U.S. patent application number 14/449630 was filed with the patent office on 2015-02-26 for benzimidazoles and related analogs as sirtuin modulators.
The applicant listed for this patent is GLAXOSMITHKLINE LLC. Invention is credited to Charles A. Blum, Jeremy S. Disch, Pui Yee Ng, Robert B. Perni, Chi B. Vu.
Application Number | 20150057272 14/449630 |
Document ID | / |
Family ID | 41056804 |
Filed Date | 2015-02-26 |
United States Patent
Application |
20150057272 |
Kind Code |
A1 |
Vu; Chi B. ; et al. |
February 26, 2015 |
BENZIMIDAZOLES AND RELATED ANALOGS AS SIRTUIN MODULATORS
Abstract
Provided herein are novel sirtuin-modulating compounds and
methods of use thereof. The sirtuin-modulating compounds may be
used for increasing the lifespan of a cell, and treating and/or
preventing a wide variety of diseases and disorders including, for
example, diseases or disorders related to aging or stress,
diabetes, obesity, neurodegenerative diseases, cardiovascular
disease, blood clotting disorders, inflammation, cancer, and/or
flushing as well as diseases or disorders that would benefit from
increased mitochondrial activity. Also provided are compositions
comprising a sirtuin-modulating compound in combination with
another therapeutic agent.
Inventors: |
Vu; Chi B.; (Arlington,
MA) ; Disch; Jeremy S.; (Natick, MA) ; Ng; Pui
Yee; (Waltham, MA) ; Blum; Charles A.;
(Waltham, MA) ; Perni; Robert B.; (Marlborough,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GLAXOSMITHKLINE LLC |
Wilmington |
DE |
US |
|
|
Family ID: |
41056804 |
Appl. No.: |
14/449630 |
Filed: |
August 1, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12999795 |
Dec 17, 2010 |
8846947 |
|
|
PCT/US2009/049498 |
Jul 2, 2009 |
|
|
|
14449630 |
|
|
|
|
61133938 |
Jul 3, 2008 |
|
|
|
Current U.S.
Class: |
514/230.5 ;
514/234.5; 514/255.05; 514/303; 514/305; 514/338; 514/371; 514/394;
544/105; 544/133; 544/405; 546/118; 546/133; 546/270.7; 548/181;
548/306.1 |
Current CPC
Class: |
A61K 31/5355 20130101;
A61P 27/02 20180101; A61P 25/28 20180101; A61P 43/00 20180101; C07D
453/02 20130101; C07D 413/14 20130101; A61P 7/04 20180101; A61K
31/4427 20130101; C07D 413/12 20130101; A61P 3/04 20180101; A61P
9/10 20180101; C07D 498/04 20130101; A61K 31/422 20130101; A61K
45/06 20130101; A61K 31/4439 20130101; C07D 513/04 20130101; A61K
31/427 20130101; A61P 29/00 20180101; A61K 31/495 20130101; C07D
405/14 20130101; A61K 31/46 20130101; A61P 3/10 20180101; A61P
35/00 20180101; C07D 403/12 20130101; C07D 401/14 20130101; A61P
25/00 20180101; A61P 21/00 20180101; C07D 403/14 20130101; C07D
401/12 20130101; C07D 471/04 20130101; A61K 31/495 20130101; C07D
417/14 20130101; A61K 31/5383 20130101; A61K 31/5355 20130101; A61K
31/497 20130101; A61P 3/00 20180101; A61K 31/5377 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 31/439 20130101; A61K
31/427 20130101; A61K 31/4427 20130101; A61K 31/4184 20130101; A61P
9/00 20180101; A61K 31/422 20130101; C07D 405/12 20130101; A61K
31/437 20130101; A61K 31/46 20130101; A61K 31/437 20130101; A61P
5/50 20180101; C07D 417/12 20130101; C07D 471/08 20130101; A61K
31/4184 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/230.5 ;
548/181; 514/371; 544/405; 514/255.05; 548/306.1; 514/394;
546/270.7; 514/338; 544/133; 514/234.5; 546/133; 514/305; 546/118;
514/303; 544/105 |
International
Class: |
C07D 513/04 20060101
C07D513/04; A61K 31/427 20060101 A61K031/427; C07D 403/12 20060101
C07D403/12; A61K 31/497 20060101 A61K031/497; A61K 31/4184 20060101
A61K031/4184; C07D 417/14 20060101 C07D417/14; A61K 31/4439
20060101 A61K031/4439; A61K 31/5377 20060101 A61K031/5377; C07D
453/02 20060101 C07D453/02; A61K 31/439 20060101 A61K031/439; C07D
471/04 20060101 C07D471/04; A61K 31/437 20060101 A61K031/437; C07D
498/04 20060101 C07D498/04; A61K 31/5383 20060101 A61K031/5383;
C07D 417/12 20060101 C07D417/12 |
Claims
1. A compound of Formula (II): ##STR00854## wherein: one of
Z.sup.11 and Z.sup.12 is CR.sup.13, and the other of Z.sup.11 and
Z.sup.12 is selected from N and CR.sup.13; wherein: R.sup.13 is
selected from hydrogen, halo, --OH, --C.ident.N, fluoro-substituted
C.sub.1-C.sub.2 alkyl, --O--(C.sub.1-C.sub.2 fluoro-substituted
alkyl), --S--(C.sub.1-C.sub.2 fluoro-substituted alkyl),
C.sub.1-C.sub.4 alkyl, --(C.sub.1-C.sub.2
alkyl)-N(R.sup.14)(R.sup.14), --O--CH.sub.2CH(OH)CH.sub.2OH,
--O--(C.sub.1-C.sub.4) alkyl, --O--(C.sub.1-C.sub.3)
alkyl-N(R.sup.14)(R.sup.14), --N(R.sup.14)(R.sup.14),
--S--(C.sub.1-C.sub.4) alkyl and C.sub.3-C.sub.7 cycloalkyl; R is
selected from hydrogen, --(C.sub.2-C.sub.4) alkyl,
--(C.sub.1-C.sub.4) fluoro-substituted alkyl, --(C.sub.1-C.sub.4)
alkyl-N(R.sup.7)(R.sup.7), --(C.sub.1-C.sub.4)
alkyl-C(O)--N(R.sup.7)(R.sup.7), --(C.sub.2-C.sub.4) alkyl
--O--R.sup.7, and --(C.sub.2-C.sub.4)
alkyl-N(R.sup.7)--C(O)--R.sup.7; wherein: each R.sup.7 is
independently selected from hydrogen, and --C.sub.1-C.sub.4 alkyl;
or two R.sup.7 bound to the same nitrogen atom are taken together
with the nitrogen atom to form a 4- to 7-membered saturated
heterocycle optionally comprising one additional heteroatom
selected from N, S, S(.dbd.O), S(.dbd.O).sub.2, and O, wherein: the
saturated heterocycle is optionally substituted at a single carbon
atom with --OH, --C.sub.1-C.sub.4 alkyl, fluoro, --NH.sub.2,
--NH(C.sub.1-C.sub.4 alkyl), --N(C.sub.1-C.sub.4 alkyl).sub.2,
--NH(CH.sub.2CH.sub.2OCH.sub.3), or
--N(CH.sub.2CH.sub.2OCH.sub.3).sub.2; R.sup.4 is selected from
hydrogen, halo, --C.ident.N, fluoro-substituted C.sub.1-C.sub.2
alkyl, --S--(C.sub.1-C.sub.2) fluoro-substituted alkyl,
C.sub.1-C.sub.4 alkyl, --S--(C.sub.1-C.sub.4) alkyl and
C.sub.3-C.sub.7 cycloalkyl; X is selected from
--NH--C(.dbd.O)-.dagger., --C(.dbd.O)--NH-.dagger.,
--NH--C(.dbd.S)-.dagger., --C(.dbd.S)--NH-.dagger.,
--NH--S(.dbd.O)-.dagger., --S(.dbd.O)--NH-.dagger.,
--S(.dbd.O).sub.2--NH-.dagger., --NH--S(.dbd.O).sub.2-.dagger.,
--NH--S(O).sub.2--NR.sup.15-.dagger.,
NR.sup.15--S(O).sub.2--NH-.dagger., --NH--C(.dbd.O)O-.dagger.,
O--C(.dbd.O)--NH-.dagger., --NH--C(.dbd.O)NH-.dagger.,
--NH--C(.dbd.O)NR.sup.15-.dagger.,
--NR.sup.15--C(.dbd.O)NH-.dagger., --NH--NR.sup.15-.dagger.,
--NR.sup.15--NH-.dagger., --O--NH-.dagger., --NH--O-.dagger.,
--NH--CR.sup.15R.sup.16-.dagger., --CR.sup.15R.sup.16--NH-.dagger.,
--NH--C(.dbd.NR.sup.15)-.dagger., --C(.dbd.NR.sup.15)--NH-.dagger.,
--C(.dbd.O)--NH--CR.sup.15R.sup.16-.dagger.,
--CR.sup.15R.sup.16--NH--C(O)-.dagger.,
--NH--C(.dbd.S)--CR.sup.15R.sup.16-.dagger.,
--CR.sup.15R.sup.16--C(.dbd.S)--NH-.dagger.,
--NH--S(O)--CR.sup.15R.sup.16-.dagger.,
--CR.sup.15R.sup.16--S(O)--NH-.dagger.,
--NH--S(O).sub.2--CR.sup.15R.sup.16-.dagger.,
--CR.sup.15R.sup.16--S(O).sub.2--NH-.dagger.,
--NH--C(.dbd.O)--O--CR.sup.15R.sup.16-.dagger.,
--CR.sup.15R.sup.16--O--C(.dbd.O)--NH-.dagger.,
--NH--C(.dbd.O)--NR.sup.14--CR.sup.15R.sup.16-.dagger.,
--NH--C(.dbd.O)--CR.sup.15R.sup.16-.dagger., and
--CR.sup.15R.sup.16--NH--C(.dbd.O)--O-.dagger.; wherein: .dagger.
represents where X is bound to R.sup.11, and: R.sup.15 and R.sup.16
are independently selected from hydrogen, C.sub.1-C.sub.4 alkyl,
CF.sub.3, and --(C.sub.1-C.sub.4 alkyl)-CF.sub.3; R.sup.11 is
selected from a carbocycle and a heterocycle; wherein: R.sup.11 is
optionally substituted with one to two substitutents independently
selected from halo, --C.ident.N, C.sub.1-C.sub.3 alkyl,
C.sub.3-C.sub.7 cycloalkyl, C.sub.1-C.sub.2 fluoro-substituted
alkyl, .dbd.O, --O--C.sub.1-C.sub.4 alkyl, --S--R.sup.14,
--(C.sub.1-C.sub.4 alkyl)-N(R.sup.14)(R.sup.14),
--N(R.sup.14)(R.sup.14), --O--(C.sub.2-C.sub.4
alkyl)-N(R.sup.14)(R.sup.14), --C(O)--N(R.sup.14)(R.sup.14),
--C(O)--O--R.sup.14, and --(C.sub.1-C.sub.4
alkyl)-C(O)--N(R.sup.14)(R.sup.14); and when R.sup.11 is phenyl,
R.sup.11 is also optionally substituted with 3,4-methylenedioxy,
fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy,
fluoro-substituted 3,4-ethylenedioxy, O-(saturated heterocycle),
fluoro-substituted --O-(saturated heterocycle), and C.sub.1-C.sub.4
alkyl-substituted O-(saturated heterocycle); wherein each R.sup.14
is independently selected from hydrogen, and --C.sub.1-C.sub.4
alkyl; or two R.sup.14 are taken together with the nitrogen atom to
which they are bound to form a 4- to 8-membered saturated
heterocycle optionally comprising one additional heteroatom
selected from N, S, S(.dbd.O), S(.dbd.O).sub.2, and O; wherein:
when R.sup.14 is alkyl, the alkyl is optionally substituted with
one or more --OH, --O--(C.sub.1-C.sub.4 alkyl), fluoro, --NH.sub.2,
--NH(C.sub.1-C.sub.4 alkyl), --N(C.sub.1-C.sub.4 alkyl).sub.2,
--NH(CH.sub.2CH.sub.2OCH.sub.3), or
--N(CH.sub.2CH.sub.2OCH.sub.3).sub.2 and when two R.sup.14 are
taken together with the nitrogen atom to which they are bound to
form a 4- to 8-membered saturated heterocycle, the saturated
heterocycle is optionally substituted at a carbon atom with --OH,
--C.sub.1-C.sub.4 alkyl, fluoro, --NH.sub.2, --NH(C.sub.1-C.sub.4
alkyl), --N(C.sub.1-C.sub.4 alkyl).sub.2,
--NH(CH.sub.2CH.sub.2OCH.sub.3), or
--N(CH.sub.2CH.sub.2OCH.sub.3).sub.2; and optionally substituted at
any substitutable nitrogen atom with --C.sub.1-C.sub.4 alkyl,
fluoro-substituted C.sub.1-C.sub.4 alkyl, or
--(CH.sub.2).sub.2--O--CH.sub.3; and R.sup.12 is selected from a
carbocycle having at least five ring atoms; wherein: R.sup.12 is
optionally substituted with one to two substitutents independently
selected from halo, --C.ident.N, C.sub.1-C.sub.4 alkyl,
C.sub.3-C.sub.7 cycloalkyl, C.sub.1-C.sub.2 fluoro-substituted
alkyl, --O--R.sup.14, --S--R.sup.14, --S(O)-e,
--S(O).sub.2--R.sup.14, --(C.sub.1-C.sub.4
alkyl)-N(R.sup.14)(R.sup.14), --N(R.sup.14)(R.sup.14),
--O--(C.sub.2-C.sub.4 alkyl)-N(R.sup.14)(R.sup.14),
--C(O)--N(R.sup.14)(R.sup.14), --(C.sub.1-C.sub.4
alkyl)-C(O)--N(R.sup.14)(R.sup.14), --O-phenyl, phenyl, and a
second heterocycle, and when R.sup.12 is phenyl, R.sup.12 is also
optionally substituted with 3,4-methylenedioxy, fluoro-substituted
3,4-methylenedioxy, 3,4-ethylenedioxy, fluoro-substituted
3,4-ethylenedioxy, or --O-(saturated heterocycle) wherein any
phenyl, saturated heterocycle or second heterocycle substituent of
R.sup.12 is optionally substituted with halo; --C.ident.N;
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.2 fluoro-substituted alkyl,
--O--(C.sub.1-C.sub.2 fluoro-substituted alkyl),
--O--(C.sub.1-C.sub.4 alkyl), --S--(C.sub.1-C.sub.4 alkyl),
--S--(C.sub.1-C.sub.2 fluoro-substituted alkyl),
--NH--(C.sub.1-C.sub.4 alkyl) and --N--(C.sub.1-C.sub.4
alkyl).sub.2; or a pharmaceutically acceptable salt thereof.
2. The compound of claim 1, wherein Formula (II) is selected from:
##STR00855##
3. The compound of claim 2, having the formula: ##STR00856##
4. The compound of claim 1, wherein R is hydrogen.
5. The compound of claim 1, wherein X is --C(O)--NH-.dagger..
6. The compound of claim 1, wherein R.sup.11 is selected from:
##STR00857## ##STR00858## ##STR00859## wherein: R.sup.11 is
optionally further substituted with substituents as defined in
claim 1.
7. The compound of claim 1, wherein R.sup.12 is selected from:
##STR00860## ##STR00861## ##STR00862## ##STR00863## ##STR00864##
wherein: R.sup.12 is optionally further substituted with
substituents as defined in claim 1.
8. A compound of Formula (IIb): ##STR00865## wherein: R.sup.13 is
hydrogen, halo, --OH, --C.ident.N, fluoro-substituted
C.sub.1-C.sub.2 alkyl, --O--(C.sub.1-C.sub.2 fluoro-substituted
alkyl), --S--(C.sub.1-C.sub.2 fluoro-substituted alkyl),
C.sub.1-C.sub.4 alkyl, --(C.sub.1-C.sub.2
alkyl)-N(R.sup.14)(R.sup.14), --O--CH.sub.2CH(OH)CH.sub.2OH,
--O--(C.sub.1-C.sub.4) alkyl, --O--(C.sub.1-C.sub.3)
alkyl-N(R.sup.14)(R.sup.14), --N(R.sup.14)(R.sup.14),
--S--(C.sub.1-C.sub.4) alkyl or C.sub.3-C.sub.7 cycloalkyl; R is
hydrogen, --(C.sub.2-C.sub.4) alkyl, --(C.sub.1-C.sub.4)
fluoro-substituted alkyl, --(C.sub.1-C.sub.4)
alkyl-N(R.sup.7)(R.sup.7), --(C.sub.1-C.sub.4)
alkyl-C(O)--N(R.sup.7)(R.sup.7), --(C.sub.2-C.sub.4) alkyl
--O--R.sup.7 or --(C.sub.2-C.sub.4)
alkyl-N(R.sup.7)--C(O)--R.sup.7; wherein: each R.sup.7 is
independently selected from hydrogen, and --C.sub.1-C.sub.4 alkyl;
or two R.sup.7 bound to the same nitrogen atom are taken together
with the nitrogen atom to form a 4- to 7-membered saturated
heterocycle optionally comprising one additional heteroatom
selected from N, S, S(.dbd.O), S(.dbd.O).sub.2, and O, wherein: the
saturated heterocycle is optionally substituted at a single carbon
atom with --OH, --C.sub.1-C.sub.4 alkyl, fluoro, --NH.sub.2,
--NH(C.sub.1-C.sub.4 alkyl), --N(C.sub.1-C.sub.4 alkyl).sub.2,
--NH(CH.sub.2CH.sub.2OCH.sub.3), or
--N(CH.sub.2CH.sub.2OCH.sub.3).sub.2; R.sup.4 is selected from
hydrogen, halo, --C.ident.N, fluoro-substituted C.sub.1-C.sub.2
alkyl, --S--(C.sub.1-C.sub.2) fluoro-substituted alkyl,
C.sub.1-C.sub.4 alkyl, --S--(C.sub.1-C.sub.4) alkyl and
C.sub.3-C.sub.7 cycloalkyl; X is selected from
--NH--C(.dbd.O)-.dagger., --C(.dbd.O)--NH-.dagger.,
--NH--C(.dbd.S)-.dagger., --C(.dbd.S)--NH-.dagger.,
--NH--S(.dbd.O)-.dagger., --S(.dbd.O)--NH-.dagger.,
--S(.dbd.O).sub.2--NH-.dagger., --NH--S(.dbd.O).sub.2-.dagger.,
--NH--S(O).sub.2--NR.sup.15-.dagger.,
--NR.sup.15--S(O).sub.2--NH-.dagger., --NH--C(.dbd.O)O-.dagger.,
O--C(.dbd.O)--NH-.dagger., --NH--C(.dbd.O)NH-.dagger.,
--NH--C(.dbd.O)NR.sup.15-.dagger.,
--NR.sup.15--C(.dbd.O)NH-.dagger., --NH--NR.sup.15-.dagger.,
--NR.sup.15--NH-.dagger., --O--NH-.dagger., --NH--O-.dagger.,
--NH--CR.sup.15R.sup.16-.dagger., --CR.sup.15R.sup.16--NH-.dagger.,
--NH--C(.dbd.NR.sup.15)-.dagger., --C(.dbd.NR.sup.15)--NH-.dagger.,
--C(.dbd.O)--NH--CR.sup.15R.sup.16-.dagger.,
--CR.sup.15R.sup.16--NH--C(O)-.dagger.,
--NH--C(.dbd.S)--CR.sup.15R.sup.16-.dagger.,
--CR.sup.15R.sup.16--C(.dbd.S)--NH-.dagger.,
--NH--S(O)--CR.sup.15R.sup.16-.dagger.,
--CR.sup.15R.sup.16--S(O)--NH-.dagger.,
--NH--S(O).sub.2--CR.sup.15R.sup.16-.dagger.,
--CR.sup.15R.sup.16--S(O).sub.2--NH-.dagger.,
--NH--C(.dbd.O)--O--CR.sup.15R.sup.16-.dagger.,
--CR.sup.15R.sup.16--O--C(.dbd.O)--NH-.dagger.,
--NH--C(.dbd.O)--NR.sup.14--CR.sup.15R.sup.16-.dagger.,
--NH--C(.dbd.O)--CR.sup.15R.sup.16-.dagger., and
--CR.sup.15R.sup.16--NH--C(.dbd.O)--O-.dagger.; wherein: .dagger.
represents where X is bound to R.sup.11, and: R.sup.15 and R.sup.16
are independently selected from hydrogen, C.sub.1-C.sub.4 alkyl,
CF.sub.3, and --(C.sub.1-C.sub.4 alkyl)-CF.sub.3; R.sup.11 is
selected from a carbocycle and a heterocycle; wherein: R.sup.11 is
optionally substituted with one to two substitutents independently
selected from halo, --C.ident.N, C.sub.1-C.sub.3 alkyl,
C.sub.3-C.sub.7 cycloalkyl, C.sub.1-C.sub.2 fluoro-substituted
alkyl, .dbd.O, --O--C.sub.1-C.sub.4 alkyl, --S--R.sup.14,
--(C.sub.1-C.sub.4 alkyl)-N(R.sup.14)(R.sup.14),
--N(R.sup.14)(R.sup.14), --O--(C.sub.2-C.sub.4
alkyl)-N(R.sup.14)(R.sup.14), --C(O)--N(R.sup.14)(R.sup.14),
--C(O)--O--R.sup.14, and --(C.sub.1-C.sub.4
alkyl)-C(O)--N(R.sup.14)(R.sup.14); and when R.sup.11 is phenyl,
R.sup.11 is also optionally substituted with 3,4-methylenedioxy,
fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy,
fluoro-substituted 3,4-ethylenedioxy, O-(saturated heterocycle),
fluoro-substituted --O-(saturated heterocycle), and C.sub.1-C.sub.4
alkyl-substituted O-(saturated heterocycle); wherein each R.sup.14
is independently selected from hydrogen, and --C.sub.1-C.sub.4
alkyl; or two R.sup.14 are taken together with the nitrogen atom to
which they are bound to form a 4- to 8-membered saturated
heterocycle optionally comprising one additional heteroatom
selected from N, S, S(.dbd.O), S(.dbd.O).sub.2, and O; wherein:
when R.sup.14 is alkyl, the alkyl is optionally substituted with
one or more --OH, --O--(C.sub.1-C.sub.4 alkyl), fluoro, --NH.sub.2,
--NH(C.sub.1-C.sub.4 alkyl), --N(C.sub.1-C.sub.4 alkyl).sub.2,
--NH(CH.sub.2CH.sub.2OCH.sub.3), or
--N(CH.sub.2CH.sub.2OCH.sub.3).sub.2 and when two R.sup.14 are
taken together with the nitrogen atom to which they are bound to
form a 4- to 8-membered saturated heterocycle, the saturated
heterocycle is optionally substituted at a carbon atom with --OH,
--C.sub.1-C.sub.4 alkyl, fluoro, --NH.sub.2, --NH(C.sub.1-C.sub.4
alkyl), --N(C.sub.1-C.sub.4 alkyl).sub.2,
--NH(CH.sub.2CH.sub.2OCH.sub.3), or
--N(CH.sub.2CH.sub.2OCH.sub.3).sub.2; and optionally substituted at
any substitutable nitrogen atom with --C.sub.1-C.sub.4 alkyl,
fluoro-substituted C.sub.1-C.sub.4 alkyl, or
--(CH.sub.2).sub.2--O--CH.sub.3; and R.sup.12 is selected from a
carbocycle having at least five ring atoms; wherein: R.sup.12 is
optionally substituted with one to two substitutents independently
selected from halo, --C.ident.N, C.sub.1-C.sub.4 alkyl,
C.sub.3-C.sub.7 cycloalkyl, C.sub.1-C.sub.2 fluoro-substituted
alkyl, --O--R.sup.14, --S--R.sup.14, --S(O)--R.sup.14,
--S(O).sub.2--R.sup.14, --(C.sub.1-C.sub.4
alkyl)-N(R.sup.14)(R.sup.14), --N(R.sup.14)(R.sup.14),
--O--(C.sub.2-C.sub.4 alkyl)-N(R.sup.14)(R.sup.14),
--C(O)--N(R.sup.14)(R.sup.14), --(C.sub.1-C.sub.4
alkyl)-C(O)--N(R.sup.14)(R.sup.14), --O-phenyl, phenyl, and a
second heterocycle, and when R.sup.12 is phenyl, R.sup.12 is also
optionally substituted with 3,4-methylenedioxy, fluoro-substituted
3,4-methylenedioxy, 3,4-ethylenedioxy, fluoro-substituted
3,4-ethylenedioxy, or --O-(saturated heterocycle) wherein any
phenyl, saturated heterocycle or second heterocycle substituent of
R.sup.12 is optionally substituted with halo; --C.ident.N;
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.2 fluoro-substituted alkyl,
--O--(C.sub.1-C.sub.2 fluoro-substituted alkyl),
--O--(C.sub.1-C.sub.4 alkyl), --S--(C.sub.1-C.sub.4 alkyl),
--S--(C.sub.1-C.sub.2 fluoro-substituted alkyl),
--NH--(C.sub.1-C.sub.4 alkyl) and --N--(C.sub.1-C.sub.4
alkyl).sub.2; or a pharmaceutically acceptable salt thereof.
9. A pharmaceutical composition comprising a compound of claim 1,
or a pharmaceutically acceptable salt thereof; and a
pharmaceutically acceptable carrier.
10. The pharmaceutical composition of claim 9, further comprising
an additional active agent.
11. A pharmaceutical composition comprising a compound of claim 8,
or a pharmaceutically acceptable salt thereof; and a
pharmaceutically acceptable carrier.
12. A method for treating a subject suffering from or susceptible
to insulin resistance, a metabolic syndrome, diabetes, or
complications thereof, or for increasing insulin sensitivity in a
subject, comprising administering to the subject in need thereof a
compound of claim 1.
13. A method for treating a subject suffering from or susceptible
to insulin resistance, a metabolic syndrome, diabetes, or
complications thereof, or for increasing insulin sensitivity in a
subject, comprising administering to the subject in need thereof a
composition of claim 9.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/133,938, filed Jul. 3, 2008, and U.S. patent
application Ser. No. 12/999,795, Filed: Dec. 17, 2010, which is a
National Stage Entry of PCT/US09/49498, which corresponds to
International Patent Appln. WO 2010/003048 A1, International Filing
Date: Jul. 2, 2009, respectively, contents of which are
incorporated by reference in their entirety.
BACKGROUND
[0002] The Silent Information Regulator (SIR) family of genes
represents a highly conserved group of genes present in the genomes
of organisms ranging from archaebacteria to higher eukaryotes. The
encoded SIR proteins are involved in diverse processes from
regulation of gene silencing to DNA repair. The proteins encoded by
members of the SIR gene family show high sequence conservation in a
250 amino acid core domain. A well-characterized gene in this
family is S. cerevisiae SIR2, which is involved in silencing HM
loci that contain information specifying yeast mating type,
telomere position effects and cell aging. The yeast Sir2 protein
belongs to a family of histone deacetylases. The Sir2 homolog,
CobB, in Salmonella typhimurium, functions as an NAD (nicotinamide
adenine dinucleotide)-dependent ADP-ribosyl transferase.
[0003] The Sir2 protein is a class III deacetylase which uses NAD
as a cosubstrate. Unlike other deacetylases, many of which are
involved in gene silencing, Sir2 is insensitive to class I and II
histone deacetylase inhibitors like trichostatin A (TSA).
[0004] Deacetylation of acetyl-lysine by Sir2 is tightly coupled to
NAD hydrolysis, producing nicotinamide and a novel acetyl-ADP
ribose compound. The NAD-dependent deacetylase activity of Sir2 is
essential for its functions which can connect its biological role
with cellular metabolism in yeast. Mammalian Sir2 homologs have
NAD-dependent histone deacetylase activity.
[0005] Biochemical studies have shown that Sir2 can readily
deacetylate the amino-terminal tails of histones H3 and H4,
resulting in the formation of 1-O-acetyl-ADP-ribose and
nicotinamide. Strains with additional copies of SIR2 display
increased rDNA silencing and a 30% longer life span. It has
recently been shown that additional copies of the C. elegans SIR2
homolog, sir-2.1, and the D. melanogaster dSir2 gene greatly extend
life span in those organisms. This implies that the SIR2-dependent
regulatory pathway for aging arose early in evolution and has been
well conserved. Today, Sir2 genes are believed to have evolved to
enhance an organism's health and stress resistance to increase its
chance of surviving adversity.
[0006] In humans, there are seven Sir2-like genes (SIRT1-SIRT5)
that share the conserved catalytic domain of Sir2. SIRT1 is a
nuclear protein with the highest degree of sequence similarity to
Sir2. SIRT1 regulates multiple cellular targets by deacetylation
including the tumor suppressor p53, the cellular signaling factor
NF-.kappa.B, and the FOXO transcription factor.
[0007] SIRT3 is a homolog of SIRT1 that is conserved in prokaryotes
and eukaryotes. The SIRT3 protein is targeted to the mitochondrial
cristae by a unique domain located at the N-terminus. SIRT3 has
NAD+-dependent protein deacetylase activity and is ubiquitously
expressed, particularly in metabolically active tissues. Upon
transfer to the mitochondria, SIRT3 is believed to be cleaved into
a smaller, active form by a mitochondrial matrix processing
peptidase (MPP).
[0008] Caloric restriction has been known for over 70 years to
improve the health and extend the lifespan of mammals. Yeast life
span, like that of metazoans, is also extended by interventions
that resemble caloric restriction, such as low glucose. The
discovery that both yeast and flies lacking the SIR2 gene do not
live longer when calorically restricted provides evidence that SIR2
genes mediate the beneficial health effects of a restricted calorie
diet. Moreover, mutations that reduce the activity of the yeast
glucose-responsive cAMP (adenosine 3',5'-monophosphate)-dependent
(PKA) pathway extend life span in wild type cells but not in mutant
sir2 strains, demonstrating that SIR2 is likely to be a key
downstream component of the caloric restriction pathway.
SUMMARY
[0009] Provided herein are novel sirtuin-modulating compounds and
methods of use thereof.
[0010] In one aspect, the invention provides sirtuin-modulating
compounds of Structural Formulas (I), (II) (e.g., IIa, IIb, and
IIc), and (III), as are described in detail below.
[0011] In another aspect, the invention provides methods for using
sirtuin-modulating compounds, or compositions comprising
sirtuin-modulating compounds. In certain embodiments,
sirtuin-modulating compounds that increase the level and/or
activity of a sirtuin protein may be used for a variety of
therapeutic applications including, for example, increasing the
lifespan of a cell, and treating and/or preventing a wide variety
of diseases and disorders including, for example, diseases or
disorders related to aging or stress, diabetes, obesity,
neurodegenerative diseases, chemotherapeutic induced neuropathy,
neuropathy associated with an ischemic event, ocular diseases
and/or disorders, cardiovascular disease, blood clotting disorders,
inflammation, and/or flushing, etc. Sirtuin-modulating compounds
that increase the level and/or activity of a sirtuin protein may
also be used for treating a disease or disorder in a subject that
would benefit from increased mitochondrial activity, for enhancing
muscle performance, for increasing muscle ATP levels, or for
treating or preventing muscle tissue damage associated with hypoxia
or ischemia. In other embodiments, sirtuin-modulating compounds
that decrease the level and/or activity of a sirtuin protein may be
used for a variety of therapeutic applications including, for
example, increasing cellular sensitivity to stress, increasing
apoptosis, treatment of cancer, stimulation of appetite, and/or
stimulation of weight gain, etc. As described further below, the
methods comprise administering to a subject in need thereof a
pharmaceutically effective amount of a sirtuin-modulating
compound.
[0012] In certain aspects, the sirtuin-modulating compounds may be
administered alone or in combination with other compounds,
including other sirtuin-modulating compounds, or other therapeutic
agents.
DETAILED DESCRIPTION
1. Definitions
[0013] As used herein, the following terms and phrases shall have
the meanings set forth below. Unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
commonly understood to one of ordinary skill in the art.
[0014] The term "agent" is used herein to denote a chemical
compound, a mixture of chemical compounds, a biological
macromolecule (such as a nucleic acid, an antibody, a protein or
portion thereof, e.g., a peptide), or an extract made from
biological materials such as bacteria, plants, fungi, or animal
(particularly mammalian) cells or tissues. The activity of such
agents may render it suitable as a "therapeutic agent" which is a
biologically, physiologically, or pharmacologically active
substance (or substances) that acts locally or systemically in a
subject.
[0015] The term "bioavailable" when referring to a compound is
art-recognized and refers to a form of a compound that allows for
it, or a portion of the amount of compound administered, to be
absorbed by, incorporated to, or otherwise physiologically
available to a subject or patient to whom it is administered.
[0016] "Biologically active portion of a sirtuin" refers to a
portion of a sirtuin protein having a biological activity, such as
the ability to deacetylate. Biologically active portions of a
sirtuin may comprise the core domain of sirtuins. Biologically
active portions of SIRT1 having GenBank Accession No.
NP.sub.--036370 that encompass the NAD+ binding domain and the
substrate binding domain, for example, may include without
limitation, amino acids 62-293 of GenBank Accession No.
NP.sub.--036370, which are encoded by nucleotides 237 to 932 of
GenBank Accession No. NM.sub.--012238. Therefore, this region is
sometimes referred to as the core domain. Other biologically active
portions of SIRT1, also sometimes referred to as core domains,
include about amino acids 261 to 447 of GenBank Accession No.
NP.sub.--036370, which are encoded by nucleotides 834 to 1394 of
GenBank Accession No. NM.sub.--012238; about amino acids 242 to 493
of GenBank Accession No. NP.sub.--036370, which are encoded by
nucleotides 777 to 1532 of GenBank Accession No. NM.sub.--012238;
or about amino acids 254 to 495 of GenBank Accession No.
NP.sub.--036370, which are encoded by nucleotides 813 to 1538 of
GenBank Accession No. NM.sub.--012238.
[0017] The term "companion animals" refers to cats and dogs. As
used herein, the term "dog(s)" denotes any member of the species
Canis familiaris, of which there are a large number of different
breeds. The term "cat(s)" refers to a feline animal including
domestic cats and other members of the family Felidae, genus
Felis.
[0018] "Diabetes" refers to high blood sugar or ketoacidosis, as
well as chronic, general metabolic abnormalities arising from a
prolonged high blood sugar status or a decrease in glucose
tolerance. "Diabetes" encompasses both the type I and type II (Non
Insulin Dependent Diabetes Mellitus or NIDDM) forms of the disease.
The risk factors for diabetes include the following factors:
waistline of more than 40 inches for men or 35 inches for women,
blood pressure of 130/85 mmHg or higher, triglycerides above 150
mg/dl, fasting blood glucose greater than 100 mg/dl or high-density
lipoprotein of less than 40 mg/dl in men or 50 mg/dl in women.
[0019] The term "ED.sub.50" is art-recognized. In certain
embodiments, ED.sub.50 means the dose of a drug which produces 50%
of its maximum response or effect, or alternatively, the dose which
produces a pre-determined response in 50% of test subjects or
preparations. The term "LD.sub.50" is art-recognized. In certain
embodiments, LD.sub.50 means the dose of a drug which is lethal in
50% of test subjects. The term "therapeutic index" is an
art-recognized term which refers to the therapeutic index of a
drug, defined as LD.sub.50/ED.sub.50.
[0020] The term "hyperinsulinemia" refers to a state in an
individual in which the level of insulin in the blood is higher
than normal.
[0021] The term "insulin resistance" refers to a state in which a
normal amount of insulin produces a subnormal biologic response
relative to the biological response in a subject that does not have
insulin resistance.
[0022] An "insulin resistance disorder," as discussed herein,
refers to any disease or condition that is caused by or contributed
to by insulin resistance. Examples include: diabetes, obesity,
metabolic syndrome, insulin-resistance syndromes, syndrome X,
insulin resistance, high blood pressure, hypertension, high blood
cholesterol, dyslipidemia, hyperlipidemia, dyslipidemia,
atherosclerotic disease including stroke, coronary artery disease
or myocardial infarction, hyperglycemia, hyperinsulinemia and/or
hyperproinsulinemia, impaired glucose tolerance, delayed insulin
release, diabetic complications, including coronary heart disease,
angina pectoris, congestive heart failure, stroke, cognitive
functions in dementia, retinopathy, peripheral neuropathy,
nephropathy, glomerulonephritis, glomerulosclerosis, nephrotic
syndrome, hypertensive nephrosclerosis some types of cancer (such
as endometrial, breast, prostate, and colon), complications of
pregnancy, poor female reproductive health (such as menstrual
irregularities, infertility, irregular ovulation, polycystic
ovarian syndrome (PCOS)), lipodystrophy, cholesterol related
disorders, such as gallstones, cholecystitis and cholelithiasis,
gout, obstructive sleep apnea and respiratory problems,
osteoarthritis, and prevention and treatment of bone loss, e.g.
osteoporosis.
[0023] The term "livestock animals" refers to domesticated
quadrupeds, which includes those being raised for meat and various
byproducts, e.g., a bovine animal including cattle and other
members of the genus Bos, a porcine animal including domestic swine
and other members of the genus Sus, an ovine animal including sheep
and other members of the genus Ovis, domestic goats and other
members of the genus Capra; domesticated quadrupeds being raised
for specialized tasks such as use as a beast of burden, e.g., an
equine animal including domestic horses and other members of the
family Equidae, genus Equus.
[0024] The term "mammal" is known in the art, and exemplary mammals
include humans, primates, livestock animals (including bovines,
porcines, etc.), companion animals (e.g., canines, felines, etc.)
and rodents (e.g., mice and rats).
[0025] "Obese" individuals or individuals suffering from obesity
are generally individuals having a body mass index (BMI) of at
least 25 or greater. Obesity may or may not be associated with
insulin resistance.
[0026] The terms "parenteral administration" and "administered
parenterally" are art-recognized and refer to 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,
intra-articulare, subcapsular, subarachnoid, intraspinal, and
intrasternal injection and infusion.
[0027] A "patient", "subject", "individual" or "host" refers to
either a human or a non-human animal.
[0028] The term "pharmaceutically acceptable carrier" is
art-recognized and refers to a pharmaceutically-acceptable
material, composition or vehicle, such as a liquid or solid filler,
diluent, excipient, solvent or encapsulating material, involved in
carrying or transporting any subject composition or component
thereof. Each carrier must be "acceptable" in the sense of being
compatible with the subject composition and its components and not
injurious to the patient. Some examples of materials which may
serve as pharmaceutically acceptable carriers include: (1) sugars,
such as lactose, glucose and sucrose; (2) starches, such as corn
starch and potato starch; (3) cellulose, and its derivatives, such
as sodium carboxymethyl cellulose, ethyl cellulose and cellulose
acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc;
(8) excipients, such as cocoa butter and suppository waxes; (9)
oils, such as peanut oil, cottonseed oil, safflower oil, sesame
oil, olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other
non-toxic compatible substances employed in pharmaceutical
formulations.
[0029] The term "prophylactic" or "therapeutic" treatment is
art-recognized and refers to administration of a drug to a host. If
it is administered prior to clinical manifestation of the unwanted
condition (e.g., disease or other unwanted state of the host
animal) then the treatment is prophylactic, i.e., it protects the
host against developing the unwanted condition, whereas if
administered after manifestation of the unwanted condition, the
treatment is therapeutic (i.e., it is intended to diminish,
ameliorate or maintain the existing unwanted condition or side
effects therefrom).
[0030] The term "pyrogen-free", with reference to a composition,
refers to a composition that does not contain a pyrogen in an
amount that would lead to an adverse effect (e.g., irritation,
fever, inflammation, diarrhea, respiratory distress, endotoxic
shock, etc.) in a subject to which the composition has been
administered. For example, the term is meant to encompass
compositions that are free of, or substantially free of, an
endotoxin such as, for example, a lipopolysaccharide (LPS).
[0031] "Replicative lifespan" of a cell refers to the number of
daughter cells produced by an individual "mother cell."
"Chronological aging" or "chronological lifespan," on the other
hand, refers to the length of time a population of non-dividing
cells remains viable when deprived of nutrients. "Increasing the
lifespan of a cell" or "extending the lifespan of a cell," as
applied to cells or organisms, refers to increasing the number of
daughter cells produced by one cell; increasing the ability of
cells or organisms to cope with stresses and combat damage, e.g.,
to DNA, proteins; and/or increasing the ability of cells or
organisms to survive and exist in a living state for longer under a
particular condition, e.g., stress (for example, heatshock, osmotic
stress, high energy radiation, chemically-induced stress, DNA
damage, inadequate salt level, inadequate nitrogen level, or
inadequate nutrient level). Lifespan can be increased by at least
about 20%, 30%, 40%, 50%, 60% or between 20% and 70%, 30% and 60%,
40% and 60% or more using methods described herein.
[0032] "Sirtuin-activating compound" refers to a compound that
increases the level of a sirtuin protein and/or increases at least
one activity of a sirtuin protein. In an exemplary embodiment, a
sirtuin-activating compound may increase at least one biological
activity of a sirtuin protein by at least about 10%, 25%, 50%, 75%,
100%, or more. Exemplary biological activities of sirtuin proteins
include deacetylation, e.g., of histones and p53; extending
lifespan; increasing genomic stability; silencing transcription;
and controlling the segregation of oxidized proteins between mother
and daughter cells.
