. 2p OH OH H H H H OH 1 49 (+/- 4) n.t. 2q OH H OH H OH
H H 1 42 (+/- 2) n.t. 2r OH H OH H H OH H 1 55 (+/- 3) n.t. 2s OH H
OH H H H OH 1 42 (+/- 3) n.t. 2t OH H H OH H OH H 0 68 (+/- 5) n.t.
2u OH H H OH H OH H 1 40 (+/- 3) n.t. 2v H H OH H H OH H 0 45 (+/-
5) n.t. Iminochromene 9TA 30 (+/- 15) Lit.sup.8: 0.118 Piceatannol
41 (+/- 2) Lit.sup.13: 66 (lck) ST-638 37 (+/- 5) Lit.sup.14: 18
Emodin.sup.d 22 (+/- 3) Lit.sup.15: 38 Tyrophostin A47 43 (+/- 3)
Table IX Footnotes: .sup.aThe previously described assay procedure
(Lai et al., 1998) was used with the following assay components,
final concentrations and conditions: 50.0 mM MOPS, 4.02 mM
MgCl.sub.2, 6.00 mM K.sub.3citrate (used as a Mg.sup.2+ buffer to
stabilize the free Mg.sup.2+ at 0.5 mM), 99.0 mM KCl, 10.0 mM
2-mercaptoethanol, 198 .mu.M ATP, 19.8 .mu.M ADP, 10 U full length
human purified recombinant pp60.sup.c-src (Upstate Biotechnology
Inc.), 2.00 mM RR-SRC, 4.0% DMSO, pH 7.2, 37.degree. C. These
overall assay conditions have been shown (Choi, 1999) to reproduce
the intracellular conditions of pH, temp., free Mg.sup.2+ (0.5 mM),
ionic strength, osmolality, ATP/ADP and reduction potential.
.sup.bAll new compounds were characterized by proton NMR, EI or
FAB(+) MS and are pure by TLC. .sup.cN/A = Not applicable, n.t. =
Not tested. .sup.dATP-competitive.
[0585] The series of 2-carbonyl, 3,6-dihydroxy naphthalene
inhibitors (1b, 2e-2h, 2m-2n, 2q-2s) were synthesized from
3,6-dihydroxy-2-naphthoic acid 6 using the methods described above.
The synthesis of intermediate 6 that was developed is shown in
Scheme 2 beginning with commercially available
2,7-dihydroxynaphthalene 3 (Aldrich).
##STR00573##
[0586] Compound 1d was synthesized from 3-amino-2-naphthoic acid
(Aldrich) by reaction with TMS-diazomethane in DCM at room
temperature. Compound 2v was synthesized from 6-hydroxy-2-naphthoic
acid (Aldrich) using the amidation method described by Froyen
(Froyen, 1997).
[0587] Kinase assay conditions have been shown to influence the
measured inhibitory activity (Lawrence et al., 1998). Consequently,
in order to accurately determine the relative potency of the newly
designed class of pp60.sup.c-src inhibitors, the inhibitory
activity of four previously published, non-ATP competitive PTK
inhibitors, was also tested. Piceatannol, ST-638, and Tyrphostin
A47 were chosen because they are commercially available (Sigma or
Calbiochem), and are representative of the spectrum of known
non-ATP competitive PTK inhibitors. Emodin (Calbiochem) is
ATP-competitive when analyzed with the tyrosine kinase p56.sup.lck.
Previously, iminochromene 9TA was the most potent non-ATP
competitive pp60.sup.c-src inhibitor reported (Huang et al., 1995).
Since iminochromene 9TA was not commercially available, it was
synthesized using a novel route by converting 3-Aminophenol to the
corresponding TBDMS ether (1.1 eq. TBDMS-Cl, 1.1 eq. DIEA, 5 mol %
DMAP, DMF, 24 h, rt, 71%). The resulting aniline was coupled using
2.0 eq. of cyanoacetic acid (1.1 eq. EDCI, 1.1 eq. TEA, DMF, 18 h,
75.degree. C., 70%). Condensation of the resulting amide with 1.2
eq. of 2,3-dihydroxybenzaldehyde (cat. piperidine, abs. EtOH, 2 h,
60.degree. C.) followed by deprotection (1.1 eq. TBAF, THF, 15 m,
43% overall) gave iminochromene 9TA with satisfactory elemental,
FAB(+)MS and .sup.1H NMR analysis after purification by flash
chromatography (10:1, DCM:MeOH).
[0588] The inhibitory activities shown in Table IX for compounds
1a-d and 2a-2v were determined using purified, full length, human
recombinant pp60.sup.c-src. Due to the number of compounds tested,
and the associated cost, their rank order potencies were first
determined at a constant inhibitor concentration (100 .mu.M). As
predicted by the modeling studies, based upon analogy to the IRTK
Tyr 1,162 hydroxyl group, a preference for positioning the
naphthalene hydroxyl group on carbon 6 vs. 5 or 7 was observed in
both the ester (1b, 47% vs. 1a, 5% & 1c, 19%) and amide (e.g.
2f, 89% vs. 2b, 51% & 2t, 68%) series. The prediction that
attaching a hydroxyl group at naphthalene carbon 3 (mimicking the
Tyr 1,162 NH) would improve potency was also confirmed (2f, 89% vs.
2v, 45%). Finally, the prediction that extending the inhibitor as
an amide at the 2 position (mimicking the peptide bond) could
further improve potency was confirmed as well (e.g. 2f, 89% vs. 1b,
47%).
[0589] The data provided in Table IX demonstrate that moving the
hydroxyl group from the optimal 6 position to the adjacent
naphthalene carbon 5 results in a different structure activity
profile with regard to the optimal concurrent positioning of the
hydroxyl group(s) in the amide side chain (e.g. 2f/2g vs. 2b/2c).
Also of note is the replacement of the amide side chain hydroxyl
group with a corresponding methoxy group in compounds 2i-2n. In the
case of the N-phenyl amides (2i-2j), their activity, relative to
the corresponding hydroxy amides (2b-2c), was not reduced as
significantly as in the case of the N-benzyl amides (2k-2n vs.
2o-2q, 2s). This suggests that in the benzyl derivatives, the amide
side chain hydroxyl groups either interact with the enzyme as
hydrogen bond donors, or the methoxy groups are too large to fit in
the binding site.
[0590] A more quantitative analysis of the selectivity for
positioning a hydroxyl group on carbon 6 vs. 5 is provided by
comparing the IC.sub.50's of 2f (16 .mu.M) vs. 2b (150 .mu.M),
respectively. These results also confirm that a drop in %
inhibition from ca. 90% to ca. 50% represents an order of magnitude
difference in potency, as expected. Similarly, a drop in %
inhibition from ca. 50% to 10% would represent another order of
magnitude difference in potency.
[0591] A direct comparison of the most potent inhibitor from this
series, compound 2f, with the five previously reported PTK
inhibitors shown in Table IX demonstrates that, under these assay
conditions, 2f is more potent by one to two orders of magnitude.
Interestingly, iminochromene 9TA was previously reported (Huang et
al., 1995) to have an IC.sub.50 of 118 nM against pp60.sup.c-src,
and was the most potent known non-ATP competitive pp60.sup.c-src
inhibitor, but under the current assay conditions only a 30%
inhibition at 100 .mu.M was observed. These results re-emphasize
(Lawrence et al., 1998) the importance of comparing protein kinase
inhibitors under identical assay conditions.
[0592] A goal of these studies was to obtain non-peptide
pp60.sup.c-src inhibitors which do not compete with ATP.
Consequently the % inhibition of pp60.sup.c-src by 2f and 2b at
constant inhibitor concentrations was monitored as a function of
increasing [ATP] up to a cellular mimetic 1 mM level. Since the
[ATP] had little effect on the % inhibition, both 2f and 2b are
non-competitive inhibitors with respect to ATP. The % inhibition
was measured using ATP concentrations of 200, 500 & 1,000 .mu.M
while holding the inhibitor concentration constant. If the
inhibitor is directly competing with ATP, then this 5-fold overall
increase in [ATP] is equivalent to decreasing the inhibitor
concentration 5-fold in terms of the effect on % inhibition.
Consequently the % inhibition should decrease to the value observed
in the IC.sub.50 dose-response curve (obtained with 200 .mu.M ATP)
for 1/5 of the set inhibitor concentration used in this experiment
if direct competition with ATP is occurring. For inhibitor 2f (set
at 25 .mu.M) a 62% (+/-5), 54% (+/-3) and 50% (+/-1) inhibition at
200 .mu.M, 500 .mu.M and 1,000 .mu.M ATP, respectively, was
obtained whereas the level of inhibition should have dropped to ca.
20% at 1,000 .mu.M ATP if direct competition with ATP were
occurring. Similarly, for inhibitor 2b (set at 300 .mu.M) an 84%
(+/-1), 81% (+/-1) and 77% (+/-2) inhibition at 200 .mu.M, 500
.mu.M and 1,000 .mu.M ATP, respectively, was obtained. The high
cost of many kinases has stimulated other researchers to monitor
inhibitor potency as a function of increasing [ATP] for the same
purpose (Saperstein et al., 1989; Burke et al., 1993; Davis et al.,
1989; Davis et al., 1992; Faltynek et al., 1995; and Sawutz et al.,
1996).
[0593] In summary, structure-based design has produced a series of
hydroxynaphthalene pp60.sup.c-src non-peptide inhibitors that do
not compete with ATP. Results with compounds from this series in
cell-based assays, as well as detailed kinetic studies under
various assay conditions, will be reported in due course. An
extension of these design concepts from the naphthalene scaffold to
an indole scaffold is reported in the following Example.
Example 4
Design, Synthesis and Activity of Non-ATP Competitive Hydroxyindole
Derivative Inhibitors of pp60.sup.c-Src Tyrosine Kinase
[0594] In the preceding example, the structure-based design of a
series of pp60.sup.c-src inhibitors utilizing a naphthalene
scaffold is described. These compounds were designed to bind in the
peptide substrate site because of the potential for greater
selectivity and efficacy in a cellular environment relative to the
alternative ATP substrate site. This example presents an extension
of these design concepts to a series of pp60.sup.c-src inhibitors
based upon an indole scaffold. Once again the crystal structure of
the autoinhibited insulin receptor PTK (IRTK) was used to carry out
qualitative molecular modeling studies, except in this case an
indole ring was superimposed upon the IRTK Tyr 1,162. This
superimposition indicated that the indole NH can mimic the Tyr
1,162 NH, that a carbonyl should be placed at the 2-position, and a
hydroxyl group at the 5 position to mimic the Tyr 1,162 carbonyl
and OH, respectively (Scheme 1).
##STR00574##
The conceptual cyclization of Tyr 1,162 to the smaller 5-membered
ring of an indole illustrated in Scheme 1, relative to a 6-membered
ring in the case of the naphthalene scaffold (Karni et al., 1999),
results in a movement of the optimal positioning of the OH from
carbon 6 in the naphthalene scaffold to carbon 5 in the indole
scaffold.
[0595] The indole amide derivatives containing hydroxy
phenyl/benzyl side chains 2d-f, 2j-l (Table X), respectively, were
selected based upon the increase in pp60.sup.c-src inhibitor
potency observed for the analogous naphthalene-based hydroxy phenyl
amides reported in the previous example. The corresponding methyl
ethers 2a-c,g-i,v are precursors in the synthesis. The additional
analogs shown in Table X were prepared to begin expanding the range
of side chains beyond the hydroxy/methoxy groups that have now been
extensively probed with both the indole and naphthalene
scaffolds.
[0596] The indole amides containing only hydroxy or methoxy side
chains were synthesized as illustrated:
##STR00575##
[0597] The 2-indolecarboxylic acid derivative, the methoxyphenyl
amine (1.1 eq, Aldrich, Lancaster or Fluka), and the coupling
reagent PyBOP
(benzotriazol-1-yloxy)tripyrrolidino-phosphonium-hexafluorophosphate)
(1 eq, Fluka) were dissolved in anhydrous DMF. The solution was
cooled to 0.degree. C. under argon and then diisopropylethylamine
(DIEA, 3 eq) was added. The reaction was stirred at 0.degree. C.
for 1 m followed by 1 hour at room temperature. After workup the
residue was purified by silica gel chromatography.
[0598] The methyl ethers were cleaved with boron tribromide (1 M in
DCM, Aldrich) when desired. The indole amide methyl ether was
suspended in dry DCM and cooled to -78.degree. C. under argon. One
equivalent of BBr.sub.3 was added for each heteroatom in the
starting material plus one excess equivalent. The resulting dark
red solution was stirred at -78.degree. for 30 m and then at room
temperature for 1-2 hours. The reaction was quenched with water (10
minutes) before workup.
