U.S. patent application number 12/640335 was filed with the patent office on 2010-04-15 for beta-carbolines useful for treating inflammatory disease.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Alfredo Castro, Julie Fields Liu, Paul E. Fleming, Michael E. Hepperle, Jeremy D. Little, Hormoz Mazdiyasni, Robert S. Murray, Raman Prakash, Dominic Reynolds, Francois Soucy, Yingchun Ye.
Application Number | 20100093713 12/640335 |
Document ID | / |
Family ID | 33299818 |
Filed Date | 2010-04-15 |
United States Patent
Application |
20100093713 |
Kind Code |
A1 |
Hepperle; Michael E. ; et
al. |
April 15, 2010 |
BETA-CARBOLINES USEFUL FOR TREATING INFLAMMATORY DISEASE
Abstract
This invention provides beta-carboline compounds of formula
##STR00001## wherein Ring A is a substituted pyridinyl,
pyrimidinyl, morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl,
pyranyl, tetrahydrofuranyl, cyclohexyl, cyclopentyl or
thiomorpholinyl ring and R.sup.1, R.sup.2 and R.sup.3 are as
described in the specification. The compounds are IKK-2 inhibitors
that are useful for treating IKK-2-mediated diseases such as
inflammatory diseases and cancer.
Inventors: |
Hepperle; Michael E.; (San
Diego, CA) ; Fields Liu; Julie; (Lexington, MA)
; Soucy; Francois; (Stoneham, MA) ; Ye;
Yingchun; (Belmont, MA) ; Murray; Robert S.;
(Framingham, MA) ; Prakash; Raman; (Acton, MA)
; Little; Jeremy D.; (Wakefield, MA) ; Castro;
Alfredo; (Winchester, MA) ; Mazdiyasni; Hormoz;
(Marlborough, MA) ; Fleming; Paul E.; (Wellesley,
MA) ; Reynolds; Dominic; (Stoneham, MA) |
Correspondence
Address: |
MILLENNIUM PHARMACEUTICALS, INC.
40 Landsdowne Street
CAMBRIDGE
MA
02139
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
Cambridge
MA
|
Family ID: |
33299818 |
Appl. No.: |
12/640335 |
Filed: |
December 17, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10821545 |
Apr 9, 2004 |
|
|
|
12640335 |
|
|
|
|
60461468 |
Apr 9, 2003 |
|
|
|
Current U.S.
Class: |
514/228.2 ;
514/232.5; 514/232.8; 514/256; 514/292; 544/126; 544/175; 544/333;
544/58.2; 544/82; 546/87 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 19/02 20180101; C07D 265/30 20130101; A61P 11/08 20180101;
A61P 1/04 20180101; A61P 17/06 20180101; C07D 471/04 20130101; A61P
35/04 20180101; A61P 35/02 20180101; A61P 11/06 20180101; A61P
29/00 20180101; A61P 37/00 20180101; A61P 25/28 20180101; A61P
35/00 20180101 |
Class at
Publication: |
514/228.2 ;
546/87; 544/333; 544/126; 544/58.2; 544/82; 514/232.5; 514/232.8;
514/256; 514/292; 544/175 |
International
Class: |
A61K 31/541 20060101
A61K031/541; C07D 471/04 20060101 C07D471/04; A61P 35/04 20060101
A61P035/04; A61P 37/00 20060101 A61P037/00; A61K 31/5377 20060101
A61K031/5377; A61P 29/00 20060101 A61P029/00; A61K 31/501 20060101
A61K031/501; A61K 31/437 20060101 A61K031/437; C07D 295/10 20060101
C07D295/10 |
Claims
1. A compound of formula I: ##STR00133## or a pharmaceutically
acceptable salt thereof, wherein: Ring A is selected from the group
consisting of: (a) a pyridinyl or pyrimidinyl ring that is
substituted by (i) --CH.sub.2C(O)-G and 0-1 R.sup.6a or (ii) 1-2
R.sup.6a, and (b) a morpholinyl, piperidinyl, piperazinyl,
pyrrolidinyl, pyranyl, tetrahydrofuranyl, cyclohexyl, cyclopentyl
or thiomorpholinyl ring that is substituted by (i)
--C(R.sup.9).sub.3, --W-G, or -G, (ii) 0-4 R.sup.6b and (iii) 0-1
oxo groups on a ring carbon or 0-2 oxo groups on a ring sulfur;
each R.sup.6a is independently selected from C.sub.1-6 aliphatic,
halo, alkoxy, or amino; each R.sup.6b is independently selected
from C.sub.1-3 aliphatic or --N(R.sup.7).sub.2, and two R.sup.6b on
the same or an adjacent carbon optionally are taken together with
the intervening carbon(s) to form a 5-6 membered ring having 1-2
ring heteroatoms selected from N, O or S; W is -Q-, -Q-C(O)--,
--C(R.sup.9).sub.2--C(R.sup.9)(R.sup.12)--, or
--C(R.sup.9).sub.2-[C(R.sup.9)(R.sup.12)].sub.2--; Q is
--C(R.sup.9).sub.2-- or --C(R.sup.9).sub.2C(R.sup.9).sub.2--; G is
--OH, --NR.sup.4R.sup.5, --N(R.sup.9)CONR.sup.4R.sup.5,
--N(R.sup.9)SO.sub.2 (C.sub.1-3 aliphatic),
--N(R.sup.9)COCF.sub.3--N(R.sup.9)CO(C.sub.1-6 aliphatic),
--N(R.sup.9)CO(heterocyclyl), --N(R.sup.9)CO(heteroaryl))
--N(R.sup.9)CO(aryl), a 3-7 membered heterocyclyl ring, or a 5-6
membered heteroaryl, wherein each of the heteroaryl, aryl and
heterocyclyl moieties of G is optionally substituted by 1-3
R.sup.10; R.sup.1 is hydrogen, halo, C.sub.1-3 aliphatic, amino,
cyano, (C.sub.1-3 alkyl).sub.1-2 amino, C.sub.1-3 alkoxy,
--CONH.sub.2, --NHCOCF.sub.3, or --CH.sub.2NH.sub.2; R.sup.2 is
hydrogen, halo, C.sub.1-3 aliphatic, --CF.sub.3; R.sup.3 is
hydrogen, halo, C.sub.1-6 aliphatic, C.sub.1-6 haloalkyl, C.sub.1-6
alkoxy, hydroxy, amino, cyano, or (C.sub.1-6 alkyl).sub.1-2 amino;
R.sup.4 is hydrogen, 3-7 membered heterocyclyl, or C.sub.1-6
aliphatic; R.sup.5 is hydrogen, C.sub.1-6 aliphatic group or a 3-7
membered heterocyclic ring having 1-2 ring heteroatoms selected
from N, O, or S, wherein R.sup.5 is optionally substituted by halo,
--OR', --CN, --SR.sup.O, --S(O).sub.2R.sup.8,
--S(O).sub.2N(R.sup.7).sub.2, --C(O)R.sup.7, --CO.sub.2R.sup.7,
--N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.13, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2; each R.sup.7 is independently
selected from hydrogen or C.sub.1-4 aliphatic, or two R.sup.7 on
the same nitrogen atom are taken together with the nitrogen to form
a 5-6 membered heteroaryl or heterocyclyl ring; each R.sup.8 is
independently selected from C.sub.1-4 aliphatic; each R.sup.9 is
independently selected from hydrogen or C.sub.1-3 aliphatic; each
R.sup.10 is independently selected from oxo, --R.sup.11,
-T-R.sup.11, or -V-T-R.sup.11; each R.sup.11 is independently
selected from C.sub.1-6 aliphatic, halo,
--S(O).sub.2N(R.sup.7).sub.2, --OR.sup.7, --CN, --SR.sup.8,
--S(O).sub.2R.sup.8, --C(O)R.sup.7, --CO.sub.2R.sup.7,
--N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.7, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2; T is a straight or branched
C.sub.1-4 alkylene chain; V is --O--, --N(R.sup.7)--, --S--,
--S(O)--, --S(O).sub.2--, --C(O)--, or --CO.sub.2--, and R.sup.12
is hydrogen or an amino acid side chain.
2. The compound of claim 1 where Ring A is a 3-pyridinyl or
5-pyrimidinyl ring substituted by 1-2 R.sup.6a groups.
3. The compound of claim 2 having formula II-C: ##STR00134##
wherein: Y is N or CH; R.sup.1 is hydrogen, halo, C.sub.1-3
aliphatic, amino, cyano, (C.sub.1-3 alkyl).sub.1-2 amino, C.sub.1-3
alkoxy, --CONH.sub.2, --NHCOCF.sub.3, or --CH.sub.2NH.sub.2;
R.sup.2 is hydrogen, halo, C.sub.1-3 aliphatic, --CF.sub.3; R.sup.3
is hydrogen, halo, C.sub.1-6 aliphatic, C.sub.1-6 haloalkyl,
C.sub.1-6 alkoxy, hydroxy, amino, cyano, or (C.sub.1-6
alkyl).sub.1-2 amino; and R.sup.6a is selected from C.sub.1-6
aliphatic or halo.
4. The compound of claim 3 where R.sup.6a is methyl.
5. The compound of claim 4 where R.sup.1 is hydrogen, methyl, amino
or fluoro; R.sup.2 is hydrogen or halo; and R.sup.3 is hydrogen,
halo or C.sub.1-4alkoxy.
6. The compound of claim 5 where Y is CH.
7. The compound of claim 5 where Y is N.
8. The compound of claim 1 where Ring A is selected from
morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, pyranyl,
tetrahydrofuranyl, cyclohexyl, cyclopentyl or thiomorpholinyl and
where Ring A is substituted by (i) --C(R.sup.9).sub.3 or --W-G,
(ii) 0-4 R.sup.6b and (iii) 0-1 oxo groups on a ring carbon or 0-2
oxo groups on a ring sulfur.
9. The compound of claim 8 where the --W-G or --C(R.sup.9).sub.3
substituent on Ring A is ortho to the position where the
beta-carboline portion is attached.
10. A compound of formula III-A: ##STR00135## or a pharmaceutically
acceptable salt thereof, wherein: Ring A is substituted by 0-4
R.sup.6b; each R.sup.6b is independently selected from C.sub.1-3
aliphatic or --N(R.sup.7).sub.2, and two R.sup.6b on the same or an
adjacent carbon optionally are taken together with the intervening
carbon(s) to form a 5-6 membered ring having 1-2 ring heteroatoms
selected from N, O or S; W is -Q-, -Q-C(O)--,
--C(R.sup.9).sub.2--C(R.sup.9)(R.sup.12)--, or
--C(R.sup.9).sub.2--[C(R.sup.9)(R.sup.12)].sub.2--; Q is
--C(R.sup.9).sub.2-- or --C(R.sup.9).sub.2C(R.sup.9).sub.2--; G is
--OH, --NR.sup.4R.sup.5, --N(R.sup.9)CONR.sup.4R.sup.5,
--N(R.sup.9)SO.sub.2 (C.sub.1-3 aliphatic), --N(R.sup.9)COCF.sub.3,
--N(R.sup.9)CO(C.sub.1-6 aliphatic),
--N(R.sup.9)CO(heterocyclyl)-N(R.sup.9)CO(heteroaryl)
--N(R.sup.9)CO(aryl), a 3-7 membered heterocyclyl ring, or a 5-6
membered heteroaryl, wherein each of the heteroaryl, aryl and
heterocyclyl moieties of G is optionally substituted by 1-3
R.sup.10; R.sup.1 is hydrogen, halo, C.sub.1-3 aliphatic, amino,
cyano, (C.sub.1-3 alkyl).sub.1-2 amino, C.sub.1-3 alkoxy,
--CONH.sub.2, --NHCOCF.sub.3, or --CH.sub.2NH.sub.2; R.sup.2 is
hydrogen, halo, C.sub.1-3 aliphatic, --CF.sub.3; R.sup.3 is
hydrogen, halo, C.sub.1-6 aliphatic, C.sub.1-6 haloalkyl, C.sub.1-6
alkoxy, hydroxy, amino, cyano, or (C.sub.1-6 alkyl).sub.1-2 amino;
R.sup.4 is hydrogen, 5-6 membered heterocyclyl, or C.sub.1-6
aliphatic; R.sup.5 is hydrogen, C.sub.1-6 aliphatic group or a 5-6
membered heterocyclic ring having 1-2 ring heteroatoms selected
from N, O, or S, wherein R.sup.5 is optionally substituted by halo,
--OR.sup.7, --CN, --SR.sup.8, --S(O).sub.2R.sup.8,
--S(O).sub.2N(R.sup.7).sub.2 (O)R.sup.7, --CO.sub.2R.sup.7,
--N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.8, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2; each R.sup.7 is independently
selected from hydrogen or C.sub.1-4 aliphatic, or two R.sup.7 on
the same nitrogen atom are taken together with the nitrogen to form
a 5-6 membered heteroaryl or heterocyclyl ring; each R.sup.8 is
independently selected from C.sub.1-4 aliphatic; each R.sup.9 is
independently selected from hydrogen or C.sub.1-3 aliphatic; each
R.sup.10 is independently selected from oxo, --R.sup.11,
-T-R.sup.11, or -V-T-R.sup.11; each R.sup.11 is independently
selected from C.sub.1-6 aliphatic, halo,
--S(O).sub.2N(R.sup.7).sub.2, --CN, --SR.sup.8,
--S(O).sub.2R.sup.8, --C(O)R.sup.7, --CO.sub.2R.sup.7,
--N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.7, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2; T is a straight or branched
C.sub.1-4 alkylene chain; V is --O--, --N(R.sup.7)--, --S--,
--S(O)--, --S(O).sub.2--, --C(O)--, or --CO.sub.2--; and R.sup.12
is hydrogen or an amino acid side chain.
11. The compound of claim 10 having the formula (S)-III-A:
##STR00136## where n is 0-4 and R.sup.1, R.sup.2, R.sup.3, W, G and
R.sup.6b are as defined in claim 10.
12. The compound of claim 11 where: R.sup.1 is hydrogen, halo,
methyl or amino; R.sup.2 is hydrogen, methyl or halo; R.sup.3 is
hydrogen, halo, alkoxy, or (C.sub.1-6 aliphatic).sub.2 amino; n is
0-2; R.sup.6b is C.sub.1-3 aliphatic; W is -Q-, -Q-C(O)--,
--C(R.sup.9).sub.2--C(R.sup.9)(R.sup.12)--, or
--C(R.sup.9).sub.2--[C(R.sup.9)(R.sup.12)].sub.2--; Q is
--C(R.sup.9).sub.2-- or --C(R.sup.9).sub.2C(R.sup.9).sub.2--; G is
--NR.sup.4R.sup.5, --N(R.sup.9)C(O)NR.sup.4R.sup.5,
--N(R.sup.9)SO.sub.2(C.sub.1-3 aliphatic),
--N(R.sup.9)C(O)CF.sub.3, --N(R.sup.9)CO(C.sub.1-6 aliphatic), and
--N(R.sup.9)CO(heterocyclyl)-N(R.sup.9)CO(heteroaryl)
--N(R.sup.9)CO(aryl), a 5-6 membered heterocyclyl ring, or a 5-6
membered heteroaryl, wherein each of the heteroaryl, aryl and
heterocyclyl moieties of G is optionally substituted by 1-3
R.sup.10; R.sup.4 is hydrogen or C.sub.1-6 aliphatic; R.sup.5 is
hydrogen or a C.sub.1-6 aliphatic group that is optionally
substituted by halo, --OR.sup.7, --CN, --SR.sup.8,
--S(O).sub.2R.sup.8, --S(O).sub.2N(R.sup.7).sub.2, --C(O)R.sup.7,
--CO.sub.2R.sup.7, --N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.8, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2; each R.sup.7 is independently
selected from hydrogen or C.sub.1-4 aliphatic, or two R.sup.7 on
the same nitrogen atom are taken together with the nitrogen to form
a 5-6 membered heteroaryl or heterocyclyl ring; each R.sup.8 is
independently selected from C.sub.1-4 aliphatic; R.sup.9 is
hydrogen; each R.sup.10 is independently selected from oxo,
R.sup.11, T-R.sup.11, or V-T-R.sup.11; each R.sup.11 is
independently selected from C.sub.1-6 aliphatic, halo,
--S(O).sub.2N(R.sup.7).sub.2, --OR.sup.7, --CN, --SR.sup.8,
--S(O).sub.2R.sup.8, --C(O)R.sup.7, --CO.sub.2R.sup.7,
--N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.7, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2; T is a straight or branched
C.sub.1-4 alkylene chain; V is --O--, --N(R.sup.7)--, --S--,
--S(O)--, --S(O).sub.2--, --C(O)--, or --CO.sub.2--; and R.sup.12
is hydrogen, C.sub.1-6 aliphatic, substituted or unsubstituted
phenyl, or substituted or unsubstituted benzyl.
13. The compound of claim 12 where: R.sup.1 is hydrogen, methyl,
fluoro or amino; R.sup.2 is chloro; R.sup.3 is hydrogen or alkoxy;
n is zero or 2; R.sup.6b is methyl; W is -Q-, -Q-C(O)-- or
--C(R.sup.9).sub.2--C(R.sup.9)(R.sup.12)--; Q is
--C(R.sup.9).sub.2-- or --C(R.sup.9).sub.2C(R.sup.9).sub.2--; G is
--NR.sup.4R.sup.5, --N(R.sup.9)C(O)NR.sup.4R.sup.5,
--N(R.sup.9)C(O)CF.sub.3, --N(R.sup.9)CO(C.sub.1-6 aliphatic), and
--N(R.sup.9)CO(heterocyclyl) --N(R.sup.9)CO(heteroaryl), a 5-6
membered heterocyclyl ring, or a 5-6 membered heteroaryl, wherein
each of the heteroaryl and heterocyclyl moieties of G is optionally
substituted by 1-3 R.sup.10; R.sup.4 is hydrogen or C.sub.1-6
aliphatic; R.sup.5 is hydrogen or C.sub.1-6 aliphatic; each R.sup.7
is independently selected from hydrogen or C.sub.1-4 aliphatic, or
two R.sup.7 on the same nitrogen atom are taken together with the
nitrogen to form a 5-6 membered heteroaryl or heterocyclyl ring;
each R.sup.8 is independently selected from C.sub.1-4 aliphatic;
R.sup.9 is hydrogen; each R.sup.10 is independently selected from
oxo, R.sup.11, T-R.sup.11, or V-T-R.sup.11; each R.sup.11 is
independently selected from C.sub.1-6 aliphatic, halo,
--S(O).sub.2N(R.sup.7).sub.2, --OR.sup.7, --CN, --SR.sup.8,
--S(O).sub.2R.sup.8, --C(O)R.sup.7, --CO.sub.2R.sup.7,
--N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.7, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2; T is a straight or branched
C.sub.1-4 alkylene chain; V is --O--, --N(R.sup.7)--, --S--,
--S(O)--, --S(O).sub.2--, --C(O)--, or --CO.sub.2--; and R.sup.12
is hydrogen, C.sub.1-6 aliphatic, phenyl, or benzyl.