[0033] "Sirtuin protein" refers to a member of the sirtuin
deacetylase protein family, or preferably to the sir2 family, which
include yeast Sir2 (GenBank Accession No. P53685), C. elegans
Sir-2.1 (GenBank Accession No. NP 501912), and human SIRT1 (GenBank
Accession No. NM.sub.--012238 and NP.sub.--036370 (or AF083106))
and SIRT2 (GenBank Accession No. NM.sub.--012237, NM.sub.--030593,
NP.sub.--036369, NP.sub.--085096, and AF083107) proteins. Other
family members include the four additional yeast Sir2-like genes
termed "HST genes" (homologues of Sir two) HST1, HST2, HST3 and
HST4, and the five other human homologues hSIRT3, hSIRT4, hSIRT5,
hSIRT6 and hSIRT7 (Brachmann et al. (1995) Genes Dev. 9:2888 and
Frye et al. (1999) BBRC 260:273). Preferred sirtuins are those that
share more similarities with SIRT1, i.e., hSIRT1, and/or Sir2 than
with SIRT2, such as those members having at least part of the
N-terminal sequence present in SIRT1 and absent in SIRT2 such as
SIRT3 has.
[0034] "SIRT1 protein" refers to a member of the sir2 family of
sirtuin deacetylases. In one embodiment, a SIRT1 protein includes
yeast Sir2 (GenBank Accession No. P53685), C. elegans Sir-2.1
(GenBank Accession No. NP 501912), human SIRT1 (GenBank Accession
No. NM.sub.--012238 or NP.sub.--036370 (or AF083106)) proteins, and
equivalents and fragments thereof. In another embodiment, a SIRT1
protein includes a polypeptide comprising a sequence consisting of,
or consisting essentially of, the amino acid sequence set forth in
GenBank Accession Nos. NP.sub.--036370, NP 501912, NP.sub.--085096,
NP.sub.--036369, or P53685. SIRT1 proteins include polypeptides
comprising all or a portion of the amino acid sequence set forth in
GenBank Accession Nos. NP.sub.--036370, NP 501912, NP.sub.--085096,
NP.sub.--036369, or P53685; the amino acid sequence set forth in
GenBank Accession Nos. NP.sub.--036370, NP 501912, NP.sub.--085096,
NP.sub.--036369, or P53685 with 1 to about 2, 3, 5, 7, 10, 15, 20,
30, 50, 75 or more conservative amino acid substitutions; an amino
acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%,
98%, or 99% identical to GenBank Accession Nos. NP.sub.--036370,
NP.sub.--501912, NP.sub.--085096, NP.sub.--036369, or P53685, and
functional fragments thereof. Polypeptides of the invention also
include homologs (e.g., orthologs and paralogs), variants, or
fragments, of GenBank Accession Nos. NP.sub.--036370, NP 501912,
NP.sub.--085096, NP.sub.--036369, or P53685.
[0035] "SIRT3 protein" refers to a member of the sirtuin
deacetylase protein family and/or to a homolog of a SIRT1 protein.
In one embodiment, a SIRT3 protein includes human SIRT3 (GenBank
Accession No. AAH01042, NP.sub.--036371, or NP.sub.--001017524) and
mouse SIRT3 (GenBank Accession No. NP.sub.--071878) proteins, and
equivalents and fragments thereof. In another embodiment, a SIRT3
protein includes a polypeptide comprising a sequence consisting of,
or consisting essentially of, the amino acid sequence set forth in
GenBank Accession Nos. AAH01042, NP.sub.--036371,
NP.sub.--001017524, or NP.sub.--071878. SIRT3 proteins include
polypeptides comprising all or a portion of the amino acid sequence
set forth in GenBank Accession AAH01042, NP.sub.--036371,
NP.sub.--001017524, or NP.sub.--071878; the amino acid sequence set
forth in GenBank Accession Nos. AAH01042, NP.sub.--036371,
NP.sub.--001017524, or NP.sub.--071878 with 1 to about 2, 3, 5, 7,
10, 15, 20, 30, 50, 75 or more conservative amino acid
substitutions; an amino acid sequence that is at least 60%, 70%,
80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to GenBank Accession
Nos. AAH01042, NP.sub.--036371, NP.sub.--001017524, or
NP.sub.--071878, and functional fragments thereof. Polypeptides of
the invention also include homologs (e.g., orthologs and paralogs),
variants, or fragments, of GenBank Accession Nos. AAH01042,
NP.sub.--036371, NP.sub.--001017524, or NP.sub.--071878. In one
embodiment, a SIRT3 protein includes a fragment of SIRT3 protein
that is produced by cleavage with a mitochondrial matrix processing
peptidase (MPP) and/or a mitochondrial intermediate peptidase
(MIP).
[0036] The terms "systemic administration," "administered
systemically," "peripheral administration" and "administered
peripherally" are art-recognized and refer to the administration of
a subject composition, therapeutic or other material other than
directly into the central nervous system, such that it enters the
patient's system and, thus, is subject to metabolism and other like
processes.
[0037] The term "therapeutic agent" is art-recognized and refers to
any chemical moiety that is a biologically, physiologically, or
pharmacologically active substance that acts locally or
systemically in a subject. The term also means any substance
intended for use in the diagnosis, cure, mitigation, treatment or
prevention of disease or in the enhancement of desirable physical
or mental development and/or conditions in an animal or human.
[0038] The term "therapeutic effect" is art-recognized and refers
to a local or systemic effect in animals, particularly mammals, and
more particularly humans caused by a pharmacologically active
substance. The phrase "therapeutically-effective amount" means that
amount of such a substance that produces some desired local or
systemic effect at a reasonable benefit/risk ratio applicable to
any treatment. The therapeutically effective amount of such
substance will vary depending upon the subject and disease
condition being treated, the weight and age of the subject, the
severity of the disease condition, the manner of administration and
the like, which can readily be determined by one of ordinary skill
in the art. For example, certain compositions described herein may
be administered in a sufficient amount to produce a desired effect
at a reasonable benefit/risk ratio applicable to such
treatment.
[0039] "Treating" a condition or disease refers to curing as well
as ameliorating at least one symptom of the condition or
disease.
[0040] The term "vision impairment" refers to diminished vision,
which is often only partially reversible or irreversible upon
treatment (e.g., surgery). Particularly severe vision impairment is
termed "blindness" or "vision loss", which refers to a complete
loss of vision, vision worse than 20/200 that cannot be improved
with corrective lenses, or a visual field of less than 20 degrees
diameter (10 degrees radius).
2. Sirtuin Modulators
[0041] In one aspect, the invention provides novel
sirtuin-modulating compounds for treating and/or preventing a wide
variety of diseases and disorders including, for example, diseases
or disorders related to aging or stress, diabetes, obesity,
neurodegenerative diseases, ocular diseases and disorders,
cardiovascular disease, blood clotting disorders, inflammation,
cancer, and/or flushing, etc. Sirtuin-modulating compounds that
increase the level and/or activity of a sirtuin protein may also be
used for treating a disease or disorder in a subject that would
benefit from increased mitochondrial activity, for enhancing muscle
performance, for increasing muscle ATP levels, or for treating or
preventing muscle tissue damage associated with hypoxia or
ischemia. Other compounds disclosed herein may be suitable for use
in a pharmaceutical composition and/or one or more methods
disclosed herein.
[0042] In one embodiment, sirtuin-modulating compounds of the
invention are represented by Structural Formula (I):
##STR00001##
or a salt thereof wherein:
[0043] one of Z.sup.1 and Z.sup.2 is CR.sup.3, and the other of
Z.sup.1 and Z.sup.2 is selected from N and CR.sup.3, wherein
[0044] R.sup.3 is selected at each occurrence from hydrogen,
hydroxy, halo, --C.ident.N, fluoro-substituted C.sub.1-C.sub.2
alkyl, --O--(C.sub.1-C.sub.2) fluoro-substituted alkyl,
--S--(C.sub.1-C.sub.2) fluoro-substituted alkyl, C.sub.1-C.sub.4
alkyl, --O--(C.sub.1-C.sub.4) alkyl, --S--(C.sub.1-C.sub.4) alkyl,
C.sub.3-C.sub.7 cycloalkyl;
[0045] R is selected from hydrogen, --(C.sub.2-C.sub.4) alkyl,
--(C.sub.1-C.sub.4) fluoro-substituted alkyl, --(C.sub.1-C.sub.4)
alkyl-N(R.sup.7)(R.sup.7), --(C.sub.1-C.sub.4)
alkyl-C(O)--N(R.sup.7)(R.sup.7), --(C.sub.2-C.sub.4) alkyl
--O--R.sup.7, and --(C.sub.2-C.sub.4)
alkyl-N(R.sup.7)--C(O)--R.sup.7, wherein:
[0046] each R.sup.7 is independently selected from hydrogen, and
--C.sub.1-C.sub.4 alkyl; or
[0047] two R.sup.7 bound to the same nitrogen atom are taken
together with the nitrogen atom to form a 4- to 7-membered
saturated heterocycle optionally comprising one additional
heteroatom selected from N, S, S(.dbd.O), S(.dbd.O).sub.2, and O,
wherein the saturated heterocycle is optionally substituted at a
single carbon atom with --OH, --C.sub.1-C.sub.4 alkyl, fluoro,
--NH.sub.2, --NH(C.sub.1-C.sub.4 alkyl), --N(C.sub.1-C.sub.4
alkyl).sub.2, --NH(CH.sub.2CH.sub.2OCH.sub.3), or
--N(CH.sub.2CH.sub.2OCH.sub.3).sub.2;
[0048] R.sup.1 is selected from a carbocycle and a heterocycle,
wherein R.sup.1 is optionally substituted with one to two
substitutents independently selected from halo, --C.ident.N,
C.sub.1-C.sub.3 alkyl, .dbd.O, C.sub.3-C.sub.7 cycloalkyl,
fluoro-substituted C.sub.1-C.sub.2 alkyl, --O--R.sup.8,
--S--R.sup.8, --(C.sub.1-C.sub.2 alkyl)-N(R.sup.8)(R.sup.8),
--N(R.sup.8)(R.sup.8) (e.g., --NH--CH.sub.2--CH(OH)--CH.sub.2OH and
--O--CH.sub.2--CH(OH)--CH(OH), --O--(C.sub.1-C.sub.2
alkyl)-N(R.sup.8)(R.sup.8), --(C.sub.1-C.sub.2
alkyl)-O--(C.sub.1-C.sub.2 alkyl)-N(R.sup.8)(R.sup.8),
--C(O)--N(R.sup.8)(R.sup.8)--(C.sub.1-C.sub.2
alkyl)-C(O)--N(R.sup.8)(R.sup.8), and when R.sup.1 is phenyl,
R.sup.1 is also optionally substituted with 3,4-methylenedioxy,
fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy, and
fluoro-substituted 3,4-ethylenedioxy; each R.sup.8 is independently
selected from hydrogen, and --C.sub.1-C.sub.4 alkyl; or two R.sup.8
are taken together with the nitrogen atom to which they are bound
to form a 4- to 8-membered saturated heterocycle optionally
comprising one additional heteroatom selected from N, S, S(.dbd.O),
S(.dbd.O).sub.2, and O, wherein the alkyl is optionally substituted
with one or more --OH, fluoro, --NH.sub.2, --NH(C.sub.1-C.sub.4
alkyl), --N(C.sub.1-C.sub.4 alkyl).sub.2,
--NH(CH.sub.2CH.sub.2OCH.sub.3), or
--N(CH.sub.2CH.sub.2OCH.sub.3).sub.2 and the saturated heterocycle
is optionally substituted at a carbon atom with --OH,
--C.sub.1-C.sub.4 alkyl, fluoro, --NH.sub.2, --NH(C.sub.1-C.sub.4
alkyl), --N(C.sub.1-C.sub.4 alkyl).sub.2,
--NH(CH.sub.2CH.sub.2OCH.sub.3), or
--N(CH.sub.2CH.sub.2OCH.sub.3).sub.2.
[0049] R.sup.2 is selected from a carbocycle having at least five
ring atoms and a heterocycle other than piperazine, wherein R.sup.2
is optionally substituted with one to two substituents
independently selected from halo, --C.ident.N, C.sub.1-C.sub.3
alkyl, C.sub.3-C.sub.7 cycloalkyl, C.sub.1-C.sub.2
fluoro-substituted alkyl, --O--R.sup.8, --S--R.sup.8,
--(C.sub.1-C.sub.2 alkyl)-N(R.sup.8)(R.sup.8),
--N(R.sup.8)(R.sup.8) (e.g., (e.g.,
--NH--CH.sub.2--CH(OH)--CH.sub.2OH and
--O--CH.sub.2--CH(OH)--CH.sub.2OH), --O--(C.sub.1-C.sub.2
alkyl)-N(R.sup.8)(R.sup.8), --(C.sub.1-C.sub.2
alkyl)-O--(C.sub.1-C.sub.2 alkyl)-N(R.sup.8)(R.sup.8),
--C(O)--N(R.sup.8)(R.sup.8), --(C.sub.1-C.sub.2
alkyl)-C(O)--N(R.sup.8)(R.sup.8), --O-phenyl, phenyl, a second
heterocycle, and when R.sup.2 is phenyl, R.sup.2 is also optionally
substituted with 3,4-methylenedioxy, fluoro-substituted
3,4-methylenedioxy, 3,4-ethylenedioxy, or fluoro-substituted
3,4-ethylenedioxy, wherein any phenyl or second heterocycle
substituent of R.sup.2 is optionally substituted with halo;
--C.ident.N; C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.2
fluoro-substituted alkyl, --O--(C.sub.1-C.sub.2) fluoro-substituted
alkyl, --O--(C.sub.1-C.sub.3) alkyl, --S--(C.sub.1-C.sub.3) alkyl,
--S--(C.sub.1-C.sub.2) fluoro-substituted alkyl,
--NH--(C.sub.1-C.sub.3) alkyl and --N--(C.sub.1-C.sub.3).sub.2
alkyl; and
[0050] R.sup.4 is selected from hydrogen, halo, --C.ident.N,
fluoro-substituted C.sub.1-C.sub.2 alkyl, --S--(C.sub.1-C.sub.2)
fluoro-substituted alkyl, C.sub.1-C.sub.4 alkyl,
--S--(C.sub.1-C.sub.4) alkyl and C.sub.3-C.sub.7 cycloalkyl;
[0051] X is selected from --NH--C(.dbd.O)-.dagger.,
--C(.dbd.O)--NH-.dagger., --NH--C(.dbd.S)-.dagger.,
--C(.dbd.S)--NH-.dagger., --NH--S(.dbd.O)-.dagger.,
--S(.dbd.O)--NH-.dagger., --S(.dbd.O).sub.2--NH-.dagger.,
--NH--S(.dbd.O).sub.2-.dagger., --NH--C(.dbd.O)O-.dagger.,
--OC(.dbd.O)NH-.dagger., --NH--C(.dbd.O)NR.sup.5-.dagger.,
--NR.sup.5--C(.dbd.O)NH-.dagger., --NH--NR.sup.5-.dagger.,
--NR.sup.5--NH-.dagger., --O--NH-.dagger., --NH--O-.dagger.,
--NH--CR.sup.5R.sup.6-.dagger., --CR.sup.5R.sup.6--NH-.dagger.,
--NH--C(.dbd.NR.sup.5)-.dagger., --C(.dbd.NR.sup.5)--NH-.dagger.,
--C(.dbd.O)--NH--CR.sup.5R.sup.6-.dagger.,
--CR.sup.5R.sup.6--NH--C(O)-.dagger.,
--NH--C(.dbd.S)--CR.sup.5R.sup.6-.dagger.,
--CR.sup.5R.sup.6--C(.dbd.S)--NH-.dagger.,
--NH--S(O)--CR.sup.5R.sup.6-.dagger.,
CR.sup.5R.sup.6--S(O)--NH.dagger.,
--NH--S(O).sub.2--CR.sup.5R.sup.6-.dagger.,
CR.sup.5R.sup.6--S(O).sub.2--NH.dagger.,
--NH--C(.dbd.O)--O--CR.sup.5R.sup.6-.dagger.,
--CR.sup.5R.sup.6--O--C(.dbd.O)--NH-.dagger.,
--NH--C(.dbd.O)--NR.sup.5--CR.sup.5R.sup.6-.dagger.,
--CR.sup.5R.sup.6--O--C(.dbd.O)--NH-.dagger.,
--NH--S(O).sub.2--N(R.sup.5)-.dagger. and
--N(R.sup.5)--S(O).sub.2--NH-.dagger., wherein:
[0052] .dagger. represents where X is bound to R.sup.1; and
[0053] each R.sup.5 and R.sup.6 is independently selected from
hydrogen, C.sub.1-C.sub.4 alkyl, --CF.sub.3, and (C.sub.1-C.sub.2
alkyl)-CF.sub.3, wherein:
[0054] when X is --C(O)--NH-.dagger., Z.sup.1 and Z.sup.2 are each
CH, and R.sup.1 is optionally substituted phenyl, then R.sup.2 is
not pyridinyl;
[0055] when X is --C(O)--NH-.dagger., Z.sup.1 and Z.sup.2 are each
CH, R.sup.1 is phenyl, and R and R.sup.4 are H, then R.sup.2 is not
quinolinyl or indazolyl;
[0056] when X is --NH--S(O).sub.2-.dagger., Z.sup.1 and Z.sup.2 are
each CH, R.sup.1 is 4-methoxy phenyl and R and R.sup.4 are H then
R.sup.2 is not pyridinyl; and
[0057] when X is --NH--CH.sub.2-.dagger., Z.sup.1 is N, Z.sup.2 is
CR.sup.3, R.sup.3 is H or CH.sub.3, R.sup.4 is H, R is --CH.sub.3
and R.sup.1 is phenyl, then R.sup.2 is not triazolyl.
[0058] In certain embodiments, the compound of Structural Formula
(I) is selected from:
##STR00002##
In certain embodiments, the compound has the formula:
##STR00003##
[0059] In certain embodiments, R is selected from hydrogen,
--(C.sub.1-C.sub.4) alkyl-N(R.sup.7)(R.sup.7), --(C.sub.1-C.sub.4)
alkyl-C(O)--N(R.sup.7)(R.sup.7), --(C.sub.2-C.sub.4) alkyl
--O--R.sup.7, and --(C.sub.2-C.sub.4)
alkyl-N(R.sup.7)--C(O)--R.sup.7. In certain such embodiments, R is
hydrogen.
[0060] In certain embodiments, Z.sup.1 and Z.sup.2 are both
CR.sup.3.
[0061] In certain embodiments R.sup.4 is selected from hydrogen,
halo, --C.ident.N, C.sub.1-C.sub.4 alkyl, and fluoro-substituted
C.sub.1-C.sub.2 alkyl. In certain embodiments, R.sup.4 is hydrogen.
In certain embodiments wherein Z.sup.1 and Z.sup.2 are both
CR.sup.3, R and R.sup.4 are both H.
[0062] In certain embodiments, X is --C(.dbd.O)--NH-.dagger.. In
certain embodiments, X is
--C(.dbd.O)--NH--CR.sup.5R.sup.6-.dagger., where, in certain such
embodiments, R.sup.5 and R.sup.6 are independently selected from
hydrogen and C.sub.1-C.sub.4 alkyl. In certain such embodiments,
R.sup.5 and R.sup.6 are both hydrogen. In an exemplary embodiment,
X is --C(.dbd.O)--NH-.dagger., Z.sup.1 and Z.sup.2 are both
CR.sup.3, and R and R.sup.4 are both H.
[0063] In certain embodiments, R.sup.1 is selected from
heterocycles comprising one or more heteroatoms selected from N, O
and S. In particular embodiments, R.sup.1 is selected from
heterocycles comprising one or two nitrogens. In particular
embodiments, R.sup.1 is selected from heterocycles comprising up to
three heteroatoms selected from S and N. In other embodiments,
R.sup.1 is selected from heterocycles comprising up to three
heteroatoms selected from O and N.
[0064] Examples of R.sup.1 include:
##STR00004## ##STR00005##
[0065] In certain such embodiments, R.sup.1 is selected from:
##STR00006##
[0066] In the embodiments above, R.sup.1 is optionally substituted
by 1 or 2 substituents independently selected from halo,
(C.sub.1-C.sub.4) alkyl and .dbd.O. In certain embodiments, R.sup.1
is triazolyl optionally substituted with one or more substituents
selected from halo and (C.sub.1-C.sub.4) alkyl. In certain
embodiments, R.sup.1 is optionally substituted 2-triazolyl. In
certain embodiments, where R.sup.1 is optionally substituted
triazolyl, X is --C(.dbd.O)--NH-.dagger..
[0067] In certain embodiments, R.sup.2 is selected from aryl and
heteroaryl. Examples of R.sup.2 include:
##STR00007## ##STR00008## ##STR00009## ##STR00010##
##STR00011##
[0068] In particular embodiments, R.sup.2 is meta-substituted
relative to the attachment of R.sup.2 to the rest of the compound,
and wherein R.sup.2 is optionally further substituted as described
above. In certain embodiments, R.sup.2 is selected from:
##STR00012##
[0069] In certain embodiments, R.sup.2 is optionally substituted
with one to two substituents independently selected from halo,
--C.ident.N, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.2
fluoro-substituted alkyl, --OR.sup.8 wherein R.sup.8 is alkyl
optionally substituted with one or more halo substituents. In
certain embodiments, R.sup.2 is phenyl optionally substituted by
one or more substituents independently selected from --Cl, --Br,
--F, --C.ident.N, --CF.sub.3 and --OCF.sub.3.
[0070] In another embodiment, the invention provides a compound
represented by Structural Formula II:
##STR00013##
or a salt thereof wherein:
[0071] one of Z.sup.11 and Z.sup.12 is CR.sup.13, and the other of
Z.sup.11 and Z.sup.12 is selected from N and CR.sup.13, wherein
[0072] R.sup.13 is selected from hydrogen, halo, --OH, --C.ident.N,
fluoro-substituted C.sub.1-C.sub.2 alkyl, --O--(C.sub.1-C.sub.2
fluoro-substituted alkyl), --S--(C.sub.1-C.sub.2 fluoro-substituted
alkyl), C.sub.1-C.sub.4 alkyl, --(C.sub.1-C.sub.2
alkyl)-N(R.sup.14)(R.sup.14), --O--CH.sub.2CH(OH)CH.sub.2OH,
--O--(C.sub.1-C.sub.4) alkyl, --O--(C.sub.1-C.sub.3)
alkyl-N(R.sup.14)(R.sup.14), --N(R.sup.14)(R.sup.14),
--S--(C.sub.1-C.sub.4) alkyl and C.sub.3-C.sub.7 cycloalkyl; [0073]
R is selected from hydrogen, --(C.sub.2-C.sub.4) alkyl,
--(C.sub.1-C.sub.4) fluoro-substituted alkyl, --(C.sub.1-C.sub.4)
alkyl-N(R.sup.7)(R.sup.7), --(C.sub.1-C.sub.4)
alkyl-C(O)--N(R.sup.7)(R.sup.7), --(C.sub.2-C.sub.4) alkyl
--O--R.sup.7, and --(C.sub.2-C.sub.4)
alkyl-N(R.sup.7)--C(O)--R.sup.7, wherein: [0074] each R.sup.7 is
independently selected from hydrogen, and --C.sub.1-C.sub.4 alkyl;
or [0075] two R.sup.7 bound to the same nitrogen atom are taken
together with the nitrogen atom to form a 4- to 7-membered
saturated heterocycle optionally comprising one additional
heteroatom selected from N, S, S(.dbd.O), S(.dbd.O).sub.2, and O,
wherein the saturated heterocycle is optionally substituted at a
single carbon atom with --OH, --C.sub.1-C.sub.4 alkyl, fluoro,
--NH.sub.2, --NH(C.sub.1-C.sub.4 alkyl), --N(C.sub.1-C.sub.4
alkyl).sub.2, --NH(CH.sub.2CH.sub.2OCH.sub.3), or
--N(CH.sub.2CH.sub.2OCH.sub.3).sub.2; [0076] R.sup.4 is selected
from hydrogen, halo, --C.ident.N, fluoro-substituted
C.sub.1-C.sub.2 alkyl, --S--(C.sub.1-C.sub.2) fluoro-substituted
alkyl, C.sub.1-C.sub.4 alkyl, --S--(C.sub.1-C.sub.4) alkyl and
C.sub.3-C.sub.7 cycloalkyl;
[0077] X is selected from --NH--C(.dbd.O)-.dagger.,
--C(.dbd.O)--NH-.dagger., --NH--C(.dbd.S)-.dagger.,
--C(.dbd.S)--NH-.dagger., --NH--S(.dbd.O)-.dagger.,
--S(.dbd.O)--NH-.dagger., --S(.dbd.O).sub.2--NH-.dagger.,
--NH--S(.dbd.O).sub.2-.dagger.,
--NH--S(O).sub.2--NR.sup.15-.dagger.,
--NR.sup.15--S(O).sub.2--NH-.dagger., --NH--C(.dbd.O)O-.dagger.,
O--C(.dbd.O)--NH-.dagger., --NH--C(.dbd.O)NH-.dagger.,
--NH--C(.dbd.O)NR.sup.15-.dagger.,
--NR.sup.15--C(.dbd.O)NH-.dagger., --NH--NR.sup.15-.dagger.,
--NR.sup.15--NH-.dagger., --O--NH-.dagger., --NH--O-.dagger.,
--NH--CR.sup.15R.sup.16-.dagger., --CR.sup.15R.sup.16--NH-.dagger.,
--NH--C(.dbd.NR.sup.15)-.dagger., --C(.dbd.NR.sup.15)--NH-.dagger.,
--C(.dbd.O)--NH--CR.sup.15R.sup.16-.dagger.,
--CR.sup.15R.sup.16--NH--C(O)-.dagger.,
--NH--C(.dbd.S)--CR.sup.15R.sup.16-.dagger.,
--CR.sup.15R.sup.16--C(.dbd.S)--NH-.dagger.,
--NH--S(O)--CR.sup.15R.sup.16-.dagger.,
--CR.sup.15R.sup.16--S(O)--NH-.dagger.,
--NH--S(O).sub.2--CR.sup.15R.sup.16-.dagger.,
--CR.sup.15R.sup.16--S(O).sub.2--NH-.dagger.,
--NH--C(.dbd.O)--O--CR.sup.15R.sup.16-.dagger.,
--CR.sup.15R.sup.16--O--C(.dbd.O)--NH-.dagger.,
--NH--C(.dbd.O)--NR.sup.14--CR.sup.15R.sup.16-.dagger.,
--NH--C(.dbd.O)--CR.sup.15R.sup.16-.dagger., and
--CR.sup.15R.sup.16--NH--C(.dbd.O)--O-.dagger., wherein
[0078] .dagger. represents where X is bound to R.sup.11, and:
[0079] R.sup.15 and R.sup.16 are independently selected from
hydrogen, C.sub.1-C.sub.4 alkyl, CF.sub.3, and --(C.sub.1-C.sub.4
alkyl)-CF.sub.3;
[0080] R.sup.11 is selected from a carbocycle and a heterocycle,
wherein R.sup.11 is optionally substituted with one to two
substitutents independently selected from halo, --C.ident.N,
C.sub.1-C.sub.3 alkyl, C.sub.3-C.sub.7 cycloalkyl, C.sub.1-C.sub.2
fluoro-substituted alkyl, .dbd.O, --O--R.sup.14, --S--R.sup.14,
--(C.sub.1-C.sub.4 alkyl)-N(R.sup.14)(R.sup.14),
--N(R.sup.14)(R.sup.14), --O--(C.sub.2-C.sub.4
alkyl)-N(R.sup.14)(R.sup.14), --C(O)--N(R.sup.14)(R.sup.14),
--C(O)--O--R.sup.14, and --(C.sub.1-C.sub.4
alkyl)-C(O)--N(R.sup.14)(R.sup.14), and when R.sup.11 is phenyl,
R.sup.11 is also optionally substituted with 3,4-methylenedioxy,
fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy,
fluoro-substituted 3,4-ethylenedioxy, O-(saturated heterocycle),
fluoro-substituted --O-(saturated heterocycle), and C.sub.1-C.sub.4
alkyl-substituted O-(saturated heterocycle), wherein
[0081] each R.sup.14 is independently selected from hydrogen, and
--C.sub.1-C.sub.4 alkyl; or
[0082] two R.sup.14 are taken together with the nitrogen atom to
which they are bound to form a 4- to 8-membered saturated
heterocycle optionally comprising one additional heteroatom
selected from N, S, S(.dbd.O), S(.dbd.O).sub.2, and O, wherein:
[0083] when R.sup.14 is alkyl, the alkyl is optionally substituted
with one or more --OH, --O--(C.sub.1-C.sub.4 alkyl), fluoro,
--NH.sub.2, --NH(C.sub.1-C.sub.4 alkyl), --N(C.sub.1-C.sub.4
alkyl).sub.2, --NH(CH.sub.2CH.sub.2OCH.sub.3), or
--N(CH.sub.2CH.sub.2OCH.sub.3).sub.2 and [0084] when two R.sup.14
are taken together with the nitrogen atom to which they are bound
to form a 4- to 8-membered saturated heterocycle, the saturated
heterocycle is optionally substituted at a carbon atom with --OH,
--C.sub.1-C.sub.4 alkyl, fluoro, --NH.sub.2, --NH(C.sub.1-C.sub.4
alkyl), --N(C.sub.1-C.sub.4 alkyl).sub.2,
--NH(CH.sub.2CH.sub.2OCH.sub.3), or
--N(CH.sub.2CH.sub.2OCH.sub.3).sub.2; and optionally substituted at
any substitutable nitrogen atom with --C.sub.1-C.sub.4 alkyl,
fluoro-substituted C.sub.1-C.sub.4 alkyl, or
--(CH.sub.2).sub.2--O--CH.sub.3; and
[0085] R.sup.12 is selected from a carbocycle having at least five
ring atoms and a heterocycle other than piperazine, indazole,
triazole or pyrazolopyridine, wherein R.sup.12 is optionally
substituted with one to two substitutents independently selected
from halo, --C.ident.N, C.sub.1-C.sub.4 alkyl, C.sub.3-C.sub.7
cycloalkyl, C.sub.1-C.sub.2 fluoro-substituted alkyl,
--O--(C.sub.1-C.sub.4 alkyl), --S--R.sup.14, --S(O)--R.sup.14,
--S(O).sub.2--R.sup.14, --(C.sub.1-C.sub.4
alkyl)-N(R.sup.14)(R.sup.14), --N(R.sup.14)(R.sup.14),
--O--(C.sub.2-C.sub.4 alkyl)-N(R.sup.14)(R.sup.14),
--C(O)--N(R.sup.14)(R.sup.14), --(C.sub.1-C.sub.4
alkyl)-C(O)--N(R.sup.14)(R.sup.14), --O-phenyl, phenyl, and a
second heterocycle, and when R.sup.12 is phenyl, R.sup.12 is also
optionally substituted with 3,4-methylenedioxy, fluoro-substituted
3,4-methylenedioxy, 3,4-ethylenedioxy, fluoro-substituted
3,4-ethylenedioxy, or --O-(saturated heterocycle) wherein any
phenyl, saturated heterocycle or second heterocycle substituent of
R.sup.12 is optionally substituted with halo; --C.ident.N;
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.2 fluoro-substituted alkyl,
--O--(C.sub.1-C.sub.2 fluoro-substituted alkyl),
--O--(C.sub.1-C.sub.4 alkyl), --S--(C.sub.1-C.sub.4 alkyl),
--S--(C.sub.1-C.sub.2 fluoro-substituted alkyl),
--NH--(C.sub.1-C.sub.4 alkyl) and --N--(C.sub.1-C.sub.4
alkyl).sub.2, wherein: [0086] when X is --C(O)--NH-.dagger.,
Z.sup.11 and Z.sup.12 are each CH, R.sup.11 is optionally
substituted phenyl, and R and R.sup.4 are H, then R.sup.12 is not
pyridinyl or quinolinyl; and [0087] when X is
--NH--S(O).sub.2-.dagger., Z.sup.11 and Z.sup.12 are each CH,
R.sup.11 is 4-methoxy phenyl and R and R.sup.4 are H then R.sup.12
is not pyridinyl. In certain embodiments of Formula II, when X is
--S(O).sub.2--NH-.dagger., Z.sup.11 and Z.sup.12 are each CH,
R.sup.11 is phenyl, and R and R.sup.4 are H, then R.sup.12 is not
optionally substituted phenyl, or pyridin-4-yl.
[0088] In certain embodiments of Formula II, R.sup.12 is not
pyrazolyl, imidazolyl, indolyl, thienopyrazolyl,
tetrahydroindazolyl, tetrahydrocyclopentapyrazolyl,
dihydrofuropyrazolyl, tetrahydropyrrolopyrazolyl,
tetrahydropyranopyrazolyl, or tetrahydropyridinopyrazolyl.
[0089] In other embodiments of Formula II, R.sup.12 is not
pyrazolyl.
[0090] In certain embodiments of Formula II, the compound is
represented by any one of Structural Formulae IIa, IIb and IIc:
##STR00014##
In a more specific embodiment of Formula II, the compound is
represented by Structural Formula IIc
##STR00015##
[0091] In certain embodiments of Formula II, R is hydrogen. In one
aspect of this embodiment, the compound is represented by
Structural Formula IIc. In another aspect of this embodiment,
R.sup.4 is hydrogen. In yet another aspect of this embodiment, any
R.sup.3 present is hydrogen. In an even more specific aspect of
this embodiment, the compound is represented by Structural Formula
IIc, and each R.sup.3 and R.sup.4 are hydrogen.
[0092] In some embodiments X is --C(O)--NH-.dagger.. In one aspect
of this embodiment, the compound is represented by Structural
Formula IIc. In an even more specific aspect of this embodiment,
the compound is represented by Structural Formula III, and R, each
R.sup.3 and R.sup.4 are hydrogen.
[0093] In certain embodiments, R.sup.11 is selected from:
##STR00016## ##STR00017## ##STR00018## ##STR00019##
wherein R.sup.11 is optionally further substituted. In a more
specific embodiment, R.sup.11 is not further substituted. In one
aspect of either of these embodiments, X is --C(O)--NH-.dagger.. In
an even more specific aspect of these embodiments X is
--C(O)--NH-.dagger., the compound is represented by Structural
Formula IIc.
[0094] In certain embodiments of Formula II R.sup.12 is selected
from:
##STR00020## ##STR00021## ##STR00022## ##STR00023##
##STR00024##
wherein R.sup.12 is optionally further substituted. In a more
specific embodiment, R.sup.12 is not further substituted. In one
aspect of either of these embodiments, X is --C(O)--NH-.dagger.. In
an even more specific aspect of these embodiments X is
--C(O)--NH-.dagger., and the compound is represented by Structural
Formula IIc.
[0095] In yet another specific embodiment, the invention provides a
compound having Structural Formula III:
##STR00025##
wherein:
[0096] R.sup.3 is selected from hydrogen, fluoro, --OCH.sub.3 and
morpholin-4-yl;
[0097] R.sup.11 is selected from:
##STR00026## ##STR00027## ##STR00028##
[0098] R.sup.12 is selected from:
##STR00029## ##STR00030## ##STR00031## ##STR00032## ##STR00033##
##STR00034##
and when R.sup.11 is
##STR00035##
R.sup.12 is not
##STR00036##
[0100] Compounds of the invention, including novel compounds of the
invention, can also be used in the methods described herein.
[0101] The compounds and salts thereof described herein also
include their corresponding hydrates (e.g., hemihydrate,
monohydrate, dihydrate, trihydrate, tetrahydrate) and solvates.
Suitable solvents for preparation of solvates and hydrates can
generally be selected by a skilled artisan.
[0102] The compounds and salts thereof can be present in amorphous
or crystalline (including co-crystalline and polymorph) forms.
[0103] Sirtuin-modulating compounds of the invention advantageously
modulate the level and/or activity of a sirtuin protein,
particularly the deacetylase activity of the sirtuin protein.
[0104] Separately or in addition to the above properties, certain
sirtuin-modulating compounds of the invention do not substantially
have one or more of the following activities: inhibition of
PI3-kinase, inhibition of aldoreductase, inhibition of tyrosine
kinase, transactivation of EGFR tyrosine kinase, coronary dilation,
or spasmolytic activity, at concentrations of the compound that are
effective for modulating the deacetylation activity of a sirtuin
protein (e.g., such as a SIRT1 and/or a SIRT3 protein).
[0105] The term "carbocycle" or "carbocyclic" means ring systems,
wherein all ring atoms are carbon and includes 5-7 membered
monocyclic and 8-12 membered bicyclic rings, as well as spyrofused
and bridged polycyclic ring systems wherein the monocyclic or each
of the bicyclic rings is independently selected from saturated,
unsaturated and aromatic. Unless otherwise specified, a carbocycle
is optionally substituted with one or more substituents selected
from halo, --C.ident.N, C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.2
fluoro-substituted alkyl, --O--(C.sub.1-C.sub.2) fluoro-substituted
alkyl, --O--(C.sub.1-C.sub.3) alkyl, --S--(C.sub.1-C.sub.3) alkyl,
--S--(C.sub.1-C.sub.2) fluoro-substituted alkyl, hydroxyl, amino,
--NH--(C.sub.1-C.sub.3) alkyl and --N--(C.sub.1-C.sub.3).sub.2
alkyl. Exemplary carbocycles include cyclopentyl, cyclohexyl,
cyclohexenyl, adamantyl, phenyl and naphthyl.
[0106] The term "heterocycle" or "heterocyclic" means ring systems
comprising one or more heteroatoms selected from, for example, N,
O, and S atoms and includes 4-7 membered monocyclic and 8-12
membered bicyclic rings, as well as spyrofused and bridged
polycyclic ring systems wherein the monocyclic ring and each of the
bicyclic rings are independently selected from saturated,
unsaturated or aromatic. When a heterocycle is polycyclic, it
should be understood that only one ring is required to contain a
heteroatom. Unless otherwise specified, a heterocycle is optionally
substituted with one or more substituents selected from halo,
--C.ident.N, C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.2
fluoro-substituted alkyl, --O--(C.sub.1-C.sub.2) fluoro-substituted
alkyl, --O--(C.sub.1-C.sub.3) alkyl, --S--(C.sub.1-C.sub.3) alkyl,
--S--(C.sub.1-C.sub.2) fluoro-substituted alkyl, hydroxyl, amino,
--NH--(C.sub.1-C.sub.3) alkyl and --N--(C.sub.1-C.sub.3).sub.2
alkyl.
[0107] Monocyclic rings include 5-7 membered aryl or heteroaryl,
3-7 membered cycloalkyl, and 5-7 membered non-aromatic
heterocyclyl. Monocyclic rings are optionally substituted with one
or more substituents selected from halo, cyano, lower alkoxy, lower
alkyl, hydroxyl, amino, lower alkylamino and lower dialkylamino.
Exemplary monocyclic groups include substituted and unsubstituted
heterocycles and carbocycles. Specific monocyclic groups include
thiazolyl, oxazolyl, oxazinyl, thiazinyl, dithianyl, dioxanyl,
isoxazolyl, isothiazolyl, triazolyl, furanyl, tetrahydrofuranyl,
dihydrofuranyl, pyranyl, tetrazolyl, pyrazolyl, pyrazinyl,
pyridazinyl, imidazolyl, pyridinyl, pyrrolyl, dihydropyrrolyl,
pyrrolidinyl, piperidinyl, piperazinyl, pyrimidinyl, morpholinyl,
tetrahydrothiophenyl, thiophenyl, cyclohexyl, cyclopentyl,
cyclopropyl, cyclobutyl, cycloheptanyl, azetidinyl, oxetanyl,
thiiranyl, oxiranyl, aziridinyl, and thiomorpholinyl.
[0108] Aromatic (aryl) groups include carbocyclic aromatic groups
such as phenyl, naphthyl, and anthracyl, and heteroaryl groups such
as imidazolyl, thienyl, furyl, pyridyl, pyrimidyl, pyranyl,
pyrazolyl, pyrrolyl, pyrazinyl, thiazolyl, oxazolyl, and
tetrazolyl. Aromatic groups also include fused polycyclic aromatic
ring systems in which a carbocyclic aromatic ring or heteroaryl
ring is fused to one or more other heteroaryl rings. Examples
include benzothienyl, benzofuryl, indolyl, quinolinyl,
benzothiazole, benzoxazole, benzimidazole, quinolinyl,
isoquinolinyl and isoindolyl.
[0109] Fluoro-substituted includes from one fluoro substituent up
to per-fluoro-substitution. Exemplary fluoro-substituted
C.sub.1-C.sub.2 alkyl includes --CFH.sub.2, CF.sub.2H, --CF.sub.3,
--CH.sub.2CH.sub.2F, --CH.sub.2CHF.sub.2, --CHFCH.sub.3,
--CF.sub.2CHF.sub.2. Per-fluoro-substituted C.sub.1-C.sub.2 alkyl,
for example, includes --CF.sub.3, and --CF.sub.2CF.sub.3.
[0110] Suitable substituents on moieties indicated as being
substituted or unsubstituted are those which do not substantially
interfere with the ability of the disclosed compounds to have one
or more of the properties disclosed herein. A substituent
substantially interferes with the properties of a compound when the
magnitude of the property is reduced by more than about 50% in a
compound with the substituent compared with a compound without the
substituent.