TABLE-US-00010 TABLE X pp60.sup.c-src INHIBITORY ACTIVITY OF
HYDROXYINDOLE DERIVATIVES..sup.a,b,c 1a-1b ##STR00576## 2a-2z
##STR00577## % Inhibition at 100 .mu.M Cmpd R.sub.1 R.sub.2 R.sub.3
R.sub.4 R.sub.5 R.sub.6 R.sub.7 (std. dev.) 1a H OH H CH.sub.3 N/A
N/A N/A 40 (+/- 5) [at 500 .mu.M] 1b H OH OH CH.sub.2CH.sub.2 N/A
N/A N/A 28 (+/- 3) 2a H OH H -- OCH.sub.3 H H 3 (+/- 1) 2b H OH H
-- H OCH.sub.3 H 21 (+/- 2) 2c H OH H -- H H OCH.sub.3 39 (+/- 9)
2d H OH H -- OH H H 43 (+/- 1) 2e H OH H -- H OH H 30 (+/- 6) 2f H
OH H -- H H OH 45 (+/- 3) 2g H OH H CH.sub.2 OCH.sub.3 H H 21 (+/-
5) 2h H OH H CH.sub.2 H OCH.sub.3 H 7 (+/- 6) 2i H OH H CH.sub.2 H
H OCH.sub.3 18 (+/- 4) 2j H OH H CH.sub.2 OH H H 24 (+/- 3) 2k H OH
H CH.sub.2 H OH H 74 (+/- 2) [IC.sub.50 = 38 .mu.M] 2l H OH H
CH.sub.2 H H OH 54 (+/- 2) 2m H OH H CH.sub.2CH.sub.2 H H OH 21
(+/- 7) 2n H OH H CH.sub.2 H H CO.sub.2H not active 2o H OH H
CH.sub.2 H H CO.sub.2CH.sub.3 11 (+/- 4) 2p H OH H -- H H
CH.sub.2CO.sub.2H 7 (+/- 6) 2q H OH H -- H H
CH.sub.2CO.sub.2CH.sub.3 32 (+/- 7) 2r H OH H -- H F H 21 (+/- 7)
2s H OH H CH.sub.2 H F H 57 (+/- 6) 2t H OH OH CH.sub.2 H OH H 26
(+/- 2) 2u H H OH CH.sub.2 H OH H 56 (+/- 6) 2v H H H CH.sub.2 H H
OCH.sub.3 4 (+/- 4) 2w H H H CH.sub.2 H H OH 36 (+/- 4) 2x OH H H
CH.sub.2 H OH H 60 (+/- 3) 2y H OH H CH(CH.sub.3) R H OH H 15 (+/-
3) 2z H OH H CH(CH.sub.3) S H OH H 13 (+/- 7) .sup.aAll compounds
were tested as described in the preceding Example..sup.5 .sup.bAll
compounds were characterized by proton NMR, FAB(+) MS and are pure
by TLC. .sup.cN/A = Not applicable.
[0599] Using this synthetic route, the series of 5-hydroxyindole
amide inhibitors 2a-m,y,z were prepared from
5-hydroxy-2-indolecarboxylic acid. The 4- and 6-hydroxyindole
amides (2x,u, respectively) were synthesized from methyl
4-methoxy-2-indolecarboxylate and methyl
6-methoxy-2-indolecarboxylate, respectively. The
5,6-dihydroxyindole amide 2t was prepared from ethyl
5,6-dimethoxyindole-2-carboxylate. Sonication of the esters in 1 N
NaOH for 1 hour provided the corresponding carboxylic acids for
coupling. The des-hydroxy indole amides 2v,w were synthesized from
indole-2-carboxylic acid. All of the indole starting materials were
commercially available (Aldrich or Lancaster).
[0600] The fluoro inhibitors 2r,s were likewise prepared from the
corresponding fluorophenyl amines (Aldrich). The inhibitors
containing esters or carboxylic acids on the amide side chain,
2n-q, were prepared from the corresponding amino carboxylic acids
(Aldrich). The side chain carboxylic acid was first protected as a
methyl ester (anh. MeOH pre-saturated with HCl, reflux, 1d),
followed by PyBOP coupling (as above), then saponification back to
the carboxylic acid when desired.
[0601] The methyl ester 1a was prepared by refluxing a solution of
the carboxylic acid overnight in anhydrous methanol pre-saturated
with HCl gas. The ethyl ester 1b was prepared by BBr.sub.3
deprotection of ethyl 5,6-dimethoxyindole-2-carboxylate as above.
All of the inhibitors listed in Table X were purified by silica gel
chromatography.
[0602] As in Marsilje 2000, the rank order activity of this series
of pp60.sup.c-src inhibitors was first determined at a constant
inhibitor concentration (Table X). The same inhibitor concentration
(100 .mu.M) was used for the current indole series of inhibitors,
the previous naphthalene series of inhibitors, and five non-ATP
competitive literature PTK inhibitors (see preceding example). This
allowed an efficient rank order comparison of 59 compounds in total
under identical assay conditions.
[0603] The modeling studies predicted that a hydroxy group at
carbon 5 of the indole scaffold would be optimal. Comparison of the
5-hydroxy indole inhibitor 2k (74%) with the analogous 6-hydroxy
indole inhibitor 2u (56%) and 4-hydroxy indole inhibitor 2x (60%)
confirms this prediction, although the preference is not strong.
The prediction that a hydroxy group at carbon 5 will improve the
activity (relative to no hydroxy group) is confirmed by comparing
the 5-hydroxy indole inhibitor 2l (54%) with the corresponding
des-hydroxy inhibitor 2w (36%).
[0604] Extending the indole inhibitors as aryl amides at carbon 2
improved potency, as expected based upon the previous naphthalene
inhibitors. For example, the meta-hydroxybenzyl amide indole 2k
gives 74% inhibition at 100 .mu.M whereas the analogous methyl
ester 1a gives only 40% inhibition at 500 .mu.M. Interestingly,
comparing the 5,6-dihydroxy ethyl ester 1b (28%) to the
corresponding aryl amide 2t (26%) shows that the simultaneous
presence of the second hydroxy at carbon 6 prevents the potency
enhancement normally provided by the otherwise preferred
meta-hydroxybenzyl amide side chain. This amide side chain was the
best of the current series when the 5-hydroxyl group is present
alone (2k, 74%) and still gave good inhibition when a 6-hydroxy
group was present alone (2u, 56%). Also, the simultaneous presence
of two hydroxy groups at carbons 5 and 6 seems well tolerated in
the absence of an amide side chain (1b vs. 1a and 2e). This data
suggests that a change in the binding orientation of the indole
scaffold may have occurred due to the presence of the second
hydroxy group and that a different amide side chain may now be
preferred. The optimal combination of side chains at carbons 4-7
(including functional group replacements for hydroxy groups (Lai et
al., 1999)) and amide side chains is currently under
investigation.
[0605] In general, the indole scaffold
structure-activity-relationships ("SARs") revealed by the data in
Table X parallels that reported in the preceding example for the
naphthalene scaffold. In both cases positioning a hydroxy group on
the scaffold analogous to the Tyr 1,162 OH, as identified by
modeling studies, provided the highest potency. Moving this hydroxy
group to one of the adjacent carbons reduced the potency, but not
dramatically, in both cases. Extending both scaffolds with aryl
amides at the position identified by the modeling studies to mimic
the Tyr 1,162 peptide bond improved the potency. With both
scaffolds, substitution of a methoxy group for the hydroxy groups
on the amide side chain usually reduced potency, and did so to a
greater extent with the longer benzylamide side chain (e.g. 2k, 74%
vs. 2h, 7% compared to 2e, 30% vs. 2b, 21%). The major difference
in the SARs for these two scaffolds is that the 5-hydroxyindole
scaffold prefers the longer m-hydroxybenzyl amide side chain (2k,
74% vs. 2e, 30%) whereas the analogous 3,6-dihydroxynaphthalene
scaffold prefers the shorter amide side chain derived from
m-hydroxyaniline. The 5-hydroxyindole scaffold showed essentially
no preference for the position of the hydroxyl group on the shorter
amide side chain (2d-f) whereas with the longer hydroxybenzyl amide
side chain a significant preference for the meta position was
observed (2j-l). In the case of the 3,6-dihydroxynaphthalene
scaffold the opposite was observed.
[0606] Additional molecular modeling studies were carried out to
further probe the preference for a longer amide side chain with the
indole scaffold. The most active naphthalene inhibitor 3 from the
previous report was used as a template upon which the analogous
indole inhibitor 2e and the homologated indole inhibitor 2k were
superimposed. The three most important side chain functional groups
in naphthalene inhibitor 3 are considered to be the 6-hydroxy group
(H-bond donor and acceptor), the hydrogen from the 3-hydroxy group
(H-bond donor), and the side chain hydroxy group (H-bond acceptor)
based upon the rational design and SAR for both series of
inhibitors. This three point pharmacophore model is identified in
both series by asterisks in Scheme 3.
##STR00578##
[0607] The "multifit" energy minimization and "fit atoms"
facilities within SYBYL.TM. (6.4, Tripos, St. Louis) were used in
sequence to superimpose 2e and 2k onto 3. This overall fitting
process was carried out with spring constants (multifit) and
weights (fit atoms) chosen such that the highest emphasis was on
optimally superimposing the scaffold pharmacophore O's and H's
(100), followed by the side chain O's (10) and then the intervening
amide bond (1). The "multifit" process adjusted the conformations
for maximum pharmacophore fit, the subsequent minimization produced
the nearest local minimum energy conformations and finally the "fit
atoms" process produced the best pharmacophore superimposition of
these minimized conformations. As expected, the scaffold
pharmacophore O's and H's of both 2e and 2k superimposed closely
and similarly upon the corresponding atoms in 3 (all within ca.
0.50 .ANG.). However, the side chain pharmacophore O's of 2e and 2k
differed significantly in their superimposition on the
corresponding O of 3, with displacements of 1.8 .ANG. vs. only 0.08
.ANG. respectively. This close fit of the three key pharmacophore
sites between 2k and 3 provides a rationalization for their potency
differing by only a factor of 2.4 (IC.sub.50's 38 .mu.M vs. 16
.mu.M, respectively).
[0608] Extending the amide side chain by another carbon atom
reduced the activity (2m, 21% vs. 2l, 54%). Adding a methyl group
to the benzylic carbon of 2k, in either stereochemistry, greatly
reduced the activity (2y, 15% & 2z, 13% vs. 2k, 74%). Replacing
the side chain hydroxy group (in the para position) with a
carboxylate anion (2n, 0% vs. 2l, 54% and 2p, 7% vs. 2f, 45%)
reduced the activity whereas the corresponding methyl esters (2o,
11% & 2q, 32%, respectively) showed a smaller loss of potency.
Importantly, replacing the side chain hydroxy group with a fluorine
maintained much of the potency (2s, 57% vs. 2k, 74% and 2r, 21% vs.
2e, 30%). Consequently, the fluoro analog 2s has only one hydroxy
group left for potential Phase II metabolism (e.g. glucuronide
formation), and this remaining hydroxy group is a current target
for replacement (Lai et al., 1998).
[0609] Using the same method as in the preceding example (Marsilje,
2000), the most potent inhibitor from the current indole series
(2k) was analyzed for ATP competition by monitoring the %
inhibition at increasing [ATP] while holding the inhibitor
concentration constant. Since the [ATP] had little effect on the %
inhibition (The % inhibition was 46% and 41% with 2k at 45 .mu.M
and [ATP] at 200 .mu.M or 1,000 .mu.M, respectively.), 2k is
non-competitive with respect to ATP under these assay
conditions.
[0610] In summary, an indole scaffold has been designed, and an
initial SAR carried out, for the development of non-ATP competitive
pp60.sup.c-src inhibitors. The potency of the best indole-based
inhibitor from the current series was found to be close to that of
the best naphthalene-based inhibitor. The % inhibition was 46% and
41% with 2k at 45 .mu.M and [ATP] at 200 .mu.M or 1,000 .mu.M,
respectively.
Example 5
Synthesis of Additional Indole Derivative Protein Kinase
Inhibitors
[0611] The following results show the synthesis and testing of
indole derived protein kinase inhibitors. Four reaction schemes are
provided and separately followed by experimental details for the
preparation of the final product of each of these reaction schemes.
These final products are examples of indole-base tyrosine kinase
inhibitors wherein the synthesis with preferred R groups is
illustrated (boronic acid, Scheme 1; OH, Scheme 2; an aliphatic
amide extension, Scheme 3; and a phosphonic acid Scheme 4).