14. A compound of formula (S)-III-A': ##STR00137## or a
pharmaceutically acceptable salt thereof, wherein: R.sup.1 is
hydrogen, methyl, fluoro or amino; R.sup.2 is chloro; R.sup.3 is
hydrogen or alkoxy; W is -Q-, -Q-C(O)-- or
--C(R.sup.8).sub.2--C(R.sup.8)(R.sup.12); Q is --C(R.sup.9).sub.2--
or --C(R.sup.8).sub.2C(R.sup.9).sub.2--; G is --NR.sup.4R.sup.5,
--N(R.sup.9)C(O)NR.sup.4R.sup.5, --N(R.sup.9)C(O)CF.sub.3,
--N(R.sup.9)CO(C.sub.1-6 aliphatic), and
--N(R.sup.8)CO(heterocyclyl) --N(R.sup.9)CO(heteroaryl), a 5-6
membered heterocyclyl ring, or a 5-6 membered heteroaryl, wherein
each of the heteroaryl and heterocyclyl moieties of G is optionally
substituted by 1-3 R.sup.10; R.sup.4 is hydrogen or C.sub.1-6
aliphatic; R.sup.5 is hydrogen or C.sub.1-6 aliphatic; each R.sup.7
is independently selected from hydrogen or C.sub.1-4 aliphatic, or
two R.sup.7 on the same nitrogen atom are taken together with the
nitrogen to form a 5-6 membered heteroaryl or heterocyclyl ring;
each R.sup.8 is independently selected from C.sub.1-4 aliphatic;
R.sup.9 is hydrogen; each R.sup.18 is independently selected from
oxo, R.sup.11, T-R.sup.11, or V-T-R.sup.11; each R.sup.11 is
independently selected from C.sub.1-6 aliphatic, halo,
--S(O).sub.2N(R.sup.7).sub.2, --CN, --SR.sup.a,
--S(O).sub.2R.sup.8, --C(O)R.sup.7, --CO.sub.2R.sup.7,
--N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.7, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2; T is a straight or branched
C.sub.1-4 alkylene chain; V is --O--, --N(R.sup.7)--, --S--,
--S(O)--, --S(O).sub.2--, --C(O)--, or --CO.sub.2--; and R.sup.12
is hydrogen, C.sub.1-6, aliphatic, phenyl, or benzyl.
15. The compound of claim 14 having the formula (S)-III-A-a:
##STR00138## where R.sup.1 is hydrogen, halo, methyl or amino;
R.sup.2 is hydrogen, methyl or halo; R.sup.3 is hydrogen, halo,
alkoxy, or (C.sub.1-6 aliphatic).sub.2 amino; Ring A is substituted
by 0-2 R.sup.6b; R.sup.6b is C.sub.1-3 aliphatic; Q is
--C(R.sup.9).sub.2-- or --C(R.sup.9).sub.2C(R.sup.9).sub.2--; G is
--NR.sup.4R.sup.5 or a substituted or unsubstituted 5-6 membered
heterocyclyl ring; R.sup.4 is hydrogen or C.sub.1-6 aliphatic;
R.sup.5 is hydrogen or a C.sub.1-6 aliphatic group that is
optionally substituted by halo, --OR.sup.7, --CN, --SR.sup.8,
--S(O).sub.2R.sup.8, --S(O).sub.2N(R.sup.7).sub.2, --C(O)R.sup.7,
--CO.sub.2R.sup.7, --N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.8, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2; each R.sup.7 is independently
selected from hydrogen or C.sub.1-4 aliphatic, or two R.sup.7 on
the same nitrogen atom are taken together with the nitrogen to form
a 5-6 membered heteroaryl or heterocyclyl ring; each R.sup.8 is
independently selected from C.sub.1-4 aliphatic; and each R.sup.9
is independently hydrogen or C.sub.1-3 aliphatic.
16. A compound selected from the group consisting of: ##STR00139##
##STR00140## ##STR00141## ##STR00142## ##STR00143## ##STR00144##
##STR00145## ##STR00146## ##STR00147## ##STR00148## ##STR00149##
##STR00150## ##STR00151## ##STR00152##
17. A pharmaceutical composition comprising a compound of claim 1
and a pharmaceutically acceptable carrier.
18. A pharmaceutical composition comprising a compound of claim 16
and a pharmaceutically acceptable carrier.
19. A method of treating an IKK-mediated disease comprising
administering to a patient in need of such treatment a
therapeutically effective amount of a compound of claim 1.
20. The method of claim 19 wherein the disease is an inflammatory
disease or an immune-related disease.
21. The method of claim 19 wherein the disease is selected from the
group consisting of rheumatoid arthritis, asthma, psoriasis,
psoriatic arthritis, chronic obstructive pulmonary disease,
inflammatory bowel disease or multiple sclerosis.
22. The method of claim 19 wherein the disease is cancer.
23. The method of claim 22 wherein the cancer is selected from
lymphoma, multiple myeloma, osteolytic bone mestasis, head or neck
cancer, lung cancer, prostate cancer or pancreatic cancer.
24. The method of claim 23 wherein the cancer is a lymphoma.
25. A method of inhibiting IRK in a patient in need thereof
comprising administering to the patient a compound of claim 1.
26. A compound of formula 3a: ##STR00153## where R.sup.13 is halo,
OH, OR.sup.15, or a carboxylic acid protecting group; R.sup.15 is
an aliphatic, aryl, heteroaryl, aralkyl, or heteroaralkyl; R.sup.14
is an amino protecting group, hydrogen or --W-G; W is -Q-,
-Q-C(O)--, --C(R.sup.9).sub.2--C(R.sup.9)(R.sup.12)--, or
--C(R.sup.9).sub.2--[C(R.sup.9)(R.sup.12)].sub.2--; Q is
--C(R.sup.9).sub.2-- or --C(R.sup.9).sub.2C(R.sup.9).sub.2--; G is
--OH, --NR.sup.4R.sup.5, --N(R.sup.9)CONR.sup.4R.sup.5,
--N(R.sup.9)SO.sub.2 (C.sub.1-3 aliphatic), --N(R.sup.9)COCF.sub.3,
--N(R.sup.9)CO(C.sub.1-6 aliphatic), --N(R.sup.9)CO(heterocyclyl),
--N(R.sup.9)CO(heteroaryl) --N(R.sup.9)CO(aryl), a 3-7 membered
heterocyclyl ring, or a 5-6 membered heteroaryl, wherein each of
the heteroaryl, aryl and heterocyclyl moieties of G is optionally
substituted by 1-3 R.sup.10; R.sup.4 is hydrogen, 3-7 membered
heterocyclyl, or C.sub.1-6 aliphatic; R.sup.5 is hydrogen,
C.sub.1-E, aliphatic group or a 3-7 membered heterocyclic ring
having 1-2 ring heteroatoms selected from N, O, or S, wherein
R.sup.5 is optionally substituted by halo, --CN, --SR.sup.8,
--S(O).sub.2O, --S(O).sub.2N(R.sup.7).sub.2, --C(O)R.sup.7,
--CO.sub.2R.sup.7, --N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.8, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2; each R.sup.7 is independently
selected from hydrogen or C.sub.1-4 aliphatic, or two R.sup.7 on
the same nitrogen atom are taken together with the nitrogen to form
a 5-6 membered heteroaryl or heterocyclyl ring; each R.sup.8 is
independently selected from C.sub.1-4 aliphatic; each R.sup.9 is
independently selected from hydrogen or C.sub.1-3 aliphatic; each
R.sup.10 is independently selected from oxo, --R.sup.11,
-T-R.sup.11, or -V-T-R.sup.11; each R.sup.11 is independently
selected from C.sub.1-6 aliphatic, halo,
--S(O).sub.2N(R.sup.7).sub.2, --OR.sup.7, --CN, --SR.sup.8,
--S(O).sub.2R.sup.8, --C(O)R.sup.7, --CO.sub.2R.sup.7,
--N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.7, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2; T is a straight or branched
C.sub.1-4 alkylene chain; V is --O--, --N(R.sup.7)--, --S--,
--S(O)--, --S(O).sub.2--, --C(O)--, or --CO.sub.2--; and R.sup.12
is hydrogen or an amino acid side chain.
27. The compound of claim 26 that is (S)-3a.
28. A compound of formula IV: ##STR00154## where R.sup.14 is an
amino protecting group or hydrogen; R.sup.1 is hydrogen, halo,
C.sub.1-3 aliphatic, amino, cyano, (C.sub.1-3 alkyl).sub.1-2 amino,
C.sub.1-3 alkoxy, --CONH.sub.2, --NHCOCF.sub.3, or
--CH.sub.2NH.sub.2; R.sup.2 is hydrogen, halo, C.sub.1-3 aliphatic,
--CF.sub.3; and R.sup.3 is hydrogen, halo, C.sub.1-6 aliphatic,
C.sub.1-6 haloalkyl, C.sub.1-6 alkoxy, hydroxy, amino, cyano, or
(C.sub.1-6 alkyl).sub.1-2 amino.
29. The compound of claim 28 that is (S)-IV.
Description
RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/821,545, filed Apr. 9, 2004 (pending),
which claims the benefit of U.S. Provisional Application Ser. No.
60/461,468, filed Apr. 9, 2003 (abandoned). The entire contents of
each of the above-referenced patent applications are incorporated
herein by this reference.
FIELD OF THE INVENTION
[0002] This invention relates to beta-carboline compounds,
pharmaceutical compositions thereof, and methods of using the
compositions for treating disease. The compounds are particularly
useful for treating inflammatory disease and cancer.
BACKGROUND OF THE INVENTION
[0003] The transcription (nuclear) factor NF-.kappa.B is a member
of the Rel protein family, and is typically a heterodimer composed
of p50 and p65 subunits. NF-.kappa.B is constitutively present in
the cytosol, and is inactivated by its association with one of the
I.kappa.B family of inhibitors. Palombella et al., WO 95/25533,
teaches that the ubiquitin-proteasome pathway plays an essential
role in the regulation of NF-.kappa.B activity, being responsible
for the processing of p105 to p50 and the degradation of the
inhibitor protein I.kappa.B-.alpha.. Chen et al., Cell 84:853
(1996), teaches that prior to degradation, I.kappa.B-.alpha.
undergoes selective phosphorylation at serine residues 32 and 36 by
the multisubunit I.kappa.B kinase complex (IKK). I.kappa.B-.alpha.
is phosphorylated by IKK, which has two catalytic subunits, IKK-1
(I.kappa.B kinase a or IKK-.alpha.) and IKK-2 (I.kappa.B kinase
.beta. or IKK-.beta.). Once phosphorylated, I.kappa.B is targeted
for ubiquitination and degradation by the 26S proteasome, allowing
translocation of NF-.kappa.B into the nucleus, where it binds to
specific DNA sequences in the promoters of target genes and
stimulates their transcription. Inhibitors of IKK can block the
phosphorylation of I.kappa.B and its further downstream effects,
particularly those associated with NF-.kappa.B transcription
factors.
[0004] The protein products of genes under the regulatory control
of NF-.kappa.B include cytokines, chemokines, cell adhesion
molecules, and proteins mediating cellular growth and control.
Importantly, many of these proinflammatory proteins also are able
to act, either in an autocrine or paracrine fashion, to further
stimulate NF-.kappa.B activation. In addition, NF-.kappa.B plays a
role in the growth of normal and malignant cells. Furthermore,
NF-.kappa.B is a heterodimeric transcription factor which can
activate a large number of genes which code, inter alia, for
proinflammatory cytokines such as IL-1, IL-2, TNF.alpha. or IL-6.
NF-.kappa.B is present in the cytosol of cells, building a complex
with its naturally occurring inhibitor I.kappa.B. The stimulation
of cells, for example by cytokines, leads to the phosphorylation
and subsequent proteolytic degradation of I.kappa.B. This
proteolytic degradation leads to the activation of NF-.kappa.B,
which subsequently migrates into the nucleus of the cell and
activates a large number of proinflammatory genes.
[0005] Rinehart et al., U.S. Pat. No. 4,631,149 (1986), discloses
beta-carboline compounds useful as antiviral, antibacterial, and
antitumor agents.
[0006] Ritzeler et al., WO 01/68648, discloses beta-carboline
compounds with I.kappa.B kinase inhibitory activity for use in the
treatment of inflammatory disorders (e.g., rheumatoid arthritis),
osteoarthritis, asthma, cardiac infarct, Alzheimer's disease,
carcinomatous disorders (potentiation of cytotoxic therapies) and
atherosclerosis.
[0007] It would be beneficial to provide novel IKK inhibitors that
possess good therapeutic properties, especially for the treatment
of inflammatory disease.
DESCRIPTION OF THE INVENTION
[0008] This invention provides compounds that are useful for
treating IKK-2-mediated diseases, such as inflammatory diseases and
cancer. The compounds are represented by formula I:
##STR00002##
wherein Ring A is selected from the group consisting of:
[0009] (a) a pyridinyl or pyrimidinyl ring that is substituted by
(i) --CH.sub.2C(O)-G and 0-1 R.sup.6a or (ii) 1-2 R.sup.6a, and
[0010] (b) a morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl,
pyranyl, tetrahydrofuranyl, cyclohexyl, cyclopentyl or
thiomorpholinyl ring that is substituted by (i) --C(R.sup.9).sub.3,
--W-G, or -G, (ii) 0-4 R.sup.6b and (iii) 0-1 oxo groups on a ring
carbon or 0-2 oxo groups on a ring sulfur;
[0011] each R.sup.6a is independently selected from C.sub.1-6
aliphatic, halo, C.sub.1-6 alkoxy, or amino;
[0012] each R.sup.6b is independently selected from C.sub.1-3
aliphatic or --N(R.sup.7).sub.2, and two R.sup.6b on the same or an
adjacent carbon optionally are taken together with the intervening
carbon(s) to form a 5-6 membered ring having 1-2 ring heteroatoms
selected from N, O or S;
[0013] W is -Q-, -Q-C(O)--,
--C(R.sup.9).sub.2--C(R.sup.9)(R.sup.12)--, or
--C(R.sup.9).sub.2-[C(R.sup.9)(R.sup.12)].sub.2--;
[0014] Q is --C(R.sup.9).sub.2-- or
--C(R.sup.9).sub.2C(R.sup.9).sub.2--;
[0015] G is --OH, --NR.sup.4R.sup.5, --N(R.sup.9)CONR.sup.4R.sup.5,
--N(R.sup.9)SO.sub.2(C.sub.1-3 aliphatic), --N(R.sup.9)COCF.sub.3,
--N(R.sup.9)CO(C.sub.1-6 aliphatic), --N(R.sup.9)CO(heterocyclyl),
--N(R.sup.9)CO(heteroaryl), --N(R.sup.9)CO(aryl), a 3-7 membered
heterocyclyl ring, or a 5-6 membered heteroaryl, wherein each of
the heteroaryl, aryl and heterocyclyl moieties of G is optionally
substituted by 1-3 R.sup.10;
[0016] R.sup.1 is hydrogen, halo, C.sub.1-3 aliphatic, amino,
cyano, (C.sub.1-3 alkyl).sub.1-2 amino, C.sub.1-3 alkoxy,
--CONH.sub.2, --NHCOCF.sub.3, or --CH.sub.2NH.sub.2;
[0017] R.sup.2 is hydrogen, halo, C.sub.1-3 aliphatic,
--CF.sub.3;
[0018] R.sup.3 is hydrogen, halo, C.sub.1-6 aliphatic, C.sub.1-6
haloalkyl, C.sub.1-6 alkoxy, hydroxy, amino, cyano, or (C.sub.1-6
alkyl).sub.1-2 amino;
[0019] R.sup.4 is hydrogen, 3-7 membered heterocyclyl, or C.sub.1-6
aliphatic;
[0020] R.sup.5 is hydrogen, C.sub.1-6 aliphatic group or a 3-7
membered heterocyclic ring having 1-2 ring heteroatoms selected
from N, O, or S, wherein R.sup.5 is optionally substituted by halo,
--OR.sup.7, --CN, --SR.sup.8, --S(O).sub.2R.sup.8,
--S(O).sub.2N(R.sup.7).sub.2, --C(O)R.sup.7, --CO.sub.2R.sup.7,
--N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.8, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2;
[0021] each R.sup.7 is independently selected from hydrogen or
C.sub.1-4 aliphatic, or two R.sup.7 on the same nitrogen atom are
taken together with the nitrogen to form a 5-6 membered heteroaryl
or heterocyclyl ring;
[0022] each R.sup.8 is independently selected from C.sub.1-4
aliphatic;
[0023] each R.sup.9 is independently selected from hydrogen or
C.sub.1-3 aliphatic;
[0024] each R.sup.10 is independently selected from oxo,
--R.sup.11, -T-R.sup.11, or -V-T-R.sup.11;
[0025] each R.sup.11 is independently selected from C.sub.1-6
aliphatic, halo, --S(O).sub.2N(R.sup.7).sub.2, --OR.sup.7, --CN,
--SR.sup.8, --S(O).sub.2R.sup.8, --C(O)R.sup.7, --CO.sub.2R.sup.7,
--N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.7, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2;
[0026] T is a straight or branched C.sub.1-4 alkylene chain;
[0027] V is --O--, --N(R.sup.7)--, --S--, --S(O)--, --S(O).sub.2--,
--C(O)--, or --CO.sub.2--; and
[0028] R.sup.12 is hydrogen or an amino acid side chain.
[0029] The term "aliphatic" as used herein means straight-chain,
branched or cyclic C.sub.1-C.sub.12 hydrocarbons which are
completely saturated or which contain one or more units of
unsaturation but which are not aromatic. For example, suitable
aliphatic groups include substituted or unsubstituted linear,
branched or cyclic alkyl, alkenyl, alkynyl groups and hybrids
thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or
(cycloalkyl)alkenyl. The terms "alkyl", "alkoxy", "hydroxyalkyl",
"alkoxyalkyl", and "alkoxycarbonyl", used alone or as part of a
larger moiety, include both straight and branched chains containing
one to twelve carbon atoms. The terms "alkenyl" and "alkynyl", used
alone or as part of a larger moiety, include both straight and
branched chains containing two to twelve carbon atoms. The term
"cycloalkyl, used alone or as part of a larger moiety, includes
cyclic C.sub.3-C.sub.12 hydrocarbons which are completely saturated
or which contain one or more units of unsaturation, but which are
not aromatic.
[0030] The terms "haloalkyl", "haloalkenyl" and "haloalkoxy", mean
alkyl, alkenyl or alkoxy, as the case may be, substituted with one
or more halogen atoms. The term "halogen" means F, Cl, Br, or
I.
[0031] The term "heteroatom" means nitrogen, oxygen, or sulfur and
includes any oxidized form of nitrogen and sulfur, and the
quaternized form of any basic nitrogen. Also the term "nitrogen"
includes a substitutable nitrogen of a heterocyclic ring. As an
example, in a saturated or partially unsaturated ring having 0-3
heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen
may be N (as in 3,4-dihydro-2-pyrrolyl), NH (as in pyrrolidinyl) or
NR.sup.+ (as in N-substituted pyrrolidinyl).
[0032] The terms "carbocycle", "carbocyclyl", "carbocyclo", or
"carbocyclic" as used herein means an aliphatic ring system having
three to fourteen members.
[0033] The terms "carbocycle", "carbocyclyl", "carbocyclo", or
"carbocyclic" whether saturated or partially unsaturated, also
refers to rings that are optionally substituted. The terms
"carbocycle", "carbocyclyl", "carbocyclo", or "carbocyclic" also
include aliphatic rings that are fused to one or more aromatic or
nonaromatic rings, such as in a decahydronaphthyl or
tetrahydronaphthyl, where the radical or point of attachment is on
the aliphatic ring.
[0034] The term "aryl" used alone or as part of a larger moiety as
in "aralkyl", "aralkoxy", or "aryloxyalkyl", refers to aromatic
ring groups having five to fourteen members, such as phenyl,
benzyl, phenethyl, 1-naphthyl, 2-naphthyl, 1-anthracyl and
2-anthracyl. The term "aryl" also refers to rings that are
optionally substituted. The term "aryl" may be used interchangeably
with the term "aryl ring". "Aryl" also includes fused polycyclic
aromatic ring systems in which an aromatic ring is fused to one or
more rings. Examples include 1-naphthyl, 2-naphthyl, 1-anthracyl
and 2-anthracyl. Also included within the scope of the term "aryl",
as it is used herein, is a group in which an aromatic ring is fused
to one or more non-aromatic rings, such as in an indanyl,
phenanthridinyl, or tetrahydronaphthyl, where the radical or point
of attachment is on the aromatic ring.