[0111] Combinations of substituents and variables envisioned by
this invention are only those that result in the formation of
stable compounds. As used herein, the term "stable" refers to
compounds that possess stability sufficient to allow manufacture
and that maintain the integrity of the compound for a sufficient
period of time to be useful for the purposes detailed herein.
[0112] The compounds disclosed herein also include partially and
fully deuterated variants. In certain embodiments, one or more
deuterium atoms are present for kinetic studies. One of ordinary
skill in the art can select the sites at which such deuterium atoms
are present.
[0113] Also included in the present invention are salts,
particularly pharmaceutically acceptable salts, of the
sirtuin-modulating compounds described herein. The compounds of the
present invention that possess a sufficiently acidic, a
sufficiently basic, or both functional groups, can react with any
of a number of inorganic bases, and inorganic and organic acids, to
form a salt. Alternatively, compounds that are inherently charged,
such as those with a quaternary nitrogen, can form a salt with an
appropriate counterion (e.g., a halide such as bromide, chloride,
or fluoride, particularly bromide).
[0114] Acids commonly employed to form acid addition salts are
inorganic acids such as hydrochloric acid, hydrobromic acid,
hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and
organic acids such as p-toluenesulfonic acid, methanesulfonic acid,
oxalic acid, p-bromophenyl-sulfonic acid, carbonic acid, succinic
acid, citric acid, benzoic acid, acetic acid, and the like.
Examples of such salts include the sulfate, pyrosulfate, bisulfate,
sulfite, bisulfite, phosphate, monohydrogenphosphate,
dihydrogenphosphate, metaphosphate, pyrophosphate, chloride,
bromide, iodide, acetate, propionate, decanoate, caprylate,
acrylate, formate, isobutyrate, caproate, heptanoate, propiolate,
oxalate, malonate, succinate, suberate, sebacate, fumarate,
maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate,
chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate,
methoxybenzoate, phthalate, sulfonate, xylenesulfonate,
phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,
gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate,
propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,
mandelate, and the like.
[0115] Base addition salts include those derived from inorganic
bases, such as ammonium or alkali or alkaline earth metal
hydroxides, carbonates, bicarbonates, and the like. Such bases
useful in preparing the salts of this invention thus include sodium
hydroxide, potassium hydroxide, ammonium hydroxide, potassium
carbonate, and the like.
[0116] According to another embodiment, the present invention
provides methods of producing the above-defined sirtuin-modulating
compounds. The compounds may be synthesized using conventional
techniques. Advantageously, these compounds are conveniently
synthesized from readily available starting materials.
[0117] Synthetic chemistry transformations and methodologies useful
in synthesizing the sirtuin-modulating compounds described herein
are known in the art and include, for example, those described in
R. Larock, Comprehensive Organic Transformations (1989); T. W.
Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis,
2d. Ed. (1991); L. Fieser and M. Fieser, Fieser and Fieser's
Reagents for Organic Synthesis (1994); and L. Paquette, ed.,
Encyclopedia of Reagents for Organic Synthesis (1995).
[0118] In an exemplary embodiment, a sirtuin-modulating compound
may traverse the cytoplasmic membrane of a cell. For example, a
compound may have a cell-permeability of at least about 20%, 50%,
75%, 80%, 90% or 95%.
[0119] Sirtuin-modulating compounds described herein may also have
one or more of the following characteristics: the compound may be
essentially non-toxic to a cell or subject; the sirtuin-modulating
compound may be an organic molecule or a small molecule of 2000 amu
or less, 1000 amu or less; a compound may have a half-life under
normal atmospheric conditions of at least about 30 days, 60 days,
120 days, 6 months or 1 year; the compound may have a half-life in
solution of at least about 30 days, 60 days, 120 days, 6 months or
1 year; a sirtuin-modulating compound may be more stable in
solution than resveratrol by at least a factor of about 50%, 2
fold, 5 fold, 10 fold, 30 fold, 50 fold or 100 fold; a
sirtuin-modulating compound may promote deacetylation of the DNA
repair factor Ku70; a sirtuin-modulating compound may promote
deacetylation of RelA/p65; a compound may increase general turnover
rates and enhance the sensitivity of cells to TNF-induced
apoptosis.
[0120] In certain embodiments, a sirtuin-modulating compound does
not have any substantial ability to inhibit a histone deacetylase
(HDACs) class I, a HDAC class II, or HDACs I and II, at
concentrations (e.g., in vivo) effective for modulating the
deacetylase activity of the sirtuin. For instance, in preferred
embodiments the sirtuin-modulating compound is a sirtuin-activating
compound and is chosen to have an EC.sub.50 for activating sirtuin
deacetylase activity that is at least 5 fold less than the
EC.sub.50 for inhibition of an HDAC I and/or HDAC II, and even more
preferably at least 10 fold, 100 fold or even 1000 fold less.
Methods for assaying HDAC I and/or HDAC II activity are well known
in the art and kits to perform such assays may be purchased
commercially. See e.g., BioVision, Inc. (Mountain View, Calif.;
world wide web at biovision.com) and Thomas Scientific (Swedesboro,
N.J.; world wide web at tomassci.com).
[0121] In certain embodiments, a sirtuin-modulating compound does
not have any substantial ability to modulate sirtuin homologs. In
one embodiment, an activator of a human sirtuin protein may not
have any substantial ability to activate a sirtuin protein from
lower eukaryotes, particularly yeast or human pathogens, at
concentrations (e.g., in vivo) effective for activating the
deacetylase activity of human sirtuin. For example, a
sirtuin-activating compound may be chosen to have an EC.sub.50 for
activating a human sirtuin, such as SIRT1 and/or SIRT3, deacetylase
activity that is at least 5 fold less than the EC.sub.50 for
activating a yeast sirtuin, such as Sir2 (such as Candida, S.
cerevisiae, etc.), and even more preferably at least 10 fold, 100
fold or even 1000 fold less. In another embodiment, an inhibitor of
a sirtuin protein from lower eukaryotes, particularly yeast or
human pathogens, does not have any substantial ability to inhibit a
sirtuin protein from humans at concentrations (e.g., in vivo)
effective for inhibiting the deacetylase activity of a sirtuin
protein from a lower eukaryote. For example, a sirtuin-inhibiting
compound may be chosen to have an IC.sub.50 for inhibiting a human
sirtuin, such as SIRT1 and/or SIRT3, deacetylase activity that is
at least 5 fold less than the IC.sub.50 for inhibiting a yeast
sirtuin, such as Sir2 (such as Candida, S. cerevisiae, etc.), and
even more preferably at least 10 fold, 100 fold or even 1000 fold
less.
[0122] In certain embodiments, a sirtuin-modulating compound may
have the ability to modulate one or more sirtuin protein homologs,
such as, for example, one or more of human SIRT1, SIRT2, SIRT3,
SIRT4, SIRT5, SIRT6, or SIRT7. In one embodiment, a
sirtuin-modulating compound has the ability to modulate both a
SIRT1 and a SIRT3 protein.
[0123] In other embodiments, a SIRT1 modulator does not have any
substantial ability to modulate other sirtuin protein homologs,
such as, for example, one or more of human SIRT2, SIRT3, SIRT4,
SIRT5, SIRT6, or SIRT7, at concentrations (e.g., in vivo) effective
for modulating the deacetylase activity of human SIRT1. For
example, a sirtuin-modulating compound may be chosen to have an
ED.sub.50 for modulating human SIRT1 deacetylase activity that is
at least 5 fold less than the ED.sub.50 for modulating one or more
of human SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7, and even more
preferably at least 10 fold, 100 fold or even 1000 fold less. In
one embodiment, a SIRT1 modulator does not have any substantial
ability to modulate a SIRT3 protein.
[0124] In other embodiments, a SIRT3 modulator does not have any
substantial ability to modulate other sirtuin protein homologs,
such as, for example, one or more of human SIRT1, SIRT2, SIRT4,
SIRT5, SIRT6, or SIRT7, at concentrations (e.g., in vivo) effective
for modulating the deacetylase activity of human SIRT3. For
example, a sirtuin-modulating compound may be chosen to have an
ED.sub.50 for modulating human SIRT3 deacetylase activity that is
at least 5 fold less than the ED.sub.50 for modulating one or more
of human SIRT1, SIRT2, SIRT4, SIRT5, SIRT6, or SIRT7, and even more
preferably at least 10 fold, 100 fold or even 1000 fold less. In
one embodiment, a SIRT3 modulator does not have any substantial
ability to modulate a SIRT1 protein.
[0125] In certain embodiments, a sirtuin-modulating compound may
have a binding affinity for a sirtuin protein of about 10.sup.-9M,
10.sup.-10M, 10.sup.-11M, 10.sup.-12M or less. A sirtuin-modulating
compound may reduce (activator) or increase (inhibitor) the
apparent Km of a sirtuin protein for its substrate or NAD+ (or
other cofactor) by a factor of at least about 2, 3, 4, 5, 10, 20,
30, 50 or 100. In certain embodiments, Km values are determined
using the mass spectrometry assay described herein. Preferred
activating compounds reduce the Km of a sirtuin for its substrate
or cofactor to a greater extent than caused by resveratrol at a
similar concentration or reduce the Km of a sirtuin for its
substrate or cofactor similar to that caused by resveratrol at a
lower concentration. A sirtuin-modulating compound may increase the
Vmax of a sirtuin protein by a factor of at least about 2, 3, 4, 5,
10, 20, 30, 50 or 100. A sirtuin-modulating compound may have an
ED50 for modulating the deacetylase activity of a SIRT1 and/or
SIRT3 protein of less than about 1 nM, less than about 10 nM, less
than about 100 nM, less than about 1 .mu.M, less than about 10
.mu.M, less than about 100 .mu.M, or from about 1-10 nM, from about
10-100 nM, from about 0.1-1 .mu.M, from about 1-10 .mu.M or from
about 10-100 .mu.M. A sirtuin-modulating compound may modulate the
deacetylase activity of a SIRT1 and/or SIRT3 protein by a factor of
at least about 5, 10, 20, 30, 50, or 100, as measured in a cellular
assay or in a cell based assay. A sirtuin-activating compound may
cause at least about 10%, 30%, 50%, 80%, 2 fold, 5 fold, 10 fold,
50 fold or 100 fold greater induction of the deacetylase activity
of a sirtuin protein relative to the same concentration of
resveratrol. A sirtuin-modulating compound may have an ED50 for
modulating SIRT5 that is at least about 10 fold, 20 fold, 30 fold,
50 fold, or greater than that for modulating SIRT1 and/or
SIRT3.
3. Exemplary Uses
[0126] In certain aspects, the invention provides methods for
modulating the level and/or activity of a sirtuin protein and
methods of use thereof.
[0127] In certain embodiments, the invention provides methods for
using sirtuin-modulating compounds wherein the sirtuin-modulating
compounds activate a sirtuin protein, e.g., increase the level
and/or activity of a sirtuin protein. Sirtuin-modulating compounds
that increase the level and/or activity of a sirtuin protein may be
useful for a variety of therapeutic applications including, for
example, increasing the lifespan of a cell, and treating and/or
preventing a wide variety of diseases and disorders including, for
example, diseases or disorders related to aging or stress,
diabetes, obesity, neurodegenerative diseases, cardiovascular
disease, blood clotting disorders, inflammation, cancer, and/or
flushing, etc. The methods comprise administering to a subject in
need thereof a pharmaceutically effective amount of a
sirtuin-modulating compound, e.g., a sirtuin-activating
compound.
[0128] While Applicants do not wish to be bound by theory, it is
believed that activators of the instant invention may interact with
a sirtuin at the same location within the sirtuin protein (e.g.,
active site or site affecting the Km or Vmax of the active site).
It is believed that this is the reason why certain classes of
sirtuin activators and inhibitors can have substantial structural
similarity.
[0129] In certain embodiments, the sirtuin-modulating compounds
described herein may be taken alone or in combination with other
compounds. In one embodiment, a mixture of two or more
sirtuin-modulating compounds may be administered to a subject in
need thereof. In another embodiment, a sirtuin-modulating compound
that increases the level and/or activity of a sirtuin protein may
be administered with one or more of the following compounds:
resveratrol, butein, fisetin, piceatannol, or quercetin. In an
exemplary embodiment, a sirtuin-modulating compound that increases
the level and/or activity of a sirtuin protein may be administered
in combination with nicotinic acid. In another embodiment, a
sirtuin-modulating compound that decreases the level and/or
activity of a sirtuin protein may be administered with one or more
of the following compounds: nicotinamide (NAM), suramin; NF023 (a
G-protein antagonist); NF279 (a purinergic receptor antagonist);
Trolox (6-hydroxy-2,5,7,8,tetramethylchroman-2-carboxylic acid);
(-)-epigallocatechin (hydroxy on sites 3,5,7,3',4',5');
(-)-epigallocatechin gallate (Hydroxy sites 5,7,3',4',5' and
gallate ester on 3); cyanidin chloride
(3,5,7,3',4'-pentahydroxyflavylium chloride); delphinidin chloride
(3,5,7,3',4',5'-hexahydroxyflavylium chloride); myricetin
(cannabiscetin; 3,5,7,3',4',5'-hexahydroxyflavone);
3,7,3',4',5'-pentahydroxyflavone; gossypetin
(3,5,7,8,3',4'-hexahydroxyflavone), sirtinol; and splitomicin. In
yet another embodiment, one or more sirtuin-modulating compounds
may be administered with one or more therapeutic agents for the
treatment or prevention of various diseases, including, for
example, cancer, diabetes, neurodegenerative diseases,
cardiovascular disease, blood clotting, inflammation, flushing,
obesity, ageing, stress, etc. In various embodiments, combination
therapies comprising a sirtuin-modulating compound may refer to (1)
pharmaceutical compositions that comprise one or more
sirtuin-modulating compounds in combination with one or more
therapeutic agents (e.g., one or more therapeutic agents described
herein); and (2) co-administration of one or more
sirtuin-modulating compounds with one or more therapeutic agents
wherein the sirtuin-modulating compound and therapeutic agent have
not been formulated in the same compositions (but may be present
within the same kit or package, such as a blister pack or other
multi-chamber package; connected, separately sealed containers
(e.g., foil pouches) that can be separated by the user; or a kit
where the sirtuin modulating compound(s) and other therapeutic
agent(s) are in separate vessels). When using separate
formulations, the sirtuin-modulating compound may be administered
at the same, intermittent, staggered, prior to, subsequent to, or
combinations thereof, with the administration of another
therapeutic agent.
[0130] In certain embodiments, methods for reducing, preventing or
treating diseases or disorders using a sirtuin-modulating compound
may also comprise increasing the protein level of a sirtuin, such
as human SIRT1, SIRT2 and/or SIRT3, or homologs thereof. Increasing
protein levels can be achieved by introducing into a cell one or
more copies of a nucleic acid that encodes a sirtuin. For example,
the level of a sirtuin can be increased in a mammalian cell by
introducing into the mammalian cell a nucleic acid encoding the
sirtuin, e.g., increasing the level of SIRT1 by introducing a
nucleic acid encoding the amino acid sequence set forth in GenBank
Accession No. NP.sub.--036370 and/or increasing the level of SIRT3
by introducing a nucleic acid encoding the amino acid sequence set
forth in GenBank Accession No. AAH01042.
[0131] A nucleic acid that is introduced into a cell to increase
the protein level of a sirtuin may encode a protein that is at
least about 80%, 85%, 90%, 95%, 98%, or 99% identical to the
sequence of a sirtuin, e.g., SIRT1 and/or SIRT3 protein. For
example, the nucleic acid encoding the protein may be at least
about 80%, 85%, 90%, 95%, 98%, or 99% identical to a nucleic acid
encoding a SIRT1 (e.g. GenBank Accession No. NM.sub.--012238)
and/or SIRT3 (e.g., GenBank Accession No. BC001042) protein. The
nucleic acid may also be a nucleic acid that hybridizes, preferably
under stringent hybridization conditions, to a nucleic acid
encoding a wild-type sirtuin, e.g., SIRT1 and/or SIRT3 protein.
Stringent hybridization conditions may include hybridization and a
wash in 0.2.times.SSC at 65.degree. C. When using a nucleic acid
that encodes a protein that is different from a wild-type sirtuin
protein, such as a protein that is a fragment of a wild-type
sirtuin, the protein is preferably biologically active, e.g., is
capable of deacetylation. It is only necessary to express in a cell
a portion of the sirtuin that is biologically active. For example,
a protein that differs from wild-type SIRT1 having GenBank
Accession No. NP.sub.--036370, preferably contains the core
structure thereof. The core structure sometimes refers to amino
acids 62-293 of GenBank Accession No. NP.sub.--036370, which are
encoded by nucleotides 237 to 932 of GenBank Accession No.
NM.sub.--012238, which encompasses the NAD binding as well as the
substrate binding domains. The core domain of SIRT1 may also refer
to about amino acids 261 to 447 of GenBank Accession No.
NP.sub.--036370, which are encoded by nucleotides 834 to 1394 of
GenBank Accession No. NM.sub.--012238; to about amino acids 242 to
493 of GenBank Accession No. NP.sub.--036370, which are encoded by
nucleotides 777 to 1532 of GenBank Accession No. NM.sub.--012238;
or to about amino acids 254 to 495 of GenBank Accession No.
NP.sub.--036370, which are encoded by nucleotides 813 to 1538 of
GenBank Accession No. NM.sub.--012238. Whether a protein retains a
biological function, e.g., deacetylation capabilities, can be
determined according to methods known in the art.
[0132] In certain embodiments, methods for reducing, preventing or
treating diseases or disorders using a sirtuin-modulating compound
may also comprise decreasing the protein level of a sirtuin, such
as human SIRT1, SIRT2 and/or SIRT3, or homologs thereof. Decreasing
a sirtuin protein level can be achieved according to methods known
in the art. For example, an siRNA, an antisense nucleic acid, or a
ribozyme targeted to the sirtuin can be expressed in the cell. A
dominant negative sirtuin mutant, e.g., a mutant that is not
capable of deacetylating, may also be used. For example, mutant
H363Y of SIRT1, described, e.g., in Luo et al. (2001) Cell 107:137
can be used. Alternatively, agents that inhibit transcription can
be used.
[0133] Methods for modulating sirtuin protein levels also include
methods for modulating the transcription of genes encoding
sirtuins, methods for stabilizing/destabilizing the corresponding
mRNAs, and other methods known in the art.
Aging/Stress
[0134] In one embodiment, the invention provides a method extending
the lifespan of a cell, extending the proliferative capacity of a
cell, slowing aging of a cell, promoting the survival of a cell,
delaying cellular senescence in a cell, mimicking the effects of
calorie restriction, increasing the resistance of a cell to stress,
or preventing apoptosis of a cell, by contacting the cell with a
sirtuin-modulating compound of the invention that increases the
level and/or activity of a sirtuin protein. In an exemplary
embodiment, the methods comprise contacting the cell with a
sirtuin-activating compound.
[0135] The methods described herein may be used to increase the
amount of time that cells, particularly primary cells (i.e., cells
obtained from an organism, e.g., a human), may be kept alive in a
cell culture. Embryonic stem (ES) cells and pluripotent cells, and
cells differentiated therefrom, may also be treated with a
sirtuin-modulating compound that increases the level and/or
activity of a sirtuin protein to keep the cells, or progeny
thereof, in culture for longer periods of time. Such cells can also
be used for transplantation into a subject, e.g., after ex vivo
modification.
[0136] In one embodiment, cells that are intended to be preserved
for long periods of time may be treated with a sirtuin-modulating
compound that increases the level and/or activity of a sirtuin
protein. The cells may be in suspension (e.g., blood cells, serum,
biological growth media, etc.) or in tissues or organs. For
example, blood collected from an individual for purposes of
transfusion may be treated with a sirtuin-modulating compound that
increases the level and/or activity of a sirtuin protein to
preserve the blood cells for longer periods of time. Additionally,
blood to be used for forensic purposes may also be preserved using
a sirtuin-modulating compound that increases the level and/or
activity of a sirtuin protein. Other cells that may be treated to
extend their lifespan or protect against apoptosis include cells
for consumption, e.g., cells from non-human mammals (such as meat)
or plant cells (such as vegetables).
[0137] Sirtuin-modulating compounds that increase the level and/or
activity of a sirtuin protein may also be applied during
developmental and growth phases in mammals, plants, insects or
microorganisms, in order to, e.g., alter, retard or accelerate the
developmental and/or growth process.
[0138] In another embodiment, sirtuin-modulating compounds that
increase the level and/or activity of a sirtuin protein may be used
to treat cells useful for transplantation or cell therapy,
including, for example, solid tissue grafts, organ transplants,
cell suspensions, stem cells, bone marrow cells, etc. The cells or
tissue may be an autograft, an allograft, a syngraft or a
xenograft. The cells or tissue may be treated with the
sirtuin-modulating compound prior to administration/implantation,
concurrently with administration/implantation, and/or post
administration/implantation into a subject. The cells or tissue may
be treated prior to removal of the cells from the donor individual,
ex vivo after removal of the cells or tissue from the donor
individual, or post implantation into the recipient. For example,
the donor or recipient individual may be treated systemically with
a sirtuin-modulating compound or may have a subset of cells/tissue
treated locally with a sirtuin-modulating compound that increases
the level and/or activity of a sirtuin protein. In certain
embodiments, the cells or tissue (or donor/recipient individuals)
may additionally be treated with another therapeutic agent useful
for prolonging graft survival, such as, for example, an
immunosuppressive agent, a cytokine, an angiogenic factor, etc.
[0139] In yet other embodiments, cells may be treated with a
sirtuin-modulating compound that increases the level and/or
activity of a sirtuin protein in vivo, e.g., to increase their
lifespan or prevent apoptosis. For example, skin can be protected
from aging (e.g., developing wrinkles, loss of elasticity, etc.) by
treating skin or epithelial cells with a sirtuin-modulating
compound that increases the level and/or activity of a sirtuin
protein. In an exemplary embodiment, skin is contacted with a
pharmaceutical or cosmetic composition comprising a
sirtuin-modulating compound that increases the level and/or
activity of a sirtuin protein. Exemplary skin afflictions or skin
conditions that may be treated in accordance with the methods
described herein include disorders or diseases associated with or
caused by inflammation, sun damage or natural aging. For example,
the compositions find utility in the prevention or treatment of
contact dermatitis (including irritant contact dermatitis and
allergic contact dermatitis), atopic dermatitis (also known as
allergic eczema), actinic keratosis, keratinization disorders
(including eczema), epidermolysis bullosa diseases (including
pemphigus), exfoliative dermatitis, seborrheic dermatitis,
erythemas (including erythema multiforme and erythema nodosum),
damage caused by the sun or other light sources, discoid lupus
erythematosus, dermatomyositis, psoriasis, skin cancer and the
effects of natural aging. In another embodiment, sirtuin-modulating
compounds that increase the level and/or activity of a sirtuin
protein may be used for the treatment of wounds and/or burns to
promote healing, including, for example, first-, second- or
third-degree burns and/or thermal, chemical or electrical burns.
The formulations may be administered topically, to the skin or
mucosal tissue.
[0140] Topical formulations comprising one or more
sirtuin-modulating compounds that increase the level and/or
activity of a sirtuin protein may also be used as preventive, e.g.,
chemopreventive, compositions. When used in a chemopreventive
method, susceptible skin is treated prior to any visible condition
in a particular individual.
[0141] Sirtuin-modulating compounds may be delivered locally or
systemically to a subject. In one embodiment, a sirtuin-modulating
compound is delivered locally to a tissue or organ of a subject by
injection, topical formulation, etc.
[0142] In another embodiment, a sirtuin-modulating compound that
increases the level and/or activity of a sirtuin protein may be
used for treating or preventing a disease or condition induced or
exacerbated by cellular senescence in a subject; methods for
decreasing the rate of senescence of a subject, e.g., after onset
of senescence; methods for extending the lifespan of a subject;
methods for treating or preventing a disease or condition relating
to lifespan; methods for treating or preventing a disease or
condition relating to the proliferative capacity of cells; and
methods for treating or preventing a disease or condition resulting
from cell damage or death. In certain embodiments, the method does
not act by decreasing the rate of occurrence of diseases that
shorten the lifespan of a subject. In certain embodiments, a method
does not act by reducing the lethality caused by a disease, such as
cancer.
[0143] In yet another embodiment, a sirtuin-modulating compound
that increases the level and/or activity of a sirtuin protein may
be administered to a subject in order to generally increase the
lifespan of its cells and to protect its cells against stress
and/or against apoptosis. It is believed that treating a subject
with a compound described herein is similar to subjecting the
subject to hormesis, i.e., mild stress that is beneficial to
organisms and may extend their lifespan.
[0144] Sirtuin-modulating compounds that increase the level and/or
activity of a sirtuin protein may be administered to a subject to
prevent aging and aging-related consequences or diseases, such as
stroke, heart disease, heart failure, arthritis, high blood
pressure, and Alzheimer's disease. Other conditions that can be
treated include ocular disorders, e.g., associated with the aging
of the eye, such as cataracts, glaucoma, and macular degeneration.
Sirtuin-modulating compounds that increase the level and/or
activity of a sirtuin protein can also be administered to subjects
for treatment of diseases, e.g., chronic diseases, associated with
cell death, in order to protect the cells from cell death.
Exemplary diseases include those associated with neural cell death,
neuronal dysfunction, or muscular cell death or dysfunction, such
as Parkinson's disease, Alzheimer's disease, multiple sclerosis,
amniotropic lateral sclerosis, and muscular dystrophy; AIDS;
fulminant hepatitis; diseases linked to degeneration of the brain,
such as Creutzfeldt-Jakob disease, retinitis pigmentosa and
cerebellar degeneration; myelodysplasia such as aplastic anemia;
ischemic diseases such as myocardial infarction and stroke; hepatic
diseases such as alcoholic hepatitis, hepatitis B and hepatitis C;
joint-diseases such as osteoarthritis; atherosclerosis; alopecia;
damage to the skin due to UV light; lichen planus; atrophy of the
skin; cataract; and graft rejections. Cell death can also be caused
by surgery, drug therapy, chemical exposure or radiation
exposure.
[0145] Sirtuin-modulating compounds that increase the level and/or
activity of a sirtuin protein can also be administered to a subject
suffering from an acute disease, e.g., damage to an organ or
tissue, e.g., a subject suffering from stroke or myocardial
infarction or a subject suffering from a spinal cord injury.
Sirtuin-modulating compounds that increase the level and/or
activity of a sirtuin protein may also be used to repair an
alcoholic's liver.
Cardiovascular Disease
[0146] In another embodiment, the invention provides a method for
treating and/or preventing a cardiovascular disease by
administering to a subject in need thereof a sirtuin-modulating
compound that increases the level and/or activity of a sirtuin
protein.
[0147] Cardiovascular diseases that can be treated or prevented
using the sirtuin-modulating compounds that increase the level
and/or activity of a sirtuin protein include cardiomyopathy or
myocarditis; such as idiopathic cardiomyopathy, metabolic
cardiomyopathy, alcoholic cardiomyopathy, drug-induced
cardiomyopathy, ischemic cardiomyopathy, and hypertensive
cardiomyopathy. Also treatable or preventable using compounds and
methods described herein are atheromatous disorders of the major
blood vessels (macrovascular disease) such as the aorta, the
coronary arteries, the carotid arteries, the cerebrovascular
arteries, the renal arteries, the iliac arteries, the femoral
arteries, and the popliteal arteries. Other vascular diseases that
can be treated or prevented include those related to platelet
aggregation, the retinal arterioles, the glomerular arterioles, the
vasa nervorum, cardiac arterioles, and associated capillary beds of
the eye, the kidney, the heart, and the central and peripheral
nervous systems. The sirtuin-modulating compounds that increase the
level and/or activity of a sirtuin protein may also be used for
increasing HDL levels in plasma of an individual.
[0148] Yet other disorders that may be treated with
sirtuin-modulating compounds that increase the level and/or
activity of a sirtuin protein include restenosis, e.g., following
coronary intervention, and disorders relating to an abnormal level
of high density and low density cholesterol.
[0149] In one embodiment, a sirtuin-modulating compound that
increases the level and/or activity of a sirtuin protein may be
administered as part of a combination therapeutic with another
cardiovascular agent. In one embodiment, a sirtuin-modulating
compound that increases the level and/or activity of a sirtuin
protein may be administered as part of a combination therapeutic
with an anti-arrhythmia agent. In another embodiment, a
sirtuin-modulating compound that increases the level and/or
activity of a sirtuin protein may be administered as part of a
combination therapeutic with another cardiovascular agent.
Cell Death/Cancer
[0150] Sirtuin-modulating compounds that increase the level and/or
activity of a sirtuin protein may be administered to subjects who
have recently received or are likely to receive a dose of radiation
or toxin. In one embodiment, the dose of radiation or toxin is
received as part of a work-related or medical procedure, e.g.,
administered as a prophylactic measure. In another embodiment, the
radiation or toxin exposure is received unintentionally. In such a
case, the compound is preferably administered as soon as possible
after the exposure to inhibit apoptosis and the subsequent
development of acute radiation syndrome.
[0151] Sirtuin-modulating compounds may also be used for treating
and/or preventing cancer. In certain embodiments,
sirtuin-modulating compounds that increase the level and/or
activity of a sirtuin protein may be used for treating and/or
preventing cancer. Calorie restriction has been linked to a
reduction in the incidence of age-related disorders including
cancer. Accordingly, an increase in the level and/or activity of a
sirtuin protein may be useful for treating and/or preventing the
incidence of age-related disorders, such as, for example, cancer.
Exemplary cancers that may be treated using a sirtuin-modulating
compound are those of the brain and kidney; hormone-dependent
cancers including breast, prostate, testicular, and ovarian
cancers; lymphomas, and leukemias. In cancers associated with solid
tumors, a modulating compound may be administered directly into the
tumor. Cancer of blood cells, e.g., leukemia, can be treated by
administering a modulating compound into the blood stream or into
the bone marrow. Benign cell growth, e.g., warts, can also be
treated. Other diseases that can be treated include autoimmune
diseases, e.g., systemic lupus erythematosus, scleroderma, and
arthritis, in which autoimmune cells should be removed. Viral
infections such as herpes, HIV, adenovirus, and HTLV-1 associated
malignant and benign disorders can also be treated by
administration of sirtuin-modulating compound. Alternatively, cells
can be obtained from a subject, treated ex vivo to remove certain
undesirable cells, e.g., cancer cells, and administered back to the
same or a different subject.
[0152] Chemotherapeutic agents may be co-administered with
modulating compounds described herein as having anti-cancer
activity, e.g., compounds that induce apoptosis, compounds that
reduce lifespan or compounds that render cells sensitive to stress.
Chemotherapeutic agents may be used by themselves with a
sirtuin-modulating compound described herein as inducing cell death
or reducing lifespan or increasing sensitivity to stress and/or in
combination with other chemotherapeutics agents. In addition to
conventional chemotherapeutics, the sirtuin-modulating compounds
described herein may also be used with antisense RNA, RNAi or other
polynucleotides to inhibit the expression of the cellular
components that contribute to unwanted cellular proliferation.
[0153] Combination therapies comprising sirtuin-modulating
compounds and a conventional chemotherapeutic agent may be
advantageous over combination therapies known in the art because
the combination allows the conventional chemotherapeutic agent to
exert greater effect at lower dosage. In a preferred embodiment,
the effective dose (ED.sub.50) for a chemotherapeutic agent, or
combination of conventional chemotherapeutic agents, when used in
combination with a sirtuin-modulating compound is at least 2 fold
less than the ED.sub.50 for the chemotherapeutic agent alone, and
even more preferably at 5 fold, 10 fold or even 25 fold less.
Conversely, the therapeutic index (TI) for such chemotherapeutic
agent or combination of such chemotherapeutic agent when used in
combination with a sirtuin-modulating compound described herein can
be at least 2 fold greater than the TI for conventional
chemotherapeutic regimen alone, and even more preferably at 5 fold,
10 fold or even 25 fold greater.
Neuronal Diseases/Disorders
[0154] In certain aspects, sirtuin-modulating compounds that
increase the level and/or activity of a sirtuin protein can be used
to treat patients suffering from neurodegenerative diseases, and
traumatic or mechanical injury to the central nervous system (CNS),
spinal cord or peripheral nervous system (PNS). Neurodegenerative
disease typically involves reductions in the mass and volume of the
human brain, which may be due to the atrophy and/or death of brain
cells, which are far more profound than those in a healthy person
that are attributable to aging. Neurodegenerative diseases can
evolve gradually, after a long period of normal brain function, due
to progressive degeneration (e.g., nerve cell dysfunction and
death) of specific brain regions. Alternatively, neurodegenerative
diseases can have a quick onset, such as those associated with
trauma or toxins. The actual onset of brain degeneration may
precede clinical expression by many years. Examples of
neurodegenerative diseases include, but are not limited to,
Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's
disease (HD), amyotrophic lateral sclerosis (ALS; Lou Gehrig's
disease), diffuse Lewy body disease, chorea-acanthocytosis, primary
lateral sclerosis, ocular diseases (ocular neuritis),
chemotherapy-induced neuropathies (e.g., from vincristine,
paclitaxel, bortezomib), diabetes-induced neuropathies and
Friedreich's ataxia. Sirtuin-modulating compounds that increase the
level and/or activity of a sirtuin protein can be used to treat
these disorders and others as described below.
[0155] AD is a CNS disorder that results in memory loss, unusual
behavior, personality changes, and a decline in thinking abilities.
These losses are related to the death of specific types of brain
cells and the breakdown of connections and their supporting network
(e.g. glial cells) between them. The earliest symptoms include loss
of recent memory, faulty judgment, and changes in personality. PD
is a CNS disorder that results in uncontrolled body movements,
rigidity, tremor, and dyskinesia, and is associated with the death
of brain cells in an area of the brain that produces dopamine. ALS
(motor neuron disease) is a CNS disorder that attacks the motor
neurons, components of the CNS that connect the brain to the
skeletal muscles.
[0156] HD is another neurodegenerative disease that causes
uncontrolled movements, loss of intellectual faculties, and
emotional disturbance. Tay-Sachs disease and Sandhoff disease are
glycolipid storage diseases where GM2 ganglioside and related
glycolipid substrates for .beta.-hexosaminidase accumulate in the
nervous system and trigger acute neurodegeneration.
[0157] It is well-known that apoptosis plays a role in AIDS
pathogenesis in the immune system. However, HIV-1 also induces
neurological disease, which can be treated with sirtuin-modulating
compounds of the invention.
[0158] Neuronal loss is also a salient feature of prion diseases,
such as Creutzfeldt-Jakob disease in human, BSE in cattle (mad cow
disease), Scrapie Disease in sheep and goats, and feline spongiform
encephalopathy (FSE) in cats. Sirtuin-modulating compounds that
increase the level and/or activity of a sirtuin protein may be
useful for treating or preventing neuronal loss due to these prior
diseases.
[0159] In another embodiment, a sirtuin-modulating compound that
increases the level and/or activity of a sirtuin protein may be
used to treat or prevent any disease or disorder involving
axonopathy. Distal axonopathy is a type of peripheral neuropathy
that results from some metabolic or toxic derangement of peripheral
nervous system (PNS) neurons. It is the most common response of
nerves to metabolic or toxic disturbances, and as such may be
caused by metabolic diseases such as diabetes, renal failure,
deficiency syndromes such as malnutrition and alcoholism, or the
effects of toxins or drugs. Those with distal axonopathies usually
present with symmetrical glove-stocking sensori-motor disturbances.
Deep tendon reflexes and autonomic nervous system (ANS) functions
are also lost or diminished in affected areas.
[0160] Diabetic neuropathies are neuropathic disorders that are
associated with diabetes mellitus. Relatively common conditions
which may be associated with diabetic neuropathy include third
nerve palsy; mononeuropathy; mononeuritis multiplex; diabetic
amyotrophy; a painful polyneuropathy; autonomic neuropathy; and
thoracoabdominal neuropathy.
[0161] Peripheral neuropathy is the medical term for damage to
nerves of the peripheral nervous system, which may be caused either
by diseases of the nerve or from the side-effects of systemic
illness. Major causes of peripheral neuropathy include seizures,
nutritional deficiencies, and HIV, though diabetes is the most
likely cause.
[0162] In an exemplary embodiment, a sirtuin-modulating compound
that increases the level and/or activity of a sirtuin protein may
be used to treat or prevent multiple sclerosis (MS), including
relapsing MS and monosymptomatic MS, and other demyelinating
conditions, such as, for example, chromic inflammatory
demyelinating polyneuropathy (CIDP), or symptoms associated
therewith.
[0163] In yet another embodiment, a sirtuin-modulating compound
that increases the level and/or activity of a sirtuin protein may
be used to treat trauma to the nerves, including, trauma due to
disease, injury (including surgical intervention), or environmental
trauma (e.g., neurotoxins, alcoholism, etc.).
[0164] Sirtuin-modulating compounds that increase the level and/or
activity of a sirtuin protein may also be useful to prevent, treat,
and alleviate symptoms of various PNS disorders. The term
"peripheral neuropathy" encompasses a wide range of disorders in
which the nerves outside of the brain and spinal cord--peripheral
nerves--have been damaged. Peripheral neuropathy may also be
referred to as peripheral neuritis, or if many nerves are involved,
the terms polyneuropathy or polyneuritis may be used.
[0165] PNS diseases treatable with sirtuin-modulating compounds
that increase the level and/or activity of a sirtuin protein
include: diabetes, leprosy, Charcot-Marie-Tooth disease,
Guillain-Barre syndrome and Brachial Plexus Neuropathies (diseases
of the cervical and first thoracic roots, nerve trunks, cords, and
peripheral nerve components of the brachial plexus.
[0166] In another embodiment, a sirtuin activating compound may be
used to treat or prevent a polyglutamine disease. Exemplary
polyglutamine diseases include Spinobulbar muscular atrophy
(Kennedy disease), Huntington's Disease (HD),
Dentatorubral-pallidoluysian atrophy (Haw River syndrome),
Spinocerebellar ataxia type 1, Spinocerebellar ataxia type 2,
Spinocerebellar ataxia type 3 (Machado-Joseph disease),
Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7, and
Spinocerebellar ataxia type 17.
[0167] In certain embodiments, the invention provides a method to
treat a central nervous system cell to prevent damage in response
to a decrease in blood flow to the cell. Typically the severity of
damage that may be prevented will depend in large part on the
degree of reduction in blood flow to the cell and the duration of
the reduction. In one embodiment, apoptotic or necrotic cell death
may be prevented. In still a further embodiment, ischemic-mediated
damage, such as cytoxic edema or central nervous system tissue
anoxemia, may be prevented. In each embodiment, the central nervous
system cell may be a spinal cell or a brain cell.
[0168] Another aspect encompasses administrating a sirtuin
activating compound to a subject to treat a central nervous system
ischemic condition. A number of central nervous system ischemic
conditions may be treated by the sirtuin activating compounds
described herein. In one embodiment, the ischemic condition is a
stroke that results in any type of ischemic central nervous system
damage, such as apoptotic or necrotic cell death, cytoxic edema or
central nervous system tissue anoxia. The stroke may impact any
area of the brain or be caused by any etiology commonly known to
result in the occurrence of a stroke.
[0169] In one alternative of this embodiment, the stroke is a brain
stem stroke. In another alternative of this embodiment, the stroke
is a cerebellar stroke. In still another embodiment, the stroke is
an embolic stroke. In yet another alternative, the stroke may be a
hemorrhagic stroke. In a further embodiment, the stroke is a
thrombotic stroke.
[0170] In yet another aspect, a sirtuin activating compound may be
administered to reduce infarct size of the ischemic core following
a central nervous system ischemic condition. Moreover, a sirtuin
activating compound may also be beneficially administered to reduce
the size of the ischemic penumbra or transitional zone following a
central nervous system ischemic condition.
[0171] In one embodiment, a combination drug regimen may include
drugs or compounds for the treatment or prevention of
neurodegenerative disorders or secondary conditions associated with
these conditions. Thus, a combination drug regimen may include one
or more sirtuin activators and one or more anti-neurodegeneration
agents.
Blood Coagulation Disorders
[0172] In other aspects, sirtuin-modulating compounds that increase
the level and/or activity of a sirtuin protein can be used to treat
or prevent blood coagulation disorders (or hemostatic disorders).
As used interchangeably herein, the terms "hemostasis", "blood
coagulation," and "blood clotting" refer to the control of
bleeding, including the physiological properties of
vasoconstriction and coagulation. Blood coagulation assists in
maintaining the integrity of mammalian circulation after injury,
inflammation, disease, congenital defect, dysfunction or other
disruption. Further, the formation of blood clots does not only
limit bleeding in case of an injury (hemostasis), but may lead to
serious organ damage and death in the context of atherosclerotic
diseases by occlusion of an important artery or vein. Thrombosis is
thus blood clot formation at the wrong time and place.
[0173] Accordingly, the present invention provides anticoagulation
and antithrombotic treatments aiming at inhibiting the formation of
blood clots in order to prevent or treat blood coagulation
disorders, such as myocardial infarction, stroke, loss of a limb by
peripheral artery disease or pulmonary embolism.
[0174] As used interchangeably herein, "modulating or modulation of
hemostasis" and "regulating or regulation of hemostasis" includes
the induction (e.g., stimulation or increase) of hemostasis, as
well as the inhibition (e.g., reduction or decrease) of
hemostasis.