##STR00579##
Methyl 5-hydroxy-2-indolecarboxylate (1)
[0612] Dissolved 3.50 g 5-hydroxy-2-indolecarboxylic acid in anh.
MeOH presaturated with HCl gas. Refluxed for 48 hours. Concentrated
in vacuo and triturated with AcCN.times.3 to remove residual acid.
Filtered through silica plug with EtOAc to remove baseline
contamination. Recovered 4.32 g (quant. yield) TLC R.sub.f=0.78
(EtOAc) 1H NMR (DMSO-d.sub.6): 3.82 (s, 3H), 6.78 (d, J=8.8 Hz,
1H), 6.88 (s, 1H), 6.93 (s, 1H), 7.23 (d, J=8.8 Hz, 1H), 8.90 (s,
1H) 11.62 (s, 1H) FAB(+) MS m/e 191.9 (M+1)
Methyl 5-[(trifluoromethyl)sulfonyloxy]indole-2-carboxylate (2)
[0613] Added 150 ml anh. DCM to 3.24 g (17 mmol) methyl
5-hydroxy-2-indolecarboxylate (1) and 6.67 g (18.7 mm) n-phenyl
trifluoromethane sulfonamide at 0.degree. C. Added 2.6 ml
triethylamine dropwise at which point clear yellow solution formed.
Stirred at 0.degree. C. for 1 hour. Warmed to room temperature and
stirred for 2 hours. Concentrated in vacuo and purified through
silica gel column (1/1 EtOAc/hexanes). Recovered 4.69 g (86%). TLC
R.sub.f=0.63 (1/1 EtOAc/hexanes). HPLC R.sub.f=20.879 1H NMR
(DMSO-d.sub.6): 3.87 (s, 3H), 7.25 (s, 1H), 7.31 (d, J=9.2 Hz, 1H),
7.55 (d, J=9.2 Hz, 1H), 7.80 (s, 1H), 12.34 s, 1H) FAB(+) MS m/e
323.1 (M+1).
Methyl 5-methylindole-2-carboxylate,
4,4,5,5-tetramethyl-1,3,2-dioxaborolanemethyl (3)
[0614] 500 mg 1.55 mmol methyl
5-[(trifluoromethyl)sulfonyloxy]indole-2-carboxylate (2), 37.9 mg
(0.05 mmol) PdCl.sub.2 (dppf), 432 mg (1.7 mmol)
bispinacolatodiboron, 454.8 mg (4.65 mmol) potassium acetate, and
25.7 mg (0.05 mmol) dppf were added to a flask and vacuum dried at
40.degree. C. for 2 hours. Added 20 ml anh dioxane and heated to
80.degree. C. overnight. Reaction turned black as Pd black
precipitated out. Filtered off catalyst and ran silica plug to
remove baseline impurities. TLC R.sub.f=0.51 (1/4 EtOAc/Hexane)
Crude product was taken through to next reaction.
Methyl 5-boronyl indole-2-carboxylate (4)
[0615] 391.2 mg (1.3 mmol) methyl 5-methylindole-2-carboxylate,
4,4,5,5-tetramethyl-1,3,2-dioxaborolanemethyl (3) was dissolved in
EtOAc. 0.25 ml (2.6 mmol) diethanolamine was added, and the
reaction was stirred at room temperature overnight. The white ppt
which formed was filtered and sonicated in 1 N HCl. The resulting
white ppt was filtered, dissolved in MeOH, and concentrated in
vacuo. Recovered 36.6 mg (13%). HPLC R.sub.f=13.912, 1H NMR
(DMSO-d.sub.6): 3.85 (s, 3H), 7.15 s, (1H), 7.36 (d, J=8.4 Hz, 1H),
7.67 (d, J=8.4 Hz, 1H), 7.87 (s, 1H), 8.14 (s, 1H), 11.91 (s,
1H).
##STR00580##
(5-hydroxyindol-2-yl)-N-[(3-methoxyphenyl)methyl]carboxyamide
(5)
[0616] Dissolved 2.00 g (11.3 mmol) 5-hydroxy-2-indolecarboxylic
acid, 1.6 ml (12.4 mmol) 3 methoxybenzylamine, and 5.87 g (11.3
mmol) PyBOP in 10 ml anh. DMF. Cooled to 0.degree. C. and added 5.9
ml (33.9 mmol) DIEA. Stirred for 5 minutes at 0.degree. C. and
allowed to warm to room temperature for 1 hour. Recovered 2.83 g
(85% yield) TLC R.sub.f=0.34 (1/1 EtOAc/hexanes) 1H NMR
(DMSO-d.sub.6): 3.70 (s, 3H), 4.43 (d, J=4.4 Hz, 2H) 6.69 (d, J=8.8
Hz, 1H), 6.78 (d, J=7.7 Hz, 1H), 6.83 (s, 1H), 6.86 (s, 1H), 6.94
(s, 1H), 7.20 (m, 3H), 8.92 (t, J=4.4 Hz, 1H), 11.36 (s, 1H) FAB(+)
MS m/e 297.3 (M+1)
(5-hydroxyindol-2-yl)-N-[(3-hydroxyphenyl)methyl]carboxyamide
(6)
[0617] Added 20 ml anh. DCM to 200 mg (0.67 mmol)
(5-hydroxyindol-2-yl)-N-[(3-methoxyphenyl)methyl]carboxyamide (5)
and cooled to -78.degree. C. under argon. Added 4.0 ml (4.0 mmol, 6
eq) BBr.sub.3. Held at -78.degree. C. for 5 minutes and warmed to
rt. After 90 minutes at room temperature, quenched with H.sub.2O
and stirred for 10 minutes. Diluted reaction mix with EtOAc and
washed with NaHCO.sub.3 and brine. Dried organic layer over
MgSO.sub.4 and concentrated in vacuo. Ran through silica plug to
remove baseline contamination. Recovered X mg. (80% yield.) TLC
R.sub.f=0.21 (1/1 EtOAc/hexanes). .sup.1H NMR (DMSO-d.sub.6): 4.38
(d, J=4.8 Hz, 2H), 6.59 (d, J=8.8 Hz, 1H), 6.71 (m, 3H) 6.83 (d,
J=1.8 Hz, 1H), 6.94 (s, 1H), 7.08 (dd, J=7.7 Hz, 1H), 7.19 (d,
J=8.8 Hz, 1H), 8.84 (t, J=5.9 Hz), 11.28, (s, 1H). FAB(+) MS m/e
283.2 (M+1)
##STR00581##
N-(1-carbamoyl-2-methylbutyl)(5-hydroxyindol-2-yl)carboxyamide
(7)
[0618] 100 mg (0.56 mmol) 5-hydroxy-2-indolecarboxylic acid, 103.4
mg (0.62 mmol, 1.1 eq) L-isoleucinamide, and 291 mg (0.56 mmol, 1
eq) PyBOP were all dissolved in 1 ml anh DMF. The solution was
cooled to 0.degree. C. and 0.3 ml (1.68 mmol, 3 eq) DIEA was added.
The reaction mixture was stirred for 1 minute at 0.degree. C. and
at room temperature for 1 hour. The reaction was then diluted with
EtOAc and washed with 1 N HCl.times.3 and sated NaHCO3.times.3. The
organic layer was dried over MgSO4, and concentrated in vacuo to
give 166.7 mg (91% yield.) TLC R.sub.f=0.08 (1/1 EtOAc/hexanes).
.sup.1H NMR (DMSO-d.sub.6): 0.83 (m, 6H), 1.15 (m, 2H), 1.68 (m,
1H), 1.83 (m, 1H), 4.29 (t, J=8.8 Hz, 1H), 6.69 (d, J=8.5 Hz, 1H),
6.83 (s, 1H), 7.01, (s, 1H), 7.06 (s, 1H), 7.19 (d, J=8.4 Hz, 1H),
7.48, (s, 1H), 8.00 (d, 9.2 Hz, 1H), 8.76 (s, 1H), 11.3, (s, 1H).
FAB(+) MS m/e 290.1 (M+1)
##STR00582##
Methyl 5-dibenzylphosphorylindole-2-carboxylate (8)
[0619] 200 mg (0.62 mmol) methyl
5-[(trifluoromethyl)sulfonyloxy]indole-2-carboxylate (2), 195.8 mg
(0.74 mmol, 1.2 eq) dibenzylphosphite, 0.14 ml (0.81 mmol, 1.3 eq)
DIEA, and 35.7 mg (0.03 mmol, 5 mol %) Pd(PPh.sub.3).sub.4 were all
dissolved in anh AcCN under argon. The reaction mix was heated to
80.degree. C. overnight. The solvent was removed under reduced
pressure, and the title compound was isolated by silica gel
chromatography. 130 mg (50% yield). TLC R.sub.f=0.28 (1/1
EtOAc/hexanes) .sup.1H NMR (DMSO-d.sub.6): 3.85 (s, 3H), 4.98-5.01
(m, 4H), 7.28-7.32 (m, 11H), 7.53-7.55 (m, 2H), 8.17 (d, J=14.6 Hz,
1H) .sup.31P NMR (DMSO-d.sub.6): 23.89.
Methyl 5-phosphonolindole-2-carboxylate
[0620] Methyl 5-dibenzylphosphorylindole-2-carboxylate (8) (125 mg)
was dissolved in 10 ml MeOH. 20 mg Pd--C was added and the mixture
was hydrogenated in a Parr apparatus overnight. Filtered off
catalyst and removed solvent under reduced pressure. Obtained 72.5
mg (73% yield). TLC R.sub.f=baseline in EtOAc. .sup.1H NMR
(DMSO-d.sub.6): 3.84 (s, 3H), 7.24 (s, 1H), 7.44-7.49 (m, 2H), 8.01
(d, J=14.3 Hz, 1H) 12.11 (s, 1H) .sup.31P NMR (DMSO-d.sub.6):
17.22.
[0621] The ester compounds in this example could be increased in
potency by converting the ester to an amide and/or adding
additional specificity elements.
Example 6
Synthesis of Further Indole Derivative Protein Kinase
Inhibitors
[0622] The synthesis of some further elaborated indole inhibitors
is illustrated in below. These syntheses could result in compounds
with greater potency against pp60c-src and other tyrosine kinases.
The methyl ester group can be subsequently converted into various
amide derivatives to increase potency.
##STR00583##
##STR00584##
##STR00585##
Example 7
Toxicity of Src inhibitors
[0623] There is considerable recent literature support for
targeting pp60.sup.c-src (Src) as a broadly useful approach to
cancer therapy without resulting in serious toxicity. For example,
tumors that display enhanced EGF receptor PTK signaling, or
overexpress the related Her-2/neu receptor, have constitutively
activated Src and enhanced tumor invasiveness. Inhibition of Src in
these cells induces growth arrest, triggers apoptosis, and reverses
the transformed phenotype (Karni et al., 1999). It is known that
abnormally elevated Src activity allows transformed cells to grow
in an anchorage-independent fashion. This is apparently caused by
the fact that extracellular matrix signaling elevates Src activity
in the FAK/Src pathway, in a coordinated fashion with mitogenic
signaling, and thereby blocks an apoptotic mechanism which would
normally have been activated. Consequently FAK/Src inhibition in
tumor cells may induce apoptosis because the apoptotic mechanism
which would have normally become activated upon breaking free from
the extracellular matrix would be induced (Hisano et al., 1997).
Additionally, reduced VEGF mRNA expression was noted upon Src
inhibition and tumors derived from these Src-inhibited cell lines
showed reduced angiogenic development (Ellis et al., 1998).
[0624] The issue of potential toxicity of Src inhibition has been
addressed with very promising results. For example, a knock-out of
the Src gene in mice led to only one defect, namely osteoclasts
that fail to form ruffled borders and consequently do not resorb
bone. However, the osteoclast bone resorb function was rescued in
these mice by inserting a kinase defective Src gene (Schwartzberg
et al., 1997). This suggested that Src kinase activity can be
inhibited in vivo without triggering the only known toxicity
because the presence of the Src protein is apparently sufficient to
recruit and activate other PTKs (which are essential for
maintaining osteoclast function) in an osteoclast essential
signaling complex.
[0625] Src has been proposed to be a "universal" target for cancer
therapy since it has been found to be overactivated in a growing
number of human tumors, in addition to those noted above (Levitzki,
1996). The potential benefits of Src inhibition for cancer therapy
appear to be four-fold based upon the cited, and additional,
literature. They are: 1) Inhibition of uncontrolled cell growth
caused by autocrine growth factor loop effects, etc. 2) Inhibition
of metastasis due to triggering apoptosis upon breaking free from
the cell matrix. 3) Inhibition of tumor angiogenesis via reduced
VEGF levels. 4) Low toxicity.