[0035] The term "heterocycle", "heterocyclyl", or "heterocyclic" as
used herein includes non-aromatic ring systems having five to
fourteen members, preferably five to ten, in which one or more ring
carbons, preferably one to four, are each replaced by a heteroatom
such as N, O, or S. Examples of heterocyclic rings include
3-1H-benzimidazol-2-one, (1-substituted)-2-oxo-benzimidazol-3-yl,
2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahydropyranyl,
3-tetrahydropyranyl, 4-tetrahydropyranyl, [1,3]-dioxalanyl,
[1,3]-dithiolanyl, [1,3]-dioxanyl, 2-tetrahydrothiophenyl,
3-tetrahydrothiophenyl, 2-morpholinyl, 3-morpholinyl,
4-morpholinyl, 2-thiomorpholinyl, 3-thiomorpholinyl,
4-thiomorpholinyl, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl,
1-piperazinyl, 2-piperazinyl, 1-piperidinyl, 2-piperidinyl,
3-piperidinyl, 4-piperidinyl, 4-thiazolidinyl, diazolonyl,
N-substituted diazolonyl, 1-phthalimidinyl, benzoxanyl,
benzopyrrolidinyl, benzopiperidinyl, benzoxolanyl, benzothiolanyl,
and benzothianyl. Also included within the scope of the term
"heterocyclyl" or "heterocyclic", as it is used herein, is a group
in which a non-aromatic heteroatom-containing ring is fused to one
or more aromatic or non-aromatic rings, such as in an indolinyl,
chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where the
radical or point of attachment is on the non-aromatic
heteroatom-containing ring. The term "heterocycle", "heterocyclyl",
or "heterocyclic" whether saturated or partially unsaturated, also
refers to rings that are optionally substituted.
[0036] The term "heteroaryl", used alone or as part of a larger
moiety as in "heteroaralkyl" or "heteroarylalkoxy", refers to
heteroaromatic ring groups having five to fourteen members.
Examples of heteroaryl rings include 2-furanyl, 3-furanyl,
3-furazanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl,
5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl,
2-oxadiazolyl, 5-oxadiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl,
1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 1-pyrazolyl, 2-pyrazolyl,
3-pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,
4-pyrimidyl, 5-pyrimidyl, 3-pyridazinyl, 2-thiazolyl, 4-thiazolyl,
5-thiazolyl, 5-tetrazolyl, 2-triazolyl, 5-triazolyl, 2-thienyl,
3-thienyl, carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl,
indolyl, quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl,
benzimidazolyl, isoquinolinyl, indazolyl, isoindolyl, acridinyl, or
benzoisoxazolyl. Also included within the scope of the term
"heteroaryl", as it is used herein, is a group in which a
heteroatomic ring is fused to one or more aromatic or nonaromatic
rings where the radical or point of attachment is on the
heteroaromatic ring. Examples include tetrahydroquinolinyl,
tetrahydroisoquinolinyl, and pyrido[3,4-d]pyrimidinyl. The term
"heteroaryl" also refers to rings that are optionally substituted.
The term "heteroaryl" may be used interchangeably with the term
"heteroaryl ring" or the term "heteroaromatic".
[0037] An aryl (including aralkyl, aralkoxy, aryloxyalkyl and the
like) or heteroaryl (including heteroaralkyl and heteroarylalkoxy
and the like) group may contain one or more substituents. Examples
of suitable substituents on the unsaturated carbon atom of an aryl,
heteroaryl, aralkyl, or heteroaralkyl group include a halogen,
--R.sup.O, --OR.sup.O, --SR.sup.O, 1,2-methylene-dioxy,
1,2-ethylenedioxy, protected OH (such as acyloxy), phenyl (Ph),
substituted Ph, --O(Ph), substituted --O(Ph), --CH.sub.2(Ph),
substituted --CH.sub.2(Ph), --CH.sub.2CH.sub.2(Ph), substituted
--CH.sub.2CH.sub.2(Ph), --NO.sub.2, --CN, --N(R.sup.O).sub.2,
--NR.sup.OC(O)R.sup.O, --NR.sup.OC(O)N(R.sup.O).sub.2,
--NR.sup.OCO.sub.2R.sup.O, --NR.sup.ONR.sup.OC(O)R.sup.O,
--NR.sup.ONR.sup.OC(O)N(R.sup.O).sub.2,
--NR.sup.ONR.sup.OCO.sub.2R.sup.O, --C(O)C(O)R.sup.O,
--C(O)CH.sub.2C(O)R.sup.O, --CO.sub.2R.sup.O, --C(O)R.sup.O,
---C(O)N(R.sup.O).sub.2, --OC(O)N(R.sup.O).sub.2,
--S(O).sub.2R.sup.O, --SO.sub.2N)(R.sup.O).sub.2, --S(O)R.sup.O,
--NR.sup.OSO.sub.2N)(R.sup.O).sub.2, --NR.sup.OSO.sub.2R.sup.O,
--C(.dbd.S)N(R.sup.O--C(.dbd.NH)--N(R.sup.O).sub.2,
--(CH.sub.2).sub.yNHC(O)R.sup.O, --(CH.sub.2).sub.yNHC(O)
CH(V--R.sup.O)(R.sup.O); wherein each R.sup.O is independently
selected from hydrogen, a substituted or unsubstituted aliphatic
group, an unsubstituted heteroaryl or heterocyclic ring, phenyl
(Ph), substituted Ph, --O(Ph), substituted --O(Ph), --CH.sub.2(Ph),
or substituted --CH.sub.2(Ph); y is 0-6; and V is a linker group.
Examples of substituents on the aliphatic group or the phenyl ring
of R.sup.O include amino, alkylamino, dialkylamino, aminocarbonyl,
halogen, alkyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, nitro,
cyano, carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy, haloalkoxy,
or haloalkyl.
[0038] An aliphatic group or a non-aromatic heterocyclic ring may
contain one or more substituents. Examples of suitable substituents
on the saturated carbon 20 of an aliphatic group or of a
non-aromatic heterocyclic ring include those listed above for the
unsaturated carbon of an aryl or heteroaryl group and the
following: .dbd.O, .dbd.S, .dbd.NNHR*, .dbd.NN(R*).sub.2, .dbd.N--,
.dbd.NNHC(O)R*, .dbd.NNHCO.sub.2(alkyl), .dbd.NNHSO.sub.2(alkyl),
or .dbd.NR*, where each R* is independently selected from hydrogen,
an unsubstituted aliphatic group or a substituted aliphatic group.
Examples of substituents on the aliphatic group include amino,
alkylamino, dialkylamino, aminocarbonyl, halogen, alkyl,
alkylaminocarbonyl, dialkylaminocarbonyl, alkylaminocarbonyloxy,
dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy,
alkoxycarbonyl, alkylcarbonyl, hydroxy, haloalkoxy, or
haloalkyl.
[0039] Suitable substituents on the nitrogen of a non-aromatic
heterocyclic ring include --R.sup.+, --N(R.sup.+).sub.2,
--C(O)R.sup.+, --CO.sub.2R.sup.+, --C(O)C(O)R.sup.+,
--C(O)CH.sub.2C(O)R.sup.+, --SO.sub.2R.sup.+,
--SO.sub.2N(R.sup.+).sub.2, --C(.dbd.S)N(R.sup.+).sub.2,
--C(.dbd.NH)--N(R.sup.+).sub.2, and --NR.sup.+SO.sub.2R.sup.+;
wherein each R.sup.+ is independently selected from hydrogen, an
aliphatic group, a substituted aliphatic group, phenyl (Ph),
substituted Ph, --O(Ph), substituted --O(Ph), CH.sub.2(Ph),
substituted CH.sub.2(Ph), or an unsubstituted heteroaryl or
heterocyclic ring. Examples of substituents on the aliphatic group
or the phenyl ring include amino, alkylamino, dialkylamino,
aminocarbonyl, halogen, alkyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylaminocarbonyloxy,
dialkylaminocarbonyloxy, alkoxy, nitro, cyano, carboxy,
alkoxycarbonyl, alkylcarbonyl, hydroxy, haloalkoxy, or
haloalkyl.
[0040] The term "linker group" or "linker" means an organic moiety
that connects two parts of a compound. Linkers are typically
comprised of an atom such as oxygen or sulfur, a unit such as
--NH--, --CH.sub.2--, --C(O)--, --C(O)NH--, or a chain of atoms,
such as an alkylidene chain. The molecular mass of a linker is
typically in the range of about 14 to 200, preferably in the range
of 14 to 96 with a length of up to about six atoms. Examples of
linkers include a saturated or unsaturated C.sub.1-6 alkylidene
chain which is optionally substituted, and wherein one or two
saturated carbons of the chain are optionally replaced by --C(O)--,
--C(O)C(O)--, --CONH--, --CONHNH--, --CO.sub.2--, --OC(O)--,
--NHCO.sub.2--, --O--, --NHCONH--, --OC(O)NH--, --NHNH--, --NHCO--,
--S--, --SO--, --SO.sub.2--, --NH--, --SO.sub.2NH--, or
--NHSO.sub.2--.
[0041] The term "alkylidene chain" or "alkylene chain" refers to an
optionally substituted, straight or branched carbon chain that may
be fully saturated or have one or more units of unsaturation. The
optional substituents are as described above for an aliphatic
group.
[0042] A combination of substituents or variables is permissible
only if such a combination results in a stable or chemically
feasible compound. A stable compound or chemically feasible
compound is one in which the chemical structure is not
substantially altered when kept at a temperature of 40.degree. C.
or less, in the absence of moisture or other chemically reactive
conditions, for at least a week.
[0043] Unless otherwise stated, structures depicted herein are also
meant to include all stereochemical forms of the structure; i.e.,
the R and S configurations for each asymmetric center. Therefore,
single stereochemical isomers as well as enantiomeric and
diastereomeric mixtures of the present compounds are within the
scope of the invention. Unless otherwise stated, structures
depicted herein are also meant to include compounds which differ
only in the presence of one or more isotopically enriched atoms.
For example, compounds having the present structures except for the
replacement of a hydrogen atom by a deuterium or tritium, or the
replacement of a carbon by a .sup.13C- or .sup.14C-enriched carbon
are within the scope of this invention.
[0044] In one embodiment of the invention, Ring A is selected from
a pyridinyl or a pyrimidinyl ring that is substituted by 1-2
R.sup.6a groups. In this embodiment, preferred Ring A include a
3-pyridinyl or a 5-pyrimidinyl ring, shown below by the compounds
of formula II-A and II-B, respectively.
##STR00003##
[0045] Preferably, R.sup.6a is selected from halo or a C.sub.1-6
aliphatic, such as chloro or methyl. When R.sup.6a is an aliphatic
group such as methyl, a favorable position for the R.sup.6a group
is at the 2-position of the pyridinyl ring or the 4-position of the
pyrimidinyl ring, as shown in II-C above. Particular Ring A
moieties are 2-methyl-3-pyridinyl and 4-methyl-5-pyrimidinyl. It
has been found that compounds of formula II-C where R.sup.6a is a
methyl group are surprisingly more potent in biological testing for
IKK inhibition than analogous compounds that have an unsubstituted
Ring A pyridine, such as those described in the aforementioned
Ritzeler et al. PCT application WO 01/68648.
[0046] Preferred R.sup.1 groups are small groups such as hydrogen,
methyl, amino and fluoro.
[0047] Preferred R.sup.2 groups include hydrogen and halo. Chloro
is a preferred R.sup.2 halo group.
[0048] Preferred R.sup.3 groups include hydrogen, halo (especially
chloro) and alkoxy. Examples of suitable alkoxy groups include
C.sub.1-d alkoxy groups such as methoxy, ethoxy, propoxy and
cyclopropylmethoxy.
[0049] In another embodiment, Ring A is selected from a 5-6
membered non-aromatic ring having 0-2 ring heteroatoms selected
from nitrogen, oxygen and sulfur. These are designated generally as
compounds of formula III. Examples of non-aromatic Ring A groups
include a morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl,
pyranyl, tetrahydrofuranyl, cyclohexyl, cyclopentyl and a
thiomorpholinyl ring. Preferably, such non-aromatic rings are
substituted by (i) --C(R.sup.9).sub.3 or --W-G, (ii) 0-4 R.sup.6b
and (iii) 0-1 oxo groups on a ring carbon or 0-2 oxo groups on a
ring sulfur. More preferably, such non-aromatic rings are
substituted by (i) --W-G, (ii) 0-2 R.sup.6b and (iii) 0-1 oxo
groups on a ring carbon or 0-2 oxo groups on a ring sulfur.
[0050] A preferred G is --NR.sup.4R.sup.5 or a 3-7 membered
heterocyclyl ring. More preferably G is --NR.sup.4R.sup.5 or a 5-6
membered heterocyclyl ring, where G is substituted by 1-2
R.sup.10.
[0051] A preferred R.sup.4 is a hydrogen, 5-6 membered heterocyclyl
ring, or C.sub.1-6 aliphatic, more preferably hydrogen or C.sub.1-6
aliphatic. R.sup.4 may also be a C.sub.1-6alkoxy.
[0052] A preferred R.sup.5 is a hydrogen, 5-6 membered heterocyclyl
ring, or C.sub.1-6 aliphatic, more preferably hydrogen or C.sub.1-6
aliphatic.
[0053] Various formula III compounds where Ring A is a non-aromatic
ring are shown in Table 2. For ease of viewing, substituents on
these non-aromatic Ring A compounds, except for the oxo group in
some cases, are not shown.
TABLE-US-00001 TABLE 2 Compounds where Ring A is Non-Aromatic III-A
##STR00004## III-B ##STR00005## III-C ##STR00006## III-D
##STR00007## III-E ##STR00008## III-F ##STR00009## III-G
##STR00010## III-H ##STR00011## III-J ##STR00012## III-K
##STR00013## III-L ##STR00014## III-M ##STR00015##
[0054] When Ring A is a non-aromatic, six-membered heterocylic
ring, a favorable position for the --W-G and --C(R.sup.9).sub.3
substituents on Ring A is ortho to the position where the
beta-carboline portion is attached. For example, in compounds
III-A, III-B, III-D, III-H, III-J and III-M, a preferred position
for attachment of --W-G and --C(R.sup.9).sub.3 is at the Ring A
nitrogen or at N-1 in the case of compound
[0055] Preferably, W is -Q-, -Q-C(O)--,
--C(R.sup.9).sub.2--C(R.sup.9)(R.sup.12)--, or
--C(R.sup.9).sub.2--[C(R.sup.9)(R.sup.12)].sub.2-- where R.sup.9 is
hydrogen. More preferably, W is -Q-, -Q-C(O)--, or
--C(R.sup.9).sub.2--C(R.sup.9)(R.sup.12)--. R.sup.12 is hydrogen,
C.sub.1-6 aliphatic, substituted or unsubstituted phenyl,
substituted or unsubstituted benzyl or an amino acid side chain,
particularly the side chain of a natural amino acid. Examples of
particular natural amino acids include alanine, phenylalanine,
valine, leucine, isoleucine, serine, tyrosine, aspartic acid and
glutamic acid.
[0056] In one embodiment, W is Q-C(O)--. In this embodiment, a
preferred Q is --CH.sub.2-- or --CH.sub.2--CH.sub.2--, more
preferably --CH.sub.2--.
[0057] In another embodiment, Ring A is substituted by 0-2
R.sup.6b. A preferred R.sup.6b group is methyl. When Ring A is a
non-aromatic six membered ring, one embodiment provides compounds
of formula III where there are two methyl groups on the Ring A
position para to the position where the beta carboline portion is
attached. An example of this embodiment is a compound where Ring A
is a 6,6-dimethyl-morpholinyl ring. Preferably, such compounds are
further substituted by --W-G as described above.
[0058] When Ring A is a morpholinyl ring, it has been found that
compounds having the "S" stereochemistry at position 3 of the
morpholine ring are preferred, as shown below by compounds of
formula (S)-III-A.
##STR00016##
where n is 0-4 and R.sup.1, R.sup.2, R.sup.3, W, G and R.sup.6b are
as defined above. By analogy, it is expected that "S"
stereochemistry is also preferred for other six-membered
non-aromatic Ring A compounds of formula III.
[0059] One embodiment relates to compounds of formula III-A or
(S)-III-A where R.sup.1 is hydrogen, halo, methyl or amino;
[0060] R.sup.2 is hydrogen, methyl or halo;
[0061] R.sup.3 is hydrogen, halo, alkoxy, or (C.sub.1-6
aliphatic).sub.2 amino;
[0062] Ring A is substituted by 0-2 R.sup.6b;
[0063] R.sup.6b is C.sub.1-3 aliphatic;
[0064] W is -Q-, -Q-C(O)--,
--C(R.sup.9).sub.2--C(R.sup.9)(R.sup.12) or
--C(R.sup.9).sub.2--[C(R.sup.9)(R.sup.12)].sub.2--;
[0065] Q is --C(R.sup.9).sub.2-- or
--C(R.sup.9).sub.2C(R.sup.9).sub.2--;
[0066] G is --NR.sup.4R.sup.5, --N(R.sup.9)C(O)NR.sup.4R.sup.5,
--N(R.sup.9)SO.sub.2(C.sub.1-3 aliphatic),
--N(R.sup.9)C(O)CF.sub.3, --N(R.sup.9)CO(C.sub.1-6 aliphatic), and
--N(R.sup.9)CO(heterocyclyl), --N(R.sup.9)CO(heteroaryl),
--N(R.sup.9)CO(aryl), a 5-6 membered heterocyclyl ring, or a 5-6
membered heteroaryl, wherein each of the heteroaryl, aryl and
heterocyclyl moieties of G is optionally substituted by 1-3
R.sup.10;
[0067] R.sup.4 is hydrogen or C.sub.1-6 aliphatic;
[0068] R.sup.5 is hydrogen or a C.sub.1-6 aliphatic group that is
optionally substituted by halo, --OR.sup.7, --CN, --SR.sup.8,
--S(O).sub.2R.sup.8, --S(O).sub.2N(R.sup.7).sub.2, --C(O)R.sup.7,
--CO.sub.2R.sup.7, --N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.8, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2;
[0069] each R.sup.7 is independently selected from hydrogen or
C.sub.1-4 aliphatic, or two R.sup.7 on the same nitrogen atom are
taken, together with the nitrogen to form a 5-6 membered heteroaryl
or heterocyclyl ring;
[0070] each R.sup.8 is independently selected from C.sub.1-4
aliphatic;
[0071] R.sup.9 is hydrogen;
[0072] each R.sup.10 is independently selected from oxo, R.sup.11,
T-R.sup.11, or V-T-R.sup.11;
[0073] each R.sup.11 is independently selected from C.sub.1-6
aliphatic, halo, --S(O).sub.2N(R.sup.7).sub.2, --OR.sup.7, --CN,
--SR.sup.8, --S(O).sub.2R.sup.8, --C(O)R.sup.7, --CO.sub.2R.sup.7,
--N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.7, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2;
[0074] T is a straight or branched C.sub.1-4 alkylene chain;
[0075] V is --O--, --N(R.sup.7)--, --S--, --S(O)--, --S(O).sub.2--,
--C(O)--, or --CO.sub.2--; and
[0076] R.sup.12 is hydrogen, C.sub.1-6 aliphatic, substituted or
unsubstituted phenyl, or substituted or unsubstituted benzyl.