[0175] In one aspect, the invention provides a method for reducing
or inhibiting hemostasis in a subject by administering a
sirtuin-modulating compound that increases the level and/or
activity of a sirtuin protein. The compositions and methods
disclosed herein are useful for the treatment or prevention of
thrombotic disorders. As used herein, the term "thrombotic
disorder" includes any disorder or condition characterized by
excessive or unwanted coagulation or hemostatic activity, or a
hypercoagulable state. Thrombotic disorders include diseases or
disorders involving platelet adhesion and thrombus formation, and
may manifest as an increased propensity to form thromboses, e.g.,
an increased number of thromboses, thrombosis at an early age, a
familial tendency towards thrombosis, and thrombosis at unusual
sites.
[0176] In another embodiment, a combination drug regimen may
include drugs or compounds for the treatment or prevention of blood
coagulation disorders or secondary conditions associated with these
conditions. Thus, a combination drug regimen may include one or
more sirtuin-modulating compounds that increase the level and/or
activity of a sirtuin protein and one or more anti-coagulation or
anti-thrombosis agents.
Weight Control
[0177] In another aspect, sirtuin-modulating compounds that
increase the level and/or activity of a sirtuin protein may be used
for treating or preventing weight gain or obesity in a subject. For
example, sirtuin-modulating compounds that increase the level
and/or activity of a sirtuin protein may be used, for example, to
treat or prevent hereditary obesity, dietary obesity, hormone
related obesity, obesity related to the administration of
medication, to reduce the weight of a subject, or to reduce or
prevent weight gain in a subject. A subject in need of such a
treatment may be a subject who is obese, likely to become obese,
overweight, or likely to become overweight. Subjects who are likely
to become obese or overweight can be identified, for example, based
on family history, genetics, diet, activity level, medication
intake, or various combinations thereof.
[0178] In yet other embodiments, sirtuin-modulating compounds that
increase the level and/or activity of a sirtuin protein may be
administered to subjects suffering from a variety of other diseases
and conditions that may be treated or prevented by promoting weight
loss in the subject. Such diseases include, for example, high blood
pressure, hypertension, high blood cholesterol, dyslipidemia, type
2 diabetes, insulin resistance, glucose intolerance,
hyperinsulinemia, coronary heart disease, angina pectoris,
congestive heart failure, stroke, gallstones, cholecystitis and
cholelithiasis, gout, osteoarthritis, obstructive sleep apnea and
respiratory problems, some types of cancer (such as endometrial,
breast, prostate, and colon), complications of pregnancy, poor
female reproductive health (such as menstrual irregularities,
infertility, irregular ovulation), bladder control problems (such
as stress incontinence); uric acid nephrolithiasis; psychological
disorders (such as depression, eating disorders, distorted body
image, and low self esteem). Finally, patients with AIDS can
develop lipodystrophy or insulin resistance in response to
combination therapies for AIDS.
[0179] In another embodiment, sirtuin-modulating compounds that
increase the level and/or activity of a sirtuin protein may be used
for inhibiting adipogenesis or fat cell differentiation, whether in
vitro or in vivo. Such methods may be used for treating or
preventing obesity.
[0180] In other embodiments, sirtuin-modulating compounds that
increase the level and/or activity of a sirtuin protein may be used
for reducing appetite and/or increasing satiety, thereby causing
weight loss or avoidance of weight gain. A subject in need of such
a treatment may be a subject who is overweight, obese or a subject
likely to become overweight or obese. The method may comprise
administering daily or, every other day, or once a week, a dose,
e.g., in the form of a pill, to a subject. The dose may be an
"appetite reducing dose."
[0181] In an exemplary embodiment, sirtuin-modulating compounds
that increase the level and/or activity of a sirtuin protein may be
administered as a combination therapy for treating or preventing
weight gain or obesity. For example, one or more sirtuin-modulating
compounds that increase the level and/or activity of a sirtuin
protein may be administered in combination with one or more
anti-obesity agents.
[0182] In another embodiment, sirtuin-modulating compounds that
increase the level and/or activity of a sirtuin protein may be
administered to reduce drug-induced weight gain. For example, a
sirtuin-modulating compound that increases the level and/or
activity of a sirtuin protein may be administered as a combination
therapy with medications that may stimulate appetite or cause
weight gain, in particular, weight gain due to factors other than
water retention.
Metabolic Disorders/Diabetes
[0183] In another aspect, sirtuin-modulating compounds that
increase the level and/or activity of a sirtuin protein may be used
for treating or preventing a metabolic disorder, such as
insulin-resistance, a pre-diabetic state, type II diabetes, and/or
complications thereof. Administration of a sirtuin-modulating
compounds that increases the level and/or activity of a sirtuin
protein may increase insulin sensitivity and/or decrease insulin
levels in a subject. A subject in need of such a treatment may be a
subject who has insulin resistance or other precursor symptom of
type II diabetes, who has type II diabetes, or who is likely to
develop any of these conditions. For example, the subject may be a
subject having insulin resistance, e.g., having high circulating
levels of insulin and/or associated conditions, such as
hyperlipidemia, dyslipogenesis, hypercholesterolemia, impaired
glucose tolerance, high blood glucose sugar level, other
manifestations of syndrome X, hypertension, atherosclerosis and
lipodystrophy.
[0184] In an exemplary embodiment, sirtuin-modulating compounds
that increase the level and/or activity of a sirtuin protein may be
administered as a combination therapy for treating or preventing a
metabolic disorder. For example, one or more sirtuin-modulating
compounds that increase the level and/or activity of a sirtuin
protein may be administered in combination with one or more
anti-diabetic agents.
Inflammatory Diseases
[0185] In other aspects, sirtuin-modulating compounds that increase
the level and/or activity of a sirtuin protein can be used to treat
or prevent a disease or disorder associated with inflammation.
Sirtuin-modulating compounds that increase the level and/or
activity of a sirtuin protein may be administered prior to the
onset of, at, or after the initiation of inflammation. When used
prophylactically, the compounds are preferably provided in advance
of any inflammatory response or symptom. Administration of the
compounds may prevent or attenuate inflammatory responses or
symptoms.
[0186] In another embodiment, sirtuin-modulating compounds that
increase the level and/or activity of a sirtuin protein may be used
to treat or prevent allergies and respiratory conditions, including
asthma, bronchitis, pulmonary fibrosis, allergic rhinitis, oxygen
toxicity, emphysema, chronic bronchitis, acute respiratory distress
syndrome, and any chronic obstructive pulmonary disease (COPD). The
compounds may be used to treat chronic hepatitis infection,
including hepatitis B and hepatitis C.
[0187] Additionally, sirtuin-modulating compounds that increase the
level and/or activity of a sirtuin protein may be used to treat
autoimmune diseases and/or inflammation associated with autoimmune
diseases such as arthritis, including rheumatoid arthritis,
psoriatic arthritis, and ankylosing spondylitis, as well as
organ-tissue autoimmune diseases (e.g., Raynaud's syndrome),
ulcerative colitis, Crohns Disease, oral mucositis, scleroderma,
myasthenia gravis, transplant rejection, endotoxin shock, sepsis,
psoriasis, eczema, dermatitis, multiple sclerosis, autoimmune
thyroiditis, uveitis, systemic lupus erythematosis, Addison's
disease, autoimmune polyglandular disease (also known as autoimmune
polyglandular syndrome), and Grave's disease.
[0188] In certain embodiments, one or more sirtuin-modulating
compounds that increase the level and/or activity of a sirtuin
protein may be taken alone or in combination with other compounds
useful for treating or preventing inflammation.
Flushing
[0189] In another aspect, sirtuin-modulating compounds that
increase the level and/or activity of a sirtuin protein may be used
for reducing the incidence or severity of flushing and/or hot
flashes which are symptoms of a disorder. For instance, the subject
method includes the use of sirtuin-modulating compounds that
increase the level and/or activity of a sirtuin protein, alone or
in combination with other agents, for reducing incidence or
severity of flushing and/or hot flashes in cancer patients. In
other embodiments, the method provides for the use of
sirtuin-modulating compounds that increase the level and/or
activity of a sirtuin protein to reduce the incidence or severity
of flushing and/or hot flashes in menopausal and post-menopausal
woman.
[0190] In another aspect, sirtuin-modulating compounds that
increase the level and/or activity of a sirtuin protein may be used
as a therapy for reducing the incidence or severity of flushing
and/or hot flashes which are side-effects of another drug therapy,
e.g., drug-induced flushing. In certain embodiments, a method for
treating and/or preventing drug-induced flushing comprises
administering to a patient in need thereof a formulation comprising
at least one flushing inducing compound and at least one
sirtuin-modulating compound that increases the level and/or
activity of a sirtuin protein. In other embodiments, a method for
treating drug induced flushing comprises separately administering
one or more compounds that induce flushing and one or more
sirtuin-modulating compounds, e.g., wherein the sirtuin-modulating
compound and flushing inducing agent have not been formulated in
the same compositions. When using separate formulations, the
sirtuin-modulating compound may be administered (1) at the same as
administration of the flushing inducing agent, (2) intermittently
with the flushing inducing agent, (3) staggered relative to
administration of the flushing inducing agent, (4) prior to
administration of the flushing inducing agent, (5) subsequent to
administration of the flushing inducing agent, and (6) various
combination thereof. Exemplary flushing inducing agents include,
for example, niacin, raloxifene, antidepressants, anti-psychotics,
chemotherapeutics, calcium channel blockers, and antibiotics.
[0191] In one embodiment, sirtuin-modulating compounds that
increase the level and/or activity of a sirtuin protein may be used
to reduce flushing side effects of a vasodilator or an antilipemic
agent (including anticholesteremic agents and lipotropic agents).
In an exemplary embodiment, a sirtuin-modulating compound that
increases the level and/or activity of a sirtuin protein may be
used to reduce flushing associated with the administration of
niacin.
[0192] In another embodiment, the invention provides a method for
treating and/or preventing hyperlipidemia with reduced flushing
side effects. In another representative embodiment, the method
involves the use of sirtuin-modulating compounds that increase the
level and/or activity of a sirtuin protein to reduce flushing side
effects of raloxifene. In another representative embodiment, the
method involves the use of sirtuin-modulating compounds that
increase the level and/or activity of a sirtuin protein to reduce
flushing side effects of antidepressants or anti-psychotic agent.
For instance, sirtuin-modulating compounds that increase the level
and/or activity of a sirtuin protein can be used in conjunction
(administered separately or together) with a serotonin reuptake
inhibitor, or a 5HT2 receptor antagonist.
[0193] In certain embodiments, sirtuin-modulating compounds that
increase the level and/or activity of a sirtuin protein may be used
as part of a treatment with a serotonin reuptake inhibitor (SRI) to
reduce flushing. In still another representative embodiment,
sirtuin-modulating compounds that increase the level and/or
activity of a sirtuin protein may be used to reduce flushing side
effects of chemotherapeutic agents, such as cyclophosphamide and
tamoxifen.
[0194] In another embodiment, sirtuin-modulating compounds that
increase the level and/or activity of a sirtuin protein may be used
to reduce flushing side effects of calcium channel blockers, such
as amlodipine.
[0195] In another embodiment, sirtuin-modulating compounds that
increase the level and/or activity of a sirtuin protein may be used
to reduce flushing side effects of antibiotics. For example,
sirtuin-modulating compounds that increase the level and/or
activity of a sirtuin protein can be used in combination with
levofloxacin.
Ocular Disorders
[0196] One aspect of the present invention is a method for
inhibiting, reducing or otherwise treating vision impairment by
administering to a patient a therapeutic dosage of sirtuin
modulator selected from a compound disclosed herein, or a
pharmaceutically acceptable salt, prodrug or a metabolic derivative
thereof.
[0197] In certain aspects of the invention, the vision impairment
is caused by damage to the optic nerve or central nervous system.
In particular embodiments, optic nerve damage is caused by high
intraocular pressure, such as that created by glaucoma. In other
particular embodiments, optic nerve damage is caused by swelling of
the nerve, which is often associated with an infection or an immune
(e.g., autoimmune) response such as in optic neuritis.
[0198] In certain aspects of the invention, the vision impairment
is caused by retinal damage. In particular embodiments, retinal
damage is caused by disturbances in blood flow to the eye (e.g.,
arteriosclerosis, vasculitis). In particular embodiments, retinal
damage is caused by disruptor of the macula (e.g., exudative or
non-exudative macular degeneration).
[0199] Exemplary retinal diseases include Exudative Age Related
Macular Degeneration, Nonexudative Age Related Macular
Degeneration, Retinal Electronic Prosthesis and RPE Transplantation
Age Related Macular Degeneration, Acute Multifocal Placoid Pigment
Epitheliopathy, Acute Retinal Necrosis, Best Disease, Branch
Retinal Artery Occlusion, Branch Retinal Vein Occlusion, Cancer
Associated and Related Autoimmune Retinopathies, Central Retinal
Artery Occlusion, Central Retinal Vein Occlusion, Central Serous
Chorioretinopathy, Eales Disease, Epimacular Membrane, Lattice
Degeneration, Macroaneurysm, Diabetic Macular Edema, Irvine-Gass
Macular Edema, Macular Hole, Subretinal Neovascular Membranes,
Diffuse Unilateral Subacute Neuroretinitis, Nonpseudophakic Cystoid
Macular Edema, Presumed Ocular Histoplasmosis Syndrome, Exudative
Retinal Detachment, Postoperative Retinal Detachment, Proliferative
Retinal Detachment, Rhegmatogenous Retinal Detachment, Tractional
Retinal Detachment, Retinitis Pigmentosa, CMV Retinitis,
Retinoblastoma, Retinopathy of Prematurity, Birdshot Retinopathy,
Background Diabetic Retinopathy, Proliferative Diabetic
Retinopathy, Hemoglobinopathies Retinopathy, Purtscher Retinopathy,
Valsalva Retinopathy, Juvenile Retinoschisis, Senile Retinoschisis,
Terson Syndrome and White Dot Syndromes.
[0200] Other exemplary diseases include ocular bacterial infections
(e.g. conjunctivitis, keratitis, tuberculosis, syphilis,
gonorrhea), viral infections (e.g. Ocular Herpes Simplex Virus,
Varicella Zoster Virus, Cytomegalovirus retinitis, Human
Immunodeficiency Virus (HIV)) as well as progressive outer retinal
necrosis secondary to HIV or other HIV-associated and other
immunodeficiency-associated ocular diseases. In addition, ocular
diseases include fungal infections (e.g. Candida choroiditis,
histoplasmosis), protozoal infections (e.g. toxoplasmosis) and
others such as ocular toxocariasis and sarcoidosis.
[0201] One aspect of the invention is a method for inhibiting,
reducing or treating vision impairment in a subject undergoing
treatment with a chemotherapeutic drug (e.g., a neurotoxic drug, a
drug that raises intraocular pressure such as a steroid), by
administering to the subject in need of such treatment a
therapeutic dosage of a sirtuin modulator disclosed herein.
[0202] Another aspect of the invention is a method for inhibiting,
reducing or treating vision impairment in a subject undergoing
surgery, including ocular or other surgeries performed in the prone
position such as spinal cord surgery, by administering to the
subject in need of such treatment a therapeutic dosage of a sirtuin
modulator disclosed herein. Ocular surgeries include cataract,
iridotomy and lens replacements.
[0203] Another aspect of the invention is the treatment, including
inhibition and prophylactic treatment, of age related ocular
diseases include cataracts, dry eye, age-related macular
degeneration (AMD), retinal damage and the like, by administering
to the subject in need of such treatment a therapeutic dosage of a
sirtuin modulator disclosed herein.
[0204] Another aspect of the invention is the prevention or
treatment of damage to the eye caused by stress, chemical insult or
radiation, by administering to the subject in need of such
treatment a therapeutic dosage of a sirtuin modulator disclosed
herein. Radiation or electromagnetic damage to the eye can include
that caused by CRT's or exposure to sunlight or UV.
[0205] In one embodiment, a combination drug regimen may include
drugs or compounds for the treatment or prevention of ocular
disorders or secondary conditions associated with these conditions.
Thus, a combination drug regimen may include one or more sirtuin
activators and one or more therapeutic agents for the treatment of
an ocular disorder.
[0206] In one embodiment, a sirtuin modulator can be administered
in conjunction with a therapy for reducing intraocular pressure. In
another embodiment, a sirtuin modulator can be administered in
conjunction with a therapy for treating and/or preventing glaucoma.
In yet another embodiment, a sirtuin modulator can be administered
in conjunction with a therapy for treating and/or preventing optic
neuritis. In one embodiment, a sirtuin modulator can be
administered in conjunction with a therapy for treating and/or
preventing CMV Retinopathy. In another embodiment, a sirtuin
modulator can be administered in conjunction with a therapy for
treating and/or preventing multiple sclerosis.
Mitochondrial-Associated Diseases and Disorders
[0207] In certain embodiments, the invention provides methods for
treating diseases or disorders that would benefit from increased
mitochondrial activity. The methods involve administering to a
subject in need thereof a therapeutically effective amount of a
sirtuin activating compound. Increased mitochondrial activity
refers to increasing activity of the mitochondria while maintaining
the overall numbers of mitochondria (e.g., mitochondrial mass),
increasing the numbers of mitochondria thereby increasing
mitochondrial activity (e.g., by stimulating mitochondrial
biogenesis), or combinations thereof. In certain embodiments,
diseases and disorders that would benefit from increased
mitochondrial activity include diseases or disorders associated
with mitochondrial dysfunction.
[0208] In certain embodiments, methods for treating diseases or
disorders that would benefit from increased mitochondrial activity
may comprise identifying a subject suffering from a mitochondrial
dysfunction. Methods for diagnosing a mitochondrial dysfunction may
involve molecular genetic, pathologic and/or biochemical analyses.
Diseases and disorders associated with mitochondrial dysfunction
include diseases and disorders in which deficits in mitochondrial
respiratory chain activity contribute to the development of
pathophysiology of such diseases or disorders in a mammal. Diseases
or disorders that would benefit from increased mitochondrial
activity generally include for example, diseases in which free
radical mediated oxidative injury leads to tissue degeneration,
diseases in which cells inappropriately undergo apoptosis, and
diseases in which cells fail to undergo apoptosis.
[0209] In certain embodiments, the invention provides methods for
treating a disease or disorder that would benefit from increased
mitochondrial activity that involves administering to a subject in
need thereof one or more sirtuin activating compounds in
combination with another therapeutic agent such as, for example, an
agent useful for treating mitochondrial dysfunction or an agent
useful for reducing a symptom associated with a disease or disorder
involving mitochondrial dysfunction.
[0210] In exemplary embodiments, the invention provides methods for
treating diseases or disorders that would benefit from increased
mitochondrial activity by administering to a subject a
therapeutically effective amount of a sirtuin activating compound.
Exemplary diseases or disorders include, for example, neuromuscular
disorders (e.g., Friedreich's Ataxia, muscular dystrophy, multiple
sclerosis, etc.), disorders of neuronal instability (e.g., seizure
disorders, migraine, etc.), developmental delay, neurodegenerative
disorders (e.g., Alzheimer's Disease, Parkinson's Disease,
amyotrophic lateral sclerosis, etc.), ischemia, renal tubular
acidosis, age-related neurodegeneration and cognitive decline,
chemotherapy fatigue, age-related or chemotherapy-induced menopause
or irregularities of menstrual cycling or ovulation, mitochondrial
myopathies, mitochondrial damage (e.g., calcium accumulation,
excitotoxicity, nitric oxide exposure, hypoxia, etc.), and
mitochondrial deregulation.
[0211] Muscular dystrophy refers to a family of diseases involving
deterioration of neuromuscular structure and function, often
resulting in atrophy of skeletal muscle and myocardial dysfunction,
such as Duchenne muscular dystrophy. In certain embodiments,
sirtuin activating compounds may be used for reducing the rate of
decline in muscular functional capacities and for improving
muscular functional status in patients with muscular dystrophy.
[0212] In certain embodiments, sirtuin modulating compounds may be
useful for treatment mitochondrial myopathies. Mitochondrial
myopathies range from mild, slowly progressive weakness of the
extraocular muscles to severe, fatal infantile myopathies and
multisystem encephalomyopathies. Some syndromes have been defined,
with some overlap between them. Established syndromes affecting
muscle include progressive external ophthalmoplegia, the
Kearns-Sayre syndrome (with ophthalmoplegia, pigmentary
retinopathy, cardiac conduction defects, cerebellar ataxia, and
sensorineural deafness), the MELAS syndrome (mitochondrial
encephalomyopathy, lactic acidosis, and stroke-like episodes), the
MERFF syndrome (myoclonic epilepsy and ragged red fibers),
limb-girdle distribution weakness, and infantile myopathy (benign
or severe and fatal).
[0213] In certain embodiments, sirtuin activating compounds may be
useful for treating patients suffering from toxic damage to
mitochondria, such as, toxic damage due to calcium accumulation,
excitotoxicity, nitric oxide exposure, drug induced toxic damage,
or hypoxia.
[0214] In certain embodiments, sirtuin activating compounds may be
useful for treating diseases or disorders associated with
mitochondrial deregulation.
Muscle Performance
[0215] In other embodiments, the invention provides methods for
enhancing muscle performance by administering a therapeutically
effective amount of a sirtuin activating compound. For example,
sirtuin activating compounds may be useful for improving physical
endurance (e.g., ability to perform a physical task such as
exercise, physical labor, sports activities, etc), inhibiting or
retarding physical fatigues, enhancing blood oxygen levels,
enhancing energy in healthy individuals, enhance working capacity
and endurance, reducing muscle fatigue, reducing stress, enhancing
cardiac and cardiovascular function, improving sexual ability,
increasing muscle ATP levels, and/or reducing lactic acid in blood.
In certain embodiments, the methods involve administering an amount
of a sirtuin activating compound that increase mitochondrial
activity, increase mitochondrial biogenesis, and/or increase
mitochondrial mass.
[0216] Sports performance refers to the ability of the athlete's
muscles to perform when participating in sports activities.
Enhanced sports performance, strength, speed and endurance are
measured by an increase in muscular contraction strength, increase
in amplitude of muscle contraction, shortening of muscle reaction
time between stimulation and contraction. Athlete refers to an
individual who participates in sports at any level and who seeks to
achieve an improved level of strength, speed and endurance in their
performance, such as, for example, body builders, bicyclists, long
distance runners, short distance runners, etc. Enhanced sports
performance in manifested by the ability to overcome muscle
fatigue, ability to maintain activity for longer periods of time,
and have a more effective workout.
[0217] In the arena of athlete muscle performance, it is desirable
to create conditions that permit competition or training at higher
levels of resistance for a prolonged period of time.
[0218] It is contemplated that the methods of the present invention
will also be effective in the treatment of muscle related
pathological conditions, including acute sarcopenia, for example,
muscle atrophy and/or cachexia associated with burns, bed rest,
limb immobilization, or major thoracic, abdominal, and/or
orthopedic surgery.
[0219] In certain embodiments, the invention provides novel dietary
compositions comprising sirtuin modulators, a method for their
preparation, and a method of using the compositions for improvement
of sports performance. Accordingly, provided are therapeutic
compositions, foods and beverages that have actions of improving
physical endurance and/or inhibiting physical fatigues for those
people involved in broadly-defined exercises including sports
requiring endurance and labors requiring repeated muscle exertions.
Such dietary compositions may additional comprise electrolytes,
caffeine, vitamins, carbohydrates, etc.
Other Uses
[0220] Sirtuin-modulating compounds that increase the level and/or
activity of a sirtuin protein may be used for treating or
preventing viral infections (such as infections by influenza,
herpes or papilloma virus) or as antifungal agents. In certain
embodiments, sirtuin-modulating compounds that increase the level
and/or activity of a sirtuin protein may be administered as part of
a combination drug therapy with another therapeutic agent for the
treatment of viral diseases. In another embodiment,
sirtuin-modulating compounds that increase the level and/or
activity of a sirtuin protein may be administered as part of a
combination drug therapy with another anti-fungal agent.
[0221] Subjects that may be treated as described herein include
eukaryotes, such as mammals, e.g., humans, ovines, bovines,
equines, porcines, canines, felines, non-human primate, mice, and
rats. Cells that may be treated include eukaryotic cells, e.g.,
from a subject described above, or plant cells, yeast cells and
prokaryotic cells, e.g., bacterial cells. For example, modulating
compounds may be administered to farm animals to improve their
ability to withstand farming conditions longer.
[0222] Sirtuin-modulating compounds that increase the level and/or
activity of a sirtuin protein may also be used to increase
lifespan, stress resistance, and resistance to apoptosis in plants.
In one embodiment, a compound is applied to plants, e.g., on a
periodic basis, or to fungi. In another embodiment, plants are
genetically modified to produce a compound. In another embodiment,
plants and fruits are treated with a compound prior to picking and
shipping to increase resistance to damage during shipping. Plant
seeds may also be contacted with compounds described herein, e.g.,
to preserve them.
[0223] In other embodiments, sirtuin-modulating compounds that
increase the level and/or activity of a sirtuin protein may be used
for modulating lifespan in yeast cells. Situations in which it may
be desirable to extend the lifespan of yeast cells include any
process in which yeast is used, e.g., the making of beer, yogurt,
and bakery items, e.g., bread. Use of yeast having an extended
lifespan can result in using less yeast or in having the yeast be
active for longer periods of time. Yeast or other mammalian cells
used for recombinantly producing proteins may also be treated as
described herein.
[0224] Sirtuin-modulating compounds that increase the level and/or
activity of a sirtuin protein may also be used to increase
lifespan, stress resistance and resistance to apoptosis in insects.
In this embodiment, compounds would be applied to useful insects,
e.g., bees and other insects that are involved in pollination of
plants. In a specific embodiment, a compound would be applied to
bees involved in the production of honey. Generally, the methods
described herein may be applied to any organism, e.g., eukaryote,
that may have commercial importance. For example, they can be
applied to fish (aquaculture) and birds (e.g., chicken and
fowl).
[0225] Higher doses of sirtuin-modulating compounds that increase
the level and/or activity of a sirtuin protein may also be used as
a pesticide by interfering with the regulation of silenced genes
and the regulation of apoptosis during development. In this
embodiment, a compound may be applied to plants using a method
known in the art that ensures the compound is bio-available to
insect larvae, and not to plants.
[0226] At least in view of the link between reproduction and
longevity, sirtuin-modulating compounds that increase the level
and/or activity of a sirtuin protein can be applied to affect the
reproduction of organisms such as insects, animals and
microorganisms.
4. Assays
[0227] Yet other methods contemplated herein include screening
methods for identifying compounds or agents that modulate sirtuins.
An agent may be a nucleic acid, such as an aptamer. Assays may be
conducted in a cell based or cell free format. For example, an
assay may comprise incubating (or contacting) a sirtuin with a test
agent under conditions in which a sirtuin can be modulated by an
agent known to modulate the sirtuin, and monitoring or determining
the level of modulation of the sirtuin in the presence of the test
agent relative to the absence of the test agent. The level of
modulation of a sirtuin can be determined by determining its
ability to deacetylate a substrate. Exemplary substrates are
acetylated peptides which can be obtained from BIOMOL (Plymouth
Meeting, Pa.). Preferred substrates include peptides of p53, such
as those comprising an acetylated K382. A particularly preferred
substrate is the Fluor de Lys-SIRT1 (BIOMOL), i.e., the acetylated
peptide Arg-His-Lys-Lys. Other substrates are peptides from human
histones H3 and H4 or an acetylated amino acid. Substrates may be
fluorogenic. The sirtuin may be SIRT1, Sir2, SIRT3, or a portion
thereof. For example, recombinant SIRT1 can be obtained from
BIOMOL. The reaction may be conducted for about 30 minutes and
stopped, e.g., with nicotinamide. The HDAC fluorescent activity
assay/drug discovery kit (AK-500, BIOMOL Research Laboratories) may
be used to determine the level of acetylation. Similar assays are
described in Bitterman et al. (2002) J. Biol. Chem. 277:45099. The
level of modulation of the sirtuin in an assay may be compared to
the level of modulation of the sirtuin in the presence of one or
more (separately or simultaneously) compounds described herein,
which may serve as positive or negative controls. Sirtuins for use
in the assays may be full length sirtuin proteins or portions
thereof. Since it has been shown herein that activating compounds
appear to interact with the N-terminus of SIRT1, proteins for use
in the assays include N-terminal portions of sirtuins, e.g., about
amino acids 1-176 or 1-255 of SIRT1; about amino acids 1-174 or
1-252 of Sir2.
[0228] In one embodiment, a screening assay comprises (i)
contacting a sirtuin with a test agent and an acetylated substrate
under conditions appropriate for the sirtuin to deacetylate the
substrate in the absence of the test agent; and (ii) determining
the level of acetylation of the substrate, wherein a lower level of
acetylation of the substrate in the presence of the test agent
relative to the absence of the test agent indicates that the test
agent stimulates deacetylation by the sirtuin, whereas a higher
level of acetylation of the substrate in the presence of the test
agent relative to the absence of the test agent indicates that the
test agent inhibits deacetylation by the sirtuin.
[0229] Methods for identifying an agent that modulates, e.g.,
stimulates, sirtuins in vivo may comprise (i) contacting a cell
with a test agent and a substrate that is capable of entering a
cell in the presence of an inhibitor of class I and class II HDACs
under conditions appropriate for the sirtuin to deacetylate the
substrate in the absence of the test agent; and (ii) determining
the level of acetylation of the substrate, wherein a lower level of
acetylation of the substrate in the presence of the test agent
relative to the absence of the test agent indicates that the test
agent stimulates deacetylation by the sirtuin, whereas a higher
level of acetylation of the substrate in the presence of the test
agent relative to the absence of the test agent indicates that the
test agent inhibits deacetylation by the sirtuin. A preferred
substrate is an acetylated peptide, which is also preferably
fluorogenic, as further described herein. The method may further
comprise lysing the cells to determine the level of acetylation of
the substrate. Substrates may be added to cells at a concentration
ranging from about 1 .mu.M to about 10 mM, preferably from about 10
.mu.M to 1 mM, even more preferably from about 100 .mu.M to 1 mM,
such as about 200 .mu.M. A preferred substrate is an acetylated
lysine, e.g., .epsilon.-acetyl lysine (Fluor de Lys, FdL) or Fluor
de Lys-SIRT1. A preferred inhibitor of class I and class II HDACs
is trichostatin A (TSA), which may be used at concentrations
ranging from about 0.01 to 100 .mu.M, preferably from about 0.1 to
10 .mu.M, such as 1 .mu.M. Incubation of cells with the test
compound and the substrate may be conducted for about 10 minutes to
5 hours, preferably for about 1-3 hours. Since TSA inhibits all
class I and class II HDACs, and that certain substrates, e.g.,
Fluor de Lys, is a poor substrate for SIRT2 and even less a
substrate for SIRT3-7, such an assay may be used to identify
modulators of SIRT1 in vivo.
5. Pharmaceutical Compositions
[0230] The sirtuin-modulating compounds described herein may be
formulated in a conventional manner using one or more
physiologically or pharmaceutically acceptable carriers or
excipients. For example, sirtuin-modulating compounds and their
pharmaceutically acceptable salts and solvates may be formulated
for administration by, for example, injection (e.g. SubQ, IM, IP),
inhalation or insufflation (either through the mouth or the nose)
or oral, buccal, sublingual, transdermal, nasal, parenteral or
rectal administration. In one embodiment, a sirtuin-modulating
compound may be administered locally, at the site where the target
cells are present, i.e., in a specific tissue, organ, or fluid
(e.g., blood, cerebrospinal fluid, etc.).
[0231] Sirtuin-modulating compounds can be formulated for a variety
of modes of administration, including systemic and topical or
localized administration. Techniques and formulations generally may
be found in Remington's Pharmaceutical Sciences, Meade Publishing
Co., Easton, Pa. For parenteral administration, injection is
preferred, including intramuscular, intravenous, intraperitoneal,
and subcutaneous. For injection, the compounds can be formulated in
liquid solutions, preferably in physiologically compatible buffers
such as Hank's solution or Ringer's solution. In addition, the
compounds may be formulated in solid form and redissolved or
suspended immediately prior to use. Lyophilized forms are also
included.
[0232] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets, lozenges, or capsules
prepared by conventional means with pharmaceutically acceptable
excipients such as binding agents (e.g., pregelatinized maize
starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose);
fillers (e.g., lactose, microcrystalline cellulose or calcium
hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or
silica); disintegrants (e.g., potato starch or sodium starch
glycolate); or wetting agents (e.g., sodium lauryl sulphate). The
tablets may be coated by methods well known in the art. Liquid
preparations for oral administration may take the form of, for
example, solutions, syrups or suspensions, or they may be presented
as a dry product for constitution with water or other suitable
vehicle before use. Such liquid preparations may be prepared by
conventional means with pharmaceutically acceptable additives such
as suspending agents (e.g., sorbitol syrup, cellulose derivatives
or hydrogenated edible fats); emulsifying agents (e.g., lecithin or
acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl
alcohol or fractionated vegetable oils); and preservatives (e.g.,
methyl or propyl-p-hydroxybenzoates or sorbic acid). The
preparations may also contain buffer salts, flavoring, coloring and
sweetening agents as appropriate. Preparations for oral
administration may be suitably formulated to give controlled
release of the active compound.
[0233] For administration by inhalation (e.g., pulmonary delivery),
sirtuin-modulating compounds may be conveniently delivered in the
form of an aerosol spray presentation from pressurized packs or a
nebuliser, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g., gelatin, for use in an inhaler or insufflator
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0234] Sirtuin-modulating compounds may be formulated for
parenteral administration by injection, e.g., by bolus injection or
continuous infusion. Formulations for injection may be presented in
unit dosage form, e.g., in ampoules or in multi-dose containers,
with an added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0235] Sirtuin-modulating compounds may also be formulated in
rectal compositions such as suppositories or retention enemas,
e.g., containing conventional suppository bases such as cocoa
butter or other glycerides.
[0236] In addition to the formulations described previously,
sirtuin-modulating compounds may also be formulated as a depot
preparation. Such long acting formulations may be administered by
implantation (for example subcutaneously or intramuscularly) or by
intramuscular injection. Thus, for example, sirtuin-modulating
compounds may be formulated with suitable polymeric or hydrophobic
materials (for example as an emulsion in an acceptable oil) or ion
exchange resins, or as sparingly soluble derivatives, for example,
as a sparingly soluble salt. Controlled release formula also
includes patches.
[0237] In certain embodiments, the compounds described herein can
be formulated for delivery to the central nervous system (CNS)
(reviewed in Begley, Pharmacology & Therapeutics 104: 29-45
(2004)). Conventional approaches for drug delivery to the CNS
include: neurosurgical strategies (e.g., intracerebral injection or
intracerebroventricular infusion); molecular manipulation of the
agent (e.g., production of a chimeric fusion protein that comprises
a transport peptide that has an affinity for an endothelial cell
surface molecule in combination with an agent that is itself
incapable of crossing the BBB) in an attempt to exploit one of the
endogenous transport pathways of the BBB; pharmacological
strategies designed to increase the lipid solubility of an agent
(e.g., conjugation of water-soluble agents to lipid or cholesterol
carriers); and the transitory disruption of the integrity of the
BBB by hyperosmotic disruption (resulting from the infusion of a
mannitol solution into the carotid artery or the use of a
biologically active agent such as an angiotensin peptide).
[0238] Liposomes are a further drug delivery system which is easily
injectable. Accordingly, in the method of invention the active
compounds can also be administered in the form of a liposome
delivery system. Liposomes are well-known by a person skilled in
the art. Liposomes can be formed from a variety of phospholipids,
such as cholesterol, stearylamine of phosphatidylcholines.
Liposomes being usable for the method of invention encompass all
types of liposomes including, but not limited to, small unilamellar
vesicles, large unilamellar vesicles and multilamellar
vesicles.
Another way to produce a formulation, particularly a solution, of a
sirtuin modulator such as resveratrol or a derivative thereof, is
through the use of cyclodextrin. By cyclodextrin is meant .alpha.-,
.beta.-, or .gamma.-cyclodextrin. Cyclodextrins are described in
detail in Pitha et al., U.S. Pat. No. 4,727,064, which is
incorporated herein by reference. Cyclodextrins are cyclic
oligomers of glucose; these compounds form inclusion complexes with
any drug whose molecule can fit into the lipophile-seeking cavities
of the cyclodextrin molecule.
[0239] Rapidly disintegrating or dissolving dosage forms are useful
for the rapid absorption, particularly buccal and sublingual
absorption, of pharmaceutically active agents. Fast melt dosage
forms are beneficial to patients, such as aged and pediatric
patients, who have difficulty in swallowing typical solid dosage
forms, such as caplets and tablets. Additionally, fast melt dosage
forms circumvent drawbacks associated with, for example, chewable
dosage forms, wherein the length of time an active agent remains in
a patient's mouth plays an important role in determining the amount
of taste masking and the extent to which a patient may object to
throat grittiness of the active agent.
[0240] Pharmaceutical compositions (including cosmetic
preparations) may comprise from about 0.00001 to 100% such as from
0.001 to 10% or from 0.1% to 5% by weight of one or more
sirtuin-modulating compounds described herein. In another
embodiment, the pharmaceutical composition comprises: (i) 0.05 to
1000 mg of the compounds of the invention, or a pharmaceutically
acceptable salt thereof, and (ii) 0.1 to 2 grams of one or more
pharmaceutically acceptable excipients.
[0241] In one embodiment, a sirtuin-modulating compound described
herein, is incorporated into a topical formulation containing a
topical carrier that is generally suited to topical drug
administration and comprising any such material known in the art.
The topical carrier may be selected so as to provide the
composition in the desired form, e.g., as an ointment, lotion,
cream, microemulsion, gel, oil, solution, or the like, and may be
comprised of a material of either naturally occurring or synthetic
origin. It is preferable that the selected carrier not adversely
affect the active agent or other components of the topical
formulation. Examples of suitable topical carriers for use herein
include water, alcohols and other nontoxic organic solvents,
glycerin, mineral oil, silicone, petroleum jelly, lanolin, fatty
acids, vegetable oils, parabens, waxes, and the like.
[0242] Formulations may be colorless, odorless ointments, lotions,
creams, microemulsions and gels.
[0243] Sirtuin-modulating compounds may be incorporated into
ointments, which generally are semisolid preparations which are
typically based on petrolatum or other petroleum derivatives. The
specific ointment base to be used, as will be appreciated by those
skilled in the art, is one that will provide for optimum drug
delivery, and, preferably, will provide for other desired
characteristics as well, e.g., emolliency or the like. As with
other carriers or vehicles, an ointment base should be inert,
stable, nonirritating and nonsensitizing.
[0244] Sirtuin-modulating compounds may be incorporated into
lotions, which generally are preparations to be applied to the skin
surface without friction, and are typically liquid or semiliquid
preparations in which solid particles, including the active agent,
are present in a water or alcohol base. Lotions are usually
suspensions of solids, and may comprise a liquid oily emulsion of
the oil-in-water type.
[0245] Sirtuin-modulating compounds may be incorporated into
creams, which generally are viscous liquid or semisolid emulsions,
either oil-in-water or water-in-oil. Cream bases are
water-washable, and contain an oil phase, an emulsifier and an
aqueous phase. The oil phase is generally comprised of petrolatum
and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous
phase usually, although not necessarily, exceeds the oil phase in
volume, and generally contains a humectant. The emulsifier in a
cream formulation, as explained in Remington's, supra, is generally
a nonionic, anionic, cationic or amphoteric surfactant.
[0246] Sirtuin-modulating compounds may be incorporated into
microemulsions, which generally are thermodynamically stable,
isotropically clear dispersions of two immiscible liquids, such as
oil and water, stabilized by an interfacial film of surfactant
molecules (Encyclopedia of Pharmaceutical Technology (New York:
Marcel Dekker, 1992), volume 9).
[0247] Sirtuin-modulating compounds may be incorporated into gel
formulations, which generally are semisolid systems consisting of
either suspensions made up of small inorganic particles (two-phase
systems) or large organic molecules distributed substantially
uniformly throughout a carrier liquid (single phase gels). Although
gels commonly employ aqueous carrier liquid, alcohols and oils can
be used as the carrier liquid as well.
[0248] Other active agents may also be included in formulations,
e.g., other anti-inflammatory agents, analgesics, antimicrobial
agents, antifungal agents, antibiotics, vitamins, antioxidants, and
sunblock agents commonly found in sunscreen formulations including,
but not limited to, anthranilates, benzophenones (particularly
benzophenone-3), camphor derivatives, cinnamates (e.g., octyl
methoxycinnamate), dibenzoyl methanes (e.g., butyl methoxydibenzoyl
methane), p-aminobenzoic acid (PABA) and derivatives thereof, and
salicylates (e.g., octyl salicylate).
[0249] In certain topical formulations, the active agent is present
in an amount in the range of approximately 0.25 wt. % to 75 wt. %
of the formulation, preferably in the range of approximately 0.25
wt. % to 30 wt. % of the formulation, more preferably in the range
of approximately 0.5 wt. % to 15 wt. % of the formulation, and most
preferably in the range of approximately 1.0 wt. % to 10 wt. % of
the formulation.