[0626] The initial non-peptide Src inhibitors have also shown very
encouraging results in four different series of cell culture
assays. 1) In the NIH 60-tumor cell panel assay, broad activity (as
one would expect for a Src inhibitor) was seen against the tumor
cell lines, including the prostate lines. For example, three of the
inhibitors gave the following growth inhibition IC.sub.50's against
the NIH prostate cancer cell lines: 45 (PC-3, 15 .mu.M; DU-145, 38
.mu.M), 43-meta (PC-3, 19 .mu.M), 49-meta (PC-3, 39 .mu.M; DU-145,
>100 .mu.M). 2) In the v-Src transformed normal rat kidney cell
line (LA25) 43-meta and 45 specifically blocked the v-Src induced
cell growth without inhibiting the normal growth of the parent
non-transformed cells. This result showed that the inhibitors do
not affect normal cells but are effective in blocking Src induced
cell transformation. 3) The Src inhibitors were compared to the
cancer drugs etoposide, taxol, doxorubicin and cisplatin in ovarian
tumors from three different patients and an abdominal carcinoma
from another patient. In all cases, the Src inhibitors were at
least as effective, and typically more effective, than the known
cancer drugs, with full efficacy seen at the lowest dose tested (3
.mu.M). As a representative example, a comparison of taxol and
doxorubicin (they were more effective than etoposide &
cisplatin in this particular tumor cell culture) with the three Src
inhibitors mentioned above (structures shown in FIG. 16E) utilizing
ovarian tumor cells from tumor N015 is shown in FIG. 16A. 4) The
Src inhibitors were also tested for inhibition of normal human
fibroblast cell growth and found no inhibition of normal cell
growth (both subconfluent and confluent; some enhanced growth was
observed instead), indicating that these inhibitors are not toxic
to normal cells even at a 10-fold higher concentration. An example
of his data is given in FIG. 16B. 5) Two of the Src inhibitors were
also tested for inhibition of ts v-Src stimulated LA25 cell growth.
The results are shown in FIG. 16C. These results show that the
tested compounds inhibit Src stimulated cell growth. 6) Two of the
Src inhibitors were also tested for inhibition of normal rat kidney
cell growth. The results are shown in FIG. 16D and illustrate that
the inhibitors are cytoprotective for normal cells.
[0627] Overall, the cell data obtained thus far shows what one
might expect for Src inhibitors, i.e. broad activity against many
cancer cell lines with little or no normal cell toxicity.
[0628] The preliminary Src inhibitors are lead structures from
which it is possible to design more potent and selective
inhibitors. In addition to utilizing the tyrosine kinase crystal
structures, molecular modeling studies can be carried out with the
natural product tyrosine kinase inhibitor damnacanthal (Faltynek et
al., 1995) to investigate its peptide-competitive binding mode.
These additional modeling studies are enable one to design further
analogs of Src inhibitors wherein the key pharmacophore elements of
damnacanthal are incorporated into the new inhibitors. Their
syntheses will be undertaken and the isolated Src testing done as
reported (Marsilje 2000).
Example 8
Protection Against Noise-Induced Hearing Loss Using PTK
Inhibitors
[0629] Chinchillas (N=8) were used in studies of noise-induced
hearing loss. The animals' hearing sensitivity was measured using
standard electrophysical techniques before the experimental
manipulation. In particular, hearing thresholds were measured
through evoked potentials from recording electrodes chronically
implanted in the inferior colliculus, following standard laboratory
procedures. Animals were anesthetized, the auditory bullae were
opened, and the left and right cochleas were visualized. The round
window leading to the scala tympani of the cochlea was used as the
access point for drug application. Four animals were treated with
30 .mu.l of 3 mM TOM 2-32, emulsified in DMSO, to 1000 mM of saline
solution, which was placed on the round window of one ear, and a
control solution of 3 mM DMSO to 1000 mM of saline solution, which
was placed on the round window of the other ear. Five animals were
treated with 30 .mu.l of 3 mM compound 1a, emulsified in DMSO, to
1000 mM of saline solution, which was placed on the round window of
one ear, and a control solution of 3 mM DMSO to 1000 mM of saline
solution, which was placed on the round window of the other ear
(one animal was lost prior to the end of the experiments). In each
case, the solution was allowed to set on the round window for 30
minutes, then the auditory bullae were closed. Subsequently, the
animals were exposed to 4 kHz band noise at 105 dB SPL for four
hours. Following the noise exposure, the animals' hearing was
tested at day 1, day 3, day 7, and day 20 to determine evoked
potential threshold shifts. Permanent threshold shift was assessed
at day 20. The cochleas were harvested at day 20 to allow for
morphological examination of the cochleas. Data for TOM 2-32 is
shown in Tables XI-XIII and data for compound 1a is shown in Table
XIV, below.
TABLE-US-00011 TABLE XI 0.5 kHz 1 kHz 2 kHz 4 kHz 8 kHz Pretest
Control 20 20 15 15 15 25 20 25 20 10 Control 22.5 20 20 17.5 12.5
TOM2 25 25 20 15 10 30 30 30 20 10 TOM2 27.5 27.5 25 17.5 10 TOM2
6778 Day 0 55 70 75 85 80 Day 1 35 30 65 75 75 Day 3 15 5 40 45 60
Day 20 5 5 25 30 0 Control 6778 Day 0 60 75 80 80 75 Day 1 45 40 75
75 75 Day 3 10 10 50 55 55 Day 20 5 10 40 35 25 TOM 6679 Day 0 55
60 65 75 75 Day 1 30 50 60 70 75 Day 3 20 25 40 55 45 Day 20 -5 0
10 23 -5 Control 6679 Day 0 55 70 70 75 80 Day 1 40 60 65 70 80 Day
3 35 60 65 75 80 Day 20 0 10 25 35 10 Control Day 0 55 70 70 75 80
Day 0 70 75 80 80 75 Average Day 0 57.5 72.5 75 77.5 77.5 Day 1 40
60 65 70 80 Day 1 45 40 75 75 75 Average Day 1 42.5 50 70 72.5 77.5
Day 3 35 60 65 75 80 Day 3 10 10 50 55 55 Average Day 3 22.5 35
57.5 65 67.5 Day 20 0 10 25 35 10 Day 20 5 10 40 35 25 Average Day
20 2.5 10 32.5 35 17.5 TOM2 Day 0 55 70 75 85 80 Day 0 55 60 65 75
75 Average day 0 55 65 70 80 77.5 Day 1 35 30 65 75 75 Day 1 30 50
60 70 75 Average Day 1 32.5 40 62.5 72.5 75 Day 3 15 5 40 45 60 Day
3 20 25 40 55 45 Average Day 3 17.5 15 40 50 52.5 Day 20 5 5 25 30
0 Day 20 -5 0 10 23 -5 Average Day 20 0 2.5 17.5 26.5 -2.5 Control
Day 0 57.5 72.5 75 77.5 77.5 TOM2 Day 0 55 65 70 80 77.5 Control
Day 1 42.5 50 70 72.5 77.5 TOM2 Day 1 32.5 40 62.5 72.5 75 Control
Day 3 22.5 35 57.5 65 67.5 TOM2 Day 3 17.5 15 40 50 52.5 Control
Day 20 2.5 10 32.5 35 17.5 TOM2 Day 20 0 2.5 17.5 26.5 -2.5 Control
Day 0 57.5 72.5 75 77.5 77.5 Control Day 1 42.5 50 70 72.5 77.5
Control Day 3 22.5 35 57.5 65 67.5 Control Day 20 2.5 10 32.5 35
17.5 TOM2 Day 0 55 65 70 80 77.5 TOM2 Day 1 32.5 40 62.5 72.5 75
TOM2 Day 3 17.5 15 40 50 52.5 TOM2 Day 20 0 2.5 17.5 26.5 -2.5 TOM2
Day 20 0 2.5 17.5 26.5 -2.5 Control Day 20 2.5 10 32.5 35 17.5
TABLE-US-00012 TABLE XII 0.5 kHz 1 kHz 2 kHz 4 kHz 8 kHz TOM2 6696
day 0 38 50 70 75 80 day 1 27 20 70 75 65 day 3 10 15 55 53 55 day
7 13 10 45 50 50 day 20 Control 6696 day 0 35 45 75 80 90 day 1 30
40 75 80 80 day 3 7 15 50 60 70 day 7 5 15 45 50 60 day 20 TOM2
6698 day 0 30 45 65 70 80 day 1 0 15 45 65 60 day 3 -5 5 25 40 40
day 7 -5 5 25 25 0 day 20 -5 0 0 0 -5 Control 6698 day 0 55 40 65
70 80 day 1 10 15 45 60 70 day 3 15 15 45 40 55 day 7 5 5 25 35 10
day 20 10 10 20 30 5 TOM2 6699 day 0 70 65 70 75 85 day 1 60 65 70
75 85 day 3 25 20 70 70 75 day 7 10 5 45 40 45 day 20 10 10 45 40
45 Control 6699 day 0 70 70 65 70 85 day 1 60 70 65 70 85 day 3 50
65 60 65 70 day 7 38 55 50 45 65 day 20 28 35 50 45 60 Pretest
Controls 25 25 30 25 10 25 30 30 25 15 Controls 25 27.5 30 25 12.5
TOM2 35 30 30 25 15 25 30 25 20 10 TOM2 30 30 27.5 22.5 12.5
TABLE-US-00013 TABLE XIII Control TOM2 0.5 kHz day 0 62.5 50 day 1
35 30 day 3 32.5 10 day 7 21.5 2.5 day 20 19 2.5 1 kHz day 0 55 55
day 1 42.5 40 day 3 40 12.5 day 7 30 5 day 20 22.5 5 2 kHz day 0 65
67.5 day 1 55 57.5 day 3 52.5 47.5 day 7 37.5 35 day 20 35 22.5 4
kHz day 0 70 72.5 day 1 65 70 day 3 52.5 55 day 7 40 32.5 day 20
37.5 20 8 kHz day 0 82.5 82.5 day 1 77.5 72.5 day 3 62.5 57.5 day 7
37.5 22.5 day 20 32.5 20
TABLE-US-00014 TABLE XIV 0.5 kHz 1 kHz 2 kHz 4 kHz 8 kHz Pretest
Control 6821 30 20 25 15 7.5 Control 6845 30 25 20 15 10 Control
6850 27 25 15 10 0 Control 6828 20 15 20 5 5 Control 6829 20 20 20
20 5 Control 25.4 21 20 13 5.5 CH65 6821 30 22.5 25 20 10 CH65 6845
35 25 25 10 0 CH65 6850 25 25 10 12 0 CH65 6828 20 25 15 5 0 CH65
6829 23 25 25 15 10 CH65 26.6 24.5 20 12.4 4 Day 1 Control 6821 5
25 50 60 72.5 Control 6845 15 15 40 75 80 Control 6850 8 10 25 65
55 Control 6828 5 20 55 75 80 Control 8.25 17.5 42.5 68.75 71.875
Day 1 ctr1sd 4.716991 6.454972 13.22876 7.5 11.79248 CH65 6821 0
2.5 0 10 0 CH65 6845 5 20 25 35 30 CH65 6850 10 0 20 68 35 CH65
6828 0 5 55 55 25 CH65 Day 1 3.75 6.875 25 42 22.5 4.787136
8.984941 22.7303 25.28504 15.54563 Day 3 Control 6821 10 25 50 55
67.5 Control 6845 10 15 35 55 45 Control 6850 3 5 15 25 15 Control
6828 5 15 40 65 55 Control 6829 20 20 35 45 45 Control 9.6 16 35 49
45.5 Day 3 6.580274 7.416198 12.74755 15.16575 19.39716 CH65 6821 5
7.5 0 10 0 CH65 6845 5 5 0 0 15 CH65 6850 5 2 15 25 15 CH65 6828 0
0 40 55 0 CH65 6829 12 20 35 45 45 CH65 Day 3 5.4 6.9 18 27 15
4.27785 7.861298 18.90767 23.07596 18.37117 Day 7 Control 6821 0 10
20 20 45 Control 6845 10 15 25 45 45 Control 6850 6 5 15 30 10
Control 6828 10 20 37 65 60 Control 6829 20 25 45 40 55 Control 9.2
15 28.4 40 43 Day 7 7.293833 7.905694 12.36123 16.95582 19.55761
CH65 6821 0 0 0 0 0 CH65 6845 5 15 0 5 20 CH65 6850 8 0 15 15 0
CH65 6828 15 0 40 55 30 CH65 6829 12 15 20 45 40 CH65 Day 7 8 6 15
24 18 5.87367 8.215838 16.58312 24.59675 17.888854 Day 20 Control
6821 0 10 25 35 7.5 Control 6845 0 5 25 45 40 Control 6850 8 0 10
20 10 Control 6829 15 20 30 30 18 Control 5.75 8.75 22.5 32.5
18.875 Day 20 7.228416 8.539126 8.660254 10.40833 14.77822 CH65
6821 0 0 0 0 -5 CH65 6845 0 0 0 0 5 CH65 6850 5 0 15 11 0 CH65 6829
7 5 10 25 15 CH65 3 1.25 6.25 9 3.75 Day 20 3.559026 2.5 7.5
11.80603 8.539126 Control 8.25 17.5 42.5 68.75 71.875 Day 1 Control
9.6 16 35 49 45.5 Day 3 Control 9.2 15 28.4 40 43 Day 7 Control
5.75 8.75 22.5 32.5 18.875 Day 20 Ch65 Day 1 3.75 6.875 25 42 22.5
CH65 Day 3 5.4 6.9 18 27 15 CH65 Day 7 8 6 15 24 18 CH65 3 1.25
6.25 9 3.75 Day 20
[0630] FIGS. 16-22 show the average threshold shifts for animals
treated with TOM 2-32. In particular, FIG. 16 shows average
threshold shifts after exposure to 0.5 kHz, 1 kHz, 2 kHz, 4 kHz,
and 8 kHz band noise tested prior to experimental manipulation
(i.e., exposure to 4 kHz band noise at 105 dB for four hours). FIG.