[0077] Another embodiment relates to compounds of formula III-A or
(S)-III-A where:
[0078] R.sup.1 is hydrogen, methyl, fluoro or amino;
[0079] R.sup.2 is chloro;
[0080] R.sup.3 is hydrogen or alkoxy;
[0081] Ring A is substituted by --W-G and 0-2 R.sup.6b;
[0082] R.sup.6b is methyl;
[0083] W is -Q-, -Q-C(O)-- or
--C(R.sup.9).sub.2--C(R.sup.9)(R.sup.12);
[0084] Q is --C(R.sup.9).sub.2-- or
--C(R.sup.9).sub.2C(R.sup.9).sub.2--;
[0085] G is --NR.sup.4R.sup.5, --N(R.sup.9)C(O)NR.sup.4R.sup.5,
--N(R.sup.9)C(O)CF.sub.3, --N(R.sup.9)CO(C.sub.1-6 aliphatic), and
--N(R.sup.9)CO(heterocyclyl) --N(R.sup.9)CO(heteroaryl), a 5-6
membered heterocyclyl ring, or a 5-6 membered heteroaryl, wherein
each of the heteroaryl and heterocyclyl moieties of G is optionally
substituted by 1-3 R.sup.10;
[0086] R.sup.4 is hydrogen or C.sub.1-6 aliphatic;
[0087] R.sup.5 is hydrogen or C.sub.1-6 aliphatic;
[0088] each R.sup.7 is independently selected from hydrogen or
C.sub.1-4 aliphatic, or two R.sup.7 on the same nitrogen atom are
taken together with the nitrogen to form a 5-6 membered heteroaryl
or heterocyclyl ring;
[0089] each R.sup.9 is independently selected from C.sub.1-4
aliphatic;
[0090] R.sup.9 is hydrogen;
[0091] each R.sup.10 is independently selected from oxo, R.sup.11,
T-R.sup.11, or V-T-R.sup.11;
[0092] each R.sup.11 is independently selected from C.sub.1-6
aliphatic, halo, --S(O).sub.2N(R.sup.7).sub.2, --OR.sup.7--CN,
--SR.sup.8, --S(O).sub.2R.sup.8, --C(O)R.sup.7, --CO.sub.2R.sup.7,
--N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.7, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2;
[0093] T is a straight or branched C.sub.1-4 alkylene chain;
[0094] V is --O--, --N(R.sup.7)--, --S--, --S(O)--, --S(O).sub.2--,
--C(O)--, or --CO.sub.2--; and
[0095] R.sup.12 is hydrogen, C.sub.1-6 aliphatic, phenyl, or
benzyl.
[0096] Preferred compounds of formula III-A are the compounds of
formula (S)-III-A':
##STR00017##
where R.sup.1, R.sup.2, R.sup.3, W and G are as defined above for
(S)-III-A.
[0097] Another embodiment relates to compounds of formula
III-A-a:
##STR00018##
or a pharmaceutically acceptable salt thereof, wherein:
[0098] Q is --CH.sub.2--, --CH(R.sup.9)--, or
--C(R.sup.9).sub.2--;
[0099] G is --NR.sup.4R.sup.5 or a 3-7 membered heterocyclyl or
heteroaryl ring that is optionally substituted by 1-2 R.sup.10;
[0100] R.sup.1 is hydrogen, halo, C.sub.1-3 aliphatic, amino,
cyano, (C.sub.1-3 alkyl).sub.1-2 amino, C.sub.1-3 alkoxy,
(C.sub.1-3 aliphatic)-C(O)--, (C.sub.1-6 aliphatic)-CO.sub.2--, or
(C.sub.1-3 aliphatic)-C(O)NH--;
[0101] R.sup.2 is hydrogen, halo, C.sub.1-3 aliphatic, C.sub.1-3
alkoxy, C.sub.1-3 haloalkoxy, or C.sub.1-3 haloalkyl;
[0102] R.sup.3 is hydrogen, halo, C.sub.1-6 aliphatic, C.sub.1-6
haloalkyl, C.sub.1-6 alkoxy, hydroxy, amino, cyano, or (C.sub.1-6
alkyl).sub.1-2-amino;
[0103] R.sup.4 is hydrogen or C.sub.1-6 aliphatic;
[0104] R.sup.5 is a C.sub.1-6 aliphatic group that is optionally
substituted by halo, --OR.sup.7, --CN, --SR.sup.8,
--S(O).sub.2R.sup.8, --C(O)R.sup.7, --CO.sub.2R.sup.7,
--N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.8, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2;
[0105] Ring A is substituted by 0-4 R.sup.6b;
[0106] each R.sup.6b is independently selected from a C.sub.1-6
aliphatic group;
[0107] each R.sup.7 is independently selected from hydrogen or
C.sub.1-4 aliphatic, or two R.sup.7 on the same nitrogen atom are
taken together with the nitrogen to form a 5-6 membered heteroaryl
or heterocyclyl ring;
[0108] each R.sup.8 is independently selected from C.sub.1-4
aliphatic;
[0109] each R.sup.9 is independently selected from a C.sub.1-3
aliphatic;
[0110] each R.sup.10 is independently selected from R.sup.11,
T-R.sup.11, or V-T-R.sup.11;
[0111] each R.sup.11 is independently selected from C.sub.1-6
aliphatic, halo, --OR.sup.7, --CN, --SR.sup.8, --S(O)--C(O)R.sup.7,
--CO.sub.2R.sup.7, --N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.7, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2;
[0112] T is a straight or branched C.sub.1-4 alkylene chain;
and
[0113] V is --O--, --N(R.sup.7)--, --S--, --S(O)--, --S(O).sub.2--,
--C(O)--, or --CO.sub.2--.
[0114] One embodiment relates to compounds of formula III-A-a
where:
[0115] Q is --CH.sub.2--, or --CH(R.sup.9)--;
[0116] G is --NR.sup.4R.sup.5 or a 5-6 membered heterocyclyl or
heteroaryl ring that is optionally substituted by 1-2 R.sup.n;
[0117] R.sup.1 is hydrogen, halo, C.sub.1-2 alkyl, amino, or
(C.sub.1-2 alkyl).sub.1-2 amino;
[0118] R.sup.2 is hydrogen, halo, C.sub.1-2 aliphatic, C.sub.1-2
alkoxy, or C.sub.1-2 haloalkyl;
[0119] R.sup.3 is hydrogen, halo, C.sub.1-2 aliphatic, C.sub.1-2
alkoxy, or C.sub.1-2 haloalkyl;
[0120] R.sup.4 is hydrogen or C.sub.1-6 aliphatic;
[0121] R.sup.5 is a C.sub.1-6 aliphatic group that is optionally
substituted by halo, --OR.sup.7, --CN, --SR.sup.8,
--S(O).sub.2R.sup.9, --C(O)R.sup.7, --CO.sub.2R.sup.7,
--N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.8, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2;
[0122] Ring A is substituted by 0-2 R.sup.6b;
[0123] each R.sup.6b is independently selected from a C.sub.1-3
aliphatic group;
[0124] each R.sup.7 is independently selected from hydrogen or
C.sub.1-4 aliphatic, or two R.sup.7 on the same nitrogen atom are
taken together with the nitrogen to form a 5-6 membered heteroaryl
or heterocyclyl ring;
[0125] R.sup.8 is C.sub.1-4 aliphatic;
[0126] R.sup.9 is independently selected from a C.sub.1-3
aliphatic;
[0127] each R.sup.10 is independently selected from R.sup.11,
T-R.sup.11, or V-T-R.sup.11;
[0128] each R.sup.11 is independently selected from C.sub.1-6
aliphatic, halo, --OR.sup.7, --CN, --SR.sup.8, --S(O).sub.2R.sup.8,
--C(O)R.sup.7, --CO.sub.2R.sup.7, --N(R.sup.7).sub.2,
--C(O)N(R.sup.7).sub.2, --N(R.sup.7)C(O)R.sup.7,
--N(R.sup.7)CO.sub.2R.sup.7, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2;
[0129] T is a straight or branched C.sub.1-4 alkylene chain;
and
[0130] V is --O--, --N(R.sup.7)--, --S--, --S(O)--, --S(O).sub.2--,
--C(O)--, or --CO.sub.2--.
[0131] Another embodiment relates to compounds of formula III-A-a
where:
[0132] Q is --CH.sub.2--, or --CH(R.sup.9)--;
[0133] G is --NR.sup.4R.sup.5 or a 5-6 membered heterocyclyl ring,
having 1-2 ring heteroatoms selected from oxygen or nitrogen, that
is optionally substituted by 1-2 R.sup.10;
[0134] R.sup.1 is hydrogen, halo, methyl, amino, or (C.sub.1-2
alkyl).sub.1-2 amino;
[0135] R.sup.2 is hydrogen, halo, C.sub.1-2 aliphatic, or C.sub.1-2
haloalkyl;
[0136] R.sup.3 is hydrogen, halo, or C.sub.1-2 aliphatic;
[0137] R.sup.4 is hydrogen or C.sub.1-6 aliphatic;
[0138] R.sup.5 is a C.sub.1-6 aliphatic group that is optionally
substituted by halo, --OR.sup.7, --CN, --SR.sup.8,
--S(O).sub.2R.sup.8, --C(O)R.sup.7, --CO.sub.2R.sup.7,
--N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.8, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2;
[0139] Ring A is substituted by zero or two R.sup.6b;
[0140] each R.sup.6b is independently selected from a C.sub.1-3
aliphatic group;
[0141] each R.sup.7 is independently selected from hydrogen or
C.sub.1-4 aliphatic, or two R.sup.7 on the same nitrogen atom are
taken together with the nitrogen to form a 5-6 membered heteroaryl
or heterocyclyl ring;
[0142] R.sup.8 is C.sub.1-4 aliphatic;
[0143] R.sup.9 is independently selected from a C.sub.1-3
aliphatic;
[0144] each R.sup.10 is independently selected from R.sup.11,
T-R.sup.11, or V-T-R.sup.11;
[0145] each R.sup.11 is independently selected from C.sub.1-6
aliphatic, halo, --OR.sup.7, --CN, --SR.sup.8, --S(O).sub.2R.sup.8,
--C(O)R.sup.7, --CO.sub.2R.sup.7, --N(R.sup.7).sub.2,
--C(O)N(R.sup.7).sub.2, --N(R.sup.7)C(O)R.sup.7,
--N(R.sup.7)CO.sub.2O, or --N(R.sup.7)C(O)N(R.sup.7).sub.2;
[0146] T is a straight or branched C.sub.1-4 alkylene chain;
and
[0147] V is --O--, --N(R.sup.7)--, --S--, --S(O)--, --S(O).sub.2--,
--C(O)--, or --CO.sub.2--.
[0148] In preferred compounds of formula III-A-a, Q is
--CH.sub.2--; G is selected from an optionally substituted
piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, or
--NR.sup.4R.sup.5; R.sup.4 is hydrogen or C.sub.1-6 aliphatic; and
R.sup.5 is C.sub.1-6 aliphatic, 5-6 membered heterocyclyl, or
C.sub.1-6 hydroxyalkyl. More preferred are compounds where G is
unsubstituted or substituted by 1-2 groups independently selected
from the group consisting of: C.sub.1-3 alkyl, HO-alkyl,
alkoxycarbonyl, mono- or dialkylaminocarbonyl, and HO.sub.2C-alkyl.
For compounds of formula III-A-a in each of the above embodiments,
the (S) stereochemistry at the morpholine 3-position is
preferred.
[0149] Another embodiment relates to compounds of formula
III-A-aa:
##STR00019##
or a pharmaceutically acceptable salt thereof wherein,
[0150] Q is --CH.sub.2--, or --CH(R.sup.9)--;
[0151] G is --NR.sup.4R.sup.5 or a 3-7 membered heterocyclyl ring
that is optionally substituted by 1-2 R.sup.10;
[0152] R.sup.1 is hydrogen, halo, C.sub.1-2 alkyl, amino, or
(C.sub.1-2 alkyl).sub.1-2 amino;
[0153] R.sup.2 is hydrogen, halo, C.sub.1-2 aliphatic, C.sub.1-2
alkoxy, or C.sub.1-2 haloalkyl;
[0154] R.sup.3 is hydrogen, halo, C.sub.1-2 aliphatic, C.sub.1-2
alkoxy, or C.sub.1-2 haloalkyl;
[0155] R.sup.4 is hydrogen or C.sub.1-6 aliphatic;
[0156] R.sup.5 is a C.sub.1-6 aliphatic group that is optionally
substituted by halo, --OR.sup.7, --CN, --SR.sup.8,
--S(O).sub.2R.sup.8, --C(O)R.sup.7, --CO.sub.2R.sup.7,
--N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.8, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2;
[0157] each R.sup.6b is independently selected from hydrogen or a
C.sub.1-6 aliphatic;
[0158] each R.sup.7 is independently selected from hydrogen or
C.sub.1-4 aliphatic, or two R.sup.7 on the same nitrogen atom are
taken together with the nitrogen to form a 5-6 membered heteroaryl
or heterocyclyl ring;
[0159] R.sup.8 is C.sub.1-4 aliphatic;
[0160] R.sup.9 is independently selected from a C.sub.1-3
aliphatic;
[0161] each R.sup.10 is independently selected from R.sup.11,
T-R.sup.11, or V-T-R.sup.11;
[0162] each R.sup.11 is independently selected from C.sub.1-6
aliphatic, halo, --OR.sup.7, --CN, --SR.sup.8, --S(O).sub.2R.sup.8,
--C(O)R.sup.7, --CO.sub.2R.sup.7, --N(R.sup.7).sub.2,
--C(O)N(R.sup.7).sub.2, --N(R.sup.7)C(O)R.sup.7,
--N(R.sup.7)CO.sub.2R.sup.7, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2;
[0163] T is a straight or branched C.sub.1-4 alkylene chain;
and
[0164] V is --O--, --N(R.sup.7)--, --S--, --S(O)--, --S(O).sub.2--,
--C(O)--, or
[0165] Another embodiment relates to compounds of formula III-A-aa
where:
[0166] Q is --CH.sub.2--;
[0167] G is --NR.sup.4R.sup.5 or a 5-6 membered heterocyclyl ring
that is optionally substituted by 1-2 R.sup.11;
[0168] R.sup.1 is hydrogen, halo or methyl;
[0169] R.sup.2 is hydrogen, halo, C.sub.1-2 aliphatic, C.sub.1-2
alkoxy, or C.sub.1-2 haloalkyl;
[0170] R.sup.3 is hydrogen;
[0171] R.sup.4 is hydrogen or C.sub.1-6 aliphatic;
[0172] R.sup.5 is a C.sub.1-6 aliphatic group that is optionally
substituted by halo, --OR.sup.7, --CN, --SR.sup.8,
--S(O).sub.2R.sup.8, --C(O)R.sup.7, --CO.sub.2R.sup.7,
--N(R.sup.7).sub.2, --C(O)N(R.sup.7).sub.2,
--N(R.sup.7)C(O)R.sup.7, --N(R.sup.7)CO.sub.2R.sup.8, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2;
[0173] each R.sup.6b is independently selected from hydrogen or a
C.sub.1-6 aliphatic;
[0174] each R.sup.7 is independently selected from hydrogen or
C.sub.1-4 aliphatic, or two R.sup.7 on the same nitrogen atom are
taken together with the nitrogen to form a 5-6 membered heteroaryl
or heterocyclyl ring;
[0175] R.sup.8 is C.sub.1-4 aliphatic;
[0176] each R.sup.10 is independently selected from R.sup.11,
T-R.sup.11, or V-T-R.sup.11;
[0177] each R.sup.11 is independently selected from C.sub.1-6
aliphatic, halo, --OR.sup.7, --CN, --SR.sup.8, --S(O).sub.2R.sup.8,
--C(O)R.sup.7, --CO.sub.2R.sup.7, --N(R.sup.7).sub.2,
--C(O)N(R.sup.7).sub.2, --N(R.sup.7)C(O)R.sup.7,
--N(R.sup.7)CO.sub.2R.sup.7, or
--N(R.sup.7)C(O)N(R.sup.7).sub.2;
[0178] T is a straight or branched C.sub.1-4 alkylene chain;
and
[0179] V is --O--, --N(R.sup.7)--, --S--, --S(O)--, --S(O).sub.2--,
--C(O)--, or --CO.sub.2--.
[0180] Preferred compounds of III-A-aa are compounds where:
[0181] Q is --CH.sub.2--;
[0182] G is selected from an optionally substituted piperidinyl,
piperazinyl, morpholinyl, pyrrolidinyl, or --NR.sup.4R.sup.5;
[0183] R.sup.1 is hydrogen, halo or methyl;
[0184] R.sup.2 is halo;
[0185] R.sup.3 is hydrogen;
[0186] R.sup.4 is hydrogen or C.sub.1-6 aliphatic;
[0187] R.sup.5 is C.sub.1-6 alkoxy, C.sub.1-6 aliphatic, or
C.sub.1-6 hydroxyalkyl;
[0188] each R.sup.6b is independently selected from hydrogen or a
C.sub.1-3 aliphatic. Preferably, each R.sup.6b is hydrogen or
methyl. For compounds of formula III-A-aa in each of the above
embodiments, the (S) stereochemistry is preferred at the three
position of the Ring A morpholine.
[0189] Examples of specific formula Y compounds are shown in Tables
3 and 4 below.
TABLE-US-00002 TABLE 3 Specific examples of formula I compounds 1
##STR00020## 2 ##STR00021## 3 ##STR00022## 4 ##STR00023## 5
##STR00024## 6 ##STR00025## 7 ##STR00026## 8 ##STR00027## 9
##STR00028## 10 ##STR00029## 11 ##STR00030## 12 ##STR00031## 13
##STR00032## 14 ##STR00033## 15 ##STR00034## 16 ##STR00035## 17
##STR00036## 18 ##STR00037## 19 ##STR00038## 20 ##STR00039## 21
##STR00040## 22 ##STR00041## 23 ##STR00042## 24 ##STR00043## 25
##STR00044## 26 ##STR00045## 27 ##STR00046## 28 ##STR00047## 29
##STR00048## 30 ##STR00049## 31 ##STR00050## 32 ##STR00051## 33
##STR00052## 34 ##STR00053## 35 ##STR00054## 36 ##STR00055## 37
##STR00056## 38 ##STR00057## 39 ##STR00058## 40 ##STR00059## 41
##STR00060## 42 ##STR00061## 43 ##STR00062## 44 ##STR00063## 45
##STR00064## 46 ##STR00065## 47 ##STR00066## 48 ##STR00067## 49
##STR00068## 50 ##STR00069## 51 ##STR00070## 52 ##STR00071## 53
##STR00072## 54 ##STR00073## 55 ##STR00074## 56 ##STR00075## 57
##STR00076## 58 ##STR00077## 59 ##STR00078## 60 ##STR00079## 61
##STR00080## 62 ##STR00081## 63 ##STR00082##
[0190] Table 4 below shows specific examples of III-A-aa
compounds.