[0250] Conditions of the eye can be treated or prevented by, e.g.,
systemic, topical, intraocular injection of a sirtuin-modulating
compound, or by insertion of a sustained release device that
releases a sirtuin-modulating compound. A sirtuin-modulating
compound that increases the level and/or activity of a sirtuin
protein may be delivered in a pharmaceutically acceptable
ophthalmic vehicle, such that the compound is maintained in contact
with the ocular surface for a sufficient time period to allow the
compound to penetrate the corneal and internal regions of the eye,
as for example the anterior chamber, posterior chamber, vitreous
body, aqueous humor, vitreous humor, cornea, iris/ciliary, lens,
choroid/retina and sclera. The pharmaceutically-acceptable
ophthalmic vehicle may, for example, be an ointment, vegetable oil
or an encapsulating material. Alternatively, the compounds of the
invention may be injected directly into the vitreous and aqueous
humour. In a further alternative, the compounds may be administered
systemically, such as by intravenous infusion or injection, for
treatment of the eye.
[0251] Sirtuin-modulating compounds described herein may be stored
in oxygen free environment. For example, resveratrol or analog
thereof can be prepared in an airtight capsule for oral
administration, such as Capsugel from Pfizer, Inc.
[0252] Cells, e.g., treated ex vivo with a sirtuin-modulating
compound, can be administered according to methods for
administering a graft to a subject, which may be accompanied, e.g.,
by administration of an immunosuppressant drug, e.g., cyclosporin
A. For general principles in medicinal formulation, the reader is
referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy,
and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds,
Cambridge University Press, 1996; and Hematopoietic Stem Cell
Therapy, E. D. Ball, J. Lister & P. Law, Churchill Livingstone,
2000.
[0253] Toxicity and therapeutic efficacy of sirtuin-modulating
compounds can be determined by standard pharmaceutical procedures
in cell cultures or experimental animals. The LD.sub.50 is the dose
lethal to 50% of the population. The ED.sub.50 is the dose
therapeutically effective in 50% of the population. The dose ratio
between toxic and therapeutic effects (LD.sub.50/ED.sub.50) is the
therapeutic index. Sirtuin-modulating compounds that exhibit large
therapeutic indexes are preferred. While sirtuin-modulating
compounds that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0254] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds may lie within a range of
circulating concentrations that include the ED.sub.50 with little
or no toxicity. The dosage may vary within this range depending
upon the dosage form employed and the route of administration
utilized. For any compound, the therapeutically effective dose can
be estimated initially from cell culture assays. A dose may be
formulated in animal models to achieve a circulating plasma
concentration range that includes the IC.sub.50 (i.e., the
concentration of the test compound that achieves a half-maximal
inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
6. Kits
[0255] Also provided herein are kits, e.g., kits for therapeutic
purposes or kits for modulating the lifespan of cells or modulating
apoptosis. A kit may comprise one or more sirtuin-modulating
compounds, e.g., in premeasured doses. A kit may optionally
comprise devices for contacting cells with the compounds and
instructions for use. Devices include syringes, stents and other
devices for introducing a sirtuin-modulating compound into a
subject (e.g., the blood vessel of a subject) or applying it to the
skin of a subject.
[0256] In yet another embodiment, the invention provides a
composition of matter comprising a sirtuin modulator of this
invention and another therapeutic agent (the same ones used in
combination therapies and combination compositions) in separate
dosage forms, but associated with one another. The term "associated
with one another" as used herein means that the separate dosage
forms are packaged together or otherwise attached to one another
such that it is readily apparent that the separate dosage forms are
intended to be sold and administered as part of the same regimen.
The agent and the sirtuin modulator are preferably packaged
together in a blister pack or other multi-chamber package, or as
connected, separately sealed containers (such as foil pouches or
the like) that can be separated by the user (e.g., by tearing on
score lines between the two containers).
[0257] In still another embodiment, the invention provides a kit
comprising in separate vessels, a) a sirtuin modulator of this
invention; and b) another therapeutic agent such as those described
elsewhere in the specification.
[0258] The practice of the present methods will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, transgenic biology, microbiology,
recombinant DNA, and immunology, which are within the skill of the
art. Such techniques are explained fully in the literature. See,
for example, Molecular Cloning A Laboratory Manual, 2.sup.nd Ed.,
ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor
Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N.
Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed.,
1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid
Hybridization (B. D. Hames & S. J. Higgins eds. 1984);
Transcription And Translation (B. D. Hames & S. J. Higgins eds.
1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc.,
1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal,
A Practical Guide To Molecular Cloning (1984); the treatise,
Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer
Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,
1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols.
154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And
Molecular Biology (Mayer and Walker, eds., Academic Press, London,
1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.
Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse
Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1986).
EXEMPLIFICATION
[0259] The invention now being generally described, it will be more
readily understood by reference to the following examples which are
included merely for purposes of illustration of certain aspects and
embodiments of the present invention, and are not intended to limit
the invention in any way.
Example 1
General procedure to prepare
2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid (3)
##STR00037##
[0261] 2,3-diaminobenzoic acid (1; 50 mg, 0.33 mmol),
2-(trifluoromethyl)benzaldehyde (2; 57 mg, 0.33 mmol), and sodium
metabisulfite (Na.sub.2S.sub.2O.sub.5, 82 mg, 0.43 mmol) were taken
up in DMF (5 mL) and stirred at 100.degree. C. for 17 h. The
reaction mixture was cooled to room temperature and diluted with
H.sub.2O. The resulting mixture was stirred at room temperature for
1 h and filtered. The collected solids were washed with water and
dried to afford
2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid 3 as a pale brown solid (75 mg, yield: 74%). MS (ESI) calcd
for C.sub.15H.sub.9F.sub.3N.sub.2O.sub.2: 306.06. found: 307
[M+H].
[0262] This general procedure is used to prepare any 4-carboxy
derivatives of 1H-benzo[d]imidazole using the appropriate aldehyde
component as a replacement for 2-(trifluoromethyl)benzaldehyde 2 in
the above scheme.
Example 2
Preparation of
N-(thiazol-2-yl)-2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-car-
boxamide (Compound 208)
##STR00038##
[0264]
2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid (3; 75 mg, 0.24 mmol) was taken up in DMF (2 mL) along with
thiazol-2-amine (4; 24 mg, 0.24 mmol) and HATU (186 mg, 0.49 mmol).
DIEA (81 .mu.L, 0.49 mmol) was added. The reaction mixture was
stirred at room temperature for 18 h. The reaction mixture was
diluted with water and filtered. The collected solids were further
purified by silica gel chromatography to afford the desired
product, namely
N-(thiazol-2-yl)-2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-car-
boxamide (Compound 208), as a white solid (35 mg, yield: 38%). MS
(ESI) calcd for C.sub.18H.sub.11F.sub.3N.sub.4OS: 388.06. found:
389 [M+H].
[0265] This general procedure is used for any of the amide
derivatives shown in Table 1 by substituting the appropriate
carboxylic acid and amine components for 3 and 4, respectively.
Example 3
Synthesis of
2-(6-(piperazin-1-yl)pyridin-3-yl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole--
4-carboxamide hydrochloride (Compound 251)
Step 1) Preparation of
2-(6-chloropyridin-3-yl)-1H-benzo[d]imidazole-4-carboxylic acid
(6)
##STR00039##
[0267] 2,3-Diaminobenzoic acid (1; 200 mg, 1.31 mmol),
6-chloronicotinaldehyde (5; 186 mg, 1.31 mmol), and
Na.sub.2S.sub.2O.sub.5 (324 mg, 1.7 mmol) were taken up in DMF (10
mL) and stirred at 100.degree. C. for 18 h. The reaction mixture
was cooled to room temperature and diluted with H.sub.2O. The
resulting mixture was stirred for 1 h at room temperature and
filtered. The collected solids were washed with water, MeOH, and
dried to afford
2-(6-chloropyridin-3-yl)-1H-benzo[d]imidazole-4-carboxylic acid 6
as a pale solid (340 mg, yield: 95%).
Step 2) Preparation of
2-(6-chloropyridin-3-yl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole-4-carboxam-
ide (7)
##STR00040##
[0269] 2-(6-Chloropyridin-3-yl)-1H-benzo[d]imidazole-4-carboxylic
acid (6; 340 mg, 1.24 mmol) was taken up in DMF (5 mL) along with
thiazol-2-amine (4; 125 mg, 1.24 mmol) and HATU (943 mg, 2.48
mmol). DIEA (0.41 mL, 2.48 mmol) was added. The reaction mixture
was stirred at room temperature overnight. The reaction mixture was
diluted with water and the resulting solids were collected by
filtration. Purification by silica gel chromatography
(pentane/EtOAc) afforded
2-(6-chloropyridin-3-yl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole-4-carboxam-
ide 7 as a pale yellow solid (215 mg, yield: 52%). MS (ESI) calcd
for C.sub.16H.sub.10ClN.sub.5OS: 355.03. found: 356 [M+H].
Step 3) Preparation of tert-butyl
4-(5-(4-(thiazol-2-ylcarbamoyl)-1H-benzo[d]imidazol-2-yl)pyridin-2-yl)pip-
erazine-1-carboxylate (8)
##STR00041##
[0271] A mixture containing
2-(6-chloropyridin-3-yl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole-4-carboxam-
ide (7; 30 mg, 0.084 mmol) and tert-butyl piperazine-1-carboxylate
(170; 156.5 mg, 0.84 mmo) in DMSO (1.2 mL) was stirred at
140.degree. C. in a microwave reactor for 25 min. The reaction
mixture was cooled to room temperature and diluted with water. The
resulting solids were collected by filtration, washed with water
and dried under vacuum to afford tert-butyl
4-(5-(4-(thiazol-2-ylcarbamoyl)-1H-benzo[d]imidazol-2-yl)pyridin-2-yl)pip-
erazine-1-carboxylate 8 as a pale yellow solid (20 mg, 71%). MS
(ESI) calcd for C.sub.25H.sub.27N.sub.7O.sub.3S: 505.19. found: 506
[M+H].
Step 4) Preparation of
2-(6-(piperazin-1-yl)pyridin-3-yl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole--
4-carboxamide hydrochloride (Compound 251)
##STR00042##
[0273] A mixture containing tert-butyl
4-(5-(4-(thiazol-2-ylcarbamoyl)-1H-benzo[d]imidazol-2-yl)pyridin-2-yl)pip-
erazine-1-carboxylate (8; 20 mg, 0.039 mmol) in methanolic HCl (3N,
2 mL) was stirred at room temperature for 18 h. The precipitated
solids were collected by filtration, washed with MeOH and dried to
afford
2-(6-(piperazin-1-yl)pyridin-3-yl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole--
4-carboxamide hydrochloride (Compound 251) as a pale yellow solid
(21 mg). MS (ESI) calcd for C.sub.20H.sub.19N.sub.7OS (m/z):
405.14. found: 406 [M+H].
Example 4
Preparation of
2-(6-morpholinopyridin-3-yl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole-4-carb-
oxamide (Compound 247)
##STR00043##
[0275] A mixture containing
2-(6-chloropyridin-3-yl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole-4-carboxam-
ide (7; 20 mg, 0.056 mmol) and morpholine (48; 49 mg, 0.56 mmol) in
DMSO (1.5 mL) was stirred at 140.degree. C. in a microwave reactor
for 25 min. The reaction mixture was cooled to room temperature and
diluted with water. The precipitated solids were collected by
filtration and dried. The solids were redissolved in EtOAc and
enough petroleum ether was added to precipitate the product out
again. The solids were collected by filtration, washed with MeOH
and dried to afford
2-(6-morpholinopyridin-3-yl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole-4-carb-
oxamide (Compound 247) as a pale yellow solid (13 mg, 57%). MS
(ESI) calcd for C.sub.20H.sub.18N.sub.6O.sub.2S: 406.12. found: 407
[M+H].
Example 5
Preparation of
2-(2-morpholinopyridin-3-yl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole-4-carb-
oxamide (Compound 250)
##STR00044##
[0277]
2-(2-Chloropyridin-3-yl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole-4-ca-
rboxamide 9 was prepared according to the general procedure
outlined above using the appropriate aldehyde component. A mixture
containing this material, namely
2-(2-chloropyridin-3-yl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole-4-carboxam-
ide (9; 19 mg, 0.053 mmo) and morpholine (48; 46 mg, 0.53 mmol) in
DMSO (1.2 mL) were stirred at 140.degree. C. in a microwave reactor
for 25 min. The reaction mixture was cooled to room temperature and
diluted with water. The precipitated solids were collected by
filtration and dried. The solids were redissolved in EtOAc and
enough petroleum ether was added to precipitate the product out
again. The solids were collected by filtration, washed with MeOH
and dried to afford
2-(2-morpholinopyridin-3-yl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole-4-carb-
oxamide (Compound 250) as a white solid (13 mg, 60%). MS (ESI)
calcd for C.sub.20H.sub.18N.sub.6O.sub.2S (m/z): 406.12. found: 407
[M+H].
Example 6
Preparation of
2-(2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxamido)pyrid-
ine 1-oxide (Compound 329)
##STR00045##
[0279] To a solution of
N-(pyridin-2-yl)-2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-car-
boxamide (10; 100 mg, 0.26 mmol) in CH.sub.2Cl.sub.2 (10 mL) was
added mCPBA (140 mg, 0.81 mmol) under nitrogen. The mixture was
stirred at room temperature for 12 h. A saturated aqueous solution
of K.sub.2CO.sub.3 (10 mL) was added and the mixture was extracted
with CH.sub.2Cl.sub.2 (3.times.10 mL). The combined organic layers
were dried (Na.sub.2SO.sub.4), concentrated under reduced pressure.
The resulting residue was purified by chromatography
(CH.sub.2Cl.sub.2:methanol=20:1) to give
2-(2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxami-
do)pyridine 1-oxide (Compound 329) (60.0 mg, 58%) as a colorless
solid. MS (ESI) calcd for C.sub.20H.sub.13F.sub.3N.sub.4O.sub.2:
398.10. found: 399 [M+H].
Example 7
Synthesis of
N-(2-(3-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-4-yl)thiazole-4-carb-
oxamide (Compound 332)
Step 1) Preparation of benzyl
2-(3-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-4-ylcarbamate
(12)
##STR00046##
[0281] A solution of
2-(3-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid (11; 1.53 g, 5.0 mmol), diphenylphosphoryl azide (DPPA, 1.39
g, 5.0 mmol) and Et.sub.3N (0.75 g, 7.4 mmol) in toluene (15 mL)
was stirred at room temperature for 2 h and then stirred under
reflux for 2 h. Benzyl alcohol (0.54 g, 5.0 mmol) was added and the
resulting reaction mixture was stirred under reflux for another 12
h. After cooling to room temperature, the mixture was washed
successively with aqueous citric acid (10% w/w), saturated
NaHCO.sub.3 and brine. The organic layer was (Na.sub.2SO.sub.4),
and concentrated under reduced pressure. The resulting residue was
purified by chromatography (petroleum ether:ethyl acetate=10:1) to
give benzyl
2-(3-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-4-ylcarbamate 12
(1.45 g, 70%) as a solid. MS (ESI) calcd for
C.sub.22H.sub.16F.sub.3N.sub.3O.sub.2: 411.12. found: 412
[M+H].
Step 2) Preparation of
2-(3-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-4-amine (13)
##STR00047##
[0283] To a stirred solution of benzyl
2-(3-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-4-ylcarbamate
(12; 1.45 g, 3.5 mmol) in methanol (20 mL) was added 5% Pd/C (0.20
g). The reaction mixture was thoroughly purged with nitrogen and
then stirred under 1 atm of hydrogen at room temperature for 18 h.
The mixture was filtered through a pad of Celite and the filtrate
was concentrated under reduced pressure to give
2-(3-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-4-amine 13 (0.90
g, 3.25 mmol, 93%) as a solid. MS (ESI) calcd for
C.sub.14H.sub.10F.sub.3N.sub.3: 277.08. found: 278 [M+H].
Step 3) Preparation of
N-(2-(3-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-4-yl)thiazole-4-carb-
oxamide (Compound 332)
##STR00048##
[0285] A mixture of
2-(3-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-4-amine (13; 0.28
g, 1.0 mmol), thiazole-4-carboxylic acid (14; 0.15 g, 1.2 mmol),
DIEA (0.42 mL, 2.4 mmol) and HATU (0.79 g, 2.07 mmol) in DMF (10
mL) was stirred at room temperature for 12 h. A saturated aqueous
solution of NaHCO.sub.3 (5 mL) was added and the mixture was
filtered. The residue was washed with ethyl acetate (3.times.15 mL)
and the aqueous layer was extracted with ethyl acetate (3.times.15
mL). The combined organic layers were dried (MgSO.sub.4) and
concentrated under reduced pressure. The resulting residue was
purified by chromatography (petroleum ether:ethyl acetate=10:1) to
give
N-(2-(3-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-4-yl)thiazole-4-carb-
oxamide (Compound 332) (0.30 g, 0.77 mmol, 77%) as a colorless
solid. MS (ESI) calcd for C.sub.18H.sub.11F.sub.3N.sub.4OS: 388.06.
found: 389 [M+H].
Example 8
Preparation of
N-(pyridin-2-yl)-2-(3-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-4-amin-
e (Compound 581)
##STR00049##
[0287] A mixture containing
2-(3-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-4-amine (13; 332
mg, 1.20 mmol), 2-chloropyridine (15; 138 g, 1.22 mmol),
Pd(OAc).sub.2 (120 mg, 0.53 mmol), BINAP (336 mg, 0.53 mmol) and
t-BuOK (150 mg, 1.34 mmol) in toluene (10 mL) was stirred under
reflux for 12 h under an inert atmosphere of N.sub.2. The reaction
mixture was concentrated under reduced pressure and the resulting
residue was extracted with ethyl acetate (3.times.15 mL). The
combined organic layers were dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure. The resulting residue was
purified by chromatography (petroleum ether:ethyl acetate=1:1) to
afford
N-(pyridin-2-yl)-2-(3-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-4-amin-
e (Compound 581) (30 mg, 0.085 mmol, 7.1%) as a colorless solid. MS
(ESI) calcd for C.sub.19H.sub.13F.sub.3N.sub.4: 354.11. found: 355
[M+H].
Example 9
Preparation of
N-(pyridin-3-ylmethyl)-2-(3-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol--
4-amine (Compound)
##STR00050##
[0289] A mixture containing
2-(3-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-4-amine (13; 120
mg, 0.43 mmol), nicotinaldehyde (16; 51 mg, 0.47 mmol) and
NaBH(OAc).sub.3 (143 mg, 0.67 mmol) in 1,2-dichloroethane (10 mL)
was stirred at room temperature for 12 h under an inert atmosphere
of N.sub.2. Saturated aqueous NaHCO.sub.3 (10 mL) was added and the
mixture was extracted with ethyl acetate (3.times.15 mL). The
combined organic layers were dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure. The resulting residue was
purified by chromatography (gradient elution, petroleum ether:ethyl
acetate=1:1 to 1:3) to afford
N-(pyridin-3-ylmethyl)-2-(3-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol--
4-amine (Compound) (70 mg, 0.198 mmol, 46%) as a pale yellow solid.
MS (ESI) calcd for C.sub.20H.sub.15F.sub.3N.sub.4: 368.12. found:
369 [M+H].
Example 10
Synthesis of
2-(4-morpholino-2-(trifluoromethyl)phenyl)-N-(pyridin-3-yl)-1H-benzo[d]im-
idazole-4-carboxamide (Compound 430)
Step 1) Preparation of
4-chloro-1-(dimethoxymethyl)-2-(trifluoromethyl)benzene (18)
##STR00051##
[0291] To a stirred solution of
4-chloro-2-(trifluoromethyl)benzaldehyde (17; 4.20 g, 20.0 mmol) in
methanol (25 mL) was added triethoxymethane (3.60 g, 24.0 mmol) at
0.degree. C. The resulting reaction mixture was warmed to room
temperature and stirred for 12 h. A solution of MeONa in methanol
was added to adjust the pH=10. The solvent was removed under
reduced pressure and the resulting residue was diluted with water.
The mixture was extracted with ethyl acetate (3.times.50 mL) and
the combined organic layers were dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure to give
4-chloro-1-(dimethoxymethyl)-2-(trifluoromethyl)benzene 18 (3.20 g,
12.6 mmol, 63%).
Step 2. Preparation of
4-(4-(dimethoxymethyl)-3-(trifluoromethyl)phenyl)morpholine
(19)
##STR00052##
[0293] A mixture of
4-chloro-1-(dimethoxymethyl)-2-(trifluoromethyl)benzene (18; 3.20
g, 12.6 mmol), Pd.sub.2(dba).sub.3 (0.30 g, 0.33 mmol), t-BuOK
(3.50 g, 31.2 mmol) and morpholine (48; 1.09 g, 12.6 mmol) in
anhydrous toluene (50 mL) was stirred under reflux for 12 h under
an inert atmosphere of nitrogen. Upon cooling to room temperature,
the mixture was filtered and the filtrate was concentrated under
reduced pressure. The resulting residue was purified by
chromatography (petroleum ether:ethyl acetate=10:1) to give
4-(4-(dimethoxymethyl)-3-(trifluoromethyl)phenyl)morpholine 19
(0.40 g, 10.4%).
Step 3. Preparation of
2-(4-morpholino-2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxy-
lic acid (20)
##STR00053##
[0295] A mixture of
4-(4-(dimethoxymethyl)-3-(trifluoromethyl)phenyl)morpholine (19;
0.40 g, 1.31 mmol), 2,3-diaminobenzolic acid (1; 0.20 g, 1.31
mmol), Na.sub.2S.sub.2O.sub.5 (0.33 g, 1.74 mmol) in DMF (10 mL)
was stirred at 110.degree. C. (oil bath) for 18 h. Upon cooling to
room temperature, the mixture was diluted with water (20 mL) and
stirred for 30 min. The mixture was filtered; the filter cake was
washed with water (3.times.10 mL) and dried to give
2-(4-morpholino-2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxy-
lic acid 20 (0.37 g, 72%) as a grey solid.
Step 4. Preparation of
2-(4-morpholino-2-(trifluoromethyl)phenyl)-N-(pyridin-3-yl)-1H-benzo[d]im-
idazole-4-carboxamide (Compound 430)
##STR00054##
[0297] A mixture of
2-(4-morpholino-2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxy-
lic acid (20; 157 mg, 0.40 mmol), HATU (304 mg, 0.80 mmol), DIEA
(207 mg, 1.60 mmol) and 3-aminopyridine (173; 37 mg, 0.40 mmol) in
DMF (2 mL) was stirred at room temperature for 12 h. Water (20 mL)
was added and stirred for 30 min. The mixture was extracted with
ethyl acetate (3.times.10 mL). The combined organic layers were
dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure.
The resulting residue was purified by chromatography to give
2-(4-morpholino-2-(trifluoromethyl)phenyl)-N-(pyridin-3-yl)-1H-benzo[d]im-
idazole-4-carboxamide (Compound 430) (64 mg, 34%) as a pale yellow
solid. MS (ESI) calcd for C.sub.24H.sub.20F.sub.3N.sub.5O.sub.2:
467.16. found: 468 [M+H].
Example 11
Preparation of
2-(5-morpholino-2-(trifluoromethyl)phenyl)-N-(pyridin-3-yl)-1H-benzo[d]im-
idazole-4-carboxamide (Compound 450)
##STR00055##
[0299] The same general procedure outlined in Example 10, above for
the preparation of
2-(4-morpholino-2-(trifluoromethyl)phenyl)-N-(pyridin-3-yl)-1H-benzo[d]im-
idazole-4-carboxamide was used, except that
5-chloro-2-(trifluoromethyl)benzaldehyde 21 was used as the
starting material to produce
2-(5-morpholino-2-(trifluoromethyl)phenyl)-N-(pyridin-3-yl)-1H-benzo[d]im-
idazole-4-carboxamide (Compound 450). MS (ESI) calcd for
C.sub.24H.sub.20F.sub.3N.sub.5O.sub.2: 467.16. found: 468
[M+H].
Example 12
Synthesis of
2-(2-(piperidin-4-yloxy)phenyl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole-4-c-
arboxamide (Compound 428)
Step 1) Preparation of tert-butyl
4-(methylsulfonyloxy)piperidine-1-carboxylate (24)
##STR00056##
[0301] To a solution of N-Boc-4-hydroxypiperidine (22; 25 g, 124
mmol) and Et.sub.3N (20.7 mL) in CH.sub.2Cl.sub.2 (100 mL) was
added a solution of methanesulfonyl chloride 23 in CH.sub.2Cl.sub.2
(20 mL) dropwise in an ice-water bath. The mixture was stirred at
room temperature for 18 h. It was then diluted with 100 mL of
CH.sub.2Cl.sub.2 and washed successively with saturated aqueous
Na.sub.2CO.sub.3 (50 mL) and brine (50 mL). The organic layer was
dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure to
afford tert-butyl 4-(methylsulfonyloxy)piperidine-1-carboxylate 24
(45 g, crude yield: >100%). This material was then used in the
next step without further purification.
Step 2) Preparation of tert-butyl
4-(2-formylphenoxy)piperidine-1-carboxylate (26)
##STR00057##
[0303] A mixture containing 2-hydroxybenzaldehyde (25; 4.2 g, 34.5
mmol), tert-butyl 4-(methylsulfonyloxy)piperidine-1-carboxylate
(24; 15.0 g, .about.41 mmol), K.sub.2CO.sub.3 (7.15 g, 51.7 mmol)
and tetrabutyl ammonium iodide (500 mg, cat) in DMF (100 mL) was
stirred at 70-80.degree. C. for 18 h. The solvent DMF was removed
by concentration under reduced pressure. The resulting residue was
diluted with EtOAc (100 mL) and washed with water (50 mL). The
organic layer was dried (MgSO.sub.4) and concentrated under reduced
pressure. The resulting residue was purified by chromatography
(pentane/EtOAc=1:10) to give tert-butyl
4-(2-formylphenoxy)piperidine-1-carboxylate 26 as a yellow oil
(2.89 g, 27%).
Step 3) Preparation of
2-(2-(1-(tert-butoxycarbonyl)piperidin-4-yloxy)phenyl)-1H-benzo[d]imidazo-
le-4-carboxylic acid (27)
##STR00058##
[0305] The same general procedure outlined above for the
preparation of benzimidazole derivatives was used, employing
2,3-diaminobenzoic acid 1 and tert-butyl
4-(2-formylphenoxy)piperidine-1-carboxylate 26 to produce
2-(2-(1-(tert-butoxycarbonyl)piperidin-4-yloxy)phenyl)-1H-benzo[d]imidazo-
le-4-carboxylic acid 27. MS (ESI) calcd for
C.sub.24H.sub.27N.sub.3O.sub.5: 437.20. found: 438 [M+H].
Step 4) Preparation of
2-(2-(piperidin-4-yloxy)phenyl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole-4-c-
arboxamide (Compound 428)
##STR00059##
[0307] The same general amide coupling procedure outlined above was
used, employing
2-(2-(1-(tert-butoxycarbonyl)piperidin-4-yloxy)phenyl)-1H-benzo-
[d]imidazole-4-carboxylic acid 27 and 2-aminothiazole 4 as the
starting materials. The resulting crude amide coupling product (0.2
mmol) was treated with 4 mL of 25% TFA in CH.sub.2Cl.sub.2 and
allowed to stand at room temperature for 8 h. The mixture was
concentrated under reduced pressure and the resulting residue was
purified by preparative HPLC using aqueous acetonitrile that has
buffered with 0.1% TFA to afford
2-(2-(piperidin-4-yloxy)phenyl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole-4-c-
arboxamide (Compound 428). MS (ESI) calcd for
C.sub.22H.sub.21N.sub.5O.sub.2S: 419.14. found: 420 [M+H].
Example 13
Synthesis of
2-(2-(pyrrolidin-3-yloxy)phenyl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole-4--
carboxamide (Compound 459)
Step 1) Preparation of tert-butyl
3-(2-formylphenoxy)pyrrolidine-1-carboxylate (30)
##STR00060##
[0309] To a solution of N-Boc-3-hydroxypyrrolidine (28; 2 g, 10.7
mmol) and Et.sub.3N (2.2 ml, 16.0 mmol) in CH.sub.2Cl.sub.2 (5 mL)
was added a solution of methanesulfonyl chloride (1.84 g, 2.52 ml,
16.0 mmol) in CH.sub.2Cl.sub.2 (5 mL) dropwise in an ice-water
bath. The mixture was stirred at room temperature for 18 h. It was
then diluted with 50 mL of CH.sub.2Cl.sub.2, washed with saturated
Na.sub.2CO.sub.3 (20 mL), brine (20 mL), dried (Na.sub.2SO.sub.4)
and concentrated under reduced pressure to afford
N-Boc-3-(methylsulfonyloxy)pyrrolidine 29 (2.6 g, 92%). This
material was used in the following step without further
purification.
[0310] 2-Hydroxybenzaldehyde (25; 1.2 g, 9.8 mmol),
N-Boc-3-(methylsulfonyloxy)pyrrolidine (29; 2.6 g, 9.8 mmol), K2CO3
(2.0 g, 14.7 mmol) and tetrabutyl ammonium iodide (300 mg, cat) in
CH.sub.2Cl.sub.2 (20 mL) was stirred at 70-80.degree. C. for 18 h.
DMF was removed by concentration under reduced pressure. The
resulting residue was diluted with EtOAc (100 mL), washed with
water (50 mL), dried (MgSO.sub.4) and concentrated under reduced
pressure. The resulting residue was purified by chromatography
(pentane:EtOAc=1:10) to afford 676 mg of tert-butyl
3-(2-formylphenoxy)pyrrolidine-1-carboxylate 30 as an oil (yield:
23%).
Step 2) Preparation of
2-(2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)phenyl)-1H-benzo[d]imidaz-
ole-4-carboxylic acid (31)
##STR00061##
[0312] A mixture of 2,3-diaminobenzoic acid (1; 350 mg, 2.3 mmol),
tert-butyl 3-(2-formylphenoxy)pyrrolidine-1-carboxylate (30; 670
mg, 2.3 mmol) and Na.sub.2S.sub.2O.sub.5 (568 mg, 1.3 mmol) in DMF
(30 mL) was stirred at 90.degree. C. for 18 h. The mixture was
cooled to room temperature. The mixture was diluted with water (100
mL), stirred for 1 h, filtered, and washed with water. The filter
cake was dried under vacuum to afford 822 mg of the titled compound
31 (84%). MS (ESI) calcd for C.sub.23H.sub.25N.sub.3O.sub.5:
423.18. found: 424 [M+H].
Step 3) Preparation of tert-butyl
3-(2-(4-(thiazol-2-ylcarbamoyl)-1H-benzo[d]imidazol-2-yl)phenoxy)pyrrolid-
ine-1-carboxylate (32)
##STR00062##
[0314] To a solution of
2-(2-(1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)phenyl)-1H-benzo[d]imidaz-
ole-4-carboxylic acid (31; 150 mg, 0.36 mmol), thiazol-2-amine (4;
35.5 mg, 0.36 mmol), and HATU (202 mg, 0.53 mmol) in DMF (5 mL) was
added DIEA (137 mg, 1.06 mmol). The mixture was stirred at room
temperature for 18 h. It was then diluted with water (20 mL) and
stirred for 30 min. The precipitate was collected by filtration,
and then purified by column chromatography to give 85 mg of the
title compound 32 (yield: 48%). MS (ESI) calcd for
C.sub.26H.sub.27N.sub.5O.sub.4S: 505.18. found: 506 [M+H].
Step 4) Preparation of
2-(2-(pyrrolidin-3-yloxy)phenyl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole-4--
carboxamide (Compound 459)
##STR00063##
[0316] A solution of tert-butyl
3-(2-(4-(thiazol-2-ylcarbamoyl)-1H-benzo[d]imidazol-2-yl)phenoxy)pyrrolid-
ine-1-carboxylate 32 in 8 mL of 4M HCl/MeOH was stirred at room
temperature for 2 h. The precipitate formed was collected by
filtration and washed with methanol to give 72 mg of
2-(2-(pyrrolidin-3-yloxy)phenyl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole-4--
carboxamide (Compound 459) (yield: 96%). MS (ESI) calcd for
C.sub.21H.sub.19N.sub.5O.sub.2S: 405.13. found: 406 [M+H].
Example 14
Preparation of
2-(3-(2,3-dihydroxypropoxy)phenyl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole--
4-carboxamide (Compound 324)
Step 1) Preparation of (2,2-dimethyl-1,3-dioxolan-4-yl)methyl
4-methylbenzenesulfonate (34)
##STR00064##
[0318] (2,2-Dimethyl-1,3-dioxolan-4-yl)methanol (33; 7.56 mmol, 1.0
g) was taken up in 20 mL of dichloromethane. Triethylamine (26.46
mmol, 3.687 mL) was then added, followed by p-toluensulfonyl
chloride (15.12 mmol, 2.88 g) and catalytic amount of
4-dimethylamino pyridine. The reaction was allowed to stir at room
temperature for 18 h. The crude reaction mixture was diluted with
dichloromethane, washed successively with saturated aqueous sodium
bicarbonate solution, water, dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure. The resulting residue 34 was
used for the next step without further purification (89% crude
yield).
Step 2) Preparation of
2-(3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)phenyl)-N-(thiazol-2-yl)-1H-
-benzo[d]imidazole-4-carboxamide (36)
##STR00065##
[0320] To a solution of (2,2-dimethyl-1,3-dioxolan-4-yl)methyl
4-methylbenzenesulfonate (34; 4.02 mmol, 1.15 g) and
3-hydroxybenzaldehyde (35; 4.824 mmol, 0.614 g) in 20 mL of
dimethylformamide was added potassium carbonate (10.05 mmol, 1.39
g). The reaction was stirred at 50.degree. C. for 18 h. To the
crude reaction mixture was diluted with water (10 mL) and extracted
with dichloromethane (3.times.15 mL). The combined organic layers
were dried (Na.sub.2SO.sub.4) and concentrated under reduced
pressure. The crude material was purified by flash chromatography
(Pentanes/EtOAc). The desired product 36 was obtained in 84%
yield.
Step 3) Preparation of
2-(3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)phenyl)-N-(thiazol-2-yl)-1H-
-benzo[d]imidazole-4-carboxamide (37)
##STR00066##
[0322] The same general procedure outlined above for the
preparation of benzimidazoles using 2,3-diaminobenzoic acid was
used to prepare
2-(3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)phenyl)-N-(thiazol-2-yl)-1H-
-benzo[d]imidazole-4-carboxamide 37. MS (ESI) calcd for
C.sub.23H.sub.22N.sub.4O.sub.4S: 450.14. found: 451 [M+H].
Step 4) Preparation of
2-(3-(2,3-dihydroxypropoxy)phenyl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole--
4-carboxamide (Compound 324)
##STR00067##
[0324] To a solution of
2-(3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)phenyl)-N-(thiazol-2-yl)-1H-
-benzo[d]imidazole-4-carboxamide (37; 50 mg, 0.111 mmol) in THF was
added 4N HCl dropwise until precipitation was observed. The
precipitate was collected via vacuum filtration and washed with
anhydrous ethanol. The product (Compound 324) was dried overnight
under vacuum. (Product was pale yellowish in color and yield=86%).
MS (ESI) calcd for C.sub.23H.sub.22N.sub.4O.sub.4S: 410.10. found:
411 [M+H].
Example 15
Preparation of
2-(2-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-N-(thiazol-2-yl)-1H-benzo[d]imida-
zole-4-carboxamide (Compound 482)
Step 1) Preparation of 2-chloroethyl 4-methylbenzenesulfonate
(39)
##STR00068##
[0326] In a typical run, p-toluenesulfonyl chloride (TsCl, 70.5 g,
0.37 mol) was added slowly to a solution of 2-chloroethanol (38;
25.0 g, 0.31 mol) in pyridine (100 mL) at 0.degree. C. The reaction
mixture was stirred at 0-5.degree. C. for 18 h. Ice water (300 mL)
was added and the resulting mixture was extracted with ethyl
acetate (3.times.100 mL). The combined organic layers were washed
with 1 N HCl (3.times.100 mL), water (100 mL.times.3), dried
(Na.sub.2SO.sub.4) and concentrated under reduced pressure to
afford 2-chloroethyl 4-methylbenzenesulfonate 39 (64.3 g, 88%).
Step 2) Preparation of 2-(2-chloroethoxy)benzaldehyde (40)
##STR00069##
[0328] A mixture of 2-chloroethyl 4-methylbenzenesulfonate (39;
27.0 g, 0.115 mol), 2-hydroxybenzaldehyde (25; 14.0 g, 0.115 mol)
and K.sub.2CO.sub.3 (31.7 g, 0.23 mol) in DMF (100 mL) was stirred
at 45.degree. C. for 12 h. Water (200 mL) was added and the mixture
was extracted with ethyl acetate (3.times.150 mL). The combined
organic layers were washed saturated brine (3.times.50 mL), dried
(Na.sub.2SO.sub.4) and concentrated under reduced pressure. The
resulting residue was purified by chromatography (petroleum
ether:ethyl acetate=10:1) to give 2-(2-chloroethoxy)benzaldehyde 40
(16.8 g, 0.091 mol, 79%) as a pale yellow solid.
Step 3) Preparation of 2-(2-(pyrrolidin-1-yl)ethoxy)benzaldehyde
(41)
##STR00070##
[0330] A solution of 2-(2-chloroethoxy)benzaldehyde (40; 2.5 g,
13.5 mmol), pyrrolidine (172; 1.9 g, 26.8 mmol), K.sub.2CO.sub.3
(3.8 g, 27.2 mmol) and NaI (0.6 g, 4.2 mmol) in DMF (50 mL) was
stirred at 80.degree. C. for 12 h. Water (200 mL) was added and the
mixture was extracted with ethyl acetate (3.times.150 mL). The
combined organic layers were washed brine (3.times.50 mL), dried
(Na.sub.2SO.sub.4) and concentrated under reduced pressure. The
resulting residue was purified by chromatography (dichloromethane:
methanol=100:1) to give 2-(2-(pyrrolidin-1-yl)ethoxy)benzaldehyde
41 (1.63 g, 7.43 mmol, 55%) as a pale yellow solid.
Step 4) Preparation of
2-(2-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid (42)
##STR00071##
[0332] A mixture of 2-(2-(pyrrolidin-1-yl)ethoxy)benzaldehyde (41;
1.6 g, 7.3 mmol), 2,3-diaminobenzolic acid (1; 1.10 g, 7.3 mmol),
Na.sub.2S.sub.2O.sub.5 (2.0 g, 11.0 mmol) in DMF (30 mL) was
stirred at 95.degree. C. (oil bath) for 18 h. Water (30 mL) was
added and the mixture was filtered. The solids were washed with
dichloromethane/methanol (50 mL, v/v=5/1) and dried to give
2-(2-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid 42 (1.80 g, 5.1 mmol, 70%) as a brown solid.
Step 5) Preparation of
2-(2-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-N-(thiazol-2-yl)-1H-benzo[d]imida-
zole-4-carboxamide (Compound 482)
##STR00072##
[0334] A mixture of
2-(2-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid (42; 0.30 g, 0.85 mmol), HATU (0.42 g, 1.1 mmol), DIPEA (0.22
g, 1.7 mmol) and 2-aminothiazole (4; 0.17 g, 1.7 mmol) in DMF (15
mL) was stirred at 50.degree. C. for 14 h. Water (30 mL) was added
and the mixture was stirred for 30 min. The mixture was filtered
and the filter cake was purified by chromatography
(dichloromethane:methanol:ammonia=60:1:0.3) to give
2-(2-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-N-(thiazol-2-yl)-1H-benzo[d]imida-
zole-4-carboxamide (Compound 482) (70 mg, 0.16 mmol, 18.8%) as a
colorless solid. MS (ESI) calcd for
C.sub.23H.sub.23N.sub.5O.sub.2S: 433.16. found: 434 [M+H].
Example 16
Synthesis of
2-(2-(difluoromethyl)-4-fluorophenyl)-1H-benzo[d]imidazole-4-carboxylic
acid (Compound 559)
Step 1) Preparation of 1-bromo-2-(difluoromethyl)-4-fluorobenzene
(44)
##STR00073##
[0336] To a solution of 2-bromo-5-fluorobenzaldehyde (43; 10 g,
43.5 mmol) in CH.sub.2Cl.sub.2 (100 mL) was added a solution of
(diethylamino)sulfur trifluoride ("DAST", 10.5 g, 65.2 mmol) in
CH.sub.2Cl.sub.2 (50 mL) at 0.degree. C. The reaction mixture was
then warmed to room temperature and stirred for 18 h. The mixture
was poured into aqueous NaHCO.sub.3 slowly and the layers were
separated. The aqueous layer was extracted with CH.sub.2Cl.sub.2.
The combined organic layers were dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure. The crude material was
purified by vacuum distillation to afford
1-bromo-2-(difluoromethyl)-4-fluorobenzene 44 (7.9 g, 71.3%).