17 shows average threshold shifts after exposure to 0.5 kHz band
noise at day 0, day 1, day 3, and day 20 after experimental
manipulation. FIG. 18 shows average threshold shifts after exposure
to 1 kHz band noise at day 0, day 1, day 3, and day 20 after
experimental manipulation. FIG. 19 shows average threshold shifts
after exposure to 2 kHz band noise at day 0, day 1, day 3, and day
20 after experimental manipulation. FIG. 20 shows average threshold
shifts after exposure to 4 kHz band noise at day 0, day 1, day 3,
and day 20 after experimental manipulation. FIG. 21 shows average
threshold shifts after exposure to 8 kHz band noise at day 0, day
1, day 3, and day 20 after experimental manipulation. FIG. 22 shows
the average dB threshold shift at day 20 for control and treated
ears. As shown in FIGS. 17-22 the average dB threshold shifts for
treated ears were lower, indicating less hearing loss.
[0631] FIGS. 23-28 show the average threshold shifts for animals
treated with compound 1a. In particular, FIG. 23 shows average
threshold shifts after exposure to 0.5 kHz, 1 kHz, 2 kHz, 4 kHz,
and 8 kHz band noise tested prior to experimental manipulation.
FIG. 24 shows average threshold shifts after exposure to 0.5 kHz, 1
kHz, 2 kHz, 4 kHz, and 8 kHz band noise on day 1 after experimental
manipulation. FIG. 25 shows average threshold shifts after exposure
to 0.5 kHz, 1 kHz, 2 kHz, 4 kHz, and 8 kHz band noise on day 3
after experimental manipulation. FIG. 26 shows average threshold
shifts after exposure to 0.5 kHz, 1 kHz, 2 kHz, 4 kHz, and 8 kHz
band noise on day 7 after experimental manipulation. FIG. 27 shows
average threshold shifts after exposure to 0.5 kHz, 1 kHz, 2 kHz, 4
kHz, and 8 kHz band noise on day 20 after experimental
manipulation. FIG. 18 shows average threshold shifts after exposure
to 8000 Hz on day 1, day 3, day 7, and day 20. As shown in FIGS.
24-28, the average dB threshold shifts for treated ears were lower,
indicating less hearing loss.
[0632] As shown in FIGS. 16-28, both TOM 2-32 and CH-65 provided
protection against the noise exposure. However, compound 1a
provided the greatest level of protection. In particular, the PTK
inhibitor treated ears had, on average, 15 to 25 dB less hearing
loss than the control ears and the animals showed no side effects
of the experimental manipulation.
Example 9
Inhibition of Noise-Induced Apoptosis in Cochlear Hair Cells Using
Inhibitors of the PTKs
[0633] Chinchillas (N=3) were exposed to 75-pairs of impulse noise
at 155 dB pSPL. The animals were sacrificed 5 minutes after the
noise exposure. The cochleas were examined for activation of the
focal adhesion complex using an antibody against focal adhesion
kinase, which is an intrinsic member of the complex.
[0634] FIGS. 29A-F show the effect of high level impulse noise on
chinchilla cochleas without treatment with a PTK inhibitor. In
particular, FIG. 29A is an electron micrograph which shows the
cochlear damage following high level impulse noise (155 dB). FIG.
29A shows a spilt at the reticular lamina (S). The split appears to
be between the second and third rows of outer hair cells. FIG. 29B
depicts a cochlea stained immunohistochemically for focal adhesion
kinase (FAK) following a moderately high level octave band noise
(105 dB). The staining observed in FIG. 29B is relatively low level
and approximates that observed without noise. The staining appears
to be localized primarily at the pharangeal processes of the Deiter
cells and not at the hair cells. Upon elevating the noise level to
110 dB OBN, apoptotic cells appeared, as shown in FIG. 29C. These
apoptotic cells are located in two regions of the upper left
quadrant of the figure and the nuclei appear bright and highly
condensed, whereas the normal nuclei are large and more diffuse in
color. FIG. 29D is a photo of the same cells stained with focal
adhesion kinase (FAK) antibody (as in FIG. 29B; however, here the
pharangeal processes appear to surround a lesion where cells are
missing). These lesions correspond to the areas in FIG. 29C where
the cells underwent apoptosis. FIG. 29E shows the same region but
at a lower vertical plane, demonstrating that the lesion extends
well below the cuticular plate and into the cell body. FIG. 29F
shows cochleas exposed to impulse noise at 155 dB SPL. The cochleas
lost their integrity at the cuticular plate and were heavily
stained throughout. Many dark areas are seen, which represent areas
where hair cells have died.
[0635] FIG. 30A shows a cochlea pretreated with compound 1a,
whereas FIG. 30B shows an untreated cochlea following exposure to
high level noise (155 dB). In the treated cochlea (FIG. 30A), there
is a high level of FAK staining that extends beyond the pharangeal
processes of the Deiter cells and well into the cuticular plate.
The punctate nature of the staining is indicative of the formation
of focal adhesion complexes of which FAK is an intrinsic member.
Furthermore, the three rows of hair cell nuclei (labeled OHC1-3)
appear both in order and intact and without any indication of
apoptosis taking place. Since FAK is known to be active within
focal adhesion complexes, this data strongly suggests that FAK is
active following a high noise exposure. It is hypothesized that it
is the inhibition of the kinase function that is prevented through
the treatment with compound 1a and results in the survival of
cochlear hair cells.
[0636] In contrast to FIG. 30A, FIG. 30B demonstrates a somewhat
lower level of FAK staining, but also shows a remarkably high level
of cell death. In this figure, nearly half of the cells have died
by apoptosis, as indicated by the number of condensed nuclei. This
contrasts with FIG. 30A, where no apoptotic nuclei were observed
with treatment. Since compound 1a can inhibit phosphorylation of
several FAK substrates, including paxillin and pp130cas, it is
believed that FAK kinase function in the cochlea is playing a
protective role in response to high level noise exposure.
[0637] As described above, the PTK inhibitor treated ears showed
less outer hair cell loss than controls. This indicates that
anoikis (detachment from the cell's matrix, resulting in apoptosis)
may play a significant role in noise-induced hair cell loss, and
that blockage of apoptotic signals generated at the cell matrix can
prevent hair cell loss.
[0638] More specifically, using the above chinchilla animal model,
it has been demonstrated that focal adhesion complexes are formed
in response to extremely high level noise. FAK is activated upon
formation of these complexes and is known to initiate several
signaling cascades, first through a series of autophosphorylation
events and subsequently through phosphorylation of downstream
peptide substrates. It has been demonstrated that apoptotic cells
are seen within the lesion surrounded by focal adhesion complexes.
Furthermore, addition of the pp60.sup.c-src inhibitor prevents the
apoptotic response without preventing the formation of the focal
adhesion complex. These data suggest that the downstream signaling
through tyrosine phosphorylation by FAK may be an early step in the
apoptotic signaling of hair cells. Since FAK is activated by shear
stress in other organic systems, these observations may represent
the first signaling pathway identified in the ear to be activated
by mechanical stress.
Example 10
Inhibition of Isolated Kinases
[0639] It is believed that the conformation of Src outside cells
vs. inside cells is markedly different, because inside cells, Src
is embedded in multiprotein signaling complexes. Thus, because the
peptide substrate binding site is not well formed in isolated Src
(as shown by Src x-ray structures), it is believed that the
activity against isolated Src for a peptide substrate binding
inhibitor would be weak. Binding to this site will require the
inhibitor to capture the very small percentage of total Src protein
in the isolated enzyme assay that is in the same conformation that
exists inside cells. This requires a large excess of the inhibitor
to drain significant amounts of the enzyme from the catalytic cycle
in the assay.
[0640] However, inside cells this large inhibitor excess is not
needed because the SH2 & SH3 domain binding proteins have
already shifted the Src conformation so that the peptide substrate
binding site is fully formed. Now, low concentrations of the
inhibitor can remove the enzyme from the catalytic cycle since all
of the enzyme is in the tight binding conformation.
[0641] KX2-328 is AstraZeneca's published ATP-competitive Src
inhibitor (AZ28) and is used as a positive control in many of the
experiments described herein. Note that KX2-391 has weak activity
against isolated kinases because the peptide binding site is not
well formed outside of cells (a close analog, KX2-394 is a little
more potent against isolated Src), but have very potent activity
inside whole cells. Without wishing to be bound by theory, it is
thought that the difference in activity is attributed to the fact
that the peptide binding site is now fully formed in cells due to
the allosteric effects of the binding protein partners in the
multi-protein signaling complexes, relative to isolated kinase
assays.
[0642] Table XV illustrates percent activity of isolated kinases in
the presence of the AstraZeneca ATP-competitive inhibitor (KX-328,
AZ-28) or KX2-391 relative to control (untreated) isolated
kinases.
TABLE-US-00015 TABLE XV Target AZ28 @ 10 .mu.M KX2-391 @ 10 .mu.M
Abl(h) 1 120 CHK1(h) NT 105 EGFR(h) 3 134 FGFR2(h) 94 94 Fyn(h) 2
85 IGF-1R(h) 110 101 IR(h) 125 112 Lck(h) 1 109 Lyn(h) 0 113
MAPK2(h) 105 112 PDGFR.beta.(h) 98 110 PKC.alpha.(h) 111 111
Pyk2(h) 43 97 Yes(h) 1 92 ZAP-70(h) 97 108 PI3 kinase 99 100
[0643] The AstraZeneca ATP competitive inhibitor shows the typical
off target kinase inhibition activity for ATP-competitive
inhibitors, poor selectivity as evidenced by strong inhibition of
Abl, EGFRTK, Fyn, Lck, Lyn & Yes. In contrast, poor inhibition
of these off-target kinases is seen with KX2-391.
[0644] However, KX2-391 is a more potent inhibitor of Src-driven
cell growth, assayed as described above. In the c-Src/NIH-3T3
engineered cell line, the GI.sub.50 for AZ28 is 99 nM, vs. 13 nm
for KX2-391, and in the NCI human colon cancer cell line HT29, the
GI.sub.50 for AZ28 is 794 nM, vs. 23 nm for KX2-391. Similar to
KX2-391, the GI.sub.50 for KX2-394 in the c-Src/NIH-3T3 engineered
cell line is 13 nM, and in the NCI human colon cancer cell line
HT29, the GI.sub.50 for KX2-394 is 794 nM, vs. 33 nm.
[0645] In separate examples, titration data indicate that AZ28 is a
potent inhibitor of isolated Src (IC50=8 nM). The titration data
with FAK shows that AZ28 is at least ca. 100.times. less potent
against isolated FAK (IC50>500 nM). Whereas, titration data
indicate that KX2-391 and KX2-394 are less potent inhibitors of
isolated Src (IC50=46 .mu.M and 5 .mu.M, respectively). The
titration data with FAK shows that KX2-391 and KX2-394 are
similarly potent against isolated FAK (IC50>48 .mu.M).