TABLE-US-00003 TABLE 4 Specific examples of formula III-A-a
compounds III-A-aa ##STR00083## No. R.sup.1 R.sup.6b/R.sup.6b Q G
64 H CH.sub.3/CH.sub.3 CH.sub.2 ##STR00084## 65 H CH.sub.3/CH.sub.3
CH.sub.2 ##STR00085## 66 H CH.sub.3/CH.sub.3 CH.sub.2 ##STR00086##
67 H CH.sub.3/CH.sub.3 CH.sub.2 HO--CH.sub.2CH.sub.2N(CH.sub.3)--
68 H CH.sub.3/CH.sub.3 CH.sub.2 ##STR00087## 69 H CH.sub.3/CH.sub.3
CH.sub.2 ##STR00088## 70 H CH.sub.3/CH.sub.3 CH.sub.2 ##STR00089##
71 H CH.sub.3/CH.sub.3 CH.sub.2 ##STR00090## 72 H CH.sub.3/CH.sub.3
CH.sub.2 Et.sub.2N-- 73 H CH.sub.3/CH.sub.3 CH.sub.2 ##STR00091##
74 H CH.sub.3/CH.sub.3 CH.sub.2 ##STR00092## 75 H CH.sub.3/CH.sub.3
CH.sub.2 ##STR00093## 76 H CH.sub.3/CH.sub.3 CH.sub.2 ##STR00094##
77 H CH.sub.3/CH.sub.3 CH.sub.2 ##STR00095## 78 H CH.sub.3/CH.sub.3
CH.sub.2 ##STR00096## 79 H CH.sub.3/CH.sub.3 CH.sub.2 ##STR00097##
80 H CH.sub.3/CH.sub.3 CH.sub.2 ##STR00098## 81 CH.sub.3
CH.sub.3/CH.sub.3 CH.sub.2 ##STR00099## 82 H CH.sub.3/CH.sub.3
CH.sub.2 ##STR00100## 83 H CH.sub.3/CH.sub.3 CH.sub.2 ##STR00101##
84 CH.sub.3 H/H CH.sub.2 ##STR00102## 85 CH.sub.3 H/H CH.sub.2
##STR00103## 86 H CH.sub.3/CH.sub.3 CH.sub.2 ##STR00104## 87
CH.sub.3 CH.sub.3/CH.sub.3 CH.sub.2 ##STR00105## 88 CH.sub.3
CH.sub.3/CH.sub.3 CH.sub.2 ##STR00106## 89 F CH.sub.3/CH.sub.3
CH.sub.2 ##STR00107## 90 F CH.sub.3/CH.sub.3 CH.sub.2 ##STR00108##
91 H CH.sub.3/CH.sub.3 R--CH (CH.sub.3) ##STR00109## 92 H
CH.sub.3/CH.sub.3 CH.sub.2 ##STR00110## 93 H CH.sub.3/CH.sub.3
CH.sub.2 ##STR00111## 94 H CH.sub.3/CH.sub.3 CH.sub.2 Me.sub.2N--
95 H CH.sub.3/CH.sub.3 CH.sub.2 (CH.sub.3O)(CH.sub.3)N-- 96 H
CH.sub.3/CH.sub.3 CH.sub.2 ##STR00112## 97 H CH.sub.3/CH.sub.3
CH.sub.2 ##STR00113## 98 H CH.sub.3/CH.sub.3 CH.sub.2 ##STR00114##
99 H CH.sub.3/CH.sub.3 CH.sub.2 ##STR00115## 100 H
CH.sub.3/CH.sub.3 CH.sub.2 ##STR00116## 101 H CH.sub.3/CH.sub.3
CH.sub.2 ##STR00117## 102 H CH.sub.3/CH.sub.3 CH.sub.2 ##STR00118##
103 H CH.sub.3/CH.sub.3 CH.sub.2 ##STR00119## 104 H
CH.sub.3/CH.sub.3 CH.sub.2 ##STR00120## 105 H CH.sub.3/CH.sub.3
CH.sub.2 ##STR00121## 106 H CH.sub.3/CH.sub.3 CH.sub.2
##STR00122##
[0191] Based on their I.kappa.B kinase inhibitory properties and
other pharmacological properties, compound example numbers 1-30,
39-62 and 64-106 are preferred. More preferred are compound example
numbers 1, 2, 7, 10, 11, 13, 16, 17, 19-27, 39-62, 64-106.
[0192] The compounds of the present invention may be administered
to humans or other mammals by a variety of routes, including oral
dosage forms and injections (intravenous, intramuscular,
intraperitoneal, subcutaneous, and the like). Numerous other dosage
forms containing compounds of the invention can be readily
formulated by one skilled in the art, utilizing the suitable
pharmaceutical excipients (or carriers) as defined below.
[0193] Examples of pharmaceutically acceptable excipients (or
carriers) and methods of manufacture for various compositions can
be found in A. Gennaro (ed.), Remington: The Science and Practice
of Pharmacy, 20.sup.th Edition, Lippincott Williams & Wilkins,
Baltimore, Md. (2000), which is incorporated in its entirety by
reference herein. Pharmaceutically acceptable excipients (or
carriers) include flavoring agents, pharmaceutical-grade dyes or
pigments, solvents, co-solvents, buffer systems, surfactants,
preservatives, sweetener agents, viscosity agents, fillers,
lubricants, glidants, disintegrants, binders and resins.
[0194] Conventional flavoring agents may be used, such as those
described in Remington's Pharmaceutical Sciences, 18.sup.th Ed.,
Mack Publishing Co., pp. 1288-1300 (1990), which is incorporated in
its entirety by reference herein. The pharmaceutical compositions
of the invention generally contain from about 0 to 2%, of flavoring
agents.
[0195] Conventional dyes and/or pigments may also be used, such as
those described in the Handbook of Pharmaceutical Excipients, by
the American Pharmaceutical Association & the Pharmaceutical
Society of Great Britain, pp. 81-90 (1986), which is incorporated
in its entirety by reference herein. The pharmaceutical
compositions of the invention generally contain from about 0 to 2%
of dyes and/or pigments.
[0196] The pharmaceutical compositions of the invention generally
contain from about 0.1 to 99.9% of solvent(s). A preferred solvent
is water. Preferred co-solvents include ethanol, glycerin,
propylene glycol, polyethylene glycol, and the like. The
pharmaceutical compositions of the invention may include from about
0 to 50% of co-solvents.
[0197] Preferred buffer systems include acetic, boric, carbonic,
phosphoric, succinic, maleic, tartaric, citric, acetic, benzoic,
lactic, glyceric, gluconic, glutaric and glutamic acids and their
sodium, potassium and ammonium salts. Particularly preferred
buffers are phosphoric, tartaric, citric and acetic acids and salts
thereof. The pharmaceutical compositions of the invention generally
contain from about 0 to 5% of a buffer.
[0198] Preferred surfactants include polyoxyethylene sorbitan fatty
acid esters, polyoxyethylene monoalkyl ethers, sucrose monoesters
and lanolin esters and ethers, alkyl sulfate salts and sodium,
potassium and ammonium salts of fatty acids. The pharmaceutical
compositions of the invention generally contain from about 0 to 2%
of surfactants.
[0199] Preferred preservatives include phenol, alkyl esters of
parahydroxybenzoic acid, o-phenylphenol benzoic acid and salts
thereof, boric acid and salts thereof, sorbic acid and salts
thereof, chlorobutanol, benzyl alcohol, thimerosal, phenylmercuric
acetate and nitrate, nitromersol, benzalkonium chloride,
cetylpyridinium chloride, methyl paraben and propyl paraben.
Particularly preferred preservatives are the salts of benzoic acid,
cetylpyridinium chloride, methyl paraben and propyl paraben. The
pharmaceutical compositions of the invention generally contain from
about 0 to 2% of preservatives.
[0200] Preferred sweeteners include sucrose, glucose, saccharin,
sorbitol, mannitol and aspartame. Particularly preferred sweeteners
are sucrose and saccharin. Pharmaceutical compositions of the
invention generally contain from about 0 to 5% of sweeteners.
[0201] Preferred viscosity agents include methylcellulose, sodium
carboxymethylcellulose, hydroxypropyl-methylcellulose,
hydroxypropylcellulose, sodium alginate, carbomer, povidone,
acacia, guar gum, xanthan gum and tragacanth. Particularly
preferred viscosity agents are methylcellulose, carbomer, xanthan
gum, guar gum, povidone, sodium carboxymethylcellulose, and
magnesium aluminum silicate. Pharmaceutical compositions of the
invention generally contain from about 0 to 5% of viscosity
agents.
[0202] Preferred fillers include lactose, mannitol, sorbitol,
tribasic calcium phosphate, dibasic calcium phosphate, compressible
sugar, starch, calcium sulfate, dextro and microcrystalline
cellulose. Pharmaceutical compositions of the invention generally
contain from about 0 to 75% of fillers.
[0203] Preferred lubricants/glidants include magnesium stearate,
stearic acid and talc. Pharmaceutical compositions of the invention
generally contain from about 0 to 7%, preferably, about 1 to 5% of
lubricants/glidants.
[0204] Preferred disintegrants include starch, sodium starch
glycolate, crospovidone and croscarmelose sodium and
microcrystalline cellulose. Pharmaceutical compositions of the
invention generally contain from about 0 to 20%, preferably, about
4 to 15% of disintegrants.
[0205] Preferred binders include acacia, tragacanth,
hydroxypropylcellulose, pregelatinized starch, gelatin, povidone,
hydroxypropylcellulose, hydroxypropylmethylcellulose,
methylcellulose, sugar solutions, such as sucrose and sorbitol, and
ethylcellulose. Pharmaceutical compositions of the invention
generally contain from about 0 to 12%, preferably, about 1 to 10%
of binders.
[0206] Additional agents known to a skilled formulator may be
combined with the compounds of the invention to create a single
dosage form. Alternatively, additional agents may be separately
administered to a mammal as part of a multiple dosage form.
[0207] For preparing pharmaceutical compositions containing the
inventive compounds, inert, pharmaceutically acceptable excipients
or carriers can be either solid or liquid. Solid form preparations
include powders, tablets, dispersible granules, capsules, cachets
and suppositories.
[0208] Generally, the powders and tablets may be comprised of from
about 5 to 95 weight percent of active ingredient. Suitable solid
carriers are known in the art, for example, magnesium carbonate,
magnesium stearate, talc, sugar and lactose. Tablets, powders,
cachets and capsules can be used as solid dosage forms suitable for
oral administration. Examples of pharmaceutically acceptable
carriers and methods of manufacture for various compositions may be
found in Remington's Pharmaceutical Sciences, 18.sup.th Ed., Mack
Publishing Co. (1990).
[0209] Liquid form preparations include solutions, suspensions and
emulsions. Common liquid form preparations include water and
water-propylene glycol solutions for parenteral injection or
addition of sweeteners and opacifiers for oral solutions,
suspensions and emulsions. Liquid form preparations may also
include solutions for intranasal administration.
[0210] Aerosol preparations suitable for inhalation include
solutions and solids in powder form, which may be combined with a
pharmaceutically acceptable carrier, such as an inert compressed
gas (e.g., nitrogen).
[0211] Also included are solid form preparations that may be
converted, shortly before use, to liquid form preparations for
either oral or parenteral administration. Such liquid forms include
solutions, suspensions and emulsions.
[0212] The compounds of the invention may also be delivered
transdermally. The transdermal compositions can take the form of
creams, lotions, aerosols and emulsions and may be included in a
transdermal patch of a matrix or reservoir type as is conventional
in the art for this purpose.
[0213] The preferred mode of administering the compounds of the
invention is oral. Preferably, the pharmaceutical preparation is in
a unit dosage form. In such a form, the preparation is subdivided
into suitable sized unit doses containing appropriate quantities of
the active component, for example, an effective amount to achieve
the desired purpose.
[0214] The quantity of active ingredient (compound) in a unit dose
of preparation may be varied or adjusted from about 0.01 to 4,000
mg, preferably, from about 0.01 to 1,000 mg, more preferably, from
about 0.01 to 500 mg, and even more preferably, from about 0.01 to
250 mg, according to the particular application. A typical
recommended daily dosage regimen for oral administration will
usually range from about 0.02 to 2,000 mg/day, in one to four
divided doses. For convenience, the total daily dosage may be
divided and administered in portions during the day as required.
Typically, pharmaceutical compositions of the invention will be
administered from about 1 to 5 times per day, or alternatively, as
a continuous infusion. Such administration can be used for chronic
or acute therapy. The amount of active ingredient that may be
combined with excipient or carrier materials to produce a single
dosage form will vary depending upon the host treated and the
particular mode of administration. A typical preparation will
usually contain from about 5 to 95% of active compound (w/w).
Preferably, such preparations will contain from about 20 to 80 wt.
% of active compound.
[0215] The pharmaceutically acceptable excipients or carriers
employed in conjunction with the compounds of the invention are
used at a concentration sufficient to provide a practical size to
dosage relationship. The pharmaceutically acceptable excipients or
carriers, in total, can comprise from about 0.1 to 99.9% by weight
of the pharmaceutical compositions of the invention, preferably,
from about 20 to 80% by weight.
[0216] Upon improvement of a patient's condition, a maintenance
dose of a compound, composition or combination of the invention may
be administered, if applicable. Subsequently, the dosage or
frequency of administration, or both, may be reduced, as a function
of the symptoms, to a level at which the improved condition is
retained. When the symptoms have been alleviated to the desired
level, treatment should cease. Patients may, however, require
intermittent treatment on a long-term basis upon any recurrence of
disease symptoms.
[0217] Specific dosage and treatment regimens for any particular
patient may be varied and will depend upon a variety of factors,
including the activity of the specific compound employed, the age,
body weight, general health status, sex and diet of the patient,
the time of administration, the rate of excretion, the specific
drug combination, the severity and course of the symptoms being
treated, the patient's disposition to the condition being treated
and the judgment of the treating physician. Determination of the
proper dosage regimen for a particular situation is within the
skill of the art. The amount and frequency of the administration of
compounds of the invention or their pharmaceutically acceptable
salts may be regulated according to the judgment of the attending
clinician, based on the factors recited above. As a skilled artisan
will appreciate, lower or higher doses than those recited above may
be required.
[0218] The inventive compounds are understood to provide
efficacious treatment of a variety of diseases, symptoms and
disorders, particularly, those which are inflammatory or
immune-related in nature, including a reasonable time of onset upon
administration, and a reasonable duration after administration.
While food, diet, pre-existing conditions, alcohol and other
systemic conditions could lengthen the time delay for an inventive
drug to work after its administration, it is understood that
optimum dosages will result in an efficacious drug treatment within
and for a reasonable amount of time.
[0219] The inventive compounds can exist in unsolvated as well as
solvated forms, including hydrated forms. In general, the solvated
forms, with pharmaceutically acceptable solvents, such as water,
ethanol and the like, are equivalent to the unsolvated forms for
purposes of this invention.
[0220] The inventive compounds may form pharmaceutically acceptable
salts with organic and inorganic acids. Examples of suitable acids
for salt formation are hydrochloric, sulfuric, phosphoric, acetic,
citric, malonic, salicylic, malic, fumaric, succinic, ascorbic,
maleic, methanesulfonic and other mineral and carboxylic acids well
known to those skilled in the art. The salts are prepared by
contacting the free base forms with a sufficient amount of the
desired acid to produce a salt in a conventional manner. The free
base forms may be regenerated by treating the salt with a suitable
dilute aqueous base solution, such as dilute aqueous sodium
hydroxide, potassium carbonate, ammonia or sodium bicarbonate. The
free base forms may differ somewhat from their respective salt
forms in certain physical properties, such as solubility in polar
solvents, but the salts are otherwise equivalent to their
respective free base forms for purposes of the invention,
[0221] On account of their pharmacological properties, the
compounds according to the invention are suitable for the
prophylaxis treatment and therapy of diseases, disorders and
symptoms that involve increased activity of IkB kinase. These
include, for example, joint inflammation (e.g., rheumatoid
arthritis (RA), rheumatoid spondylitis, gouty arthritis, traumatic
arthritis, rubella arthritis, psoriatic arthritis, osteoarthritis,
and other arthritic conditions), acute synovitis, tuberculosis,
atherosclerosis, muscle degeneration, cachexia, Reiter's syndrome,
endotoxaemia, sepsis, septic shock, endotoxic shock, gram negative
sepsis, gout, toxic shock syndrome, pulmonary inflammatory diseases
(e.g., asthma, acute respiratory distress syndrome, chronic
obstructive pulmonary disease, silicosis, pulmonary sarcoidosis,
and the like), bone resorption diseases, reperfusion injuries,
carcinoses, leukemia, sarcomas, lymph node tumors, skin carcinoses,
lymphoma, apoptosis, graft versus host reaction, graft versus host
disease (GVHD), allograft rejection and leprosy.
[0222] Furthermore, the inventive compounds may be used in the
treatment of immune-related diseases, symptoms and disorders, for
example, infections, such as viral infections (e.g., HIV,
cytomegalovirus (CMV), influenza, adenovirus, the Herpes group of
viruses, and the like), parasitic infections (e.g., malaria, such
as cerebral malaria), and yeast and fungal infections (e.g., fungal
meningitis). In addition, the inventive compounds can be useful for
treating fever and myalgias due to infection, acquired immune
deficiency syndrome (AIDS), AIDS related complex (ARC), cachexia
secondary to infection or malignancy, cachexia secondary to AIDS or
cancer, keloid and scar tissue formation, pyresis, diabetes, and
inflammatory bowel diseases (IBD) (e.g., Crohn's disease and
ulcerative colitis). The compounds of the invention are also useful
in the treatment of diseases or injuries to the brain in which
over-expression of TNF-.alpha. has been implicated, such as
multiple sclerosis (MS), ischemic brain injury, e.g. cerebral
infarction (stroke) and head trauma. The compounds of the invention
are also useful in the treatment of psoriasis, Alzheimer's disease,
carcinomatous disorders (potentiation of cytotoxic therapies),
cardiac infarct, chronic obstructive pulmonary disease (COPD) and
acute respiratory distress syndrome (ARDS).
[0223] In one embodiment the compounds of this invention are useful
for treating cancer, especially for treating cancers where IKK
activity is abnormally high. The cancer types that may be treated
include lymphoma, such as diffuse large B-cell, primary mediastinal
B-cell, and mantle cell; multiple myeloma; osteolytic bone
metastasis; head and neck squamous cell cancer; prostate cancer;
pancreatic cancer and non-small cell lung cancer. In one
embodiment, the compounds are useful for ABC lymphoma. For the
treatment of cancer, the compounds may be used as a single agent or
in combination with other agents known to be useful for the
treatment of cancer. Examples of such other agents include
bortezomib; capecitibine; gemcitabine; irinotecan; fludarabine;
5-fluorouricil or 5-fluorouricil/leucovorin; taxanes, including,
e.g., paclitaxel and docetaxel; platinum agents, including, e.g.,
cisplatin, carboplatin, and oxaliplatin; anthracyclins, including,
e.g., doxorubicin and pegylated liposomal doxorubicin;
mitoxantrone; dexamethasone; vincristine; etoposide; prednisone;
thalidomide; Herceptin; temozolomide; and alkylating agents such as
melphalan, chlorambucil, and cyclophosphamide.
[0224] The compounds of formula (I) are especially useful for
treating inflammatory and immune-related diseases, disorders and
symptoms, more especially, inflammatory ones such as RA, asthma,
IBD, psoriasis, COPD and MS. It will be appreciated that the
present compounds are useful for treating diseases, disorders or
symptoms related to the activity of NF-.kappa.B, TNF-.alpha., and
other enzymes in pathways where IKK is known to modulate
activity.
[0225] The compounds of this invention are also useful for treating
a bone associated disease, symptom or disorder in which there is a
deficit or deficiency of bone--either as a result of decreased new
bone formation or an increase in bone resorption or a combination
of both. Specific examples include osteoporosis, periodontal
disease, osteomyelitis, rheumatoid arthritis, aseptic joint
loosening and osteolytic lesions (typically cancer related). It is
known that rheumatoid arthritis, which is characterized by
inflammation of the joints, is also associated with destruction of
cartilage and bone. Furthermore, it has been reported that an IKK
inhibitor provided inhibition of cartilage and bone loss in a
murine model of collagen-induced arthritis. See McIntyre et al.,
Arthritis & Rheumatism (2003), 48(9), 2652-2659.
[0226] Osteoporosis is a broad term applied to a number of distinct
diseases in which there is decreased bone mass. These include
primary osteoporosis (e.g., post-menopausal, senile osteoporosis
and juvenile osteoporosis) and secondary osteoporosis. Examples of
secondary osteoporosis would be those associated with chronic
diseases (e.g., chronic renal disease, hepatic insufficiency,
gastrointestinal malabsorption, chronic immobilization and chronic
inflammatory diseases, including rheumatoid arthritis,
osteoarthritis, periodontal disease and aseptic prosthetic joint
loosening), endocrine dysfunction related diseases (e.g., diabetes,
hyperthyroidism, hyperparathyroidism, hypogonadism and
hypopituitarism), drug and substance related symptoms (e.g.,
corticosteroid, heparin, anticonvulsants, alcohol and
immunosupressants), and hematological disorders (e.g., metastatic
disease, myeloma, leukemia, gaucher's disease and anemia).
Inhibition of either IkB directly or the NF-kB pathway indirectly
has been reported to be useful for the treatment of osteoporosis
and osteoarthritis. See, for example, PCT applications WO
2003104219, WO 2003103658, WO 2003029242, WO 2003065972, and WO
9965495. Accordingly, this invention also provides a method of
treating or preventing bone loss in a patient in need thereof,
comprising administering to the patient a compound of this
invention. Also provided is a method of generating bone formation
in a patient comprising administering a compound of this
invention.