Step 2) Preparation of 2-(difluoromethyl)-4-fluorobenzaldehyde
(46)
##STR00074##
[0338] Isopropyl magnesium bromide (30 mL, 1M in THF, 30 mmol) was
added dropwise to an ice-cooled solution of
1-bromo-2-(difluoromethyl)-4-fluorobenzene (44; 6 g, 26.7 mmol) in
THF (100 mL). The reaction mixture was then allowed to warm to room
temperature and stirred for 3 hr. Dimethylformamide (3.5 ml, 45.2
mmol) was added and the reaction stirred for 3 hr. Water was added
and the mixture was extracted with ethyl acetate. The combined
organic layers were dried (Na.sub.2SO.sub.4) and concentrated under
reduced pressure. The resulting residue was purified by
chromatography to afford 2-(difluoromethyl)-4-fluorobenzaldehyde 45
(3.2 g, 68.5%)
Step 3) Preparation of
2-(2-(difluoromethyl)-4-fluorophenyl)-1H-benzo[d]imidazole-4-carboxylic
acid (46)
##STR00075##
[0340] The same general procedure outlined above for the
preparation of benzimidazole was used employing
2-(difluoromethyl)-4-fluorobenzaldehyde 45 and 2,3-diaminobenzoic
acid 1 as the starting materials to afford
2-(2-(difluoromethyl)-4-fluorophenyl)-1H-benzo[d]imidazole-4-carboxylic
acid 46. MS (ESI) calcd for C.sub.15H.sub.9F.sub.3N.sub.2O.sub.2:
306.6. found: 307 [M+H].
Step 4) Preparation of
2-(2-(difluoromethyl)-4-fluorophenyl)-N-(thiazol-2-yl)-1H-benzo[d]imidazo-
le-4-carboxamide (Compound 559)
##STR00076##
[0342] The same general amide coupling procedure outlined above was
used to prepare
2-(2-(difluoromethyl)-4-fluorophenyl)-N-(thiazol-2-yl)-1H-benzo[d]imidazo-
le-4-carboxamide (Compound 559). MS (ESI) calcd for
C.sub.18H.sub.11F.sub.3N.sub.4OS: 388.06. found: 389 [M+H].
Example 17
Synthesis of
2-(2-(difluoromethyl)-4-morpholinophenyl)-N-(thiazol-2-yl)-1H-benzo[d]imi-
dazole-4-carboxamide (Compound 551)
Step 1) Preparation of 2-(difluoromethyl)-4-morpholinobenzaldehyde
(49)
##STR00077##
[0344] A mixture containing 2-(difluoromethyl)-4-fluorobenzaldehyde
(47; 3.50 g, 20.1 mmol), morpholine (48; 1.9 g, 22.1 mmol) and
K.sub.2CO.sub.3 (5.5 g, 40.2 mmol) in 50 mL of DMSO was stirred at
100.degree. C. for 4 h. The resulting reaction mixture was cooled
to room temperature and diluted with water (200 mL). The
precipitated solids were collected by filtration and dried to
afford 2-(difluoromethyl)-4-morpholinobenzaldehyde 49 (2.5 g,
52%).
Step 2) Preparation of
2-(2-(difluoromethyl)-4-morpholinophenyl)-1H-benzo[d]imidazole-4-carboxyl-
ic acid (50)
##STR00078##
[0346] The same general procedure outlined above for the
preparation of benzimidazole was used employing
2-(difluoromethyl)-4-morpholinobenzaldehyde 49 and
2,3-diaminobenzoic acid 1 as the starting materials to prepare
2-(2-(difluoromethyl)-4-morpholinophenyl)-1H-benzo[d]imidazole-4-carboxyl-
ic acid (50). MS (ESI) calcd for
C.sub.19H.sub.17F.sub.2N.sub.3O.sub.3: 373.12. found: 374
[M+H].
Step 3) Preparation of
2-(2-(difluoromethyl)-4-morpholinophenyl)-N-(thiazol-2-yl)-1H-benzo[d]imi-
dazole-4-carboxamide (Compound 551)
##STR00079##
[0348] The same general amide coupling procedure outlined above was
used to prepare
2-(2-(difluoromethyl)-4-morpholinophenyl)-N-(thiazol-2-yl)-1H-benzo[d]imi-
dazole-4-carboxamide (Compound 551). MS (ESI) calcd for
C.sub.22H.sub.19F.sub.2N.sub.5O.sub.2S: 455.12. found: 456
[M+H].
Example 18
Synthesis of
2-(4-chloro-2-(trifluoromethyl)phenyl)-N-(3-(pyrrolidin-1-ylmethyl)phenyl-
)-1H-benzo[d]imidazole-4-carboxamide (Compound 490)
Step 1) Preparation of 3-(pyrrolidin-1-ylmethyl)aniline (53)
##STR00080##
[0350] 1-(Bromomethyl)-3-nitrobenzene (51; 5 g, 23.1 mmol) was
taken up in 100 mL of anhydrous THF along with pyrrolidine (2.3 mL,
27.72 mmol) and K.sub.2CO.sub.3 (4.8 g, 34.6 mmol). The reaction
mixture was stirred at room temperature for 18 h and then filtered.
The filtrate was concentrated under reduced pressure to afford
1-(3-nitrobenzyl)pyrrolidine 52. This material 52 was taken up in
100 mL of absolute EtOH and 10% Pd on C (300 mg) was added. The
resulting reaction mixture was stirred at room temperature under 1
atm of hydrogen for 18 h. The mixture was then filtered through a
pad of Celite and the filtrate was concentrated under reduced
pressure to afford 2.81 g of 3-(pyrrolidin-1-ylmethyl)aniline 53
(70%).
Step 2) Preparation of
2-(4-chloro-2-(trifluoromethyl)phenyl)-N-(3-(pyrrolidin-1-ylmethyl)phenyl-
)-1H-benzo[d]imidazole-4-carboxamide (Compound 490)
##STR00081##
[0352] The same general amide coupling procedure outlined above was
used to prepare of
2-(4-chloro-2-(trifluoromethyl)phenyl)-N-(3-(pyrrolidin-1-ylmethyl)phenyl-
)-1H-benzo[d]imidazole-4-carboxamide (Compound 490) from
2-(4-chloro-2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid 54 and 3-(pyrrolidin-1-ylmethyl)aniline 53. MS (ESI) calcd for
C.sub.26H.sub.22ClF.sub.3N.sub.4O: 498.14. found: 499 [M+H].
Example 19
Synthesis of
2-(4-fluoro-2-(trifluoromethyl)phenyl)-N-(4-(morpholinomethyl)thiazol-2-y-
l)-1H-benzo[d]imidazole-4-carboxamide (Compound 681)
Step 1) Preparation of tert-butyl
4-(hydroxymethyl)thiazol-2-ylcarbamate (57)
##STR00082##
[0354] Ethyl 2-aminothiazole-4-carboxylate (55; 10.0 g, 58.1 mmol)
was taken up in 150 mL of anhydrous THF along with di-tert-butyl
carbonate (BOC.sub.2O, 12.67 g, 58.1 mmol) along with 10 mg of
4-(dimethyl)aminopyridine (DMAP). The reaction mixture was stirred
at 50.degree. C. for 4 h and then at room temperature for 18 h. It
was then concentrated under reduced pressure to obtain a thick oil.
Pentane was added and the resulting crystalline materials were
collected by filtration and dried to afford 10.5 g of ethyl
2-(tert-butoxycarbonylamino)thiazole-4-carboxylate 56. This
material 56 (10.5 g, 38.5 mmol) was dissolved in 300 mL of
anhydrous THF and cooled in Dry Ice-acetonitrile bath. A solution
of 1 M Super Hydride.TM. in THF (85 mL) was then added over a
period of 10 min. The resulting reaction mixture was stirred at
-45.degree. C. for 2 h. Another portion of 1 M Super Hydride.TM. in
THF (35 mL) was then added and the reaction mixture was stirred for
an additional 2 h at -45.degree. C. The reaction was quenched at
-45.degree. C. by the addition of 50 mL of brine. Upon warming to
room temperature, the reaction mixture was concentrated under
reduced pressure. The resulting mixture was extracted with EtOAc.
The combined organic layers were washed with brine, dried
(Na.sub.2SO.sub.4) and concentrated under reduced pressure. The
resulting residue was purified by chromatography to afford 6.39 g
of tert-butyl 4-(hydroxymethyl)thiazol-2-ylcarbamate 57 (72%).
Step 2) Preparation of 4-(morpholinomethyl)thiazol-2-amine (59)
##STR00083##
[0356] tert-Butyl 4-(hydroxymethyl)thiazol-2-ylcarbamate (57; 2.0
g, 8.7 mmol) was taken up in 25 mL of CH.sub.2Cl.sub.2 along with
Et.sub.3N (1.82 mL, 13.05 mmol) and cooled to 0.degree. C.
Methanesulfonyl chloride (0.85 mL, 10.88 mmol) was added and the
resulting reaction mixture was stirred at 0.degree. C. for 60 min.
Morpholine (48; 3.0 mL, 35 mmol) was then added and the reaction
mixture was stirred at room temperature for 18 h. The reaction
mixture was concentrated under reduced pressure. The resulting
residue was taken up in EtOAc and washed with dilute aqueous
NaHCO.sub.3, brine, dried (Na.sub.2SO.sub.4) and concentrated under
reduced pressure. This material was purified by filtering through a
short column of silica gel. The filtrate was concentrated to afford
1.88 g of tert-butyl 4-(morpholinomethyl)thiazol-2-ylcarbamate 58.
The Boc group was removed by treating tert-butyl
4-(morpholinomethyl)thiazol-2-ylcarbamate 58 with 20 mL of 25% TFA
in CH.sub.2Cl.sub.2 for 18 h at room temperature. After all the
solvent had been removed by concentrating and drying under high
vacuum, the resulting residue was treated with a mixture of
pentane/EtOAc to afford 2.17 g 4-(morpholinomethyl)thiazol-2-amine
59 as a white solid.
Step 3) Preparation of
2-(4-fluoro-2-(trifluoromethyl)phenyl)-N-(4-(morpholinomethyl)thiazol-2-y-
l)-1H-benzo[d]imidazole-4-carboxamide (Compound 681)
##STR00084##
[0358] The same general amide coupling procedure outlined above was
used to prepare
2-(4-fluoro-2-(trifluoromethyl)phenyl)-N-(4-(morpholinomethyl)thiazol-2-y-
l)-1H-benzo[d]imidazole-4-carboxamide (Compound 681). MS (ESI)
calcd for C.sub.23H.sub.19ClF.sub.3N.sub.5O.sub.2S: 521.09. found:
522 [M+H].
Example 20
Synthesis of
N-(5-(morpholinomethyl)thiazol-2-yl)-2-(2-(trifluoromethyl)phenyl)-1H-ben-
zo[d]imidazole-4-carboxamide (Compound 780)
Step 1) Preparation of 5-(morpholinomethyl)thiazol-2-amine (61)
##STR00085##
[0360] 5-(morpholinomethyl)thiazol-2-amine 61 was prepared using
the same synthetic sequence outlined above for
4-(morpholinomethyl)thiazol-2-amine employing ethyl
2-aminothiazole-5-carboxylate 60 as the starting material.
Step 2) Preparation of
N-(5-(morpholinomethyl)thiazol-2-yl)-2-(2-(trifluoromethyl)phenyl)-1H-ben-
zo[d]imidazole-4-carboxamide (Compound 780)
##STR00086##
[0362] The same general amide coupling procedure outlined above was
used to prepare of
N-(5-(morpholinomethyl)thiazol-2-yl)-2-(2-(trifluoromethyl)phenyl)-1H-ben-
zo[d]imidazole-4-carboxamide (Compound 780) from
2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid 3 and 5-(morpholinomethyl)thiazol-2-amine 61. MS (ESI) calcd
for C.sub.23H.sub.20F.sub.3N.sub.5O.sub.2S: 487.13. found: 488
[M+H].
Example 21
Synthesis of
(R)-2-(2-(difluoromethyl)-4-(2,3-dihydroxypropoxy)phenyl)-N-(thiazol-2-yl-
)-1H-benzo[d]imidazole-4-carboxamide (Compound 556)
Step 1) Preparation of
(S)-2-bromo-5-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)benzaldehyde
(64)
##STR00087##
[0364] To a solution of 2-bromo-5-hydroxybenzaldehyde (62; 5 g,
0.025 mol) in DMF (100 ml) was added
(R)-4-(chloromethyl)-2,2-dimethyl-1,3-dioxolane (63; 4.87 g, 0.032
mol) and K.sub.2CO.sub.3 (7.0 g, 0.05 mol). The resulting reaction
mixture was stirred at 150.degree. C. for 10 h, cooled to room
temperature, diluted with water, and then extracted with EtOAc. The
combined organic layers were dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure. The resulting residue was
purified by chromatography (petroleum ether/EtOAc=10:1 to afford
the desired product, namely
(S)-2-bromo-5-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)benzaldehyde
64 (4.1 g, 56%).
Step 2) Preparation of
(S)-4-((4-bromo-3-(difluoromethyl)phenoxy)methyl)-2,2-dimethyl-1,3-dioxol-
ane (65)
##STR00088##
[0366] To a solution of DAST (3.04 g, 0.019 mol) in 20 ml
CH.sub.2Cl.sub.2 was added dropwise a solution of
(S)-2-bromo-5-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)benzaldehyde
(64; 4.0 g, 0.013 mol) in 8 ml CH.sub.2Cl.sub.2 at room
temperature. The resulting reaction mixture was stirred at room
temperature for 10 h. It was then diluted with CH.sub.2Cl.sub.2 (50
mL), washed with water, dried (Na.sub.2SO.sub.4) and concentrated
under reduced pressure. Purification by chromatography (petroleum
ether/EtOAc=5:1) afforded
(S)-4-((4-bromo-3-(difluoromethyl)phenoxy)methyl)-2,2-dimethyl-1,3-dioxol-
ane 65 (3.3 g, 75%).
Step 3) Preparation of
(S)-2-(difluoromethyl)-4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)benzald-
ehyde (66)
##STR00089##
[0368] To a solution of
(S)-4-((4-bromo-3-(difluoromethyl)phenoxy)methyl)-2,2-dimethyl-1,3-dioxol-
ane (65; 3 g, 0.089 mol) in THF (30 ml) was added n-BuLi (2.5M
solution in hexane, 3.9 ml, 0.097 mol) at -78.degree. C. The
mixture was stirred at the same temperature for 1 h and DMF (0.929
g, 0.103 mol) was added dropwise. After stirring at -78.degree. C.
for an additional 20 min, saturated aqueous NH.sub.4Cl (30 ml) was
added. The resulting reaction mixture was warmed to room
temperature and extracted with Et.sub.2O (3.times.15 mL). The
combined organic layers were dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure. The resulting residue was
purified by chromatography (petroleum ether/EtOAc=5:1) to afford
the titled compound, namely
(S)-2-(difluoromethyl)-4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)benzald-
ehyde 66 (1.83 g, 72%).
Step 4) Preparation of
(S)-2-(2-(difluoromethyl)-4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)phen-
yl)-1H-benzo[d]imidazole-4-carboxylic acid (67)
##STR00090##
[0370] To a solution of
(S)-2-(difluoromethyl)-4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)benzald-
ehyde (66; 1.83 g, 6.40 mmol) in DMF (150 ml) was added
2,3-diaminobenzoic acid (1; 0.97 g, 6.40 mmol), and
Na.sub.2S.sub.2O.sub.5 (1.82 g, 9.60 mmol). The reaction mixture
was stirred at 110.degree. C. for 18 h. It was then cooled to room
temperature and diluted with water. The resulting solids were
collected by filtration and dried to afford
(S)-2-(2-(difluoromethyl)-4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)phen-
yl)-1H-benzo[d]imidazole-4-carboxylic acid 67 as dark yellow solid
(0.84 g, 51%). MS (ESI) calcd for
C.sub.21H.sub.20F.sub.2N.sub.5O.sub.5: 418.13. found: 419
[M+H].
Step 5) Preparation of
(S)-2-(2-(difluoromethyl)-4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)phen-
yl)-N-(3H-pyrrol-2-yl)-1H-benzo[d]imidazole-4-carboxamide (68)
##STR00091##
[0372]
(S)-2-(2-(difluoromethyl)-4-((2,2-dimethyl-1,3-dioxolan-4-yl)methox-
y)phenyl)-1H-benzo[d]imidazole-4-carboxylic acid (67; 100 mg, 0.239
mmol) was taken up in 10 ml of DMF along with 2-aminothiazole (4;
28.7 mg, 0.289 mmol), HATU (181.7 mg, 0.478 mmol) and DIEA (61.8
mg, 0.289 mmol). The resulting reaction mixture was stirred at room
temperature for 12 h and diluted with water. The precipitated
solids were collected by filtration and dried. Purification by
chromatography (EtOAc/Hexane=2:1) afforded
(S)-2-(2-(difluoromethyl)-4-((2,2-dimethyl-1,3-dioxolan-4-yl)met-
hoxy)phenyl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole-4-carboxamide 68
(77 mg, 65%). MS (ESI) calcd for
C.sub.24H.sub.22F.sub.2N.sub.4O.sub.4S: 500.13. found: 501
[M+H].
Step 6) Preparation of
(R)-2-(2-(difluoromethyl)-4-(2,3-dihydroxypropoxy)phenyl)-N-(thiazol-2-yl-
)-1H-benzo[d]imidazole-4-carboxamide (Compound 556)
##STR00092##
[0374] To a solution of
(S)-2-(2-(difluoromethyl)-4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)phen-
yl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole-4-carboxamide (68; 80 mg,
0.160 mmol) in MeOH (10 ml) was added concentrated HCl (1 ml) and
water (1 ml). The resulting reaction mixture was stirred under
reflux for 2 h. It was then cooled to room temperature and enough 1
N NaOH was added to adjust the pH=8-9. The resulting mixture was
extracted with EtOAc. The combined organic layers were dried
(Na.sub.2SO.sub.4) and concentrated under reduced pressure.
Purification by chromatography (petroleum ether/EtOAc=2:1) afforded
(R)-2-(2-(difluoromethyl)-4-(2,3-dihydroxypropoxy)phenyl)-N-(thiazol-2-yl-
)-1H-benzo[d]imidazole-4-carboxamide (Compound 556) (67 mg, 91%).
MS (ESI) calcd for C.sub.21H.sub.18F.sub.2N.sub.4O.sub.4S.
Example 22
Synthesis of
6-morpholino-N-(thiazol-2-yl)-2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]im-
idazole-4-carboxamide (Compound 628)
Step 1) Preparation of 3-amino-5-chloro-2-nitrobenzoic acid
(77)
##STR00093##
[0376] To a mixture of potassium tert-butoxide (38.9 g, 0.347 mol)
and copper-(II) acetate monohydrate (0.9 g, 4.96 mmol) in DMF (160
ml) was added a solution of O-methylhydroxylamine hydrochloride
(8.3 g, 99.3 mmol) and 3-chloro-6-nitrobenzoic acid (69; 10 g, 49.6
mmol) in DMF (160 ml) at 0.degree. C. The resulting reaction
mixture was stirred at that temperature for 3 h. It was then
quenched with water, acidified with 10% HCl and extracted with
EtOAc. The combined organic layers were dried (Na.sub.2SO.sub.4)
and concentrated under reduced pressure. The residue was taken up
in EtOAc and extracted with 10% aqueous NaOH. The combined aqueous
layers were acidified with concentrated HCl to a pH=3 and then
extracted with EtOAc. The combined organic layers were dried
(Na.sub.2SO.sub.4) and concentrated under reduced pressure to
afford 3-amino-5-chloro-2-nitrobenzoic acid 70 (6.5 g, 60.5%) as a
brown red solid.
Step 2) Preparation of methyl 3-amino-5-chloro-2-nitrobenzoate
(71)
##STR00094##
[0378] Concentrated H.sub.2SO.sub.4 (1.5 ml) was added to a mixture
of 3-amino-5-chloro-2-nitrobenzoic acid (70; 3.5 g, 16.2 mmol) in
MeOH (120 ml). The resulting reaction mixture was stirred under
reflux for 3 days. It was then neutralized with saturated aqueous
Na.sub.2CO.sub.3 and extracted with EtOAc. The combined organic
layers were dried (Na.sub.2SO.sub.4) and concentrated under reduced
pressure. Purification by chromatography afforded methyl
3-amino-5-chloro-2-nitrobenzoate 71 (3.3 g, 88.5%) as a yellow
solid.
Step 3) Preparation of methyl 3-amino-5-morpholino-2-nitrobenzoate
(72)
##STR00095##
[0380] To a solution of 3-amino-5-chloro-2-nitrobenzoate (71; 3.3
g, 14.35 mmol) in DMF (66 ml) was added morpholine (48; 7.12 g,
81.78 mmol) and K.sub.2CO.sub.3 (11.28 g, 81.78 mmol). The
resulting reaction mixture was stirred at 105.degree. C. for 4 h.
It was then filtered, diluted with water (500 ml), and extracted
with EtOAc. The combined organic layers were dried
(Na.sub.2SO.sub.4) and concentrated under reduced pressure. Column
chromatography afforded methyl 3-amino-5-morpholino-2-nitrobenzoate
72 (2.1 g, 52.1%) as a brown yellow solid.
Step 4) Preparation of 2,3-diamino-5-morpholin-4-yl-benzoic acid
methyl ester (73)
##STR00096##
[0382] A solution of methyl 3-amino-5-morpholino-2-nitrobenzoate
(72; 2.1 g, 7.5 mmol) in EtOH (100 ml) was hydrogenated over 5%
Pd/C (200 mg) at room temperature. After 3 h, the reaction mixture
was filtered and concentrated under reduced pressure to afford
2,3-diamino-5-morpholin-4-yl-benzoic acid methyl ester 73 (1.3 g,
69.5%) as a brown yellow solid.
Step 5) Preparation of
6-morpholin-4-yl-2-(2-trifluoromethyl-phenyl)-1H-benzoimidazole-4-carboxy-
lic acid methyl ester (75)
##STR00097##
[0384] To a mixture of 2,3-diamino-5-morpholin-4-yl-benzoic acid
methyl ester (73; 600 mg, 2.39 mmol) in EtOH (60 ml) was added
sodium metabisulfite (373 mg, 1.96 mmol), H2O (2 ml) and
2-trifluoromethylbenzaldehyde (74; 990 mg, 5.69 mmol). The
resulting reaction mixture was stirred under reflux for 3 h. It was
then cooled to room temperature and concentrated under reduced
pressure. Purification by chromatography afforded
6-morpholin-4-yl-2-(2-trifluoromethyl-phenyl)-1H-benzoimidazole-4-carboxy-
lic acid methyl ester 75 (670 mg, 69.2%) as a yellow solid.
Step 6) Preparation of
6-morpholin-4-yl-2-(2-trifluoromethyl-phenyl)-1H-benzoimidazole-4-carboxy-
lic acid (76)
##STR00098##
[0386] A mixture containing
6-morpholin-4-yl-2-(2-trifluoromethyl-phenyl)-1H-benzoimidazole-4-carboxy-
lic acid methyl ester (75; 670 mg, 1.65 mmol) in 1M NaOH (27 ml)
was stirred at 70.degree. C. for 30 min. The reaction mixture was
neutralized with 10% aqueous HCl. The resulting precipitate was
collected by filtration and dried to afford
6-morpholin-4-yl-2-(2-trifluoromethyl-phenyl)-1H-benzoimidazole-4-carboxy-
lic acid 76 (600 mg, 93%) as a pale yellow solid.
Step 7) Preparation of
6-morpholino-N-(thiazol-2-yl)-2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]im-
idazole-4-carboxamide (Compound 628)
##STR00099##
[0388] The same general amide coupling procedure detailed earlier
was used to prepare
6-morpholino-N-(thiazol-2-yl)-2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]im-
idazole-4-carboxamide (Compound 628) from
6-morpholin-4-yl-2-(2-trifluoromethyl-phenyl)-1H-benzoimidazole-4-carboxy-
lic acid 76 and 2-aminothiazole 4. MS (ESI) calcd for
C.sub.22H.sub.18F.sub.3N.sub.5O.sub.2S: 473.11. found: 474
[M+H].
Example 23
Preparation of
N-(thiazol-2-yl)-2-(2-(trifluoromethyl)phenyl)-3H-imidazo[4,5-c]pyridine--
7-carboxamide (Compound 341)
Step 1) Preparation of 4-amino-5-nitro-nicotinic acid ethyl ester
(78)
##STR00100##
[0390] Potassium nitrate (20.5 g, 200 mmol) was added to an
ice-cooled solution of 4-aminopyridine-3-carboxylic acid (77; 27.6
g, 200 mmol) in 200 mL of concentrated H.sub.2SO.sub.4. The
resulting reaction mixture was stirred at room temperature for 20
min and then at 75.degree. C. for 3 h. Upon cooling to room
temperature, ethanol (540 mL) was added with stirring. The
resulting mixture was stirred at 60.degree. C. for 18 h and then
slowly added to an ice cold potassium acetate solution (800 g in
1500 mL of H.sub.2O). The resulting precipitate was collected by
filtration, washed with water and dried to afford
4-amino-5-nitro-nicotinic acid ethyl ester 78 (14.6 g, 35%).
Step 2) Preparation of 4,5-diamino-nicotinic acid ethyl ester
(79)
##STR00101##
[0392] A mixture containing 4-amino-5-nitro-nicotinic acid ethyl
ester (78; 15 g, 0.071 mol) and 10% Pd/C (500 mg) in MeOH (500 mL)
was stirred under 1 atm of hydrogen at room temperature for 18 h.
The reaction mixture filtered through a pad of Celite and the
filtrate was concentrated under reduced pressure to afford
4,5-diamino-nicotinic acid ethyl ester 79 (10 g, 80%).
Step 3) Preparation of
2-(2-trifluoromethyl-phenyl)-3H-imidazo[4,5-c]pyridine-7-carboxylic
acid ethyl ester (80)
##STR00102##
[0394] A mixture containing 4,5-diamino-nicotinic acid ethyl ester
(79; 1.81 g, 10 mmol), 2-trifluoromethylbenzaldehyde (1.9 g, 11
mmol), Na.sub.2S.sub.2O.sub.5 (9.5 g, 50 mmol) in 50 mL of DMF was
stirred at 120.degree. C. for 18 h. Upon cooling to room
temperature, the reaction mixture was poured into 100 mL of cold
water. The resulting solids were collected by filtration, washed
with water, and dried to provide
2-(2-trifluoromethyl-phenyl)-3H-imidazo[4,5-c]pyridine-7-carboxylic
acid ethyl ester 80 (2.35 g, 70%)
Step 4) Preparation of
2-(2-trifluoromethyl-phenyl)-3H-imidazo[4,5-c]pyridine-7-carboxylic
acid (81)
##STR00103##
[0396]
2-(2-Trifluoromethyl-phenyl)-3H-imidazo[4,5-c]pyridine-7-carboxylic
acid ethyl ester (80; 2.35 g, 7 mmol) was taken up in 10% aqueous
NaOH (40 mL) and ethanol (20 mL). The reaction mixture was stirred
under reflux for 30 min. Upon cooling to room temperature, the
reaction mixture was acidified with 5N HCl. The resulting yellow
solids were collected by filtration, washed with water, and dried
to afford
2-(2-trifluoromethyl-phenyl)-3H-imidazo[4,5-c]pyridine-7-carboxylic
acid 81 (1.77 g, 85%).
Step 5) Preparation of
N-(thiazol-2-yl)-2-(2-(trifluoromethyl)phenyl)-3H-imidazo[4,5-c]pyridine--
7-carboxamide (Compound 341)
##STR00104##
[0398] The same general amide coupling procedure detailed above was
used to prepare
N-(thiazol-2-yl)-2-(2-(trifluoromethyl)phenyl)-3H-imidazo[4,5-c]pyridine--
7-carboxamide (Compound 341) from
2-(2-trifluoromethyl-phenyl)-3H-imidazo[4,5-c]pyridine-7-carboxylic
acid 81 and 2-amiothiazole 4. MS (ESI) calcd for
C.sub.17H.sub.10F.sub.3N.sub.5OS: 389.06. found: 390 [M+H].
Example 24
Synthesis of
2-(4-fluoro-2-trifluoromethyl-phenyl)-3H-imidazo[4,5-c]pyridine-7-carboxy-
lic acid thiazol-2-ylamide (Compound 693)
Step 1) Preparation of
2-(4-fluoro-2-trifluoromethyl-phenyl)-3H-imidazo[4,5-c]pyridine-7-carboxy-
lic acid ethyl ester (83)
##STR00105##
[0400] A mixture containing ethyl 4,5-diaminonicotinate (79; 0.9 g,
5 mmol) and 4-fluoro-2-(trifluoromethyl)benzaldehyde (82; 1.0 g,
5.2 mmol) in nitrobenzene (50 mL) was stirred at 220.degree. C. for
18 h. Upon cooling to room temperature, the reaction mixture was
diluted with ether. The resulting solids were collected by
filtration, washed with ether, and dried to provide
2-(4-fluoro-2-trifluoromethyl-phenyl)-3H-imidazo[4,5-c]pyridine-7-carboxy-
lic acid ethyl ester 83 (600 mg, 39%).
Step 2) Preparation of
2-(4-fluoro-2-trifluoromethyl-phenyl)-3H-imidazo[4,5-c]pyridine-7-carboxy-
lic acid thiazol-2-ylamide (Compound 693)
##STR00106##
[0402] The same general procedure outlined above was used to
prepare
2-(4-fluoro-2-trifluoromethyl-phenyl)-3H-imidazo[4,5-c]pyridine-7-carboxy-
lic acid thiazol-2-ylamide (Compound 693), employing
2-(4-fluoro-2-trifluoromethyl-phenyl)-3H-imidazo[4,5-c]pyridine-7-carboxy-
lic acid ethyl ester 83 as the appropriate starting material. MS
(ESI) calcd for C.sub.17H.sub.9F.sub.4N.sub.5OS: 407.05. found: 408
[M+H].
Example 25
Synthesis of
6-methoxy-N-(pyridin-2-yl)-2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imida-
zole-4-carboxamide (Compound 632)
Step 1) Preparation of O-methylhydroxylamine (84)
[0403] Methoxylamine hydrochloride was treated with an excess of
50% KOH in small one piece distillation apparatus. The resulting
mixture was heated to 80 degree and the fraction that distilled
over at 45-50 degree was collected in a receiving flask containing
KOH pellets.
Step 2) Preparation of 3-amino-5-methoxy-2-nitrobenzoic acid
(85)
##STR00107##
[0405] A solution of freshly distilled o-methylhydroxylamine (3.00
g, 53.6 mmol) and 3-amino-5-methoxy-2-nitrobenzoic acid (84; 5.33
g, 27.0 mmol) in DME (100 mL) was added dropwise to a stirred
suspension of tBuOH (7 eq) and Cu(OAc).sub.2 (10 mol %) in DME (50
mL) over 10 min at room temperature. After stirring for 4-8 hours
at room temperature, it was quenched with water and enough
concentrated HCl was added to adjust the pH=3. The resulting
mixture was extracted with EtOAc. The combined organic layers were
dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure.
Purification by chromatography (CHCl.sub.3-MeOH-AcOH=8:2:0.1)
afforded 3-amino-5-methoxy-2-nitrobenzoic acid as a dark yellow
solid 85 (4.62 g).
Step 3) Preparation of 2,3-diamino-5-methoxybenzoic acid (86)
##STR00108##
[0407] 3-amino-5-methoxy-2-nitrobenzoic acid (85; 1.6 g, 7.5 mmol)
was dissolved in methanol (100 mL) and 10% Pd/C (300 mg) was added.
The resulting reaction mixture was stirred under 1 atm of hydrogen
at room temperature for 18 h. The reaction mixture was filtered
through a pad of Celite and the filtrate was concentrated under
reduced pressure to afford 2,3-diamino-5-methoxybenzoic acid 86
(0.7 g).
Step 4) Preparation of
6-methoxy-2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid (87)
##STR00109##
[0409] A mixture of 2,3-diamino-5-methoxybenzoic acid 86 (1.1
equiv) and 2-(trifluoromethyl)benzaldehyde 74 (1 equiv) and sodium
metabisulfite (3 equiv) in DMF was stirred under reflux for 12 h.
Upon cooling to room temperature, the reaction mixture was diluted
with water. The resulting solids were collected by filtration and
dried to afford
6-methoxy-2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid 87 as dark yellow solid (40%).
Step 5) Preparation of
6-methoxy-N-(pyridin-2-yl)-2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imida-
zole-4-carboxamide (Compound 632)
##STR00110##
[0411] The same general amide coupling procedure detailed earlier
was used to prepare
6-methoxy-N-(pyridin-2-yl)-2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imida-
zole-4-carboxamide (Compound 632) from
6-methoxy-2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid 87 and 2-aminopyridine 89. MS (ESI) calcd for
C.sub.21H.sub.15F.sub.3N.sub.4O.sub.2: 412.11. found: 413
[M+H].
Example 26
Synthesis of
N-(6-(pyrrolidin-1-ylmethyl)pyridin-2-yl)-2-(2-(trifluoromethyl)phenyl)-1-
H-benzo[d]imidazole-4-carboxamide (Compound 620)
Step 1) Preparation of ethyl 6-aminopicolinate (91)
##STR00111##
[0413] To a solution of 2-amino-6-pyridinecarboxylic acid (90; 6.0
g, 43.5 mmol) in ethanol (150 mL) was added SOCl.sub.2 (12.0 g, 101
mmol) at 0.degree. C. The resulting reaction mixture was stirred
under reflux for 12 h. Upon cooling to room temperature, the
reaction mixture was concentrated under reduced pressure. Enough
saturated aqueous Na.sub.2CO.sub.3 solution was added to adjust the
pH=9. The mixture was concentrated under reduced pressure and
dichloromethane (150 mL) was added to the resulting residue. The
mixture was stirred vigorously at room temperature for 30 min and
then filtered. The filtrate was concentrated under reduced pressure
to afford ethyl 6-aminopicolinate 91 (5.5 g, 76%).
Step 2) Preparation of ethyl 6-(tert-butoxycarbonylamino)picolinate
(92)
##STR00112##
[0415] To a solution of ethyl 6-aminopicolinate (91; 5.5 g, 33
mmol) in t-BuOH (120 mL) and acetone (40 mL) was added DMAP (0.08
g, 0.66 mmol) and di-t-butyl dicarbonate (10.8 g, 49.5 mmol). The
reaction mixture was stirred at room temperature for 18 h. The
solvent was removed by concentration under reduced pressure and a
mixture of hexane/dichloromethane (180 mL, 3:1) was added. The
resulting mixture was cooled to -20.degree. C. for 2 h. The
resulting solids were collected by filtration and dried to afford
ethyl 6-(tert-butoxycarbonylamino)picolinate 92 (11.0 g, 91%).
Step 3) Preparation of tert-butyl
6-(hydroxymethyl)pyridin-2-ylcarbamate (93)
##STR00113##
[0417] To a stirred solution of ethyl
6-(tert-butoxycarbonylamino)picolinate (92; 11.0 g, 33 mmol) in THF
(120 mL) under nitrogen was added LiAlH.sub.4 (3.80 g, 100 mmol) in
THF (60 mL) over a period of 30 min at 0.degree. C. The reaction
mixture was stirred at 0.degree. C. for 6 h and carefully quenched
by the addition of water (2.0 mL) and 10% NaOH solution (4.0 mL) at
0.degree. C. The reaction mixture was filtered and the filtrate was
dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure.
The resulting residue purified by chromatography (1:1 petroleum
ether:ethyl acetate) to afford tert-butyl
6-(hydroxymethyl)pyridin-2-ylcarbamate 93 (3.0 g, 41%).
Step 4) Preparation of
(6-(tert-butoxycarbonylamino)pyridin-2-yl)methyl methanesulfonate
(94)
##STR00114##
[0419] To a solution of tert-butyl
6-(hydroxymethyl)pyridin-2-ylcarbamate (93; 3.0 g, 13.4 mmol) and
DIPEA (5.0 g, 40 mmol) in acetonitrile (30 mL) was added MsCl (2.0
g, 17.4 mmol) over a period of 30 min at 0.degree. C. and the
mixture was stirred for 2 h at room temperature. The reaction was
quenched by adding saturated aqueous NaHCO.sub.3 and extracted with
ethyl acetate (3.times.60 mL). The combined organic layers were
washed with brine, dried (Na.sub.2SO.sub.4) and concentrated under
reduced pressure to afford essentially quantitative yield of crude
(6-(tert-butoxycarbonylamino)pyridin-2-yl)methyl methanesulfonate
94.
Step 5) Preparation of tert-butyl
6-(pyrrolidin-1-ylmethyl)pyridin-2-ylcarbamate (96)
##STR00115##
[0421] A mixture containing
(6-(tert-butoxycarbonylamino)pyridin-2-yl)methyl methanesulfonate
(94; 1.30 g, 3.2 mmol), pyrrolidine (95; 0.46 g, 6.4 mmol) and
K.sub.2CO.sub.3 (1.30 g, 9.6 mmol) in acetonitrile (15 mL) was
stirred at room temperature for 12 h. Saturated aqueous NaHCO.sub.3
was added and the mixture was concentrated under reduced pressure.
The resulting aqueous layer was extracted with EtOAc. The combined
organic layers were dried (Na.sub.2SO.sub.4) and concentrated under
reduced pressure to afford tert-butyl
6-(pyrrolidin-1-ylmethyl)pyridin-2-ylcarbamate 96 (0.75 g, 2.7
mmol, 62% for two steps).
Step 6) Preparation of 6-(pyrrolidin-1-yl)methyl)pyridin-2-amine
(97)
##STR00116##
[0423] To a solution of tert-butyl
6-(pyrrolidin-1-ylmethyl)pyridin-2-ylcarbamate (96; 750 mg, 2.7
mmol) in dichloromethane (10 mL) was added TFA (4.0 mL) at room
temperature. The resulting reaction mixture was stirred at room
temperature for 6 h and then concentrated under reduced pressure.
Enough saturated aqueous Na.sub.2CO.sub.3 was added to the
resulting residue to adjust the pH=9. The mixture was then
extracted with ethyl acetate (3.times.25 mL). The combined organic
layers were dried (Na.sub.2SO.sub.4) and concentrated under reduced
pressure to afford 6-(pyrrolidin-1-ylmethyl)pyridin-2-amine 97 (440
mg, 92%).
Step 7) Preparation of
N-(6-(pyrrolidin-1-ylmethyl)pyridin-2-yl)-2-(2-(trifluoromethyl)phenyl)-1-
H-benzo[d]imidazole-4-carboxamide (Compound 620)
##STR00117##
[0425] The same general amide coupling procedure detailed earlier
was used to prepare
N-(6-(pyrrolidin-1-ylmethyl)pyridin-2-yl)-2-(2-(trifluoromethyl)phenyl)-1-
H-benzo[d]imidazole-4-carboxamide (Compound 620) from
2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid 3 and 6-(pyrrolidin-1-ylmethyl)pyridin-2-amine 97. MS (ESI)
calcd for C.sub.25H.sub.22F.sub.3N.sub.5O: 465.18. found: 466
[M+H].
Example 27
Synthesis of
N-(5-(piperazin-1-ylmethyl)pyridin-2-yl)-2-(2-(trifluoromethyl)phenyl)-1H-
-benzo[d]imidazole-4-carboxamide (Compound 621)
Step 1) Preparation of ethyl 6-aminonicotinate (99)
##STR00118##
[0427] To a solution of 2-amino-5-pyridinecarboxylic acid (98; 5.0
g, 36.2 mmol) in ethanol (100 mL) was added SOCl.sub.2 (8.6 g, 72.4
mmol) at 0.degree. C. The resulting reaction mixture was stirred
under reflux for 12 h. Upon cooling to room temperature, the
reaction mixture was concentrated under reduced pressure and then
enough saturated aqueous Na.sub.2CO.sub.3 solution was added to the
reaction mixture to adjust the pH=9. The resulting solids were
collected by filtration and dried to afford ethyl 6-aminonicotinate
99 (5.64 g, 94%).
Step 2) Preparation of ethyl 6-(tert-butoxycarbonylamino)nicotinate
(100)
##STR00119##
[0429] To a solution of ethyl 6-aminonicotinate (99; 11.3 g, 68.3
mmol) in t-BuOH (120 mL) and acetone (40 mL) was added DMAP (0.17
g, 1.37 mmol) and di-t-butyl dicarbonate (30.0 g, 137 mmol). The
resulting reaction was stirred at room temperature for 18 h and
then concentrated under reduced pressure. A mixture of
hexane/dichloromethane (180 mL, 3:1) was added and the reaction
mixture was cooled to -20.degree. C. for 2 h. The resulting solids
were collected by filtration and dried to afford ethyl
6-(tert-butoxycarbonylamino)nicotinate 100 (17.0 g, 68%).
Step 3) Preparation of tert-butyl
5-(hydroxymethyl)pyridin-2-ylcarbamate (101)
##STR00120##
[0431] To a stirred solution of ethyl
6-(tert-butoxycarbonylamino)nicotinate (100; 10.0 g, 27.3 mmol) in
THF (40 mL) was added LiAlH4 (1.92 g, 50.5 mmol) in THF (60 mL)
over a period of 30 min at 0.degree. C. The resulting reaction
mixture was stirred at 0.degree. C. for 6 h. The reaction was then
quenched by carefully adding water (1.0 ml) and 10% NaOH solution
(2.0 mL) at 0.degree. C. The resulting mixture was filtered and the
filtrate was dried (Na.sub.2SO.sub.4) and concentrated under
reduced pressure. The resulting residue was purified by
chromatography (dichloromethane:methanol=40:1) to afford tert-butyl
5-(hydroxymethyl)pyridin-2-ylcarbamate 101 (4.50 g, 74%).