[0646] Note that AZ28 is 10-100.times. less potent against cell
growth than against isolated Src. This is typical of ATP
competitive inhibitors since the concentration of competing ATP is
much higher in whole cells than in the isolated enzyme assays
Example 11
Effect of Compounds on Intracellular Phosphorylation Levels
[0647] HT29 (colon cancer) and c-Src527F/NIH-3T3 (Src transformed)
cell lines were treated with KX2-391 or with AstraZeneca's ATP
competitive Src inhibitor AZ28. AZ28 serves as a positive
comparator to show what a validated Src inhibitor should do in
these assays. After treatment with compound, cells were lysed,
subjected to PAGE and probed with a battery of antibodies. The
antibodies were selected to determine whether compounds caused
changes in phosphorylation of known Src substrates. In addition,
off-target protein phosphorylation was also investigated. Further,
induction of apoptosis was evaluated via Caspase 3 cleavage.
Multiple doses of each compound were tested because the trends in
response to increasing drug concentration are the most reliable
indicator of activity.
[0648] A dose response curve for KX2-391 was generated using the
GI50 for this compound in each of the two cell lines as the
1.times. concentration. Three additional doses 0.2.times., 5.times.
& 25.times. multiples the GI50's were also tested in addition
to a no drug control "C". The same range of multiples of the GI50
for AZ28 in these two cell lines was run as a comparison. As shown
in FIG. 31, the expected dose response for Src-Y416
autophosphorylation was obtained in both cell lines, and for both
compounds. This data indicates that KX2-391 is a Src inhibitor
inside cells.
[0649] FIG. 32 shows phosphorylation of FAK Tyr 925, a known Src
transphorylation substrate within cells. KX2-391 and AZ28 inhibited
Src trans-phosphorylation. This data indicates that KX2-391 is a
Src inhibitor inside cells.
[0650] FIG. 33 shows phosphorylation of Shc Y239/240, a known Src
transphorylation substrate within cells. KX2-391 and AZ28 inhibited
Src trans-phosphorylation. This data indicates that KX2-391 is a
Src inhibitor inside cells.
[0651] FIG. 34 shows phosphorylation of Paxillin Y-31, a known Src
transphorylation substrate within cells. KX2-391 and AZ28 inhibited
Src trans-phosphorylation. This data indicates that KX2-391 is a
Src inhibitor inside cells. Note: paxillin Y-31 was not detected in
HT29 cells with or without added drug.
[0652] Cleavage of Caspase-3 is a good measure of induction of
apoptosis. It is known that AZ28 is not effective in inducing
apoptosis in HT29 (colon cancer) and c-Src527F/NIH-3T3 (Src
transformed) cell lines. In contrast, as shown in FIG. 35, KX2-391
is very effective in inducing apoptosis.
[0653] Since Src activity is very high in both HT29 (colon cancer)
and c-Src527F/NIH-3T3 (Src transformed) cell lines, one would
expect to see a reduction in the total phosphotyrosine levels when
Src activity is inhibited. FIG. 36 indicates that this is true for
both AZ28 and KX2-391. This data indicates that KX2-391 is a Src
inhibitor inside cells.
[0654] PDGF receptor tyrosine kinase autophosphorylates on
Y572/574. This is thought not to be a direct Src substrate in
cells. It is known that AZ28 is not a potent inhibitor of isolated
PDGF receptor tyrosine kinase (see Table XV). Nevertheless, a dose
response reduction in PDGF receptor autophosphorylation is seen
with AZ28, as shown in FIG. 37. This suggests that this is an
indirect effect. Some effect is seen with KX2-391, however it is
somewhat less potent. Thus, KX2-391 is less active than AZ28
against indirect PDGF autophosphorylation inhibition. PDGF receptor
tyrosine kinase Y572/574 was not detected in HT29 cells with no
drug added (as well as with drug added).
[0655] FAK Y397 is mainly a FAK autophosphorylation site and only a
poor Src transphorylation site. AZ28 is not a potent FAK inhibitor
(see isolated enzyme data in Table XV). Nevertheless, some
inhibition of FAK autophosphorylation in c-Src527F/NIH3T3 cells
with AZ28 is shown in FIG. 38. However, no inhibition of FAK
autophosphorylation in c-Src527F/NIH3T3 cells is seen with KX-391.
The opposite is true in the NCI human colon cancer cell line
HT29.
[0656] The isolated enzyme data shown in Table XV demonstrated that
AZ28 is a potent EGFR tyrosine kinase inhibitor. In agreement with
this the tumor cell data in FIG. 39 shows that AZ28 potently
inhibits EGFR tyrosine kinase autophosphorylation. This site is not
a direct Src phosphorylation site. The tumor cell data in FIG. 39
also shows that KX-391 is less active against the off target
autophosphorylation of EGFRTK.
Example 12
Protection Against Noise-Induced Hearing Loss Using PTK
Inhibitors
[0657] Chinchillas (N=6) were used in studies of noise-induced
hearing loss. The animals' hearing sensitivity was measured using
standard electrophysical techniques before the experimental
manipulation. In particular, hearing thresholds were measured
through evoked potentials from recording electrodes chronically
implanted in the inferior colliculus, following standard laboratory
procedures. Animals were anesthetized, the auditory bullae were
opened, and the left and right cochleas were visualized. The round
window leading to the scala tympani of the cochlea was used as the
access point for drug application. Animals were treated with
KX1-004, KX1-141, KX1-329 or KX2-328 (a non-ATP competitive
inhibitor from Astrazeneca), emulsified in DMSO, in 1000 mM of
saline solution, which was placed on the round window of one ear. A
control solution of 3 mM DMSO in 1000 mM of saline solution was
placed on the round window of the other ear. The solution was
allowed to set on the round window for 30 minutes, then the
auditory bullae were closed. Subsequently, the animals were exposed
to 4 kHz band noise at 105 dB SPL for four hours. Following the
noise exposure, the animals' hearing was tested at day 1, day 7,
and day 21 to determine evoked potential threshold shifts.
Permanent threshold shift was assessed at day 21.
[0658] FIGS. 40-42 show the average threshold shifts for animals
treated with KX1-004, KX1-141, KX1-329 or KX2-328. In particular,
FIG. 40 shows average threshold shifts after exposure to 0.5 kHz, 1
kHz, 2 kHz, 4 kHz, and 8 kHz band noise on day 1 after experimental
manipulation. FIG. 41 shows average threshold shifts after exposure
to 0.5 kHz, 1 kHz, 2 kHz, 4 kHz, and 8 kHz band noise on day 7
after experimental manipulation. FIG. 42 shows average threshold
shifts after exposure to 0.5 kHz, 1 kHz, 2 kHz, 4 kHz, and 8 kHz
band noise on day 21 after experimental manipulation. As shown in
FIGS. 40-42, in most cases, the average dB threshold shifts for
ears treated with KX1-004, KX1-141, KX1-329 or KX2-328 were lower,
indicating that the compounds reduced the level of hearing loss in
treated animals relative to the untreated control animals.
Example 13
Protection Against Cisplatin-Induced Hearing Loss Using PTK
Inhibitors
[0659] The effects of high level noise and ototoxic drugs, such as
cisplatin or the class of aminoglycosides, share several common
features in the inner ear. First, the noise and/or drugs alter the
free radical/antioxidant levels in the cochlea (inner ear). The
increase in free radicals has been shown to be a causative factor
in the apoptotic death of the sensory cells. Guinea pigs (N=7) were
used in studies of cisplatin-induced hearing loss. The animals'
hearing sensitivity was measured using standard electrophysical
techniques before the experimental manipulation. In particular,
hearing thresholds were measured through evoked potentials from
recording electrodes chronically implanted in the inferior
colliculus, following standard laboratory procedures. Animals were
anesthetized and treated with cisplatin. Subsequently, the animals'
hearing was tested to determine evoked potential threshold
shifts.
[0660] FIG. 43 shows threshold shifts for a number of guinea pigs
after exposure to 2 kHz, 4 kHz, 8 kHz, 12 kHz, 16 kHz and 20 kHz
band noise after treatment with cisplatin. FIG. 44 shows the
threshold shifts for animals treated with KX1-004 (CH65). Animals
were treated subcutaneously with KX1-004 prior to the
cisplatin-induced hearing loss. FIG. 45 shows the median CAP
thresholds after cisplatin-induced hearing loss for both the
untreated control animals and the KX1-004 (CH65)-treated animals.
As shown in FIG. 45, KX1-004 treatment protected against
cisplatin-induced hearing loss.
Example 14
Effect of Compounds on Osteoclast Formation
[0661] To determine the effect of the compounds on osteoclast
formation, the compounds were added to osteoclast precursors
derived from spleen cells. For the generation of spleen-derived
osteoclasts, spleen cells comprising osteoclast precursors were
treated with Rapamycin, KX1-141, KX2-328 (Astrazeneca compound), or
KX1-329 for 5 days in the presence of receptor activator of nuclear
factor-.kappa.B ligand (RANKL) and macrophage colony-stimulating
factor (M-CSF). In in vitro murine or human osteoclast models,
soluble RANKL enables osteoclast precursors to differentiate in the
presence of M-CSF (Quinn, et al.; 1998, Endocrinology, 139,
4424-4427; Jimi, et al.; 1999, J. Immunol., 163, 434-442). The
untreated control cells were incubated in the presence of RANKL and
M-CSF alone. Rapamycin was used as a positive control for the
inhibition of osteoclast formation. FIG. 46 shows that increasing
concentrations of Rapamycin (0.0001 .mu.M, 0.001 .mu.M, 0.01 .mu.M,
or 0.1 .mu.M), KX1-141 (0.5 .mu.M, 2.5 .mu.M, 12.5 .mu.M, or 20
.mu.M), KX2-328 (0.02 .mu.M, 0.1 .mu.M, 0.5 .mu.M, or 2.5 .mu.M),
or KX1-329 (0.06 .mu.M, 0.3 .mu.M, 1.5 .mu.M or 7.5 .mu.M) were
added to the spleen cells. The cells were stained as shown in FIG.
46. All four compounds, including the positive control Rapamycin,
inhibited the formation of osteoclasts compared to the untreated
control.
[0662] For generating spleen-derived osteoclasts, spleen cells were
treated as described above. FIG. 47 shows that increasing
concentrations of Rapamycin (0.1 nM, 1 nM, 10 nM, or 100 nM),
KX1-141 (0.5 .mu.M, 2.5 .mu.M, 12.5 .mu.M, or 20 .mu.M), KX2-328
(0.02 .mu.M, 0.1 .mu.M, 0.5 .mu.M, or 2.5 .mu.M), or KX1-329 (0.06
.mu.M, 0.3 82 M, 1.5 .mu.M or 7.5 .mu.M) were added to the spleen
cells. Cells were then stained with the osteoclast marker,
tartrate-resistant acid phosphatase (TRAP) to visualize
differentiated cells. The numbers of TRAP-positive osteoclasts were
counted. All four compounds, including the positive control
Rapamycin, reduced the number of TRAP-positive osteoclasts compared
to the untreated control (Ctr).
Example 15
Effect of Compounds on Osteoclast Survival
[0663] To determine the effect of the compounds on osteoclast
survival, osteoclasts were treated with Rapamycin, KX1-141,
KX2-328, or KX1-329 for 48 hours in the presence of RANKL and
M-CSF. The untreated, control cells were incubated in the presence
of RANKL and M-CSF alone. Rapamycin was used as a positive control
for the inhibition of osteoclast survival. FIG. 48 shows that
increasing concentrations of Rapamycin (0.001 .mu.M, 0.01 .mu.M,
0.1 .mu.M, or 1 .mu.M), KX1-141 (0.5 .mu.M, 2.5 .mu.M, 12.5 .mu.M,
or 20 .mu.M), KX2-328 (0.02 .mu.M, 0.1 .mu.M, 0.5 .mu.M, or 2.5
.mu.M), or KX1-329 (0.06 .mu.M, 0.3 .mu.M, 1.5 .mu.M or 7.5 .mu.M)
were added to the osteoclasts. The cells were stained as shown in
FIG. 48. All four compounds, including the positive control
Rapamycin, inhibited the survival of osteoclasts compared to the
untreated control.
[0664] As described above, osteoclasts were treated with Rapamycin,
KX1-141, KX2-328, or KX1-329 for 48 hours in the presence of RANKL
and M-CSF. FIG. 49 shows that increasing concentrations of
Rapamycin (0.1 nM, 1 nM, 10 nM, or 100 nM), KX1-141 (0.5 .mu.M, 2.5
.mu.M, 12.5 .mu.M, or 20 .mu.M), KX2-328 (0.02 .mu.M, 0.1 .mu.M,
0.5 .mu.M, or 2.5 .mu.M), or KX1-329 (0.06 .mu.M, 0.3 .mu.M, 1.5
.mu.M or 7.5 .mu.M) were added to the osteoclasts. Cells were then
stained with TRAP and the number of TRAP-positive osteoclasts were
counted. All four compounds, including the positive control
Rapamycin, reduced the number of TRAP-positive osteoclasts compared
to the untreated control.