[0227] Another embodiment of the invention provides a method of
inhibiting activation of NF-.kappa.B dependent gene expression
associated with the inhibition of IKK catalytic activity and/or
I.kappa.B phosphorylation, comprising administering to a patient in
need thereof an amount of the compound according to claim 1 or the
pharmaceutically acceptable salt or solvate thereof, or a
pharmaceutical composition thereof, which is effective to inhibit
IKK catalytic activity and/or I.kappa.B phosphorylation, thereby
inhibiting activation of NF-.kappa.B dependent gene expression.
[0228] In one embodiment of the invention, there is provided a
method of treating or preventing an inflammatory or immune-related
physiological disorder, symptom or disease in a patient in need of
such treatment, comprising administering to the patient an amount
of at least one compound according to claim 1, or the
pharmaceutically acceptable salt or solvate thereof, or a
pharmaceutical composition thereof, which is effective to treat or
prevent the inflammatory or immune-related physiological disorder,
symptom or disease. Preferably, the inflammatory disease, disorder
or symptom is rheumatoid arthritis, asthma, psoriasis, psoriatic
arthritis, chronic obstructive pulmonary disease (COPD),
inflammatory bowel disease or multiple sclerosis.
[0229] The invention comprises a compound having the formula (I), a
method for making an inventive compound, a method for making a
pharmaceutical composition from at least one inventive compound and
at least one pharmaceutically acceptable carrier or excipient, and
a method of using one or more inventive compounds to treat a
variety of disorders, symptoms and diseases, particularly ones that
are inflammatory or immune-related in nature. The inventive
compounds and their pharmaceutically acceptable salt and neutral
compositions may be formulated together with a pharmaceutically
acceptable excipient or carrier and the resulting composition may
be administered in vivo to mammals, such as primates, e.g.
chimpanzees and humans (e.g. males and females) and animals (e.g.,
dogs, cats, cows, horses, and the like), to treat a variety of
disorders, symptoms and diseases. Furthermore, the inventive
compounds can be used to prepare a medicament that is useful for
treating a variety of disorders, symptoms and diseases.
[0230] While one or more of the inventive compounds may be used in
an application of monotherapy to treat a disorder, disease or
symptom, they also may be used in combination therapy, in which the
use of an inventive compound or composition (therapeutic agent) is
combined with the use of one or more other therapeutic agents for
treating the same and/or other types of disorders, symptoms and
diseases. Combination therapy includes administration of the
therapeutic agents concurrently or sequentially. Alternatively, the
therapeutic agents can be combined into one composition which is
administered to the patient.
[0231] In one embodiment, the compounds of this invention are used
in combination with other therapeutic agents, such as other
inhibitors of IKK, other agents useful in treating NF-.kappa.B and
TNF-.alpha. associated conditions, and agents useful for treating
other disorders, symptoms and diseases. In particular, agents that
induce apoptosis such as agents that disrupt cell cycle or
mitochondrial function are useful in combination with the IKK
inhibitors of this invention. Exemplary agents for combination with
the IRK inhibitors include antiproliferative agents (e.g.,
methotrexate) and the agents disclosed in U.S. Pat. Application
Publication No. US2003/0022898, p 14, para. [0173-0174], which is
incorporated herein in its entirety. In some embodiments, a
compound of the invention is administered in conjunction with a
therapeutic agent selected from the group consisting of cytotoxic
agents, radiotherapy, and immunotherapy. Non-limiting examples of
cytotoxic agents suitable for use in combination with the IKK
inhibitors of the invention include capecitibine; gemcitabine;
irinotecan; fludarabine; 5-fluorouracil or
5-fluorouracil/leucovorin; taxanes, including, e.g., paclitaxel and
docetaxel; platinum agents, including, e.g., cisplatin,
carboplatin, and oxaliplatin; anthracyclins, including, e.g.,
doxorubicin and pegylated liposomal doxorubicin; mitoxantrone;
dexamethasone; vincristine; etoposide; prednisone; thalidomide;
herceptin; temozolomide; and alkylating agents such as melphalan,
chlorambucil, and cyclophosphamide. It is understood that other
combinations may be undertaken while remaining within the scope of
the invention.
[0232] Still another aspect of this invention is to provide a kit
comprising separate containers in a single package, wherein the
inventive pharmaceutical compounds, compositions and/or salts
thereof are used in combination with pharmaceutically acceptable
carriers to treat disorders, symptoms and diseases where IkB kinase
plays a role.
[0233] The compounds of this invention may be prepared by methods
known to those skilled in the art for analogous compounds, as
illustrated by the general schemes below, and by reference to the
preparative examples shown below.
##STR00123##
[0234] Scheme I above shows a general route for obtaining compounds
of formula I. A Ring A carboxylic acid 1a may be coupled with the
desired amino beta-carboline 2a to provide 1. Many 1a intermediates
that are useful for preparing compounds of this invention are
readily available from known starting materials and chemical
methods, especially in view of the synthetic examples detailed
herein. Schemes II-IV describe routes for making the various
.beta.-carboline intermediates 2a.
##STR00124##
[0235] Scheme II above shows a route for making a beta-carboline
moiety where R.sup.1 is hydrogen, R.sup.2 is chloro and R.sup.3 is
alkoxy. While the scheme is exemplified for R.sup.3 being methoxy,
it will be appreciated by one skilled in the art that
beta-carbolines having other R.sup.3 alkoxy groups may be obtained
by replacing NaOMe in step (e) with other sodium or metal
alkoxides.
##STR00125##
[0236] Scheme III above shows a route for preparing a
beta-carboline intermediate where R.sup.1 is an alkyl such as
methyl, R.sup.2 is a halo such as chloro and R.sup.3 is hydrogen.
One skilled in the art will understand how the above scheme may be
modified to obtain an R.sup.1 alkyl group other than methyl or an
R.sup.2 halo group other than chloro.
##STR00126##
[0237] Scheme IV above shows a route for making a beta-carboline
intermediate where R.sup.1 is fluoro, R.sup.2 is chloro and R.sup.3
is hydrogen. It will be appreciated that ready modification of this
scheme will allow for the preparation of other intermediates. For
example, another R.sup.2 group may be introduced by replacing the
4-chloro-2-iodoaniline in step (b) with another 2-iodoaniline
having a substituent other than chloro in the 4-position.
[0238] A particularly useful intermediate for making compounds of
formula III-A-aa is intermediate 3a:
##STR00127##
where R.sup.13 is halo, OH, OR.sup.15, or a carboxylic acid
protecting group; R.sup.14 is an amino protecting group, hydrogen
or --W-G as defined above; and R.sup.15 is an organic radical.
Amino protecting groups are well-known in the art. Examples of
suitable amino protecting groups include alkoxycarbonyl groups such
as t-butoxycarbonyl (t-BOC) and benzyl groups such as benzyl and
para-methoxybenzyl. The carboxylic acid group at the 3-position of
the morpholine ring may be protected as any stable ester group such
as a simple alkyl or aryl ester such as a methyl, ethyl, benzyl, or
pentafluorophenyl ester. In one embodiment, R.sup.14 is --W-G and
R.sup.13 is --OH, halo, or a carboxylic acid protecting group.
Various protecting groups are described in detail in Protecting
Groups in Organic Synthesis, Theodora W. Greene and Peter G. M.
Wuts, 3.sup.rd edition, 1999, published by John Wiley and Sons.
[0239] A preferred enantiomer of intermediate 3a is (S)-3a:
##STR00128##
where R.sup.13 and R.sup.14 are as described above.
[0240] Intermediate 3a or (S)-3a, as the carboxylic acid or an
activated form thereof (such as the acid chloride), may be coupled
with an appropriate amino-beta-carboline as outlined in Scheme I
above. When R.sup.14 is an amino protecting group, the amide
coupling reaction provides further useful intermediates, shown
below as compounds of formula IV:
##STR00129##
where R.sup.14 is an amino protecting group and R.sup.1, R.sup.2
and R.sup.3 are as described above. It will be appreciated by one
of skill in the art that certain compounds of formula III-A-aa
(where R.sup.6b is each methyl) may be prepared from compounds of
formula IV by removing the R.sup.14 protecting group and then
attaching the --W-G portion using known methods. Alternatively, the
compounds of formula III-A-aa may be prepared by first constructing
intermediate 3a where R.sup.14 is --W-G and R.sup.13 is a
carboxylic acid or derivative thereof. The amide coupling reaction
with an appropriate amino-beta-carboline then provides the
compounds of formula III-A-aa directly.
##STR00130##
[0241] Scheme V above shows a route for making intermediates of
formula 3a, including the unprotected i-19. The selective
protection and deprotection of the amino and carboxylic acid groups
in i-19 to provide various 3a intermediates will be within the
knowledge of one skilled in the art.
[0242] Another useful intermediate for making compounds of formula
III-A-aa is a compound of formula V, preferably (S)-V:
##STR00131##
[0243] where R.sup.13 is halo or other leaving group, OH,
OR.sup.15, or a carboxylic acid protecting group, R.sup.15 is an
organic radical such as a C.sub.1-6 aliphatic, aryl or benzyl, Ring
A has 0-2 or 0-4 R.sup.6b, and R.sup.1, R.sup.2, R.sup.3, and
R.sup.6b are as defined above.
[0244] Another useful intermediate for making compounds of this
invention is VI, preferably (S)-VI:
##STR00132##
where one of R.sup.13 and R.sup.13a is OH or a leaving group such
as halo and the other is OR.sup.15 or a carboxylic acid protecting
group, R.sup.15 is an organic radical such as a C.sub.1-6
aliphatic, aryl or benzyl, Ring A has 0-2, and R.sup.6b is as
defined above.
SYNTHESIS EXAMPLES
[0245] The following abbreviations are used in the methods of
preparation: RT or rt is room temperature; h, hr or hrs is hour or
hours; min is minutes; TFA is trifluoroacetic acid; DMSO is
dimethylsulfoxide; NCS is N-chlorosuccinimide; EDCI is
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; EtOAc
is ethyl acetate; DIEA is diisopropylethylamine; DCM is
dichloromethane; DDQ is dichloro dicyano benzoquinone; mCPBA is
meta-chloroperbenzoic acid; MeOH is methanol; EtOH is ethanol; MeCN
is acetonitrile; TLC is thin layer chromatography; AIBN is
azobisisobutyronitrile; NH.sub.4OAc is ammonium acetate; NaOAc is
sodium acetate; Et.sub.2O is diethyl ether; AcOH is acetic acid;
and DMF is dimethylformamide. TBTU is
N,N,N',N'-tetramethyl-o-(benzotriazol-1-yl)uranium
tetrafluoroborate.
INTERMEDIATE 1:
7-fluoro-2,3,4,9-tetrahydro-1H-.beta.-carboline-1-carboxylic
acid
[0246] To 10 g (46.6 mmol) of commercially available
6-fluorotryptamine hydrochloride was added 50 ml of 1M acetate
buffer (pH 4.4) to give a suspension that was stirred at room
temperature (RT). A suspension of glyoxylic acid monohydrate (1.1
eq, 51.28 mmol, 4.72 g) in ethyl acetate was then added to the
stirred suspension in one portion. The suspension was stirred
overnight (16 h) at RT and the precipitated solid was collected by
filtration and washed with both H.sub.2O and ethyl acetate. The
sample was then dried in vacuo to give a light yellow solid in
quantitative yield.
[0247] .sup.1H-NMR (300 MHz, acetic acid-d.sub.4): .delta. 3.04 (m,
2H), 3.56 (m, 1H), 3.83 (m, 1H), 6.80 (m, 1H), 7.13 (dd, 1H), 7.34
(dd, 1H).
[0248] Retention Time (LC, method; ammonium acetate standard): 1.17
min.
[0249] MS (M+H.sup.+): 235.0.
INTERMEDIATE 2: 7-fluoro-2,3,4,9-tetrahydro-1H-.beta.-carboline
[0250] 7-fluoro-2,3,4,9-tetrahydro-1H-.beta.-carboline-1-carboxylic
acid (5 g, 21.36 mmol) was suspended in 130 ml of 3N HCl in a 500
ml round-bottom flask and refluxed overnight (16 hr) with stirring.
Upon cooling, a light brown solid precipitated out, which was
collected by filtration and washed with H.sub.2O. The salt obtained
by filtration above was then dissolved in hot methanol (200 ml) and
treated with 3M K.sub.2CO.sub.3 (5-10 ml) such that the pH is
around 9. 100 ml of H.sub.2O was added to this mixture, which was
then allowed to stir at RT. The methanol was evaporated on a rotary
evaporator to give a white aqueous suspension of the desired free
base, which was collected by filtration (3.2 g, 79% yield).
[0251] .sup.1H-NMR (300 MHz, methanol-d.sub.4): .delta. 2.73 (t,
2H), 3.11 (t, 2H), 3.94 (s, 2H), 6.73 (m, 1H), 6.94 (m, 1H), 7.30
(dd, 1H).
[0252] Retention Time (LC, method: ammonium acetate standard): 1.25
min.
[0253] MS (M+H.sup.+): 191.1.
INTERMEDIATE 3: 7-fluoro-9H-.beta.-carboline
[0254] 7-fluoro-2,3,4,9-tetrahydro-1H-.beta.-carboline (3.5 g,
18.42 mmol) was suspended in xylenes (60 ml) in a 250 ml
round-bottom flask equipped with a condenser that was open to the
atmosphere, and heated. To this hot reaction mixture was added Pd/C
(10 wt %, 0.2 eq, 700 mg) and the mixture refluxed in xylenes
overnight (12-14 hours). The solution was then filtered through a
pad of celite and the collected filtrate was then evaporated on a
rotary evaporator to give the desired product as a brown/tan solid
(3.0 g, 88% yield).
[0255] .sup.1H-NMR (300 MHz, DMSO-d.sub.5): .delta. 7.10 (m, 1H),
7.37 (dd, 1H), 8.10 (d, 1H), 8.28 (dd, 1H), 8.35 (dd, 1H), 8.89 (s,
1H), 11.74 (s, 1H).
[0256] Retention Time (LC, method: ammonium acetate standard): 1.88
min.
[0257] MS (M+H.sup.+): 187.1.
INTERMEDIATE 4: 6-chloro-7-fluoro-9H-.beta.-carboline
[0258] 7-fluoro-9H-.beta.-carboline (2.15 g, 11.58 mmol) was
suspended in 100 ml of 1N HCl. To this mixture was added NCS (1.85
g, 13.89 mmol, 1.2 eq) and the resulting mixture was stirred at RT
overnight. The reaction mixture was then filtered to give a light
yellow solid (2.1 g, 83% yield).
[0259] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 7.86 (d, 1H),
8.64 (d, 1H), 8.79 (d, 1H), 8.91 (d, 1H), 9.33 (s, 1H), 13.05 (s,
1H).
[0260] Retention Time (LC, method: ammonium acetate standard): 2.19
min.
[0261] MS (M+H.sup.+): 221.1.
INTERMEDIATE 5: 6-chloro-7-fluoro-8-nitro-9H-.beta.-carboline
[0262] 6-chloro-7-fluoro-9H-.beta.-carboline (2.1 g, 9.54 mmol) was
taken in a round-bottom flask (250 ml) and NaNO.sub.3 (1.136 g,
13.36 mmol, 1.4 eq) was added. TFA (48 ml) was then added to the
flask and the resulting mixture refluxed overnight. The TFA is then
removed on a rotary evaporator. The resulting slurry is suspended
in water (50 ml) and sonicated thoroughly. The resulting suspension
is then filtered to give a yellow solid (2.0 g, 80% yield).
[0263] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 8.21 (d, 1H),
8.46 (d, 1H), 9.04 (m, 2H), 12.55 (s, 1H).
[0264] Retention Time (LC, method: ammonium acetate standard): 2.24
min.
[0265] MS (M+H.sup.+): 266.2.
INTERMEDIATE 6: 6-chloro-7-methoxy-8-nitro-9H-.beta.-carboline
[0266] Methanol (0.462 ml, 11.4 mmol) was added to a stirring
suspension of NaH (684 mg, 17.1 mmol) in DMF (10 ml) under an argon
atmosphere. The resulting solution was allowed to stir at RT for 20
min. 6-chloro-7-fluoro-8-nitro-9H-.beta.-carboline (500 mg, 1.9
mmol) was added to the stirring solution and the resulting mixture
was allowed to stir at RT. Upon addition of H.sub.2O, a brown solid
precipitated out which was filtered to give the desired
6-chloro-7-methoxy-8-nitro-9H-.beta.-carboline (510 mg, 97%
yield).
[0267] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 4.02 (s, 3H),
8.52 (d, 1H), 8.60 (d, 1H), 9.05 (s, 1H), 9.12 (s, 1H), 12.78 (b,
1H).
[0268] Retention Time (LC, method: ammonium acetate standard): 2.28
min.
[0269] MS (M+H.sup.+): 278.
INTERMEDIATE 7:
6-chloro-7-methoxy-9H-.beta.-carboline-8-ylamine
[0270] 6-chloro-7-methoxy-8-nitro-9H-.beta.-carboline (510 mg, 1.84
mmol) was suspended in 50 ml of methanol and 100 mg of Pd/C (10%)
was added. The flask was fitted with a balloon of hydrogen and the
reaction mixture was stirred overnight at RT. Upon filtration
through a pad of celite and evaporation of the methanol, a dark
brown solid was obtained. This residue was suspended in methanol
(15 ml) and added, with vigorous stirring, to a solution of
saturated NaHCO.sub.3 (100 ml). The light brown solid that
precipitated out was collected by filtration and dried thoroughly
in vacuo to give the desired product (512 mg, quantitative
yield).
[0271] .sup.1H-NMR (300 MHz, methanol-d.sub.4): .delta. 3.90 (s,
3H), 7.63 (s, 1H), 8.11 (d, 1H), 8.27 (d, 1H), 8.84 (s, 1H).
[0272] Retention Time (LC, method: ammonium acetate standard): 1.12
min.
[0273] MS (M+H.sup.+): 248.
INTERMEDIATE 8: 6-chloro-9H-.beta.-carboline-8-ylamine
[0274] The target compound was prepared according to the procedures
outlined for Intermediate 1 to Intermediate 7 where the starting
material for Intermediate 1 was unsubstituted tryptamine. An
alternative synthesis for 6-chloro-9H-.beta.-carboline-8-ylamine is
described on page 34, example 15 of PCT Application Publication No.
WO 01/68648 A1, which is incorporated herein in its entirety.
METHOD A: COUPLING PROCEDURE FOR 6,7,8-SUBSTITUTED-8-CARBOLINES
[0275] 6,7,8-substituted-9H-.beta.-carboline (1 mmol), EDCI (1.6
mmol) and the appropriate carboxylic acid (1.2 mmol) were taken in
a round-bottom flask and suspended in pyridine (5 ml). The
resulting mixture was heated at 60.degree. C. overnight. The
pyridine was then removed by rotary evaporation and 5%
Na.sub.2CO.sub.3 solution was added. The resulting solid that
precipitated out was collected by filtration. Chromatographic
purification gave the desired product.
METHOD B: COUPLING PROCEDURE FOR
6,8-SUBSTITUTED-.beta.-CARBOLINES
[0276] 6,8-substituted-9H-.beta.-carboline (1.0 mmol), EDCI (1.6
mmol) and the carboxylic acid (1.2 mmol) to be coupled were taken
in a round-bottom flask and suspended in pyridine (5 ml). The
resulting mixture was stirred overnight. The pyridine was then
removed by rotary evaporation and 5% Na.sub.2CO.sub.3 solution was
added. The resulting solid that precipitated out was collected by
filtration. Chromatographic purification gave the desired
product.