Step 4) Preparation of
(6-(tert-butoxycarbonylamino)pyridin-3-yl)methyl methanesulfonate
(102)
##STR00121##
[0433] To a mixture containing tert-butyl
5-(hydroxymethyl)pyridin-2-ylcarbamate (101; 4.5 g, 20.2 mmol) and
DIPEA (16.0 g, 121 mmol) in THF (45 mL) was added MsCl (6.93 g,
60.5 mmol) over a period of 30 min at 0.degree. C. The resulting
reaction mixture was stirred at room temperature for 12 h. The
mixture was then washed with water (2.times.5 mL), dried
(Na.sub.2SO.sub.4) and concentrated under reduced pressure. The
resulting residue was purified by chromatography (10:1 petroleum
ether/ethyl acetate) to afford
(6-(tert-butoxycarbonylamino)pyridin-3-yl)methyl methanesulfonate
102 (3.0 g, 61%).
Step 5) Preparation of benzyl
446-(tert-butoxycarbonylamino)pyridin-3-yl)methyl)piperazine-1-carboxylat-
e (104)
##STR00122##
[0435] A mixture of
(6-(tert-butoxycarbonylamino)pyridin-3-yl)methyl methanesulfonate
(102; 1.30 g, 5.4 mmol), benzyl piperazine-1-carboxylate 103; (1.42
g, 6.5 mmol), K.sub.2CO.sub.3 (2.30 g, 16.1 mmol) and NaI (0.08 g,
0.54 mmol) in DMF (20 mL) was stirred at 60.degree. C. for 2 h. The
reaction mixture was filtered and the filtrate was concentrated
under reduced pressure. Water (30 mL) was added and the mixture was
filtered. The collected solids were washed further with water and
dried to afford benzyl
4-((6-(tert-butoxycarbonylamino)pyridin-3-yl)methyl)piperazine-1-c-
arboxylate 104 (2.20 g, 96%).
Step 6) Preparation of benzyl
4-((6-aminopyridin-3-yl)methyl)piperazine-1-carboxylate (105)
##STR00123##
[0437] To a solution of benzyl
4-((6-(tert-butoxycarbonylamino)pyridin-3-yl)methyl)piperazine-1-carboxyl-
ate (104; 2.20 g, 5.20 mmol) in dichloromethane (10 mL) was added
TFA (3.4 g, 31.2 mmol) at room temperature. The resulting reaction
mixture was stirred at room temperature for 12 h and then
concentrated under reduced pressure. Enough saturated aqueous
Na.sub.2CO.sub.3 was added to adjust the pH=9. The resulting
mixture was then extracted with dichloromethane (3.times.25 mL).
The combined organic layers were dried (MgSO.sub.4) and
concentrated under reduced pressure to afford benzyl
4-((6-aminopyridin-3-yl)methyl)piperazine-1-carboxylate 105 (1.50
g, 89%).
Step 7) Preparation of benzyl
4-O-(2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxamido)pyr-
idin-3-yl)methyl)piperazine-1-carboxylate (106)
##STR00124##
[0439]
2-(2-(Trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid (3; 306 mg, 1.0 mmol) and benzyl
4-((6-aminopyridin-3-yl)methyl)piperazine-1-carboxylate 105 were
subjected to the same general amide coupling procedure detailed
earlier to prepare benzyl
4-((6-(2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxamido)p-
yridin-3-yl)methyl)piperazine-1-carboxylate 106 (280 mg,
45.6%).
Step 8) Preparation of
N-(5-(piperazin-1-ylmethyl)pyridin-2-yl)-2-(2-(trifluoromethyl)phenyl)-1H-
-benzo[d]imidazole-4-carboxamide (Compound 621)
##STR00125##
[0441] To a solution of benzyl
4-((6-(2-(2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxamido)p-
yridin-3-yl)methyl)piperazine-1-carboxylate (106; 280 mg, 0.46
mmol) in methanol (20 mL) was added Pd/C (0.1 g, 5% on carbon). The
reaction mixture was stirred under 1 atm of H.sub.2 for 12 h and
then filtered through a pad of Celite. The filtrate was
concentrated under reduced pressure. The resulting residue was
purified by chromatography (dichloromethane:methanol=5:1) to afford
N-(5-(piperazin-1-ylmethyl)pyridin-2-yl)-2-(2-(trifluoromethyl)phenyl)-1H-
-benzo[d]imidazole-4-carboxamide (Compound 621) (28.0 mg, 13%). MS
(ESI) calcd for C.sub.25H.sub.23F.sub.3N.sub.6O: 480.19. found: 481
[M+H].
Example 28
Synthesis of
2-(5-fluoro-2-(trifluoromethyl)phenyl)-N-(5-(morpholinomethyl)pyridin-2-y-
l)-1H-benzo[d]imidazole-4-carboxamide (Compound 624)
Step 1) Preparation of tert-butyl
5-(morpholinomethyl)pyridin-2-ylcarbamate (107)
##STR00126##
[0443] A mixture containing
(6-(tert-butoxycarbonylamino)pyridin-3-yl)methyl methanesulfonate
(102; 1.21 g, 5.0 mmol), morpholine (48; 1.31 g, 15.0 mmol),
K.sub.2CO.sub.3 (2.10 g, 15.0 mmol) and NaI (0.12 g, 0.75 mmol) in
DMF (30 mL) was stirred at 80.degree. C. for 4 h. Upon cooling to
room temperature, water (30 mL) was added and the mixture was
extracted with ethyl acetate (3.times.30 mL). The combined organic
layers were dried (MgSO.sub.4) and concentrated under reduced
pressure. The resulting residue was purified by chromatography
(petroleum ether: ethyl acetate=10:1 to 1:1) to afford tert-butyl
5-(morpholinomethyl)pyridin-2-ylcarbamate 107 (1.40 g, 95%).
Step 2) Preparation of 5-(morpholinomethyl)pyridin-2-amine
(108)
##STR00127##
[0445] To a solution of tert-butyl
5-(morpholinomethyl)pyridin-2-ylcarbamate (107; 1.40 g, 4.77 mmol)
in dichloromethane (10 mL) was added TFA (10.0 mL). The mixture was
stirred at room temperature for 12 h. The reaction mixture was
concentrated under reduced pressure. Enough saturated aqueous
Na.sub.2CO.sub.3 was added to the resulting residue to adjust the
pH=9. The mixture was extracted with dichloromethane (3.times.25
mL). The combined organic layers were dried (MgSO.sub.4) and
concentrated under reduced pressure to afford
5-(morpholinomethyl)pyridin-2-amine 108 (0.85 g, 92%).
Step 3) Preparation of
2-(5-fluoro-2-(trifluoromethyl)phenyl)-N-(5-(morpholinomethyl)pyridin-2-y-
l)-1H-benzo[d]imidazole-4-carboxamide (Compound 624)
##STR00128##
[0447] The same general amide coupling procedure detailed earlier
was used to prepare
2-(5-fluoro-2-(trifluoromethyl)phenyl)-N-(5-(morpholinomethyl)pyridin-2-y-
l)-1H-benzo[d]imidazole-4-carboxamide (Compound 624) from
5-(morpholinomethyl)pyridin-2-amine 108 and
2-(5-fluoro-2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid 109. MS (ESI) calcd for C.sub.25H.sub.21F.sub.4N.sub.5O.sub.2:
499.16. found: 500 [M+H].
Example 29
Synthesis of
2-(5-fluoro-2-(trifluoromethyl)phenyl)-N-(6-(morpholinomethyl)pyridin-3-y-
l)-1H-benzo[d]imidazole-4-carboxamide (Compound 686)
Step 1) Preparation of ethyl 5-aminopicolinate (111)
##STR00129##
[0449] To a solution of 5-aminopicolinic acid (110; 8.4 g, 60.8
mmol) in ethanol (100 mL) was added SOCl.sub.2 (14.5 g, 120 mmol)
at 0.degree. C. The mixture was stirred under reflux for 12 h. Upon
cooling to room temperature, the reaction mixture was concentrated
under reduced pressure. Enough saturated aqueous Na.sub.2CO.sub.3
solution was added to adjust pH=9. The resulting mixture was
filtered and the solids were dried under reduced pressure to afford
ethyl 5-aminopicolinate 111 (7.5 g, 75%).
Step 2) Preparation of ethyl 5-(tert-butoxycarbonylamino)picolinate
(112)
##STR00130##
[0451] To a mixture of ethyl 5-aminopicolinate (111; 7.5 g, 45
mmol) in t-BuOH (60 mL) and acetone (20 mL) was added DMAP (0.10 g,
0.9 mmol) and di-t-butyl dicarbonate (19.6 g, 90 mmol). The
resulting reaction mixture was stirred at room temperature for 18 h
and then concentrated under reduced pressure. Hexanes (150 mL) was
added and the mixture was cooled to -20.degree. C. for 2 h. The
precipitated solids were collected by filtration and dried to
afford ethyl 5-(tert-butoxycarbonylamino)picolinate 112 (8.9 g,
53%).
Step 3) Preparation of tert-butyl
6-(hydroxymethyl)pyridin-3-ylcarbamate (113)
##STR00131##
[0453] To a stirred solution of ethyl
5-(tert-butoxycarbonylamino)picolinate (112; 8.9 g, 24.0 mmol) in
ethyl ether (200 mL) was added LiAlH4 (1.80 g, 48 mmol) in ethyl
ether (100 mL) over a period of 30 min at 0.degree. C. The
resulting reaction mixture was stirred at 0.degree. C. for 3 h and
then carefully quenched by carefully adding water (1.0 mL) and 10%
NaOH solution (2.0 mL). The mixture was filtered; the filtrate was
dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure to
afford tert-butyl 6-(hydroxymethyl)pyridin-3-ylcarbamate 113 (4.20
g, 78%).
Step 4) Preparation of
(5-(tert-butoxycarbonylamino)pyridin-2-yl)methyl methanesulfonate
(114)
##STR00132##
[0455] To a solution of tert-butyl
6-(hydroxymethyl)pyridin-3-ylcarbamate (113; 4.20 g, 18.8 mmol) and
DIPEA (7.0 g, 56.4 mmol) in THF (20 mL) was added MsCl (2.8 g, 24.4
mmol) over a period of 30 min at 0.degree. C. The resulting
reaction mixture was stirred at 0.degree. C. for 1 h and then
quenched by adding saturated aqueous NaHCO.sub.3. The mixture was
extracted with ethyl acetate (3.times.60 mL). The combined organic
layers were washed with brine, dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure to afford
(5-(tert-butoxycarbonylamino)pyridin-2-yl)methyl methanesulfonate
114 (5.50 g). This material was used in the next step without
further purification.
Step 5) Preparation of tert-butyl
6-(morpholinomethyl)pyridin-3-ylcarbamate (115)
##STR00133##
[0457] A mixture containing
(5-(tert-butoxycarbonylamino)pyridin-2-yl)methyl methanesulfonate
(114; 1.70 g), morpholine (48; 1.0 g, 11.3 mmol) and
K.sub.2CO.sub.3 (2.30 g, 16.9 mmol) in acetonitrile (30 mL) was
stirred at room temperature for 12 h. Water (30 mL) was added and
the mixture was extracted with ethyl acetate (3.times.30 mL). The
combined organic layers were dried (MgSO.sub.4) and concentrated
under reduced pressure. The resulting residue was purified by
chromatography (petroleum ether:ethyl acetate=1:1 to 1:3) to afford
tert-butyl 6-(morpholinomethyl)pyridin-3-ylcarbamate 115 (1.20 g,
71% for two steps).
Step 6) Preparation of 6-(morpholinomethyl)pyridin-3-amine
(116)
##STR00134##
[0459] To a solution of tert-butyl
6-(morpholinomethyl)pyridin-3-ylcarbamate (115; 1.20 g, 4.1 mmol)
in dichloromethane (20 mL) was added TFA (6.0 mL). The resulting
reaction mixture was stirred at room temperature for 12 h and then
concentrated under reduced pressure. Enough saturated aqueous
Na.sub.2CO.sub.3 was added to the residue to adjust the pH=9. The
mixture was extracted with ethyl acetate (3.times.25 mL). The
combined organic layers were dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure to afford
6-(morpholinomethyl)pyridin-3-amine 116 (450 mg, 56%).
Step 7) Preparation of
2-(5-fluoro-2-(trifluoromethyl)phenyl)-N-(6-(morpholinomethyl)pyridin-3-y-
l)-1H-benzo[d]imidazole-4-carboxamide (Compound 686)
##STR00135##
[0461] The same general amide coupling procedure detailed earlier
was used to prepare
2-(5-fluoro-2-(trifluoromethyl)phenyl)-N-(6-(morpholinomethyl)pyridin-3-y-
l)-1H-benzo[d]imidazole-4-carboxamide (Compound 686) from
6-(morpholinomethyl)pyridin-3-amine 116 and
2-(5-fluoro-2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid 109. MS (ESI) calcd for C.sub.25H.sub.21F.sub.4N.sub.5O.sub.2:
499.16. found: 500 [M+H].
Example 30
Synthesis of
N-(5-methylthiazol-2-yl)-2-(4-(trifluoromethyl)pyridin-3-yl)-1H-benzo[d]i-
midazole-4-carboxamide (Compound 703)
Step 1) Preparation of methyl 2,3-diaminobenzoate (118)
##STR00136##
[0463] Methyl 2-amino-3-nitrobenzoate (117; 3.2 g) was dissolved in
EtOAc (100 mL) and 10% Pd on C (200 mg) was added. The resulting
reaction mixture was hydrogenated under 1 atm of hydrogen at room
temperature for 2 days and filtered through a pad of Celite. The
filtrate was concentrated under reduced pressure to afford
essentially quantitative yield of methyl 2,3-diaminobenzoate
118.
Step 2) Preparation of
2-(4-(trifluoromethyl)pyridin-3-yl)-1H-benzo[d]imidazole-4-carboxylic
acid (119)
##STR00137##
[0465] 4-(Trifluoromethyl)nicotinic acid (88; 1.00 g, 5.23 mmol)
was taken up in DMF (8 mL) along with methyl 2,3-diaminobenzoate
(118; 0.86 g, 5.23 mmol), HATU (2.98 g, 7.84 mmol) and DIEA (1.82
mL, 10.5 mmol). The reaction mixture was stirred at room
temperature for 18 h. It was then diluted with EtOAc, washed with
water, dried (Na.sub.2SO.sub.4) and concentrated under reduced
pressure to afford crude methyl
2-amino-3-(4-(trifluoromethyl)nicotinamido)benzoate. This material
was taken up in 5 mL of glacial acetic acid and stirred at
60.degree. C. for 18 h. The reaction mixture was stirred at
90.degree. C. for an additional 8 h. It was then cooled to room
temperature and concentrated under reduced pressure. The resulting
residue was diluted with EtOAc, washed with dilute aqueous
NaHCO.sub.3, dried (Na.sub.2SO.sub.4) and concentrated under
reduced pressure to afford crude methyl
2-(4-(trifluoromethyl)pyridin-3-yl)-1H-benzo[d]imidazole-4-carboxylate.
This material was taken up in MeOH (8 mL) and H.sub.2O (8 mL)
containing LiOH (0.4 g) and stirred at room temperature for 18 h.
The reaction mixture was concentrated under reduced pressure and
neutralized with 6 N HCl. The resulting solids were collected by
filtration and dried to afford 570 mg of
2-(4-(trifluoromethyl)pyridin-3-yl)-1H-benzo[d]imidazole-4-carboxylic
acid (35% yield from methyl 2,3-diaminobenzoate) 119. MS (ESI)
calcd for C.sub.14H.sub.8F.sub.3N.sub.3O.sub.2: 307.06. found: 308
[M+H].
Step 3) Preparation of
N-(5-methylthiazol-2-yl)-2-(4-(trifluoromethyl)pyridin-3-yl)-1H-benzo[d]i-
midazole-4-carboxamide (Compound 703)
##STR00138##
[0467]
2-(4-(Trifluoromethyl)pyridin-3-yl)-1H-benzo[d]imidazole-4-carboxyl-
ic acid 119 was coupled to 2-amino-5-methylthiazole 171 using the
same general amide coupling procedure shown above to afford
N-(5-methylthiazol-2-yl)-2-(4-(trifluoromethyl)pyridin-3-yl)-1H-benzo[d]i-
midazole-4-carboxamide (Compound 703). MS (ESI) calcd for
C.sub.18H.sub.12F.sub.3N.sub.5OS: 403.07. found: 404 [M+H].
Example 31
Synthesis of
2-(2-(difluoromethyl)-4-(2-morpholinoethoxy)phenyl)-N-(pyridin-2-yl)-1H-b-
enzo[d]imidazole-7-carboxamide (Compound 723)
Step 1) Preparation of 2-bromo-5-(2-morpholinoethoxy)benzaldehyde
(121)
##STR00139##
[0469] To a mixture of 2-bromo-5-hydroxybenzaldehyde (62; 3 g, 15
mmol) and 4-(2-chloroethyl)morpholine hydrochloride (120; 5.6 g, 30
mmol) in DMF (75 mL) was added K.sub.2CO.sub.3 (10.3 g, 74.6 mmol).
After reaction 2 h at 120.degree. C. for 3 h, the reaction mixture
was quenched by addition of water and extracted with EtOAc. The
organic layer was dried over Na.sub.2SO.sub.4 and concentrated
under reduced pressure. Column chromatography afforded
2-bromo-5-(2-morpholinoethoxy)benzaldehyde 121 (3.3 g, 72%) as a
brown solid.
Step 2) Preparation of
4-(2-(4-bromo-3-(difluoromethyl)phenoxy)ethyl)-morpholine (123)
##STR00140##
[0471] To a solution of 2-bromo-5-(2-morpholinoethoxy)benzaldehyde
(121; 4.7 g, 15 mmol) in CH.sub.2Cl.sub.2 (30 mL) was added a
solution of DAST (3.63 g, 22.5 mmol) in CH.sub.2Cl.sub.2 (15 mL) at
0.degree. C. The resulting reaction mixture was stirred under
reflux for 3 days. The reaction mixture was poured into saturated
aqueous NaHCO.sub.3, and extracted with CH.sub.2Cl.sub.2. The
combined organic layers were dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure. Column chromatography afforded
4-(2-(4-bromo-3-(difluoromethyl)phenoxy)ethyl)morpholine 123 (3.13
g, 63%) as a pale yellow oil.
Step 3) Preparation of
2-(difluoromethyl)-4-(2-morpholinoethoxy)benzaldehyde (124)
##STR00141##
[0473] To a solution of
4-(2-(4-bromo-3-(difluoromethyl)phenoxy)ethyl)morpholine (123; 2.8
g, 8.38 mmol) in THF (90 mL) was added n-BuLi (1.6 M solution in
hexanes, 7 mL, 11.2 mmol) at -78.degree. C. The resulting reaction
mixture was stirred at the same temperature for 1 h and DMF (1.24
g, 17 mmol) was added dropwise. After stirring the mixture at
-78.degree. C. for an additional 30 min, saturated aqueous
NH.sub.4Cl was added and the mixture was extracted with EtOAc. The
combined organic layers were dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure. Purification by chromatography
afforded 2-(difluoromethyl)-4-(2-morpholinoethoxy)benzaldehyde 124
(1.6 g, 67%) as a brown yellow oil.
Step 4) Preparation of
2-(2-(difluoromethyl)-4-(2-morpholinoethoxy)phenyl)-1H-benzo[d]imidazole--
7-carboxylic acid (125)
##STR00142##
[0475] 2,3-Diaminobenzoic acid (1; 222 mg, 1.5 mmol) and the
corresponding 2-(difluoromethyl)-4-(2-morpholinoethoxy)benzaldehyde
124 were subjected to the same general procedure detailed above to
afford
2-(2-(difluoromethyl)-4-(2-morpholinoethoxy)phenyl)-1H-benzo[d]imidazole--
7-carboxylic acid 125 (300 mg, 48%).
Step 5) Preparation of
2-(2-(difluoromethyl)-4-(2-morpholinoethoxy)phenyl)-N-(pyridin-2-yl)-1H-b-
enzo[d]imidazole-7-carboxamide (Compound 723)
##STR00143##
[0477] The same general amide coupling procedure detailed earlier
was used to prepare
2-(2-(difluoromethyl)-4-(2-morpholinoethoxy)phenyl)-N-(pyridin-2-yl)-1H-b-
enzo[d]imidazole-7-carboxamide (Compound 723) from
2-(2-(difluoromethyl)-4-(2-morpholinoethoxy)phenyl)-1H-benzo[d]imidazole--
7-carboxylic acid 125 and 2-aminopyridine 89. MS (ESI) calcd for
C.sub.25H.sub.21F.sub.4N.sub.5O.sub.2: 499.16. found: 500
[M+H].
Example 32
Synthesis of
(R)-2-(4-(2,3-dihydroxypropoxy)-2-(trifluoromethyl)phenyl)-N-(pyridin-2-y-
l)-1H-benzo[d]imidazole-4-carboxamide (Compound 757)
Step 1) Preparation of
(S)-4-((4-bromo-3-(trifluoromethyl)phenoxy)methyl)-2,2-dimethyl-1,3-dioxo-
lane (127)
##STR00144##
[0479] (S)-4-(Chloromethyl)-2,2-dimethyl-1,3-dioxolane (63; 3.3 g,
20.5 mmol) was added to a mixture containing
4-bromo-3-(trifluoromethyl)phenol (126; 5 g, 20 mmol) and
K.sub.2CO.sub.3 (8.30 g, 60 mmol) in 20 mL of DMF. The resulting
reaction mixture was stirred at 60.degree. C. for 6 h. Upon cooling
to room temperature, the mixture was poured into water and
extracted by EtOAc. The combined organic layers were dried
(Na.sub.2SO.sub.4) and concentrated under reduced pressure. The
resulting residue was purified by chromatography (petroleum
ether:EtOAc=50:1) to afford
(S)-4-((4-bromo-3-(trifluoromethyl)phenoxy)methyl)-2,2-dimethyl-1,-
3-dioxolane (4 g, 56%).
Step 2) Preparation of
(S)-4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-2-(trifluoromethyl)benzal-
dehyde (128)
##STR00145##
[0481] A solution of nBuLi in hexanes (6.76 mL, 1.6 M in hexanes)
was added dropwise to a solution of
(S)-4-((4-bromo-3-(trifluoromethyl)phenoxy)methyl)-2,2-dimethyl-1,3-dioxo-
lane (127; 3.20 g, 9 mmol) in THF (50 mL) at -78.degree. C. over a
period of 30 min. After stirring for additional 10 min, DMF (1.4
mL) was added dropwise over a period of 10 min. The resulting
reaction mixture was warmed to room temperature and stirred for 30
min. It was then quenched with water and extracted by EtOAc. The
combined organic layers were dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure. The resulting residue was
purified by chromatography (petroleum ether:EtOAc=50:1) to afford
(S)-4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-2-(trifluoromethyl-
)benzaldehyde 128 (2.3 g, 84%).
Step 3) Preparation of
(S)-2-(4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-2-(trifluoromethyl)phe-
nyl)-1H-benzo[d]imidazole-4-carboxylic acid (129)
##STR00146##
[0483]
(S)-4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-2-(trifluoromethyl)-
benzaldehyde 128 was reacted with 2,3-diaminobenzoic acid 1
following the same general procedure outlined earlier in order to
prepare
(S)-2-(4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-2-(trifluoromethyl)phe-
nyl)-1H-benzo[d]imidazole-4-carboxylic acid 129.
Step 4) Preparation
(R)-2-(4-(2,3-dihydroxypropoxy)-2-(trifluoromethyl)phenyl)-N-(pyridin-2-y-
l)-1H-benzo[d]imidazole-4-carboxamide (Compound 757)
##STR00147##
[0485]
(S)-2-(4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-2-(trifluorometh-
yl)phenyl)-1H-benzo[d]imidazole-4-carboxylic acid 129 was reacted
with 2-amionopyridine 89 using the same reaction sequence outlined
in Example 25, step 5 to prepare
(R)-2-(4-(2,3-dihydroxypropoxy)-2-(trifluoromethyl)phenyl)-N-(pyridin-2-y-
l)-1H-benzo[d]imidazole-4-carboxamide (Compound 757). MS (ESI)
calcd for C.sub.23H.sub.19F.sub.3N.sub.5O.sub.4: 472.14. found: 473
[M+H].
Example 33
Synthesis of
(S)--N-(3-(2,3-dihydroxypropoxy)phenyl)-2-(2-(trifluoromethyl)phenyl)-1H--
benzo[d]imidazole-4-carboxamide (Compound 725)
Step 1) Preparation of
(R)-2,2-dimethyl-4-((3-nitrophenoxy)methyl)-1,3-dioxolane (132)
##STR00148##
[0487] 3-Nitrophenol (130; 2.0 g, 14.37 mmol) was taken up in 20 mL
of anhydrous DMF along with anhydrous potassium carbonate (4.96 g,
35.93 mmol) and (R)-4-(chloromethyl)-2,2-dimethyl-1,3-dioxolane
(131; 2.55 mL, 18.68 mmol). The resulting reaction mixture was
heated in the microwave reactor, with stirring, at 160.degree. C.
for 4 h. The crude reaction mixture was rinsed with water, filtered
and extracted with dichloromethane (3.times.15 mL). The combined
organic layers were dried (Na.sub.2SO.sub.4) and concentrated under
reduced pressure. The resulting residue was purified by
chromatography using ethyl acetate: pentanes to obtain the desired
product 132 as an amber-colored oil (52%).
Step 2) Preparation of
(R)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)aniline (133)
##STR00149##
[0489] Under nitrogen, Fe powder (2.38 g, 42.54 mmol) and
NH.sub.4Cl (2.38 g, 42.54 mmol) were combined, followed by addition
of (R)-2,2-dimethyl-4-((3-nitrophenoxy)methyl)-1,3-dioxolane (132;
1.8 g, 7.09 mmol) and a 4:1 mixture of isopropanol:water (30 mL:10
mL). The reaction mixture was stirred under reflux for 18 h. The
crude material was filtered through a pad of Celite and the
filtrate was concentrated under reduced pressure. The resulting
aqueous layer was extracted with dichloromethane (3.times.15 mL).
The combined organic layers were dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure to afford
(R)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)aniline 133 (1.2 g,
79% yield). The material was used in the next step without any
further purification.
Step 3) Preparation of
(R)--N-(3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)phenyl)-2-(2-(trifluor-
omethyl)phenyl)-1H-benzo[d]imidazole-4-carboxamide (134)
##STR00150##
[0491]
2-(2-(Trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid (3; 75 mg, 0.24 mmol) was taken up in NMP (2 mL) along with
(R)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)aniline (133; 64.3
mg, 0.288 mmol) and HATU (186 mg, 0.49 mmol). DIEA (81 .mu.L, 0.49
mmol) was added and the resulting reaction mixture was stirred at
room temperature for 12 h. The temperature was then raised to
60.degree. C. and the reaction mixture was allowed to stir for an
additional 1 h. The reaction mixture was diluted with water and
filtered. The collected solids were further purified by silica gel
chromatography to afford the desired product 134, as a yellow solid
(51.5 mg, 42%).
Step 4) Preparation of
(S)--N-(3-(2,3-dihydroxypropoxy)phenyl)-2-(2-(trifluoromethyl)phenyl)-1H--
benzo[d]imidazole-4-carboxamide (Compound 725)
##STR00151##
[0493] To a solution of
(R)--N-(3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)phenyl)-2-(2-(trifluor-
omethyl)phenyl)-1H-benzo[d]imidazole-4-carboxamide (134; 51.5 mg,
0.1 mmol) in 3 mL of THF was added 4 N HCl dropwise until observe
color change, solution darkens, at which point an additional 1 mL
of HCL is added and the solution is allowed to stir at room
temperature for 18 h. The reaction mixture was diluted with MeOH
and then concentrated under reduced pressure. The resulting
yellowish suspension is purified by preparatory HPLC. The desired
product (Compound 725) was obtained as a white solid (32.5 mg,
64%). MS (ESI) calcd for C.sub.24H.sub.20F.sub.3N.sub.3O.sub.4:
471.14. found: 472 [M+H].
Example 34
Synthesis of
2-(3-(2,6-dimethylmorpholino)phenyl)-N-(thiazol-2-yl)-1H-benzo[d]imidazol-
e-4-carboxamide (Compound 576)
Step 1) Preparation of 2-(3-bromophenyl)-1,3-dioxane (136)
##STR00152##
[0495] A mixture containing 3-bromobenzaldehyde (135; 18.5 g, 100
mmol), propane-1,3-diol (9.1 g, 120 mmol) and TsOH (0.1 g) in
toluene (200 mL) was stirred under reflux in a Dean-Stark water
separator for 18 h. Upon cooling to room temperature, the mixture
was washed with 5% aqueous Na.sub.2CO.sub.3 (200 mL), dried
(Na2SO.sub.4) and concentrated under reduced pressure to afford
essentially quantitative yield of crude
2-(3-bromophenyl)-1,3-dioxane 136 as a yellow oil (25 g).
Step 2) Preparation of 3-(2,6-dimethylmorpholino)benzaldehyde
(137)
##STR00153##
[0497] A mixture containing 2-(3-bromophenyl)-1,3-dioxane (136; 2.0
g, 8.23 mmol), 2,6-dimethylmorpholine (122; 1.14 g, 9.88 mmol),
Pd.sub.2(dba).sub.3 (50 mg), NaOt-Bu (1.39 g, 14.0 mmol) and BINAP
(100 mg) in 20 mL of toluene was stirred at reflux for 5 h. Upon
cooling to room temperature, enough cold 1N HCl (50 mL) was added
to adjust the pH=1. The resulting reaction mixture was stirred at
room temperature for 1 h. NaOH(aq) was added to adjust the pH=11.
The aqueous mixture was extracted with CH.sub.2Cl.sub.2 (3.times.50
mL). The combined organic layers were dried (MgSO.sub.4) and
concentrated under reduced pressure. Purification by chromatography
(petroleum ether:EtOAc=10:1) afforded
3-(2,6-dimethylmorpholino)benzaldehyde 137 as a yellow oil (1.36 g,
yield: 72%).
Step 3) Preparation of
2-(3-((2,6-dimethylmorpholino)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid (138)
##STR00154##
[0499] 2,3-Diaminobenzoic acid (1; 0.94 g, 6.2 mmol) and
3-(2,6-dimethylmorpholino)-benzaldehyde (137; 1.36 g, 6.2 mmol)
were subjected to the same general conditions outlined earlier to
prepare
2-(3-((2,6-Dimethylmorpholino)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid 138 in an 87% yield (1.9 g).
Step 4) Preparation of
2-(3-(2,6-dimethylmorpholino)phenyl)-N-(thiazol-2-yl)-1H-benzo[d]imidazol-
e-4-carboxamide (Compound 576)
##STR00155##
[0501] The same general amide coupling procedure detailed earlier
was used to prepare
2-(3-(2,6-dimethylmorpholino)phenyl)-N-(thiazol-2-yl)-1H-benzo[d]imidazol-
e-4-carboxamide (Compound 576) from
2-(3-((2,6-Dimethylmorpholino)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid 138 and 2-aminothiazole 4. MS (ESI) calcd for
C.sub.23H.sub.25F.sub.2N.sub.5O.sub.3: 433.16. found: 434
[M+H].
Example 35
Synthesis of
(R)-2-(2-(difluoromethyl)phenyl)-N-(3-(2,3-dihydroxypropoxy)phenyl)-6-flu-
oro-1H-benzo[d]imidazole-4-carboxamide (Compound 811)
Step 1) Preparation of 5-fluoro-2-(2,2,2-trifluoroacetamido)benzoic
acid (140)
##STR00156##
[0503] 2-Amino-5-fluorobenzoic acid (139; 1.0 g, 6.4 mmol) was
taken up into 10 mL of anhydrous THF and the resulting mixture was
cooled to 0.degree. C. 2,2,2-Trifluoroacetic anhydride (3.6 mL) was
then added dropwise at 0.degree. C. The resulting reaction mixture
was stirred at room temperature for 16 h and then concentrated
under reduced pressure. The residue was treated with ice-cold, and
the resulting precipitate was collected by filtration, washed with
water and dried to afford
5-fluoro-2-(2,2,2-trifluoroacetamido)benzoic acid 140 (1.3 g,
80%).
Step 2) Preparation of
5-fluoro-3-nitro-2-(2,2,2-trifluoroacetamido)benzoic acid (141)
##STR00157##
[0505] 5-Fluoro-2-(2,2,2-trifluoroacetamido) benzoic acid (140;
1.29 g, 5.1 mmol) was added, in small portions, to fuming nitric
acid (7 mL) at 0.degree. C. The resulting reaction mixture was
stirred at 0.degree. C. for 1 hand then poured into ice-water (10
mL). The resulting precipitate was collected by filtration, washed
with water and dried to afford
5-fluoro-3-nitro-2-(2,2,2-trifluoroacetamido)benzoic acid 141 (1.2
g, 80%).
Step 3) Preparation of 2-amino-5-fluoro-3-nitrobenzoic acid
(142)
##STR00158##
[0507] To a solution of
5-fluoro-3-nitro-2-(2,2,2-trifluoroacetamido)benzoic acid (141; 1.2
g, 4.1 mmol) in ethanol (20 mL) was added 10% aqueous sodium
hydroxide solution (20 mL). The resulting reaction mixture was
stirred at 80.degree. C. for 3 h. Upon cooling to room temperature,
the reaction mixture was concentrated under reduced pressure. The
resulting orange solids were acidified with dilute HCl, filtered
and dried to afford 2-amino-5-fluoro-3-nitrobenzoic acid 142 (0.73
g, 90%).
Step 4) Preparation of 2,3-diamino-5-fluorobenzoic acid (143)
##STR00159##
[0509] 2-Amino-5-fluoro-3-nitrobenzoic acid (142; 0.73 g) was
dissolved into methanol (10 mL). To this solution was added 5% Pd/C
(0.1 g). The resulting reaction mixture was stirred at room
temperature under 1 atm of hydrogen for 18 h and then filtered
through a pad of Celite. The filtrate was concentrated under
reduced pressure to afford 2,3-diamino-5-fluorobenzoic acid 143 as
a brown solid (0.59 g, 95%).
Step 5) Preparation of
2-(2-(difluoromethyl)phenyl)-5-fluoro-1H-benzo[d]imidazole-7-carboxylic
acid (144)
##STR00160##
[0511] 2,3-Diamino-5-fluorobenzoic acid (143; 0.826 g, 5.29 mmol)
and 2-(difluoromethyl)benzaldehyde 144 were subjected to the same
general conditions outlined earlier to prepare
2-(2-(difluoromethyl)phenyl)-5-fluoro-1H-benzo[d]imidazole-7-carboxylic
acid 145, which was obtained in 65% yield (0.7 g). MS (ESI) calcd
for C.sub.15H.sub.9F.sub.3N.sub.2O.sub.2: 306.6. found: 307
[M+H].
Step 6) Preparation of
(S)-2-(2-(difluoromethyl)phenyl)-N-(3-((2,2-dimethyl-1,3-dioxolan-4-yl)me-
thoxy)phenyl)-6-fluoro-1H-benzo[d]imidazole-4-carboxamide (147)
##STR00161##
[0513]
2-(2-(difluoromethyl)phenyl)-5-fluoro-1H-benzo[d]imidazole-7-carbox-
ylic acid (145; 250 mg, 0.82 mmol) was coupled with
(S)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)aniline 146
(prepared as described for 133 in Example 33, steps 1-2, except
using (S)-4-(chloromethyl)-2,2-dimethyl-1,3-dioxolane in step 1) as
outlined earlier.
(S)-2-(2-(Difluoromethyl)phenyl)-N-(3-((2,2-dimethyl-1,3-dioxola-
n-4-yl)methoxy)phenyl)-6-fluoro-1H-benzo[d]imidazole-4-carboxamide
147 was obtained in 48% yield (200 mg). MS (ESI) calcd for
C.sub.27H.sub.24F.sub.3N.sub.3O.sub.4: 511.17. found: 512
[M+H].
Step 7) Preparation of
(R)-2-(2-(difluoromethyl)phenyl)-N-(3-(2,3-dihydroxypropoxy)phenyl)-6-flu-
oro-1H-benzo[d]imidazole-4-carboxamide (Compound 811)
##STR00162##
[0515] Ten drops of concentrated HCl were added to a solution of
(S)-2-(2-(difluoromethyl)phenyl)-N-(3-((2,2-dimethyl-1,3-dioxolan-4-yl)me-
thoxy)phenyl)-6-fluoro-1H-benzo[d]imidazole-4-carboxamide (147; 200
mg, 0.391 mmol) in methanol (15 mL). The resulting reaction mixture
was stirred at room temperature for 2 h and then concentrated under
reduced pressure to afford
(R)-2-(2-(difluoromethyl)phenyl)-N-(3-(2,3-dihydroxypropoxy)phenyl)-6-flu-
oro-1H-benzo[d]imidazole-4-carboxamide (Compound 811) (160 mg,
87%). MS (ESI) calcd for C.sub.27H.sub.24F.sub.3N.sub.3O.sub.4:
511.17. found: 512 [M+H].
Example 36
Synthesis of
2-(2-methylpyridin-3-yl)-N-(6-(morpholinomethyl)pyridin-2-yl)-1H-benzo[d]-
imidazole-4-carboxamide (Compound 814)
Step 1) Preparation of 2-methylnicotinaldehyde (149)
##STR00163##
[0517] To a solution of 3-bromo-2-methylpyridine (148; 10 g, 58.1
mmol) in THF (150 mL) was added n-BuLi (2.5 M, 25.6 mL) at
-78.degree. C. The reaction mixture was stirred at this temperature
for 1 h. DMF (1.30 mL) was then added and the resulting reaction
mixture was stirred for 1 h at -78.degree. C. The reaction was
quenched by the addition of aq. NH.sub.4Cl. Upon warming to room
temperature, the mixture was extracted with EtOAc. The combined
organic layers were dried (Na.sub.2SO.sub.4) and concentrated under
reduced pressure. The resulting residue was purified by
chromatography to afford 2-methylnicotinaldehyde 149 (2.18 g,
31%).
Step 2) Preparation of
2-(2-methylpyridin-3-yl)-1H-benzo[d]imidazole-4-carboxylic acid
(150)
##STR00164##
[0519] 2-Methylnicotinaldehyde (149; 2.18 g, 18 mmol) was subjected
to the standard conditions used to prepare substituted
benzimidazoles by reaction with 2,3-diaminobenzoic acid 1. The
desired product,
2-(2-methylpyridin-3-yl)-1H-benzo[d]imidazole-4-carboxylic acid
150, was obtained by purification using chromatography, followed by
crystallization from EtOAc (0.43 g, 11%).
Step 3) Preparation of
2-(2-methylpyridin-3-yl)-N-(6-(morpholinomethyl)pyridin-2-yl)-1H-benzo[d]-
imidazole-4-carboxamide (Compound 814)
##STR00165##
[0521] 2-(2-Methylpyridin-3-yl)-1H-benzo[d]imidazole-4-carboxylic
acid 150 was coupled with 6-morpholinomethylpyridin-2-amine 172
(prepared as set forth in Example 29, steps 1-6, except using
ethyl-6-aminopicolinate in step 1) by the standard amide coupling
procedure detailed earlier to prepare
2-(2-methylpyridin-3-yl)-N-(6-(morpholinomethyl)pyridin-2-yl)-1H--
benzo[d]imidazole-4-carboxamide (Compound 814). MS (ESI) calcd for
C.sub.24H.sub.24N.sub.6O.sub.2: 428.20. found: 429 [M+H].
Example 37
Synthesis of
2-(6-methylpyridin-3-yl)-N-(6-(morpholinomethyl)pyridin-2-yl)-1H-benzo[d]-
imidazole-4-carboxamide (Compound 815)
Step 1) Preparation of 6-methylnicotinaldehyde (152)
##STR00166##
[0523] To a solution of 5-bromo-2-methylpyridine (151; 10 g, 58.1
mmol) in THF (150 mL) was added n-BuLi (2.5 M, 25.6 mL) at
-78.degree. C. The reaction mixture was stirred at this temperature
for 1 h. DMF (1.30 mL) was then added and the resulting reaction
mixture was stirred for 1 h at -78.degree. C. The reaction was
quenched by the addition of aq. NH.sub.4Cl. Upon warming to room
temperature, the mixture was extracted with EtOAc. The combined
organic layers were dried (Na.sub.2SO.sub.4) and concentrated under
reduced pressure. The resulting residue was purified by
chromatography to afford 6-methylnicotinaldehyde 152 (5.0 g,
72%).
Step 2) Preparation of
2-(6-methylpyridin-3-yl)-N-(6-(morpholinomethyl)pyridin-2-yl)-1H-benzo[d]-
imidazole-4-carboxamide (Compound 815)
##STR00167##
[0525] 6-Methylnicotinaldehyde 152 was coupled with
2,3-diaminobenzoid acid 1 using the same standard conditions
detailed above to prepare the corresponding substituted
benzimidazole 153. The resulting
2-(6-methylpyridin-3-yl)-1H-benzo[d]imidazole-4-carboxylic acid 153
was coupled with 6-morpholinomethylpyridin-2-amine 172 using the
same standard amide coupling procedure to prepare
2-(6-methylpyridin-3-yl)-N-(6-(morpholinomethyl)pyridin-2-yl)-1H-benzo[d]-
imidazole-4-carboxamide (Compound 815). MS (ESI) calcd for
C.sub.24H.sub.24N.sub.6O.sub.2: 428.20. found: 429 [M+H].