Example 16
Effect of Compounds on Bone Resorption in Vitro
[0665] To determine the effects of the compounds on osteoclast
formation on bone slices, the bone slices were treated with
Rapamycin, KX1-141, KX2-328, or KX1-329. FIG. 50A shows that
increasing concentrations of Rapamycin (0.1 nM, 1 nM, or 10 nM),
KX1-141 (2.5 .mu.M, 12.5 .mu.M, or 20 .mu.M), KX2-328 (0.1 .mu.M,
0.5 .mu.M, or 2.5 .mu.M), or KX1-329 (0.3 .mu.M, 1.5 .mu.M or 7.5
.mu.M) were added to the bone slices. The number of osteoclasts on
the bone slices were counted. All four compounds, including the
positive control Rapamycin, reduced the number of osteoclasts on
the bone slices compared to the untreated control (Ctr).
[0666] During the resorption of bone, osteoclasts form resorption
pits. To determine the effects of the compounds on resorption pit
formation on bone slices, the bone slices were treated with
Rapamycin, KX1-141, KX2-328, or KX1-329, as described above. FIG.
50B shows that increasing concentrations of Rapamycin (0.1 nM, 1
nM, or 10 nM), KX1-141 (2.5 .mu.M, 12.5 .mu.M, or 20 .mu.M),
KX2-328 (0.1 .mu.M, 0.5 .mu.M, or 2.5 .mu.M), or KX1-329 (0.3
.mu.M, 1.5 .mu.M or 7.5 .mu.M) were added to the bone slices. The
number of resorption pits on the bone slices was determined. The
compounds reduced the number of resorption pits on the bone slices
compared to the untreated control (Ctr).
[0667] Bone slices were treated as indicated above. FIG. 51A shows
that increasing concentrations of Rapamycin (0.001 .mu.M, 0.01
.mu.M, or 0.1 .mu.M), KX1-141 (2.5 .mu.M, 12.5 .mu.M, or 20 .mu.M),
KX2-328 (0.1 .mu.M, 0.5 .mu.M, or 2.5 .mu.M), or KX1-329 (0.3
.mu.M, 1.5 .mu.M or 7.5 .mu.M) were added to the bone slices. The
bone slices were then stained with TRAP. All four compounds,
including the positive control Rapamycin, reduced the number of
TRAP-positive osteoclasts on the bone slices compared to the
untreated control. Notably, 12.5 .mu.M KX1-141 significantly
reduced the number of TRAP-positive osteoclasts on the bone slices
compared to the untreated control.
[0668] Bone slices were treated as indicated above. FIG. 51B shows
that increasing concentrations of Rapamycin (0.001 .mu.M, 0.01
.mu.M, or 0.1 .mu.M), KX1-141 (2.5 .mu.M, 12.5 .mu.M, or 20 .mu.M),
KX2-328 (0.1 .mu.M, 0.5 .mu.M, or 2.5 .mu.M), or KX1-329 (0.3
.mu.M, 1.5 .mu.M or 7.5 .mu.M) were added to the bone slices. The
bone slices were stained with Toluidine Blue to reveal resorption
pits, which are indicators of osteoclast-mediated resorption of
bone. All four compounds, including the positive control Rapamycin,
reduced the number of resorption pits on the bone slices compared
to the untreated control.
Example 17
Effect of Compounds on Osteoblasts
[0669] The enzyme alkaline phosphatase has been used as an
indicator of osteoblast activity, as it is involved in making
phosphate available for calcification of bone. To determine the
effects of the compounds on osteoblast activity, osteoblasts were
treated with KX1-141 (0.5 .mu.M, 2.5 .mu.M, 12.5 .mu.M, or 20
.mu.M), KX2-328 (0.02 .mu.M, 0.1 .mu.M, 0.5 .mu.M, or 2.5 .mu.M),
or KX1-329 (0.06 .mu.M, 0.3 .mu.M, 1.5 .mu.M or 7.5 .mu.M) and
alkaline phosphatase expression was determined (nM alkaline
phosphatase/.mu.g protein/min (FIG. 52). As controls, osteoblasts
were treated with medium alone, dimethyl sulfoxide (DMSO), or bone
morphogenic protein-2 (BMP2). BMPs, defined as osteoinductive by
their ability to induce osteogenesis when implanted in
extraskeletal sites, are thought to mediate the transformation of
undifferentiated mesenchymal cells into bone-producing
osteoblasts.
[0670] To determine the effects of the compounds on osteoblast
activity and protein expression, osteoblasts were treated with
medium, DMSO, BMP2, KX1-141, KX2-328, or KX1-329 as indicated
above. The protein concentration in cell lysates was determined
(.mu.g/10 .mu.l) (FIG. 53). Notably, KX1-141 increased protein
concentration when administered at 0.5 .mu.M and 2.5 .mu.M, but
reduced protein concentration in cell lysates when administered at
12.5 .mu.M and 20 .mu.M. Additionally, KX1-329 increased protein
concentration when administered at 0.06 .mu.M and 0.3 .mu.M, but
reduced protein concentration when administered at 1.5 .mu.M and
7.5 .mu.M.
Example 18
Effect of Compounds on Obesity
[0671] The following example illustrates that the compounds of the
present invention could be used to treat obesity. The compounds are
tested using a method described previously (Minet-Ringuet, et al.;
2006, Psychopharmacology, Epub ahead of print, incorporated herein
by reference). Thirty male Sprague-Dawley rats initially weighing
175-200 g are housed in individual Plexiglas cages with an
artificial 12:12-h light-dark cycle (lights on at 08:00 h) in a
room maintained at 24.+-.1.degree. C. and 55.+-.5% humidity. Food
and water are available ad libitum throughout. All rats are fed
with a medium fat diet (metabolizable energy 17.50 kJ/g) composed
of 140 g/kg of whole milk protein, 538.1 g/kg of cornstarch, 87.6
g/kg of sucrose, and 137 g/kg of soya bean oil, and this diet is
supplemented with minerals and vitamins (mineral salts 35 g/kg,
vitamins 10 g/kg, cellulose 50 g/kg, and choline 2.3 g/kg). This
food, named P14-L, which resembles the usual human diet (14%
proteins, 31% lipids, and 54% carbohydrates) is prepared in the
laboratory in the form of a powder.
[0672] Several doses of the compound of the instant invention are
tested: 0.01, 0.1, 0.5, and 2 mg/kg, in addition to the control
solution. The compound is solubilized in water and then
incorporated into the diet. The basal food intake is recorded
during the adaptation period and used to determine the daily
quantity of the compound of the instant invention incorporated into
food. The compound is mixed into the food in the laboratory. After
1 week of adaptation to the laboratory conditions, the rats are
separated into five groups (n=6 per group) with homogenous weight
and receive the compound of the instant invention in their food for
6 weeks. Weight is recorded three times per week. Body composition
is measured at the end of the study by dissection and by weighing
the main organs and tissues. Briefly, rats are deeply anesthetized
by an intraperitoneal injection of an overdose of anesthetic
(sodium pentobarbital 48 mg/kg) and heparinized (100 U heparin/100
g body weight). They are bled (to avoid coagulation in tissues) by
sectioning the vena cava and abdominal aorta before removal and
weighing of the main fresh organs (liver, spleen, kidneys, and
pancreas) and tissues (perirenal and scapular brown adipose tissue,
epididymal, retroperitoneal, visceral, and subcutaneous white
adipose tissues (WATs), and carcass defined by muscles and
skeleton). The compound of the instant invention could reduce the
body weight of the animals, indicating that the compound may be
used to treat obesity in a subject.
Example 19
Effect of Compounds on Insulin-Induced GLUT4 Translocation in
3T3-L1 Adipocytes
[0673] The following example illustrates that the compounds of the
present invention could be used to treat diabetes. The compounds
are tested using a method described previously (Nakashima, et al.;
2000, J. Biol. Chem., 275, 12889-12895). Either control IgG, or the
compound of the instant invention is injected into the nucleus of
differentiated 3T3-L1 adipocytes on coverslips. Glutathione
S-transferase fusion proteins are each coinjected with 5 mg/ml
sheep IgG for detection purposes. Prior to staining, the cells are
allowed to recover for a period of 1 h. Cells are starved for 2 hr
in serum-free medium, stimulated with or without insulin (0.5 nM or
17 nM) for 20 min and fixed.
[0674] Immunostaining is performed using rabbit polyclonal
anti-GLUT4 (F349) (1 .mu.g/ml). Each fluorescein
isothiocyanate-positive microinjected cell is evaluated for the
presence of plasma membrane-associated GLUT4 staining. Control
cells are injected with preimmune sheep IgG and then processed in
the same way as experimentally injected cells. As quantitated by
immunofluorescent GLUT4 staining, insulin leads to an increase in
GLUT4 translocation to the plasma membrane. Cells are incubated
with wortmannin as a control to block basal and insulin-induced
GLUT4 translocation. The compounds of the instant invention could
stimulate insulin-induced GLUT4 translocation, which could indicate
that administration of the compounds of the invention inhibited
kinase activity, e.g., PTEN function, resulting in an increase in
intracellular phosphatidylinositol 3,4,5-triphosphate levels, which
stimulates GLUT4 translocation.
Example 20
Effect of Compounds on Retinal Neovascularization
[0675] The following example illustrates that the compounds of the
present invention could be used to treat eye diseases, e.g.,
macular degeneration, retinopathy and macular edema. The effect of
compounds on retinal neovascularization is determined using a model
of retinal neovascularization as previously described (Aiello, et
al.; 1995, Proc. Natl. Acad. Sci., 92, 10457-10461). Briefly,
C57Bl/6J mice are exposed to 75% O.sub.2 from postnatal day 7 (P7)
to P12 along with nursing mothers. At P12, the mice are returned to
room air. Intraocular injections are performed at P12 and sometimes
P14 as described below. At P17 the mice are sacrificed by cardiac
perfusion of 4% paraformaldehyde in phosphate-buffered saline and
the eyes are enucleated and fixed in 4% paraformaldehye overnight
at 4.degree. C. before paraffin embedding.
[0676] Mice are deeply anesthetized with tribromoethanol for all
procedures. The lid fissure is opened (e.g., using a no. 11 scalpel
blade) and the eye is proptosed. Intravitreal injections are
performed by first entering the left eye with an Ethicon TG140-8
suture needle at the posterior limbus. A 32-gauge Hamilton needle
and syringe are used to deliver the compound of the instant
invention diluted in Alcon balanced salt solution through the
existing entrance site. The eye is then repositioned and the lids
are approximated over the cornea. Repeat injections are performed
through a previously unmanipulated section of limbus 2 days later.
As a control, equal amounts of saline are injected to the right
eye.
[0677] Over 50 serial 6-.mu.m paraffin-embedded axial sections are
obtained starting at the optic nerve head. After staining with
periodic acid/Schiff reagent and hematoxylin (Pierce, et al.; 1995,
Proc. Natl. Acad. Sci. USA., 92, 905-909; Smith et al.; 1994,
Invest. Ophthal. Vis. Sci., 35, 101-111), 10 intact sections of
equal length, each 30 .mu.m apart, are evaluated for a span of 300
.mu.m. Eyes exhibiting retinal detachment or endophthalmitis are
excluded from evaluation. All retinal vascular cell nuclei anterior
to the internal limiting membrane are counted in each section by a
fully masked protocol. The mean of all 10 counted sections yield
average neovascular cell nuclei per 6-.mu.m section per eye. No
vascular cell nuclei anterior to the internal limiting membrane are
observed in normal, unmanipulated animals (Smith et al.; 1994,
Invest. Ophthal. Vis. Sci., 35, 101-111). Reduced
neovascularization could be observed in the eyes treated with the
compounds of the instant invention as compared to the eyes in the
saline control group.
Example 21
Identification of Compounds that Modulate Kinase Signaling Cascade
Associated with Stroke
[0678] Many animal models for stroke have been developed and
characterized, see e.g., Andaluz, et al., Neurosurg. Clin. North
Am., vol. 13:385-393 (2002); Ashwal, S. and W. J. Pearce., Curr.
Opin. Pediatr., vol 13:506-516 (2001); De Lecinana, et al.,
Cerebrovasc. Dis., vol. 11(Suppl. 1):20-30 (2001); Ginsberg and
Busto, Stroke, vol. 20:1627-1642 (1989); Lin, et al., J. Neurosci.
Methods, vol. 123:89-97 (2003); Macrae, I. M., Br. J. Clin.
Pharmacol., vol. 34:302-308 (1992); McAuley, M. A., Cerebrovasc.