Example 1
N-(6-chloro-7-methoxy-9H-.beta.-carbolin-8-yl)-2-methyl-nicotinamide
[0277] 6-chloro-7-methoxy-9H-.beta.-carbolin-8-ylamine (100 mg, 0.4
mmol), EDCI (125 mg, 0.64 mmol) and 2-methyl nicotinic acid (66 mg,
0.48 mmol) were taken in a round-bottom flask and suspended in
pyridine (2 ml). The resulting mixture was heated at 80.degree. C.
overnight. The pyridine was then removed by rotary evaporation and
5% Na.sub.2CO.sub.3 solution was added. The resulting solid that
precipitated out was collected by filtration, Chromatographic
purification gave the desired product in 50-70% yield.
[0278] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 2.71 (s, 3H),
3.89 (s, 3H), 7.45 (dd, 1H), 8.15 (d, 1H), 8.21 (d, 1H), 8.38 (d,
1H), 8.45 (s, 1H), 8.61 (d, 1H), 8.92 (s, 1H), 10.33 (s, 1H), 11.57
(s, 1H).
[0279] Retention Time (LC, method: ammonium acetate standard): 1.77
min.
[0280] MS (M+H.sup.+): 367.1.
Example 2
4-methyl-pyrimidine-5-carboxylic acid
(6-chloro-7-methoxy-9H-beta-carbolin-8-yl)-amide
[0281] The desired compound was prepared according to Method A from
6-chloro-7-methoxy-9H-beta-carbolin-8-ylamine and
4-methyl-5-pyrimidine carboxylic acid in 80% yield.
[0282] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 2.75 (s, 3H),
3.92 (s, 3H), 8.19 (d, 1H), 8.41 (d, 1H), 8.50 (s, 1H), 8.96 (s,
1H), 9.20 (s, 1H), 9.25 (s, 1H), 10.63 (s, 1H), 11.67 (s, 1H).
[0283] Retention Time (LC, method: formic acid standard): 0.95
min.
[0284] MS (M+H.sup.+): 368.
INTERMEDIATE 9: 6-chloro-7-ethoxy-8-nitro-9H-.beta.-carboline
[0285] Sodium ethoxide (232 mg, 3.4 mmol) was added to a solution
of 6-chloro-7-fluoro-8-nitro-9H-.beta.-carboline (200 mg, 0.76
mmol) in DMSO (4 ml) and the reaction mixture allowed to stir
overnight. The reaction mixture was diluted with water and the pH
of the solution was adjusted to about 4 by adding 1N HCl. The
aqueous solution was extracted (3.times.) with EtOAc. The combined
EtOAc layers were dried and evaporated. The crude product was
purified by flash chromatography to give the desired product in
40-60% yield.
[0286] .sup.1H-NMR (300 MHz, DMSO-d.sub.5): .delta. 1.44 (t, 3H),
4.24 (q, 2H), 8.21 (d, 1H), 8.46 (d, 1H), 8.91 (s, 1H), 9.02 (s,
1H).
[0287] Retention Time (LC, method: ammonium acetate standard): 2.37
min.
[0288] MS (M+H.sup.+): 291.9.
INTERMEDIATE 10:
6-chloro-7-ethoxy-9H-.beta.-carboline-8-ylamine
[0289] 6-chloro-7-ethoxy-8-nitro-9H-.beta.-carboline (160 mg, 0.55
mmol) was suspended in 4 ml of methanol and 25 mg of Pd/C (10%) was
added. The flask was fitted with a balloon of hydrogen and the
reaction mixture was stirred overnight at RT. Upon filtration
through a pad of celite and evaporation of the methanol, a dark
brown solid was obtained and determined to be the desired
6-chloro-7-cyclopropylmethoxy-9H-.beta.-carbolin-8-ylamine (80 mg,
55%).
[0290] .sup.1H-NMR (300 MHz, Methanol-d.sub.4/CDCl.sub.3): .+-.1.26
(t, 3H), 3.91 (q, 2H), 7.34 (s, 1H), 7.71 (d, 1H), 8.02 (s, 1H),
8.56 (s, 1H).
[0291] Retention Time (LC, method: formic acid standard): 1.16
min.
[0292] MS (M+H.sup.+): 262.0.
Example 3
N-(6-chloro-7-ethoxy-9H-.beta.-carbolin-8-yl)-2-methyl-nicotinamide
[0293] The desired compound was prepared according to Method A from
6-chloro-7-ethoxy-9H-.beta.-carbolin-8-ylamine and
2-methylnicotinic acid in 40% yield.
[0294] .sup.1H-NMR (300 MHz, MeOH-d.sub.4): .beta. 1.39 (t, 3H),
2.76 (s, 3H), 4.14 (q, 2H), 7.43 (dd, 1H), 8.05 (d, 1H), 8.26 (m,
3H), 8.55 (d, 1H), 8.79 (s, 1H).
[0295] Retention Time (LC, method: ammonium acetate standard): 1.98
min.
[0296] MS (M+H.sup.+): 381.3.
INTERMEDIATE 11:
6-chloro-7-(N,N)-dimethylamino-8-nitro-9H-.beta.-carboline
[0297] N,N-dimethylamine hydrochloride (278 mg, 3.4 mmol) was added
to a stirring solution of
6-chloro-7-fluoro-8-nitro-9H-.beta.-carboline (300 mg, 1.13 mmol)
in DMSO (8 ml) under an argon atmosphere. This was followed by the
addition of DIEA (0.83 ml, 4.65 mmol) and the reaction mixture was
heated at 60.degree. C. overnight. After allowing the reaction
mixture to cool to RT, water was added and a dark orange solid
precipitated out. The solid was filtered, washed with water and
dried to give the desired product (230 mg, 70% yield).
[0298] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 4.06 (s, 6H),
7.23 (d, 1H), 7.53 (d, 1H), 7.66 (s, 1H), 8.05 (s, 1H),
[0299] Retention Time (LC, method: ammonium acetate standard): 2.41
min.
[0300] MS (M+H.sup.+): 290.9.
INTERMEDIATE 12:
6-chloro-7-(N,N)-dimethylamino-9H-.beta.-carboline-8-ylamine
[0301] 6-chloro-7-(N,N)-dimethylamino-8-nitro-9H-.beta.-carboline
(828 mg, 2.86 mmol) was suspended in 30 ml of methanol and 166 mg
of Pd/C (10%) was added. The flask was fitted with a balloon of
hydrogen and the reaction mixture was stirred overnight at ambient
temperature. Upon filtration through a pad of celite and
evaporation of the methanol, a dark brown solid was obtained and
determined to be the desired
6-chloro-7-(N,N)-dimethylamino-9H-.beta.-carboline-8-ylamine (500
mg, 67% yield).
[0302] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 2.80 (s, 6H),
5.42 (s, 2H), 7.45 (s, 1H), 7.95 (d, 1H), 8.23 (d, 1H), 8.86 (d,
1H).
[0303] Retention Time (LC, method: ammonium acetate standard): 2.32
min.
[0304] MS (M+H.sup.+): 261.1.
Example 4
N-(6-chloro-7-7-(N,N)-dimethylamino-9H-.beta.-carbolin-8-yl)-2-methyl-nico-
tinamide
[0305] The desired compound was prepared according to Method A from
6-chloro-7-(N,N)-dimethylamino-9H-.beta.-carboline-8-ylamine and
2-methylnicotinic acid in 40-60% yield.
[0306] .sup.1H-NMR (300 MHz, methanol-d.sub.4/CDCl.sub.3): .delta.
2.82 (s, 3H), 2.94 (s, 6H), 7.33 (m, 1H), 8.02 (m, 3H), 8.34 (d,
1H), 8.61 (d, 1H), 8.95 (s, 1H).
[0307] Retention Time (LC, method: ammonium acetate standard): 2.29
min.
[0308] MS (M+H.sup.+): 380.3.
INTERMEDIATE 13:
6-chloro-7-(4-methyl-piperazin-1-yl)-8-nitro-9H-.beta.-carboline
[0309] To a DMSO solution (4 ml) of 200 mg (0.755 mmol) of
6-chloro-7-fluoro-8-nitro-9H-.beta.-carboline was added
1-methylpiperazine (226 mg, 2.26 mmol) and DIEA (400 mg, 3.09 mmol)
via a syringe. The reaction was allowed to stir at RT overnight.
Upon addition of water, an orange solid precipitated out. The solid
was filtered, washed and dried to give 236 mg (91% yield) of the
desired
6-chloro-7-(4-methyl-piperazin-1-yl)-8-nitro-9H-.beta.-carboline.
[0310] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 2.24 (s, 3H),
2.48 (m, 4H), 3.13 (m, 4H), 8.13 (d, 1H), 8.40 (d, 1H), 8.73 (s,
1H), 8.94 (s, 1H), 12.05 (s, 1H).
[0311] Retention Time (LC, method: ammonium acetate standard): 1.72
min.
[0312] MS (M+H.sup.+): 346.
INTERMEDIATE 14:
6-chloro-7-(4-methyl-piperazin-1-yl)-8-amino-9H-.beta.-carboline
[0313] Suspended 236 mg of
6-chloro-7-(4-methyl-piperazin-1-yl)-8-nitro-9H-.beta.-carboline in
100 ml of methanol and added 10% Pd/C (48 mg) under argon. The
flask was flushed with hydrogen (3.times.) and the reaction mixture
was stirred under a hydrogen atmosphere at RT overnight. The
reaction mixture was filtered and Pd/C was removed using a pad of
celite. The reaction mixture was evaporated to remove solvent and
purified by flash chromatography to give 119 mg (55% yield) of the
desired
6-chloro-7-(4-methyl-piperazin-1-yl)-8-amino-9H-.beta.-carboline.
[0314] .sup.1H-NMR (300 MHz, methanol-d.sub.4): .delta. 2.39 (s,
3H), 2.46 (m, 2H), 2.87 (m, 4H), 3.83 (m, 2H), 7.50 (s, 1H), 7.97
(d, 1H), 8.24 (d, 1H), 8.78 (s, 1H).
[0315] Retention Time (LC, method: ammonium acetate polar): 1.32
min.
[0316] MS (M+H.sup.+): 316.
Example 5
2-chloro-N-[6-chloro-7-(4-methyl-piperazin-1-yl)-9H-.beta.-carbolin-8-yl]--
nicotinamide
[0317] The desired compound was prepared according to Method A from
6-chloro-7-(4-methyl-piperazin-1-yl)-8-amino-9H-.beta.-carboline
and 2-chloro-nicotinic acid in 25% yield.
[0318] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 2.21 (s, 3H),
2.33 (m, 2H), 2.54 (m, 2H), 3.24 (m, 4H), 7.73 (dd, 1H), 8.12 (d,
1H), 8.35 (d, 1H), 8.37 (s, 1H), 8.48 (dd, 1H), 8.61 (dd, 1H), 8.91
(s, 1H), 10.40 (s, 1H), 11.33 (s, 1H).
[0319] Retention Time (LC, method: ammonium acetate standard): 1.46
min.
[0320] MS (M+H.sup.+): 455.
INTERMEDIATE 15: Resolution of rac-terebic acid
[0321] Terebic acid was dissolved in a 10:1 mixture of EtOAc-MeOH
[10 g of terebic acid (17.7 g of salt) in 550 ml] and heated to
50-55.degree. C., followed by addition of
(S)-(-)-.alpha.-methyl-benzylamine. The reaction mixture was
stirred for 2 minutes and then left at RT for 15 minutes. The
reaction mixture was then seeded with enriched salt (prepared on a
smaller scale using 3 recrystallization cycles), sonicated for
10-15 seconds and left at RT overnight. The solid was filtered off,
washed with EtOAc and dried under vacuum. Recrystallization was
done in the same solvent mixture by re-dissolving the salt (24
mg/ml). This mixture was then heated to a gentle reflux for a short
period of time (few crystals remained in suspension). The mixture
was left at RT over night. The solid was processed as previously
described.
[0322] Enantiomeric excess was determined in a crude fashion using
proton NMR of the corresponding amide obtained from TBTU
coupling.
[0323] Regeneration of (R)-(+)-terebic acid: The salt was dissolved
in water (320 mg/ml), heated to 65.degree. C. and 1.2 equivalent of
aqueous 6M HCl was added. The reaction mixture was then left at
4.degree. C. overnight. The solid was filtered off, washed with
small portions of cold water and dried under a high vacuum (yield
of 25-30%).
[0324] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 1.30 (s, 3H),
1.52 (s, 3H), 2.74 (dd, 1H), 2.85 (dd, 1H), 3.25 (t, 1H).
[0325] Retention Time (LC, method: ammonium acetate standard): 0.33
min.
[0326] MS (M+H.sup.+): 159.0.
Example 6
2,2-dimethyl-5-oxo-tetrahydro-furan-3-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide
[0327] The desired compound was prepared according to Method B from
6-chloro-9H-.beta.-carboline-8-ylamine and (R)-terebic acid
(Intermediate 15) in 80-90% yield.
[0328] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 1.44 (s, 3H),
1.59 (S, 3H), 2.91 (dd, 1H), 3.08 (dd, 1H), 3.38 (dd, 1H), 7.85 (m,
1H), 8.17 (d, 1H), 8.25 (d, 1H), 8.39 (d, 1H), 9.05 (s, 1H), 10.26
(s, 1H), 11.72 (s, 1H).
[0329] Retention Time (LC, method: ammonium acetate standard): 1.22
min.
[0330] MS (M+H.sup.+): 358.3.
Example 7
1,2,2-trimethyl-5-oxo-pyrrolidine-3-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide
[0331] The desired compound was prepared according to Method B from
6-chloro-9H-.beta.-carboline-8-ylamine and
1,2,2-trimethyl-5-oxo-pyrrolidine-3-carboxylic acid in 68%
yield.
[0332] .sup.1H-NMR (DMSO-d.sub.6, 300 MHz) .delta. 1.23 (s, 3H),
1.45 (s, 3H), 2.59 (dd, 1H), 2.66 (s, 3H), 2.70 (dd, 1H), 3.17 (t,
1H), 7.91 (m, 1H), 8.18 (d, 1H), 8.24 (d, 1H), 8.40 (d, 1H), 9.07
(s, 1H), 10.16 (s, 1H), 11.32 (s, 1H).
[0333] Retention Time (LC, method: ammonium acetate standard): 1.17
min.
[0334] MS (M+H.sup.+): 371.3.
INTERMEDIATE 16: N-benzyl-serine benzyl ester
[0335] To a mixture of L-serine-benzyl ester-HCl (2.3 g),
benzaldehyde (1.05 eq.) and sodium acetate (1 eq.) in methanol was
added sodium cyanoborohydride (1.0 eq.). The resulting mixture was
stirred at ambient temperature for 15 hrs, then partitioned into
ether and aqueous saturated sodium bicarbonate. The separated
organic phase was extracted with 1M HCl (3.times.). Combined
aqueous extracts were washed with ether, basified with aqueous 4.5M
K.sub.2CO.sub.3 and extracted with ether. Combined organic extracts
were washed with brine, dried over sodium sulfate and concentrated
to dryness to give 2.32 g of the desired compound (waxy solid, 81%
yield).
[0336] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. 3.48 (dd, 1H),
3.64 (dd, 1H), 3.74 (d, 1H), 3.80 (dd, 1H), 3.88 (d, 1H), 5.18 (s,
2H), 7.25-7.39 (m, 10H).
[0337] MS (M+H.sup.+): 286.
INTERMEDIATE 17:
4-benzyl-6-iodomethyl-6-methyl-morpholine-3-carboxylic acid benzyl
ester
[0338] To a solution of N-benzyl-serine benzyl ester (Intermediate
16, 6.35 g) in 90 ml of MeCN at ambient temperature was added
3-bromo-2-methyl-propene (5.6 ml), KI (0.740 mg) and
K.sub.2CO.sub.3 (7.7 g). The reaction mixture was stirred at
ambient temperature for 72 hrs. 1 ml of 3-bromo-2-methyl-propene
was added and the reaction mixture was stirred for another 15 hrs.
Only a small amount of starting material remained based on TLC
(1:1; EtOAc-hexane). To the resulting mixture was added 11.2 g of
iodine. After 4 hrs of stirring, TLC (10% EtOAc/hexane) showed
complete conversion. The reaction mixture was partitioned into
ether (300 ml) and 0.5 M Na.sub.2S.sub.2O.sub.3 (100 ml). The
separated organic phase was washed successively with water,
saturated NaHCO.sub.3 and brine, dried over MgSO.sub.4, and
concentrated to dryness. The residue was purified on silica (5%
EtOAc/Hexane) to give 6.65 g (yellowish oil, 64% yield, about 4:1
mixture) of compound
4-benzyl-6-iodomethyl-6-methyl-morpholine-3-carboxylic acid benzyl
ester. Major Component: .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta.
1.22 (s, 3H), 2.50 (d, 1H), 3.16-3.36 (m, 3H), 3.75-3.95 (m, 4H),
4.06 (dd, 1H), 5.16 (d, 1H), 5.21 (d, 1H), 7.28-7.36 (m, 10H).
[0339] MS (M+H.sup.+): 466.
INTERMEDIATE 18: 4-benzyl-6,6-dimethyl-morpholine-3-carboxylic acid
benzyl ester
[0340] To a solution of
4-benzyl-6-iodomethyl-6-methyl-morpholine-3-carboxylic acid benzyl
ester (Intermediate 17, 1.23 g) and tributyltin hydride (1.8 ml,
2.5 eq.) in 11 ml of toluene under gentle reflux was added over 1.5
hr a solution of AIBN in toluene (25 mg/l ml). The mixture was
allowed to cool down and was concentrated to dryness. The residue
was partitioned into 15% 1M HCl in acetonitrile and hexane. The
separated hexane phase was extracted two times with the
acetonitrile solution. The combined acetonitrile solutions were
washed with hexane two times and concentrated. The residue was
partitioned into ether and 1M K.sub.2CO.sub.3. The separated ether
phase was washed successively with 0.4M Na.sub.2S.sub.2O.sub.3 and
brine, dried over MgSO.sub.4 and concentrated. The residue was
purified on silica (7.5% EtOAc/hexane) to give 760 mg (oil, 85%
yield) of compound 4-benzyl-6,6-dimethyl-morpholine-3-carboxylic
acid benzyl ester.
[0341] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. 1.21 (s, 3H),
1.24 (s, 3H), 2.12 (d, 1H), 2.84 (d, 1H), 3.29 (t, 1H), 3.58 (d,
1H), 3.95-4.05 (m, 2H), 5.15 (d, 1H), 5.21 (d, 1H), 7.28-7.36 (m,
10H).
[0342] MS (M+H.sup.+): 340.
INTERMEDIATE 19: 6,6-dimethyl-morpholine-3-carboxylic acid
[0343] To a solution of
4-benzyl-6,6-dimethyl-morpholine-3-carboxylic acid benzyl ester
(Intermediate 18, 1.25 g) in 40 ml of 10% AcOH/MeOH (under
nitrogen) was added 250 mg of 20% Pd(OH).sub.2 on charcoal. The
reaction mixture was purged with hydrogen (balloon) and was stirred
at ambient temperature for 72 hrs. To the resulting gray mixture
was added 4 ml of water to help dissolution. The catalyst was
removed by filtration and the filtrate was concentrated to dryness.
The residue was co-evaporated with ethanol (2.times.) and then
triturated with EtOAc. The generated white solid was filtered off
and dried under high vacuum to give 559 mg of
6,6-dimethyl-morpholine-3-carboxylic acid (95% yield).
[0344] .sup.1H-NMR (300 MHz, D.sub.2O): .delta. 1.35 (s, 3H), 1.38
(s, 3H), 3.11 (d, 1H), 3.32 (d, 1H), 3.81-3.87 (m, 1H), 4.05 (bt,
1H), 4.17 (bd, 1H).
[0345] MS (M+H.sup.+): 160.