Example 38
Synthesis of
2-(2-methylpyridin-4-yl)-N-(6-(morpholinomethyl)pyridin-2-yl)-1H-benzo[d]-
imidazole-4-carboxamide (Compound 816)
Step 1) Preparation of 2-methylisonicotinaldehyde (156)
##STR00168##
[0527] To a solution of 2,4-lutidine (154; 10 g, 93.3 mmol) in THF
(150 mL) was added n-BuLi (2.5 M, 41.1 mL) at -78.degree. C.
Diethylamine (8.19 g, 112 mmol) was then added at this temperature,
followed by DMF (10 mL). The resulting reaction mixture was stirred
at -78.degree. C. for 1 h. The reaction was quenched by the
addition of aq. NH.sub.4Cl. Upon warming to room temperature, the
mixture was extracted with CH.sub.2Cl.sub.2. The combined organic
layers were dried (Na.sub.2SO.sub.4) and concentrated under reduced
pressure to afford the intermediate enamine 155.
[0528] To a solution of NaIO.sub.4 (40 g) in water (200 mL) was
added the above enamine intermediate 155 in CH.sub.2Cl.sub.2 (200
mL). The reaction mixture was stirred at room temperature for 18 h.
Enough 2 N NaOH was then added to adjust the pH of the mixture to
8. The mixture was then filtered, separated and extracted with
CH.sub.2Cl.sub.2. The combined organic layers were dried
(Na.sub.2SO.sub.4) and concentrated under reduced pressure.
Purification by chromatography afforded 2-methylisonicotinaldehyde
156 (4 g, 35%).
Step 2) Preparation of
2-(2-methylpyridin-4-yl)-N-(6-(morpholinomethyl)pyridin-2-yl)-1H-benzo[d]-
imidazole-4-carboxamide (Compound 816)
##STR00169##
[0530] 2-Methylisonicotinaldehyde 156 was coupled with
2,3-diaminobenzoic acid 1 using subjected same standard conditions
detailed above to prepare
2-(2-methylpyridin-4-yl)-1H-benzo[d]imidazole-4-carboxylic acid
157, which was then coupled with 6-morpholinomethylpyridin-2-amine
172 using the same standard amide coupling procedure to prepare
2-(2-methylpyridin-4-yl)-N-(6-(morpholinomethyl)pyridin-2-yl)-1H-benzo[d]-
imidazole-4-carboxamide (Compound 816). MS (ESI) calcd for
C.sub.24H.sub.24N.sub.6O.sub.2: 428.20. found: 429 [M+H].
Example 39
Synthesis of
2-(4-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)-N-(thiazol-2-yl)--
1H-benzo[d]imidazole-4-carboxamide (Compound 828)
Step 1) Preparation of
2-(4-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)-1H-benzo[d]imidaz-
ole-4-carboxylic acid (159)
##STR00170##
[0532]
2-(4-Chloro-2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carbo-
xylic acid (54; 500 mg, 1.46 mmol) was suspended in
CH.sub.2Cl.sub.2 (15 mL). Methanol (2 mL) was added to solubilize.
To the reaction solution was added dropwise oxalyl chloride (130
.mu.L, 1.46 mmol). After having stirred the reaction at room
temperature overnight, the reaction was concentrated. The residue
was taken up in EtOAc and washed successively with saturated
NaHCO.sub.3 and then brine. The organic layer was dried over
MgSO.sub.4, filtered, and concentrated. Purification by silica gel
chromatography (EtOAc/pentane) afforded methyl
2-(4-chloro-2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxylate
as an orange solid (structure not shown) (390 mg, yield: 75%).
[0533] A sealed tube containing the mixture of methyl
2-(4-chloro-2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxylate
(390 mg, 1.10 mmol), potassium 1-trifluoroboratom-ethylpyrrolidine
(158; 212 mg, 1.11 mmol), Pd (OAc).sub.2 (22 mg, 0.03 mmol), XPhos
(31 mg, 0.06 mmol), and cesium carbonate (1.0 g, 3.3 mmol) was
evacuated and filled with nitrogen twice. To the mixture was added
THF/H.sub.2O (10:1, 4.4 mL). The suspension was heated at
80.degree. C. overnight and cooled to room temperature. Water (2
mL) was added and the reaction was extracted with CH.sub.2Cl.sub.2
twice. The combined organic layers were washed with brine, dried
over MgSO.sub.4, filtered, and concentrated. Purification by silica
gel chromatography (MeOH/CH.sub.2Cl.sub.2+1% Et.sub.3N) afforded
methyl
2-(4-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)-1H-benzo[d-
]imidazole-4-carboxylate (structure not shown) as a yellow solid
(544 mg).
[0534] Methyl
2-(4-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)-1H-benzo[d]imidaz-
ole-4-carboxylate (544 mg) and LiOH (95 mg, 4 mmol) were dissolved
in THF/H.sub.2O (1:1, 24 mL). The reaction was stirred overnight at
room temperature, then heated at 50.degree. C. overnight. The
solvents were removed in vacuo. The residue was taken up in water
and the pH was adjusted to 7-8 with 1N HCl. After allowing the
mixture to stirred for 1 hour, the solids were collected by
filtration to afford
2-(4-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)-1H-benzo[d]imidaz-
ole-4-carboxylic acid 159 as a pale solid (170 mg, 2-step yield:
40%). MS (ESI) calcd for C.sub.20H.sub.18F.sub.3N.sub.3O.sub.2:
389.14. found: 390 [M+H].
Step 2) Preparation of
2-(4-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)-N-(thiazol-2-yl)--
1H-benzo[d]imidazole-4-carboxamide (Compound 828)
##STR00171##
[0536] To a solution of
2-(4-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)-1H-benzo[d]imidaz-
ole-4-carboxylic acid (159; 60 mg, 0.154 mmol), 2-aminothiazole (4;
17 mg, 0.169 mmol), and HATU (117 mg, 0.308 mmol) in DMF (1.5 mL)
was added diisopropylethylamine (107 .mu.L, 0.616 mmol). The
reaction was heated by microwave at 140.degree. C. for 15 minutes.
The reaction mixture was diluted with EtOAc and washed successively
with water and brine. The organic layer was dried over MgSO.sub.4,
filtered, and concentrated. Purification by prep HPLC and
subsequent lyophilization with dilute aq. HCl afforded the HCl salt
of
2-(4-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)-N-(thiazol-2-yl)--
1H-benzo[d]imidazole-4-carboxamide (Compound 828) as a tan-colored
solid (24.3 mg, yield: 31%). MS (ESI) calcd for
C.sub.23H.sub.20F.sub.3N.sub.5OS: 471.13. found: 472 [M+H].
Example 40
Synthesis of
N-(pyridin-2-yl)-2-(4-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)--
1H-benzo[d]imidazole-4-carboxamide (Compound 838)
##STR00172##
[0538]
2-(4-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)-1H-benzo[d]-
imidazole-4-carboxylic acid 159 was coupled with 2-aminopyridine 89
using the same general procedure outlined above in Example 39 to
prepare
N-(pyridin-2-yl)-2-(4-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)--
1H-benzo[d]imidazole-4-carboxamide (Compound 838). MS (ESI) calcd
for C.sub.25H.sub.22F.sub.3N.sub.5O: 465.18. found: 466 [M+H].
Example 41
Synthesis of
2-(4-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)-N-(4-(pyrrolidin--
1-ylmethyl)phenyl)-1H-benzo[d]imidazole-4-carboxamide (Compound
869)
##STR00173##
[0540]
2-(4-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)-1H-benzo[d]-
imidazole-4-carboxylic acid 159 was coupled with
4-(pyrrolidin-1-ylmethyl)aniline 160 to produce
2-(4-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)-N-(4-(pyrrolidin--
1-ylmethyl)phenyl)-1H-benzo[d]imidazole-4-carboxamide (Compound
869) using
[0541] The same general procedure outlined in Example 39. MS (ESI)
calcd for C.sub.31H.sub.32F.sub.3N.sub.5O: 547.26. found: 548
[M+H].
Example 42
Synthesis of
2-(4-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)-N-p-tolyl-1H-benz-
o[d]imidazole-4-carboxamide (Compound 870)
##STR00174##
[0543]
2-(4-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)-1H-benzo[d]-
imidazole-4-carboxylic acid 159 was coupled with p-toluidine 161 to
produce
2-(4-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)-N-p-tolyl-
-1H-benzo[d]imidazole-4-carboxamide (Compound 870) using the same
general procedure outlined in Example 39. as the starting material.
MS (ESI) calcd for C.sub.27H.sub.25F.sub.3N.sub.4O: 478.20. found:
479 [M+H].
Example 43
Synthesis of
2-(5-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)-N-(thiazol-2-yl)--
1H-benzo[d]imidazole-4-carboxamide (Compound 871)
##STR00175##
[0545] The same general procedure outlined above in Example 39,
steps 1 and 2, except for using
2-(5-chloro-2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid 163 as the starting material, was used to prepare
2-(5-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)-N-(thiazol-2-yl)--
1H-benzo[d]imidazole-4-carboxamide (Compound 871). MS (ESI) calcd
for C.sub.23H.sub.20F.sub.3N.sub.5OS: 471.13. found: 472 [M+H].
Example 44
Synthesis of
N-(pyridin-2-yl)-2-(5-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)--
1H-benzo[d]imidazole-4-carboxamide (Compound 872)
##STR00176##
[0547] The same general procedure outlined above in Example 39,
step 1 and Example 40, except for using
2-(5-chloro-2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid 163 as the starting material, was used to prepare
N-(pyridin-2-yl)-2-(5-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)--
1H-benzo[d]imidazole-4-carboxamide (Compound 872). MS (ESI) calcd
for C.sub.25H.sub.22F.sub.3N.sub.5O: 465.18. found: 466 [M+H].
Example 45
Synthesis of
2-(5-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)-N-(4-(pyrrolidin--
1-ylmethyl)phenyl)-1H-benzo[d]imidazole-4-carboxamide (Compound
873)
##STR00177##
[0549] The same general procedure outlined above in Example 39,
step 1 and Example 41, except for using
2-(5-chloro-2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid 163 as the starting material, was used to prepare
2-(5-(pyrrolidin-1-ylmethyl)-2-(trifluoromethyl)phenyl)-N-(4-(pyrrolidin--
1-ylmethyl)phenyl)-1H-benzo[d]imidazole-4-carboxamide (Compound
873). MS (ESI) calcd for C.sub.31H.sub.32F.sub.3N.sub.5O: 547.26.
found: 548 [M+H].
Example 46
Synthesis of
2-(4-chloro-2-(trifluoromethyl)phenyl)-N-(6-morpholinopyridin-2-yl)-1H-be-
nzo[d]imidazole-4-carboxamide (Compound 868)
Step 1) Preparation of 6-morpholinopyridin-2-amine (165)
##STR00178##
[0551] A mixture containing 4-chloro-2-aminopyridine (164; 26 g,
0.20 mol), K.sub.2CO.sub.3 (0.40 mol) and morpholine (48; 0.6 mol)
in DMSO (150 mL) was stirred at 190.degree. C. for 10 h. Upon
cooling to room temperature, the reaction mixture was diluted with
water (300 mL) and the resulting mixture was extracted with ethyl
acetate (4.times.150 mL). The combined organic layers were washed
with water (3.times.25 mL), dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure. The resulting residue was
purified by chromatography (petroleum ether:ethyl acetate=10:1) to
afford 6-morpholinopyridin-2-amine 165 (17 g, 47%) as a white
solid.
Step 2) Preparation of
2-(4-chloro-2-(trifluoromethyl)phenyl)-N-(6-morpholinopyridin-2-yl)-1H-be-
nzo[d]imidazole-4-carboxamide (Compound 868)
##STR00179##
[0553] 6-Morpholinopyridin-2-amine 165 was coupled with
2-(4-chloro-2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-4-carboxylic
acid 54 using the same general amide coupling procedure described
above to prepare
2-(4-chloro-2-(trifluoromethyl)phenyl)-N-(6-morpholinopyridin-2-yl)-1H-be-
nzo[d]imidazole-4-carboxamide (Compound 868). MS (ESI) calcd for
C.sub.24H.sub.19ClF.sub.3N.sub.5O.sub.2: 501.12. found: 502
[M+H].
Example 47
Biological Activity
[0554] A mass spectrometry based assay was used to identify
modulators of SIRT1 activity. The mass spectrometry based assay
utilizes a peptide having 20 amino acid residues as follows:
Ac-EE-K(biotin)-GQSTSSHSK(Ac)NleSTEG-K(5TMR)-EE-NH2 (SEQ ID NO: 1)
wherein K(Ac) is an acetylated lysine residue and Nle is a
norleucine. The peptide is labeled with the fluorophore 5TMR
(excitation 540 nm/emission 580 nm) at the C-terminus. The sequence
of the peptide substrate is based on p53 with several
modifications. In addition, the methionine residue naturally
present in the sequence was replaced with the norleucine because
the methionine may be susceptible to oxidation during synthesis and
purification.
[0555] The mass spectrometry assay is conducted as follows: 0.5
.mu.M peptide substrate and 120 .mu.M .beta.NAD.sup.+ is incubated
with 10 nM SIRT1 for 25 minutes at 25.degree. C. in a reaction
buffer (50 mM Tris-acetate pH 8, 137 mM NaCl, 2.7 mM KCl, 1 mM
MgCl.sub.2, 5 mM DTT, 0.05% BSA). Test compounds may be added to
the reaction as described above. The SirT1 gene is cloned into a
T7-promoter containing vector and transformed into BL21(DE3). After
the minute incubation with SIRT1, 10 .mu.L of 10% formic acid is
added to stop the reaction. Reactions are sealed and frozen for
later mass spec analysis. Determination of the mass of the
substrate peptide allows for precise determination of the degree of
acetylation (i.e. starting material) as compared to deacetylated
peptide (product).
[0556] A control for inhibition of sirtuin activity is conducted by
adding 1 .mu.L of 500 mM nicotinamide as a negative control at the
start of the reaction (e.g., permits determination of maximum
sirtuin inhibition). A control for activation of sirtuin activity
is conducted using 10 nM of sirtuin protein, with 1 .mu.L of DMSO
in place of compound, to determine the amount of deacetylation of
the substrate at a given timepoint within the linear range of the
assay. This timepoint is the same as that used for test compounds
and, within the linear range, the endpoint represents a change in
velocity.
[0557] For the above assay, SIRT1 protein was expressed and
purified as follows. The SirT1 gene was cloned into a T7-promoter
containing vector and transformed into BL21(DE3). The protein was
expressed by induction with 1 mM IPTG as an N-terminal His-tag
fusion protein at 18.degree. C. overnight and harvested at
30,000.times.g. Cells were lysed with lysozyme in lysis buffer (50
mM Tris-HCl, 2 mM Tris[2-carboxyethyl]phosphine (TCEP), 10 .mu.M
ZnCl.sub.2, 200 mM NaCl) and further treated with sonication for 10
min for complete lysis. The protein was purified over a Ni-NTA
column (Amersham) and fractions containing pure protein were
pooled, concentrated and run over a sizing column (Sephadex S200
26/60 global). The peak containing soluble protein was collected
and run on an Ion-exchange column (MonoQ). Gradient elution (200
mM-500 mM NaCl) yielded pure protein. This protein was concentrated
and dialyzed against dialysis buffer (20 mM Tris-HCl, 2 mM TCEP)
overnight. The protein was aliquoted and frozen at -80.degree. C.
until further use.
[0558] Sirtuin modulating compounds that activated SIRT1 were
identified using the assay described above and are shown below in
Table 1. The EC.sub.1.5 values for the activating compounds are
represented by A (EC.sub.1.5<1.0 uM), B (EC.sub.1.5 1-25 uM), C
(EC.sub.1.5>25 uM). The percent maximum fold activation is
represented by A (Fold activation >200%) or B (Fold Activation
<200%).
TABLE-US-00001 TABLE 1 Compd # [M + H].sup.+ Structure EC.sub.1.5
Fold Act. 200 321 ##STR00180## B A 201 316 ##STR00181## B A 202 389
##STR00182## A A 203 384 ##STR00183## A A 204 405 ##STR00184## A A
205 400 ##STR00185## A A 206 403 ##STR00186## B A 207 372
##STR00187## B A 208 389 ##STR00188## A A 209 389 ##STR00189## C B
210 407 ##STR00190## B B 211 346 ##STR00191## B A 212 339
##STR00192## B A 213 357 ##STR00193## B A 214 357 ##STR00194## C B
215 322 ##STR00195## B A 216 407 ##STR00196## C B 217 407
##STR00197## C B 218 407 ##STR00198## B A 219 355 ##STR00199## B A
220 373 ##STR00200## B A 221 401 ##STR00201## C B 222 397
##STR00202## C B 223 397 ##STR00203## C B 224 413 ##STR00204## B A
225 404 ##STR00205## B A 226 406 ##STR00206## A A 227 322
##STR00207## B A 228 371 ##STR00208## B A 229 311 ##STR00209## B A
230 326 ##STR00210## A B 231 356 ##STR00211## A A 232 404
##STR00212## B A 233 383 ##STR00213## B A 234 383 ##STR00214## A A
235 373 ##STR00215## B A 236 390 ##STR00216## B B 237 322
##STR00217## B B 238 328 ##STR00218## B B 239 390 ##STR00219## C B
240 446 ##STR00220## C B 241 384 ##STR00221## C B 242 383
##STR00222## B B 243 383 ##STR00223## A A 244 340 ##STR00224## B A
245 356 ##STR00225## B A 246 356 ##STR00226## B B 247 407
##STR00227## A A 248 383 ##STR00228## B A 249 311 ##STR00229## B A
250 407 ##STR00230## B A 251 406 ##STR00231## A A 252 397
##STR00232## B A 253 401 ##STR00233## A A 254 387 ##STR00234## A A
255 397 ##STR00235## B A 256 406 ##STR00236## B A 257 373
##STR00237## B A 258 423 ##STR00238## B A 259 408 ##STR00239## B A
260 403 ##STR00240## B B 261 401 ##STR00241## C B 262 383
##STR00242## A A 263 407 ##STR00243## A A 264 407 ##STR00244## B A
265 357 ##STR00245## B A 266 373 ##STR00246## B A 267 420
##STR00247## B A 268 406 ##STR00248## A A 269 407 ##STR00249## A A
270 397 ##STR00250## C B 271 397 ##STR00251## C B 272 415
##STR00252## B A 273 390 ##STR00253## B A 274 417 ##STR00254## A A
275 390 ##STR00255## B A 276 374 ##STR00256## C B 277 384
##STR00257## B A 278 400 ##STR00258## B A 279 411 ##STR00259## A A
280 390 ##STR00260## B A 281 339 ##STR00261## A A 282 406
##STR00262## B A 283 465 ##STR00263## B A 284 439 ##STR00264## B A
285 417 ##STR00265## B A 286 417 ##STR00266## B A 287 400
##STR00267## A A 288 400 ##STR00268## A A 289 400 ##STR00269## A A
290 423 ##STR00270## A A 291 415 ##STR00271## A A 292 342
##STR00272## A A 293 342 ##STR00273## B A 294 356 ##STR00274## B A
295 427 ##STR00275## A A 296 390 ##STR00276## B A 297 401
##STR00277## A A 298 389 ##STR00278## B A 299 387 ##STR00279## B A
300 384 ##STR00280## A A 301 375 ##STR00281## C B 302 407
##STR00282## A A 303 325 ##STR00283## B A 304 356 ##STR00284## A A
305 405 ##STR00285## A A 306 403 ##STR00286## B B 307 417
##STR00287## B B 308 461 ##STR00288## B A 309 418 ##STR00289## C B
310 381 ##STR00290## A A 311 381 ##STR00291## B A 312 417
##STR00292## A A 313 417 ##STR00293## A A 314 406 ##STR00294## B A
315 417 ##STR00295## A A 316 401 ##STR00296## A A 317 401
##STR00297## A A 318 350 ##STR00298## A A 319 350 ##STR00299## A A
320 350 ##STR00300## B A 321 441 ##STR00301## A A 322 417
##STR00302## A A
323 401 ##STR00303## A A 324 411 ##STR00304## A A 325 411
##STR00305## A A 326 411 ##STR00306## B A 327 421 ##STR00307## A A
328 421 ##STR00308## A A 329 399 ##STR00309## B A 330 433
##STR00310## B A 331 433 ##STR00311## A A 332 389 ##STR00312## B A
333 383 ##STR00313## B A 334 383 ##STR00314## B A 335 384
##STR00315## B A 336 399 ##STR00316## A A 337 399 ##STR00317## A A
338 433 ##STR00318## A A 339 450 ##STR00319## A A 340 450
##STR00320## A A 341 390 ##STR00321## A A 342 383 ##STR00322## A A
343 403 ##STR00323## A A 344 416 ##STR00324## A A 345 411
##STR00325## A A 346 437 ##STR00326## A A 347 450 ##STR00327## B A
348 389 ##STR00328## A A 349 383 ##STR00329## A B 350 414
##STR00330## B A 351 404 ##STR00331## B A 352 398 ##STR00332## B A
353 390 ##STR00333## C B 354 400 ##STR00334## A A 355 424
##STR00335## B A 356 434 ##STR00336## A A 357 421 ##STR00337## A A
358 435 ##STR00338## A A 359 429 ##STR00339## A A 360 389
##STR00340## A A 361 383 ##STR00341## A A 362 383 ##STR00342## A A
363 389 ##STR00343## B A 364 383 ##STR00344## B A 365 383
##STR00345## B A 366 384 ##STR00346## B A 367 400 ##STR00347## B A
368 418 ##STR00348## A A 369 412 ##STR00349## A A 370 412
##STR00350## A A 371 393 ##STR00351## A A 372 387 ##STR00352## A A
373 399 ##STR00353## B A 374 408 ##STR00354## C B 375 418
##STR00355## A A 376 421 ##STR00356## A A 377 435 ##STR00357## A A
378 408 ##STR00358## C B 379 429 ##STR00359## A A 380 418
##STR00360## A A 381 401 ##STR00361## A A 382 434 ##STR00362## A A
383 391 ##STR00363## B B 384 407 ##STR00364## B A 385 401
##STR00365## B A 386 401 ##STR00366## B A 387 402 ##STR00367## B A
388 401 ##STR00368## B A 389 401 ##STR00369## B A 390 399
##STR00370## B B 391 406 ##STR00371## B A 392 387 ##STR00372## B A
393 451 ##STR00373## A A 394 445 ##STR00374## A A 395 415
##STR00375## A A 396 420 ##STR00376## A A 397 407 ##STR00377## B A
398 428 ##STR00378## A A 399 417 ##STR00379## A A 400 367
##STR00380## A A 401 385 ##STR00381## B A 402 399 ##STR00382## A A
403 393 ##STR00383## B A 404 393 ##STR00384## A A 405 399
##STR00385## B B 406 393 ##STR00386## B A 407 393 ##STR00387## C B
408 418 ##STR00388## A A 409 412 ##STR00389## A A 410 412
##STR00390## A B 411 427 ##STR00391## A A 412 421 ##STR00392## A A
413 421 ##STR00393## A A 414 474 ##STR00394## A A 415 468
##STR00395## A A 416 423 ##STR00396## B B 417 417 ##STR00397## B A
418 417 ##STR00398## B A 419 418 ##STR00399## B A 420 397
##STR00400## B A 421 390 ##STR00401## C B 422 409 ##STR00402## A A
423 398 ##STR00403## B A 424 392 ##STR00404## A B 425 418
##STR00405## B A 426 420 ##STR00406## A A 427 401 ##STR00407## A A
428 420 ##STR00408## A A 429 417 ##STR00409## C B 430 468
##STR00410## A A 431 389 ##STR00411## C B 432 418 ##STR00412## A A
433 431 ##STR00413## B B 434 424 ##STR00414## B B 435 415
##STR00415## B B 436 408 ##STR00416## B B 437 408 ##STR00417## C B
438 407 ##STR00418## C B 439 389 ##STR00419## C B 440 407
##STR00420## A A 441 ##STR00421## A A 442 400 ##STR00422## A A 443
388 ##STR00423## C B 444 404 ##STR00424## C B 445 391 ##STR00425##
B A 446 391 ##STR00426## A B 447 420 ##STR00427## A A 448 474
##STR00428## A A
449 468 ##STR00429## A A 450 468 ##STR00430## A A 451 384
##STR00431## A A 452 421 ##STR00432## A A 453 423 ##STR00433## C B
454 407 ##STR00434## C B 455 405 ##STR00435## A A 456 402
##STR00436## A A 457 388 ##STR00437## A A 458 421 ##STR00438## A A
459 406 ##STR00439## B A 460 400 ##STR00440## B A 461 415
##STR00441## A A 462 406 ##STR00442## A A 463 400 ##STR00443## B A
464 405 ##STR00444## A A 465 402 ##STR00445## A A 466 385
##STR00446## B A 467 382 ##STR00447## B A 468 395 ##STR00448## C B
469 404 ##STR00449## B A 470 401 ##STR00450## B A 471 414
##STR00451## B A 472 428 ##STR00452## B B 473 388 ##STR00453## B A
474 444 ##STR00454## B A 475 464 ##STR00455## B A 476 478
##STR00456## B A 477 393 ##STR00457## A A 478 472 ##STR00458## A A
479 506 ##STR00459## A A 480 490 ##STR00460## A A 481 387
##STR00461## A A 482 434 ##STR00462## B A 483 449 ##STR00463## B A
484 443 ##STR00464## B A 485 491 ##STR00465## B A 486 485
##STR00466## B A 487 461 ##STR00467## B A 488 445 ##STR00468## B B
489 445 ##STR00469## B B 490 499 ##STR00470## A A 491 499
##STR00471## A A 492 472 ##STR00472## A A 493 506 ##STR00473## A A
494 490 ##STR00474## A A 495 441 ##STR00475## A A 496 395
##STR00476## A A 497 412 ##STR00477## B A 498 385 ##STR00478## B B
499 385 ##STR00479## B A 500 405 ##STR00480## A A 501 484
##STR00481## A A 502 478 ##STR00482## B A 503 468 ##STR00483## B B
504 462 ##STR00484## B B 505 498 ##STR00485## B A 506 492
##STR00486## B B 507 452 ##STR00487## B B 508 446 ##STR00488## B A
509 452 ##STR00489## A B 510 446 ##STR00490## B B 511 389
##STR00491## B A 512 403 ##STR00492## A A 513 383 ##STR00493## A A
514 371 ##STR00494## A A 515 385 ##STR00495## A A 516 365
##STR00496## A A 517 399 ##STR00497## B A 518 393 ##STR00498## B A
519 393 ##STR00499## A A 520 433 ##STR00500## B A 521 427
##STR00501## B A 522 427 ##STR00502## A A 523 404 ##STR00503## C B
524 485 ##STR00504## A A 525 499 ##STR00505## A A 526 467
##STR00506## A A 527 419 ##STR00507## A A 528 413 ##STR00508## A A
529 447 ##STR00509## A A 530 441 ##STR00510## A A 531 395
##STR00511## A A 532 415 ##STR00512## A A 533 386 ##STR00513## C B
534 406 ##STR00514## A A 535 414 ##STR00515## A A 536 414
##STR00516## B A 537 434 ##STR00517## A A 538 414 ##STR00518## B A
539 414 ##STR00519## B A 540 385 ##STR00520## A A 541 466
##STR00521## A A 542 500 ##STR00522## A A 543 502 ##STR00523## B B
544 496 ##STR00524## B B 545 464 ##STR00525## A A 546 458
##STR00526## B A 547 506 ##STR00527## B B 548 500 ##STR00528## A B
549 488 ##STR00529## A A 550 469 ##STR00530## A A 551 456
##STR00531## A A 552 470 ##STR00532## A A 553 450 ##STR00533## A A
554 450 ##STR00534## A A 555 451 ##STR00535## A A 556 461
##STR00536## A A 557 475 ##STR00537## A A 558 455 ##STR00538## A A
559 389 ##STR00539## A A 560 403 ##STR00540## A A 561 383
##STR00541## A A 562 405 ##STR00542## A A 563 419 ##STR00543## A A
564 399 ##STR00544## B A 565 386 ##STR00545## B B 566 414
##STR00546## 567 414 ##STR00547## A A 568 385 ##STR00548## C B 569
398 ##STR00549## B A 570 418 ##STR00550## B B 571 496 ##STR00551##
A A 572 458 ##STR00552## B A 573 452 ##STR00553## A A
574 470 ##STR00554## A A 575 470 ##STR00555## A A 576 434
##STR00556## A A 577 428 ##STR00557## A A 578 478 ##STR00558## B B
579 484 ##STR00559## C B 580 436 ##STR00560## B A 581 355
##STR00561## C B 582 355 ##STR00562## C B 583 468 ##STR00563## B A
584 462 ##STR00564## B A 585 486 ##STR00565## B B 586 480
##STR00566## B B 587 506 ##STR00567## B B 588 500 ##STR00568## B B
589 502 ##STR00569## A A 590 496 ##STR00570## A A 591 469
##STR00571## A A 592 485 ##STR00572## A A 593 451 ##STR00573## A A
594 461 ##STR00574## A A 595 475 ##STR00575## A A 596 455
##STR00576## A A 597 481 ##STR00577## A A 598 515 ##STR00578## A A
599 501 ##STR00579## A A 600 417 ##STR00580## B A 601 441
##STR00581## A A 602 432 ##STR00582## B A 603 502 ##STR00583## A A
604 496 ##STR00584## A A 605 452 ##STR00585## B A 606 476
##STR00586## A A 607 470 ##STR00587## A A 608 476 ##STR00588## A A
609 470 ##STR00589## B A 610 472 ##STR00590## B A 611 484
##STR00591## C B 612 478 ##STR00592## C B 613 478 ##STR00593## B B
614 470 ##STR00594## C B 615 464 ##STR00595## C B 616 430
##STR00596## B A 617 369 ##STR00597## B B 618 369 ##STR00598## C B
619 386 ##STR00599## B B 620 466 ##STR00600## A A 621 481
##STR00601## A A 622 500 ##STR00602## A A 623 484 ##STR00603## A A
624 500 ##STR00604## B A 625 484 ##STR00605## A A 626 464
##STR00606## B A 627 458 ##STR00607## B A 628 474 ##STR00608## A A
629 468 ##STR00609## A A 630 468 ##STR00610## A A 631 469
##STR00611## A A 632 413 ##STR00612## A A 633 467 ##STR00613## A A
634 419 ##STR00614## A A 635 453 ##STR00615## A A 636 384
##STR00616## A A 637 384 ##STR00617## A A 638 385 ##STR00618## A A
639 404 ##STR00619## A A 640 508 ##STR00620## A A 641 502
##STR00621## A A 642 502 ##STR00622## A A 643 503 ##STR00623## A A
644 492 ##STR00624## A A 645 486 ##STR00625## A A 646 486
##STR00626## A A 647 487 ##STR00627## A A 648 424 ##STR00628## A A
649 418 ##STR00629## A A 650 418 ##STR00630## A A 651 419
##STR00631## A A 652 438 ##STR00632## A A 653 408 ##STR00633## B A
654 402 ##STR00634## B A 655 402 ##STR00635## A A 656 403
##STR00636## A A 657 422 ##STR00637## A A 658 411 ##STR00638## A A
659 411 ##STR00639## B A 660 485 ##STR00640## A A 661 499
##STR00641## A A 662 502 ##STR00642## B A 663 496 ##STR00643## C B
664 458 ##STR00644## C B 665 476 ##STR00645## A A 666 470
##STR00646## B B 667 476 ##STR00647## B A 668 470 ##STR00648## A B
669 403 ##STR00649## C B 670 387 ##STR00650## C B 671 367
##STR00651## C B 672 482 ##STR00652## A A 673 482 ##STR00653## A A
674 482 ##STR00654## A A 675 516 ##STR00655## A A 676 516
##STR00656## A A 677 500 ##STR00657## A A 678 506 ##STR00658## A A
679 490 ##STR00659## A A 680 490 ##STR00660## A A 681 506
##STR00661## A A 682 481 ##STR00662## A A 683 466 ##STR00663## A A
684 516 ##STR00664## A A 685 500 ##STR00665## A A 686 500
##STR00666## A A 687 484 ##STR00667## A A 688 452 ##STR00668## C B
689 492 ##STR00669## A A 690 486 ##STR00670## A A 691 486
##STR00671## A A 692 487 ##STR00672## A A 693 408 ##STR00673## B B
694 402 ##STR00674## B B 695 402 ##STR00675## A A 696 403
##STR00676## B A 697 422 ##STR00677## A A 698 488 ##STR00678## A A
699 488 ##STR00679## A A
700 454 ##STR00680## A A 701 506 ##STR00681## A A 702 390
##STR00682## A A 703 404 ##STR00683## A A 704 384 ##STR00684## A A
705 490 ##STR00685## A A 706 502 ##STR00686## A A 707 496
##STR00687## A A 708 502 ##STR00688## A A 709 496 ##STR00689## A A
710 463 ##STR00690## A A 711 478 ##STR00691## A A 712 472
##STR00692## B B 713 455 ##STR00693## A A 714 455 ##STR00694## A A
715 489 ##STR00695## A A 716 489 ##STR00696## A A 717 472
##STR00697## A A 718 472 ##STR00698## A A 719 472 ##STR00699## A A
720 472 ##STR00700## A A 721 454 ##STR00701## A A 722 500
##STR00702## A A 723 494 ##STR00703## A A 724 494 ##STR00704## A A
725 472 ##STR00705## A A 726 506 ##STR00706## B A 727 490
##STR00707## B A 728 481 ##STR00708## A A 729 472 ##STR00709## B A
730 490 ##STR00710## B A 731 458 ##STR00711## A A 732 472
##STR00712## A A 733 452 ##STR00713## A A 734 456 ##STR00714## A A
735 450 ##STR00715## A A 736 450 ##STR00716## A A 737 451
##STR00717## A A 738 507 ##STR00718## A A 739 507 ##STR00719## A A
740 506 ##STR00720## A A 741 490 ##STR00721## A A 742 479
##STR00722## A A 743 493 ##STR00723## A A 744 473 ##STR00724## A A
745 479 ##STR00725## A A 746 493 ##STR00726## A A 747 473
##STR00727## A A 748 473 ##STR00728## A A 749 474 ##STR00729## A A
750 468 ##STR00730## A A 751 468 ##STR00731## A A 752 469
##STR00732## A A 753 405 ##STR00733## A A 754 416 ##STR00734## A A
755 400 ##STR00735## A A 756 400 ##STR00736## A A 757 473
##STR00737## A A 758 480 ##STR00738## A A 759 494 ##STR00739## A A
760 474 ##STR00740## A A 761 480 ##STR00741## A A 762 494
##STR00742## A A 763 474 ##STR00743## A A 764 440 ##STR00744## A A
765 454 ##STR00745## A A 766 434 ##STR00746## B A 767 468
##STR00747## A A 768 474 ##STR00748## A A 769 468 ##STR00749## A A
770 469 ##STR00750## A A 771 462 ##STR00751## A A 772 476
##STR00752## A A 773 456 ##STR00753## A A 774 434 ##STR00754## A A
775 435 ##STR00755## A A 776 435 ##STR00756## A A 777 489
##STR00757## A A 778 473 ##STR00758## A A 779 559 ##STR00759## A A
780 488 ##STR00760## C B 781 422 ##STR00761## A A 782 438
##STR00762## A A 783 405 ##STR00763## A A 784 422 ##STR00764## A A
785 440 ##STR00765## A A 786 440 ##STR00766## A A 787 489
##STR00767## A A 788 473 ##STR00768## A A 789 439 ##STR00769## A A
790 454 ##STR00770## A A 791 434 ##STR00771## A A 792 434
##STR00772## A A 793 425 ##STR00773## A A 794 405 ##STR00774## A A
795 421 ##STR00775## C B 796 454 ##STR00776## B B 797 405
##STR00777## A A 798 439 ##STR00778## A A 799 441 ##STR00779## A A
800 455 ##STR00780## A A 801 435 ##STR00781## A A 802 436
##STR00782## A A 803 436 ##STR00783## A A 804 457 ##STR00784## A A
805 471 ##STR00785## A A 806 451 ##STR00786## A A 807 451
##STR00787## A A 808 462 ##STR00788## A A 809 476 ##STR00789## A A
810 456 ##STR00790## A A 811 472 ##STR00791## A A 812 435
##STR00792## A A 813 483 ##STR00793## A A 814 429 ##STR00794## A A
815 429 ##STR00795## A A 816 429 ##STR00796## A A 817 464
##STR00797## A A 818 482 ##STR00798## A A 819 498 ##STR00799## A A
820 456 ##STR00800## B A 821 423 ##STR00801## A A 822 437
##STR00802## A A 823 417 ##STR00803## A A 824 407 ##STR00804## A
A
825 421 ##STR00805## A A 826 401 ##STR00806## A A 827 472
##STR00807## A A 828 472 ##STR00808## A A 829 473 ##STR00809## A A
830 473 ##STR00810## A A 831 479 ##STR00811## A A 832 389
##STR00812## A A 833 403 ##STR00813## A A 834 383 ##STR00814## A A
835 407 ##STR00815## A A 836 421 ##STR00816## A A 837 401
##STR00817## A A 838 466 ##STR00818## A A 839 479 ##STR00819## A A
840 473 ##STR00820## A A 841 473 ##STR00821## A A 842 435
##STR00822## A A 843 435 ##STR00823## A A 844 435 ##STR00824## A A
845 470 ##STR00825## A A 846 488 ##STR00826## A A 847 449
##STR00827## A A 848 455 ##STR00828## A A 849 504 ##STR00829## A A
850 415 ##STR00830## A A 851 432 ##STR00831## A A 852 450
##STR00832## A A 853 448 ##STR00833## A A 854 432 ##STR00834## A A
855 450 ##STR00835## A A 856 528 ##STR00836## A A 857 528
##STR00837## A A 858 453 ##STR00838## B A 859 460 ##STR00839## B A
860 466 ##STR00840## A A 861 530 ##STR00841## A A 862 528
##STR00842## A A 863 446 ##STR00843## A A 864 544 ##STR00844## A A
865 446 ##STR00845## A A 866 463 ##STR00846## A A 867 453
##STR00847## A A 868 502 ##STR00848## A A 869 548 ##STR00849## A A
870 479 ##STR00850## A A 871 472 ##STR00851## B A 872 466
##STR00852## A A 873 548 ##STR00853## A A
[0559] In another embodiment, the compound of the invention is
selected from any one of Compound numbers 202, 203, 208, 226, 243,
247, 254, 262, 269, 287, 288, 289, 290, 295, 300, 302, 304, 312,
313, 315, 316, 317, 319, 321, 323, 327, 328, 340, 343, 344, 345,
346, 354, 356, 357, 358, 359, 360, 361, 362, 368, 369, 370, 371,
372, 376, 377, 379, 380, 381, 382, 394, 395, 396, 398, 399, 409,
411, 412, 413, 414, 430, 432, 440, 446, 448, 451, 452, 455, 456,
457, 458, 461, 464, 465, 478, 479, 480, 481, 492, 493, 494, 495,
514, 515, 516, 531, 540, 541, 549, 550, 551, 552, 553, 554, 555,
556, 557, 558, 559, 560, 561, 573, 574, 575, 589, 590, 591, 592,
593, 594, 595, 596, 601, 603, 604, 620, 622, 623, 628, 629, 630,
631, 640, 641, 642, 643, 644, 645, 646, 647, 672, 674, 675, 677,
678, 679, 680, 681, 689, 690, 691, 692, 698, 699, 700, 701, 706,
717, 718, 719, 720, 721, 722, 723, 724, 725, 734, 735, 736, 737,
740, 741, 742, 743, 744, 745, 746, 749, 751, 767, 768, 769, 770,
774, 775, 776, 782, 785, 786, 789, 798, 811, 817, 818, 819, 820,
824, 825, 827, 829, 831, 832, 833, 834, 836, 839, 840, 841, 845,
847, 849, 851, 852, 853, 854, 855, 862, 863, 865, 866, 867, or 868
from Table 1, above.
EQUIVALENTS
[0560] The present invention provides among other things
sirtuin-activating compounds and methods of use thereof. While
specific embodiments of the subject invention have been discussed,
the above specification is illustrative and not restrictive. Many
variations of the invention will become apparent to those skilled
in the art upon review of this specification. The full scope of the
invention should be determined by reference to the claims, along
with their full scope of equivalents, and the specification, along
with such variations.
INCORPORATION BY REFERENCE
[0561] All publications and patents mentioned herein, including
those items listed below, are hereby incorporated by reference in
their entirety as if each individual publication or patent was
specifically and individually indicated to be incorporated by
reference. In case of conflict, the present application, including
any definitions herein, will control.
[0562] Also incorporated by reference in their entirety are any
polynucleotide and polypeptide sequences which reference an
accession number correlating to an entry in a public database, such
as those maintained by The Institute for Genomic Research (TIGR)
(www.tigr.org) and/or the National Center for Biotechnology
Information (NCBI) (www.ncbi.nlm.nih.gov).
Sequence CWU 1
1
1120PRTArtificial SequenceSynthetic peptide 1Glu Glu Lys Gly Gln
Ser Thr Ser Ser His Ser Lys Xaa Ser Thr Glu1 5 10 15 Gly Lys Glu
Glu 20
* * * * *