Brain Metab. Rev., vol. 7:153-180 (1995); Megyesi, et al.,
Neurosurgery, vol. 46:448-460 (2000); Stefanovich, V. (ed.).,
Stroke: animal models. Pergamon Press, Oxford (1983); and
Traystman, R. J., ILAR J. 44:85-95 (2003), each of which is hereby
incorporated by reference in its entirety. For a review of animal
models of focal (stroke) and global (cardiac arrest) cerebral
ischemia, see e.g., Traystman, ILAR J., vol. 44(2):85-95 (2003) and
Carmichael, NeuroRx.RTM.: The Journal of the American Society for
Experimental NeuroTherapeutics, vol. 2:396-409 (2005, each Of which
is hereby incorporated by reference in its entirety.
[0679] Compounds that modulate cell death in stroke are identified
using any of the art-recognized models for stroke. In the studies
described herein, intra-arterial suture occlusion of the middle
cerebral artery (MCA), a procedure known as MCAo, through the
internal carotid artery is used as a model for cell death in
stroke. In the control and test group of rats, the external carotid
artery is transected, the common carotid artery is tied off, and
the external carotid artery is then used as a pathway to pass a
suture through the internal carotid artery, wherein the suture
lodges in the junction of the anterior and middle cerebral
arteries. To reduce subarachnoid hemorrhage and premature
reperfusion, the suture is preferably coated with an agent such as
silicone. The suture is used to occlude the MCA, e.g., for a
duration of 60, 90, or 120 minutes and to permanently occlude the
MCA.
[0680] In the test group, rats are administered a compound of the
invention at a variety of times prior to, during and after
occlusion of the MCA with the suture. The effects of the compound
on the test group is compared to the effects observed in the
control group, for example, by measuring the extent of cell death
in each MCAo group. Typically, in the control group, the pattern of
cell death follows a progression from early infarction in the
striatum to delayed infarction in the dorsolateral cortex overlying
the striatum. Striatal is mostly necrotic and occurs rapidly. The
pattern of cell-death in the test group is compared to that of the
control group to identify compounds that modulate cell death in
stroke.
Example 22
Identification of Compounds that Modulate Kinase Signaling Cascade
Associated with Atherosclerosis
[0681] Many animal models for atherosclerosis have been developed
and characterized. For a review of animal models of
atherosclerosis, restenosis and endovascular graft research, see
e.g., Narayanaswamy et al., JVIR, vol. 11(1): 5-17 (2000), which is
hereby incorporated by reference in its entirety. Atherosclerosis
is induced in a suitable animal model using a high fat/high
cholesterol (HFHC) diet. The test animal is an animal that contains
cholesterol ester transferase, such as the rabbit or the swine. The
HFHC diet is produced, e.g., using commercial chow supplemented
with fat. Cholesterol intake is between 0.5-2.0% of the diet. A
test group of animals, e.g., rabbits or swine, receives a compound
of the invention. The effect of the test compound is compared to
the effects of atherosclerosis in the untreated, control group of
animals. Effects that are compared include, for example, the degree
of plaque formation, the number and/or frequency of myocardial
infarctions observed in each group of animals, and the extent of
tissue damage secondary to myocardial infarction exhibited in
coronary tissue.
[0682] Myocardial infarction is studied using a variety of animal
models such as rats and mice. The majority of myocardial
infarctions result from acute transbotic occlusion of pre-existing
atherosclerotic plaques of coronary arteries, which is mimicked in
animal models by ligation of the left coronary artery in e.g., rats
and mice. Myocardial infarction induces global changes in the
ventricular architecture, a process called ventricular remodeling.
The infarcted heart progressively dilates and accelerates the
deterioration of ventricular dysfunction that eventually results in
heart failure.
[0683] Myocardial ischemia is induced in test and control groups of
animals, e.g., mice or rats, by ligating the left anterior
descending coronary artery. The affected heart tissue is contacted
with a compound of the invention, for example, by intraperitoneal
(i.p.) injections, after the induction of ischemia. High resolution
magnetic resonance imaging (MRI), dry weight measurements, infarct
size, heart volume, and area at risk are determined 24 hours
postoperatively. Survival rates and echocardiography are determined
at various times postoperatively in the rats receiving injections
of the compound of the invention. Other effects of the test
compound are compared to the control group of rats. For example,
changes in left ventricular geometry and function are characterized
using echocardiography to compare end-diastolic diameters, relative
wall thickness, and the percentage of fractional shortening. In
excised hearts, the infarct size calculated and expressed as a
percentage of left ventricular surface area.
Example 23
Identification of Compounds that Modulate Kinase Signaling Cascade
Associated with Neuropathic Pain
[0684] Many animal models for neuropathic pain, such as chronic
neuropathic pain, have been developed and characterized, see e.g.,
Bennett & Xie, Pain, vol. 33, 87-107 (1988); Seltzer et al.,
Pain, vol. 43, 205-18 (1990); Kim & Chung, Pain, vol. 50,
355-63 (1992); Malmberg & Basbaum, Pain, vol. 76, 215-22
(1998); Sung et al., Neurosci Lett., vol. 246, 117-9 (1998); Lee et
al., Neuroreport, vol. 11, 657-61 (2000); Decosterd & Woolf,
Pain, vol. 87, 149-58 (2000); Vadakkan et al., J Pain, vol. 6,
747-56 (2005), each of which is hereby incorporated by reference in
its entirety. For a review of animal models used for neuropathic
pain, see e.g., Eaton, J. Rehabilitation Research and Development,
vol. 40(4 Supplement):41-54 (2003), the contents of which are
hereby incorporated by reference in their entirety.
[0685] Compounds that modulate neuropathic pain are identified
using any of the art-recognized models for neuropathic pain. For
example, the models for neuropathic pain generally involve injury
to the sciatic nerve, although the method used to induce injury
varies. For example, the sciatic nerve is injured due to partial
constriction, complete transection, freezing of the nerve, and
metabolic, chemical, or immune insults to the nerve. Animals with
these types of nerve injury have been shown to develop abnormal
pain sensations similar to those reported by neuropathic pain
patients. In the studies described herein, the sciatic nerve of
test and control groups of subjects, such as mice, are injured. In
the test group, subjects are administered a compound of the
invention at a variety of times prior to, during and after injury
to the sciatic nerve. The effects of the compound on the test group
are compared to the effects observed in the control group, e.g.,
through physical observation and examination of the subjects. For
example, in mice, the subject's hindpaw is used to test the
response to non-noxious stimuli, such as tactile stimulation, or to
test the subject's response to stimuli that would be noxious in the
course of ordinary events, for example, radiant heat delivered to
the hindpaw. Evidence of allodynia, a condition in which ordinarily
nonpainful stimuli evoke pain, or a hyperalgesia, the excessive
sensitiveness or sensibility to pain, in the test subjects
indicates that test compound is not effectively modulating
neuropathic pain in the test subjects.
Example 24
Identification of Compounds that Modulate Kinase Signaling Cascade
Associated with Hepatitis B
[0686] Many animal models for hepatitis B have been developed and
characterized. For a review of animal models of hepatitis B, see
e.g., Guha et al., Lab Animal, vol. 33(7):37-46 (2004), which is
hereby incorporated by reference in its entirety. Suitable animal
models include, for example, the chimpanzee, tree shrews
(non-rodent small animals that are phylogenetically close to
primates, see Walter et al., Hepatology, vol. 24(1):1-5 (1996),
which is hereby incorporated by reference in its entirety), and
surrogate models such as the woodchuck, duck and ground squirrel.
(See e.g., Tennant and Gerin, ILAR Journal, vol. 42(2):89-102
(2001), which is hereby incorporated by reference in its
entirety).
[0687] For example, primary hepatocytes are isolated from livers of
the tree shrew species tupaia belangeri and are infected with HBV.
In vitro infection results in viral DNA and RNA synthesis in
hepatocytes and secretion hepatitis B surface antigen (HBsAg) and
hepatitis B e antigen (HBeAg) into culture medium. Tupaias can also
be infected with HBV in vivo, resulting in viral DNA replication
and gene expression in tupaia livers. Similar to acute,
self-limited hepatitis B in humans HBsAg is rapidly cleared from
serum, followed by seroconversion to anti-HBe and anti-HBs.
[0688] Compounds that modulate hepatitis B are identified using any
of the art-recognized models for hepatitis B. In the studies
described herein, test and control groups of animals, e.g.,
chimpanzees or tree shrews, are infected with HBV. In the test
group, subjects are administered a compound of the invention at a
variety of times prior to, during and after exposure to HBV. The
effects of the compound on the test group are compared to the
effects observed in the control group, e.g., through physical
observation and examination of the subjects and through blood or
serum analysis to determine at what point in time the infection is
cleared from the subject. For example, assays are run to detect the
presence and/or amount of hepatitis B virus called surface antigens
and fragments thereof. Alternatively or in addition, the subject's
liver is analyzed. Liver function tests analyze levels of certain
proteins and enzymes, such as, for example, aspartate
aminotransferase (AST, formerly serum glutamic-oxaloacetic
transaminase (SGOT)) and alanine aminotransferase (ALT, formerly
serum glutamate-pyruvate transaminase (SGPT)).
Example 25
The Effect of Compounds on Tyrosine Kinase Inhibition
[0689] The following example illustrates that the compounds of the
present invention could be used to treat autoimmune diseases. The
compounds are tested using a method described previously (Goldberg,
et al.; 2003, J. Med. Chem., 46, 1337-1349). The kinase activity is
measured using DELFIA (dissociation enhanced lanthanide
fluoroimmunoassay), which utilizes europium chelate-labeled
anti-phosphotyrosine antibodies to detect phosphate transfer to a
random polymer, poly-Glu4-Tyr1 (PGTYR). The kinase assay is
performed in a neutravidin-coated 96-well white plate in kinase
assay buffer (50 mM HEPES, pH 7.0, 25 mM MgCl2, 5 mM MnCl2, 50 mM
KCl, 100 .mu.M Na3VO4, 0.2% BSA, 0.01% CHAPS). Test samples
(compounds of the instant invention) initially dissolved in DMSO at
1 mg/mL are prediluted for dose response (10 doses with starting
final concentration of 1 .mu.g/mL, 1-3.5 serial dilutions) with the
assay buffer. A 25 .mu.L aliquot of this diluted sample and a 25
.mu.L aliquot of diluted enzyme (lck) (0.8 nM final concentration)
are sequentially added to each well. The reaction is started with a
50 .mu.L/well of a mixture of substrates containing 2 .mu.M ATP
(final ATP concentration is 1 .mu.M) and 7.2 ng/.mu.L PGTYR-biotin
in kinase buffer. Background wells are incubated with buffer and
substrates only. Following 45 mM of incubation at room temperature,
the assay plate is washed three times with 300 .mu.L/well DELFIA
wash buffer. A 100 .mu.L/well aliquot of europium-labeled
anti-phosphotyrosine (Eu.sup.3+-PT66, 1 nM, Wallac CR04-100)
diluted in DELFIA assay buffer is added to each well and incubated
for 30 min at room temperature. Upon completion of the incubation,
the plate is washed four times with 300 .mu.L/well of wash buffer
and 100 .mu.L/well of DELFIA wash buffer. Enhancement solution
(Wallac) is added to each well. After 15 min, timeresolved
fluorescence is measured on the LJL's analyst (excitation at 360
nm, emission at 620 nm, EU 400 dichroic mirror) after a delay time
of 250 .mu.s. The compound of the instant invention could inhibit
the kinase activity of lck, indicating that the compound may be
used to treat autoimmune disease in a subject.
[0690] Although preferred embodiments have been depicted and
described in detail herein, it will be apparent to those skilled in
the relevant art that various modifications, additions,
substitutions, and the like can be made without departing from the
spirit of the invention and these are therefore considered to be
within the scope of the invention as defined in the claims which
follow.
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Sequence CWU 1
1
715PRTArtificial SequenceChemically synthesized Src substrate
pentapeptide 1Ile Tyr Gly Glu Phe1 525PRTArtificial
SequenceChemically synthesized PKA pentapeptide scaffold 2Arg Arg
Gly Xaa Ile1 535PRTArtificial SequenceChemically synthesized Src
pentapeptide scaffold 3Ile Xaa Gly Glu Phe1 545PRTArtificial
SequenceChemically synthesized boronic acid-containing PKA
inhibitor 4Arg Arg Gly Xaa Ile1 557PRTArtificial SequenceChemically
synthesized Kemptamide 5Leu Arg Arg Ala Ser Leu Gly1
567PRTArtificial SequenceChemically synthesized phosphorylated
Kemptamide 6Leu Arg Arg Ala Xaa Arg Gly1 577PRTArtificial
SequenceChemically synthesized peptide substrate for Src 7Gly Ile
Tyr Trp His His Tyr1 5
* * * * *