INTERMEDIATE 20: 4,6,6-trimethyl-morpholine-3-carboxylic acid
[0346] To a suspension of 6,6-dimethyl-morpholine-3-carboxylic acid
(Intermediate 19, 540 mg) in 17 ml of ethanol (under nitrogen) was
added 100 mg of 10% Pd on charcoal and 830 ul (.about.3 eq.) of 37%
aqueous formaldehyde. The mixture was purged with hydrogen
(balloon) and stirred at ambient temperature for 5 hrs. To the
resulting gray mixture were added 4 ml of water and 4 ml of
methanol to help dissolution. The catalyst was removed by
filtration and the filtrate was concentrated to dryness. The
residue was triturated with EtOAc. The generated white solid was
filtered off and dried under high vacuum to give 574 mg of
4,6,6-trimethyl-morpholine-3-carboxylic acid (97% yield).
[0347] .sup.1H-NMR (300 MHz, D.sub.2O): .delta. 1.35 (s, 3H), 1.43
(s, 3H), 2.94 (s, 3H), 3.08 (d, 1H), 3.41 (d, 1H), 3.68 (dd, 1H),
3.96 (t, 1H), 4.14 (dd, 1H).
[0348] MS (M+H.sup.+): 174.
Example 8
4,6,6-trimethyl-morpholine-3-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide
[0349] The desired compound was prepared according to Method B from
6-chloro-9H-.beta.-carboline-8-ylamine and
4,6,6-trimethyl-morpholine-3-carboxylic acid in 61% yield.
[0350] .sup.1H-NMR (DMSO-d.sub.6, 300 MHz) .delta. 1.15 (s, 3H),
1.39 (s, 3H), 1.98 (d, 1H), 2.26 (s, 3H), 2.72 (d, 1H), 2.80 (dd,
1H), 3.79 (m, 2H), 7.91 (s, 1H), 8.03-8.08 (m, 2H), 8.22 (d, 1H),
8.97 (s, 1H).
[0351] Retention Time (LC, method: ammonium acetate standard): 1.33
min.
[0352] MS (M+H.sup.+): 373.2.
Example 9
2,2-Dimethyl-5-oxo-tetrahydro-furan-3-carboxylic acid
(6-chloro-7-methoxy-9H-.beta.-carbolin-8-yl)-amide
[0353] The desired compound was prepared according to Method A from
6-chloro-7-methoxy-9H-.beta.-carboline-8-ylamine and (R)-terebic
acid in a 60-80% yield.
[0354] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 1.48 (s, 3H),
1.61 (s, 3H), 3.03 (m, 2H), 3.51 (m, 1H), 3.86 (s, 3H), 8.16 (m,
1H), 8.37 (m, 1H), 8.43 (s, 1H), 8.94 (s, 1H), 10.10 (s, 1H), 11.33
(s, 1H).
[0355] Retention Time (LC, method: ammonium acetate standard): 1.94
min.
[0356] MS (M+H.sup.+): 388.
INTERMEDIATE 21:
6-chloro-7-cyclopropylmethoxy-8-nitro-9H-.beta.-carboline
[0357] Cyclopropylmethyl alcohol (0.921 ml, 11.4 mmol) was added to
a stirring suspension of NaH (455 mg, 11.4 mmol) in DMF (20 ml)
under an argon atmosphere. The resulting solution was allowed to
stir at RT for 20 min.
6-chloro-7-fluoro-8-nitro-9H-.beta.-carboline (500 mg, 1.9 mmol)
was added to the stirring solution and the resulting mixture was
allowed to stir at RT. Upon addition of H.sub.2O, a brown solid
precipitated out which was filtered to give the desired
6-chloro-7-cyclopropylmethyoxy-8-nitro-9H-.beta.-carboline (510 mg,
85%).
[0358] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 0.35 (m, 2H),
0.59 (m, 2H), 1.32 (m, 1H), 4.04 (d, 2H), 8.21 (d, 1H), 8.46 (d,
1H), 8.90 (s, 1H), 9.02 (s, 1H), 12.32 (b, 1H)
[0359] Retention Time (LC, method: ammonium acetate standard): 2.63
min.
[0360] MS (M+H.sup.+): 318.
INTERMEDIATE 22:
6-chloro-7-cyclopropylmethoxy-9H-.beta.-carbolin-8-ylamine
[0361] 6-chloro-7-cyclopropylmethoxy-8-nitro-9H-.beta.-carboline
(510 mg, 1.61 mmol) was suspended in 12 ml of methanol and 100 mg
of Pd/C (10%) was added. The flask was fitted with a balloon of
hydrogen and the reaction mixture was stirred overnight at RT. Upon
filtration through a pad of celite and evaporation of the methanol,
a dark brown solid was obtained. This residue was suspended in
methanol (10 ml) and added, with vigorous stirring, to a solution
of saturated NaHCO.sub.3 (100 ml). The light brown solid that
precipitated out was collected by filtration and dried thoroughly
in vacuo to give the desired
6-chloro-7-cyclopropylmethoxy-9H-.beta.-carbolin-8-ylamine (371
mgs, 80% yield).
[0362] .sup.1H-NMR (300 MHz, methanol-d.sub.4): .delta. 0.36 (m,
2H), 0.61 (m, 2H), 1.37 (m, 1H), 3.88 (d, 2H), 7.58 (s, 1H), 7.96
(d, 1H), 8.23 (d, 1H), 8.76 (s, 1H).
[0363] Retention Time (LC, method: ammonium acetate standard): 2.28
min.
[0364] MS (M+H.sup.+): 288.
Example 10
N-(6-chloro-7-cyclopropylmethoxy-9H-.beta.-carbolin-8-yl)-2-methyl-nicotin-
amide
[0365] The desired compound was prepared according to Method A from
6-chloro-7-cyclopropylmethoxy-9H-.beta.-carbolin-8-ylamine and
2-methylnicotinic acid in a 40-60% yield.
[0366] .sup.1H-NMR (300 MHz, MeOH-d.sub.4): .delta. 0.29 (m, 2H),
0.55 (m, 2H), 1.29 (m, 1H), 2.78 (s, 3H), 3.95 (m, 2H), 7.46 (dd,
1H), 8.08 (m, 1H), 8.30 (m, 3H), 8.59 (m, 1H), 8.81 (s, 1H).
[0367] Retention Time (LC, method: ammonium acetate standard): 2.18
min.
[0368] MS (M+H.sup.+): 405.
INTERMEDIATE 23:
6-chloro-7-(N,N)-dimethylaminoethoxy-8-nitro-9H-.beta.-carboline
[0369] N,N-dimethylaminoethyl alcohol (6.0 eq) was added to a
stirring suspension of NaH (6.0 eq) in DMF under an argon
atmosphere. The resulting solution was allowed to stir at RT for 20
min. 6-chloro-7-fluoro-8-nitro-9H-.beta.-carboline (1.0 eq) was
added to the stirring solution and the resulting mixture was
allowed to stir at RT. Upon addition of H.sub.2O, a brown solid
precipitated out which was filtered to give the desired
6-chloro-7-(N,N)-dimethylaminoethoxy-8-nitro-9H-.quadrature.-carboline
(quantitative yield).
[0370] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 2.23 (s, 6H),
2.74 (t, 2H), 4.28 (t, 2H), 8.21 (d, 1H), 8.46 (d, 1H), 8.90 (s,
1H), 9.02 (s, 1H).
[0371] Retention Time (LC, method: ammonium acetate standard): 1.26
min.
[0372] MS (M+H.sup.+): 335.
INTERMEDIATE 24:
6-chloro-7-(N,N)-dimethylaminoethoxy-9H-.beta.-carbolin-8-ylamine
[0373]
6-chloro-7-(N,N)-dimethylaminoethoxy-8-nitro-9H-.beta.-carboline
(500 mg, 1.5 mmol) was suspended in 12 ml of methanol and 100 mg of
Pd/C (10%) was added. The flask was fitted with a balloon of
hydrogen and the reaction mixture was stirred overnight at RT. Upon
filtration through a pad of celite and evaporation of the methanol,
a dark brown solid was obtained. The residue was suspended in
methanol (10 ml) and added, with vigorous stirring, to a solution
of saturated NaHCO.sub.3 (100 ml). The solid that precipitated out
was collected by filtration and dried thoroughly in vacuo to give
the desired
6-chloro-7-(N,N)-dimethylaminoethoxy-9H-.beta.-carboline-8-ylamine
(380 mgs, 83%).
[0374] .sup.1H-NMR (300 MHz, methanol-d.sub.4): .delta. 2.43 (s,
6H), 2.84 (t, 2H), 4.11 (t, 2H), 7.47 (s, 1H), 7.88 (d, 1H), 8.20
(d, 1H), 8.72 (s, 1H).
[0375] Retention Time (LC, method: ammonium acetate standard): 1.34
min.
[0376] MS (M+H.sup.+): 305.
Example 11
N-[6-chloro-7-(2-dimethylamino-ethoxy)-9H-.beta.-carbolin-8-yl]-2-methyl-n-
icotinamide
[0377] The desired compound was prepared according to Method A from
6-chloro-7-(N,N)-dimethylaminoethoxy-9H-.beta.-carboline-8-ylamine
and 2-methylnicotinic acid in a 40-60% yield.
[0378] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 1.92 (s, 6H),
2.49 (m, 2H), 2.68 (s, 3H), 4.25 (m, 2H), 7.50 (dd, 1H), 8.16 (m,
2H), 8.38 (d, 1H), 8.42 (s, 1H), 8.66 (m, 1H), 9.00 (s, 1H), 11.27
(s, 1H), 11.78 (s, 1H).
[0379] Retention Time (LC, method: ammonium acetate standard): 1.52
min.
[0380] MS (M+H.sup.+): 424.
Example 12
2-amino-cyclopentanecarboxylic acid
(6-chloro-7-methoxy-9H-.beta.-carbolin-8-yl)-amide
[0381] 6-chloro-7-methoxy-9H-.beta.-carboline-8-ylamine and
2-tert-butoxycarbonylamino-cyclopentanecarboxylic acid were reacted
using Method A. To this product was added .about.5 ml of 4N
HCl/dioxane and the resulting mixture was allowed to stir at RT.
The reaction was followed by LC-MS until completion. Evaporation
was allowed to remove all the solvent which gave a crude HCl salt.
The desired product was then purified by preparative HPLC.
[0382] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 1.68 (m, 1H),
1.84 (m, 2H), 2.06 (m, 2H), 2.20 (m, 1H), 3.46 (m, 1H), 3.67 (m,
1H), 3.89 (s, 3H), 8.24 (d, 2H), 8.61 (d, 1H), 8.72 (s, 1H), 8.78
(d, 1H), 9.19 (s, 1H), 10.58 (s, 1H), 13.20 (s, 1H).
[0383] Retention Time (LC, method: ammonium acetate standard): 1.54
min.
[0384] MS (M+H.sup.+): 359.
INTERMEDIATE 25:
1-(2-dimethylamino-ethyl)-5-oxo-pyrrolidine-3-carboxylic acid
[0385] A mixture of commercially available itaconic acid and
N,N-dimethylethylenediamine was heated up to 160.degree. C. for
about 20-25 minutes. The mixture was allowed to cool to 100.degree.
C. and then diluted with MeOH to prevent solidification. The
product was obtained in a 56% yield after crystallization from
MeOH/EtOAc.
[0386] .sup.1H-NMR (300 MHz, D.sub.2O): .delta. 2.63 (dd, 1H), 2.80
(dd, 1H), 2.95 (s, 6H), 3.15-3.25 (m, 1H), 3.32-3.44 (m, 1H),
3.44-3.76 (m, 4H), 3.82-3.94 (m, 1H).
[0387] Retention Time (LC, method: ammonium acetate standard): 0.13
min.
[0388] MS (M+H.sup.+): 201.0.
Example 13
1-(2-Dimethylamino-ethyl)-5-oxo-pyrrolidine-3-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide
[0389] Prepared according to Method B from
6-chloro-9H-.beta.-carboline-8-ylamine and
1-(2-dimethylamino-ethyl)-5-oxo-pyrrolidine-3-carboxylic acid in
70-80% yield as the di-HCl salt.
[0390] .sup.1H-NMR (300 MHz, D.sub.2O): .delta. 2.97 (dd, 1H), 3.03
(s, 6H), 3.08 (dd, 1H), 3.51 (t, 2H), 3.76-3.89 (m, 3H), 3.95 (dd,
1H), 4.01 (dd, 1H), 7.75 (d, 1H), 8.24 (d, 1H), 8.45 (dd, 1H), 8.52
(dd, 1H), 9.10 (bs, 1H).
[0391] Retention Time (LC, method: ammonium acetate standard): 0.99
min.
[0392] MS (M+H.sup.+): 400.
Example 14
1-(2-dimethylamino-ethyl)-5-oxo-pyrrolidine-3-carboxylic acid
(6-chloro-7-methoxy-9H-.beta.-carbolin-8-yl)-amide
[0393] Prepared according to Method A from
6-chloro-7-methoxy-9H-.beta.-carboline-8-ylamine and
1-(2-dimethylamino-ethyl)-5-oxo-pyrrolidine-3-carboxylic acid in
60% yield following purification using a semi-preparative Chiralcel
OD column with 85/7.5/7.5 hexane/EtOH/MeOH as the eluant.
[0394] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 2.44 (s, 6H),
2.75 (t, 2H), 2.88 (d, 2H), 3.56 (t, 2H), 3.66 (m, 1H), 3.85 (m,
2H), 3.90 (s, 3H), 8.03 (d, 1H), 8.21 (s, 1H), 8.28 (d, 1H), 8.79
(s, 1H).
[0395] Retention Time (LC, method: ammonium acetate standard): 1.42
min.
[0396] MS (M+H.sup.+): 430.
INTERMEDIATE 26: 6-chloro-7-fluoro-9H-.beta.-carbolin-8-ylamine
[0397] A slurry of 6-chloro-7-fluoro-8-nitro-9H-.beta.-carboline
(500 mg, 1.88 mmol) in MeOH (25 ml) was degassed with argon.
Palladium on charcoal (20% w/w on C, 50 mg) was added and the
reaction vessel was flushed with hydrogen. The slurry was stirred
under a balloon of hydrogen for 6 hr, then filtered through celite
and concentrated under reduced pressure to yield
6-chloro-7-fluoro-9H-.beta.-carbolin-8-ylamine (400 mg) as a brown
solid.
[0398] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 11.48 (br s,
1); 8.99-8.98 (m, 1); 8.37-8.35 (m, 1); 8.11-8.09 (m, 1); 7.74-7.72
(m, 1); 5.65 (br s, 2).
[0399] HCOOH standard conditions.
[0400] DAD R.sub.f=1.00 min.
[0401] MS (M+H.sup.+): 236.
Example 15
N-(6-chloro-7-fluoro-9H-.beta.-carbolin-8-yl)-2-methyl-nicotinamide
[0402] A solution of 6-chloro-7-fluoro-9H-.beta.-carbolin-8-ylamine
(100 mg, 0.424 mmol) in pyridine (2.5 ml) was stirred at RT.
2-methyl nicotinic acid (70 mg, 0.509 mmol) was added, followed by
EDCI (130 mg, 0.678 mmol). The suspension was stirred at
100.degree. C. for a day. The pyridine was removed under reduced
pressure and the resulting dark oil was triturated with saturated
aqueous NaHCO.sub.3. The precipitate which formed was filtered and
washed with MeOH. The material was treated with 2M HCl in Et.sub.2O
to yield a gray solid, the di-HCl salt
N-(6-chloro-7-fluoro-9H-.beta.-carbolin-8-yl)-2-methyl-nicotinamide
(110 mg).
[0403] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 13.15 (s, 1);
11.03 (s, 1); 9.33 (s, 1); 8.99-8.97 (m, 1); 8.92-8.89 (m, 1);
8.79-8.73 (m, 2); 8.50 (m, 1); 7.70 (m, 1); 2.80 (s, 3).
[0404] HCOOH standard conditions.
[0405] DAD R.sub.f=0.91 min.
[0406] MS (M+H.sup.+): 355.
INTERMEDIATE 27:
6-chloro-7-methylsulfanyl-8-nitro-9H-.beta.-carboline
[0407] A 250 ml round-bottom flask with magnetic stirrer was
charged with 6-chloro-7-fluoro-8-nitro-.beta.-carboline
(Intermediate 5, 3.959 g, 14.9 mmol) and 100 ml anhydrous DMF. The
resulting orange mixture was cooled to 0.degree. C. (ice and water
bath) and sodium thiomethoxide (1.809 g, 25.8 mmol) in powder form
was added slowly thereto. The reaction mixture was stirred for 1 hr
at 0.degree. C., warmed to RT, and added slowly to a stirring
mixture of 4:1 H.sub.2O/saturated aqueous sodium bicarbonate (500
ml). The precipitated solid was collected via suction filtration
and air-dried to afford 4.017 g of
6-chloro-7-methylsulfanyl-8-nitro-9H-.beta.-carboline as an orange
powder. The crude material was used directly in subsequent
steps.
[0408] .sup.1H-NMR (300 MHz, Methanol-d.sub.4): .delta. 8.91 (1H,
d) 8.63 (1H, s) 8.42 (1H, d) 8.17 (1H, dd) 2.54 (3H, s).
[0409] LCMS (formic acid standard method) retention time=1.43
min.
[0410] MS (M+H.sup.+): 294.
INTERMEDIATE 28:
6-chloro-7-methylsulfanyl-9H-.beta.-carbolin-8-ylamine
[0411] A 500 ml round-bottom flask with magnetic stirrer was
charged with 6-chloro-7-methylsulfanyl-8-nitro-9H-.beta.-carboline
(4.011 g, 13.6 mmol) and 200 ml anhydrous ethanol. To the resulting
mixture was added aqueous ammonium chloride (75 ml of 0.33 M
solution, 24.7 mmol), aqueous hydrochloric acid (10 ml of 1 M
solution, 10 mmol), and iron powder (7.734 g, 138 mmol). The
resulting mixture was heated to 60.degree. C. (oil bath) and
stirred vigorously for 3.5 hr. The reaction was cooled to RT,
diluted with EtOAc (75 ml) and activated charcoal (ca. 2.5 g) was
added. The resulting mixture was stirred at RT for an additional
1.5 hr, filtered through a pad of celite, and the resulting
filtrate concentrated (rotavap, then vacuum pump) to afford 5.153 g
of a yellowish-orange colored solid. The solid was redissolved in
MeOH (50-100 ml) and slowly added to saturated aqueous sodium
bicarbonate (500 ml) with stirring. The mixture was stirred at RT
for 45 min and the resulting solid collected via suction filtration
and air-dried to afford 3.476 g of
6-chloro-7-methylsulfanyl-9H-.beta.-carbolin-8-ylamine as a tan
solid which was used without further purification in subsequent
steps.
[0412] .sup.1H-NMR (300 MHz, Methanol-d.sub.4): .delta. 8.87 (1H,
s) 8.35-8.24 (1H, m) 8.16-8.06 (1H, m) 7.67 (1H, s) 2.32 (3H,
s).
[0413] LCMS (ammonium acetate standard method) retention time=2.12
min.
[0414] MS (M+H.sup.+): 264.
Example 16
N-(6-chloro-7-methylsulfanyl-9H-.beta.-carbolin-8-yl)-2-methyl-nicotinamid-
e
[0415] A 250 ml round-bottom flask with magnetic stirrer was
charged with 6-chloro-7-methylsulfanyl-9H-.beta.-carbolin-8-ylamine
(Intermediate 28, 2.336 g, 8.86 mmol) and 2-methylnicotinic acid
(3.219 g, 23.4 mmol) in 80 ml anhydrous pyridine. To the resulting
reaction mixture at RT was added solid
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (7.080
g, 36.9 mmol) and the reaction mixture was heated to 100.degree. C.
(oil bath) for 2 days. The resulting mixture was cooled to RT and
concentrated (rotavap) to afford a brown residue. The residue was
redissolved in MeOH (50 ml), slowly added to a stirring mixture of
5:1 H.sub.2O/saturated aqueous sodium bicarbonate (600 ran) and
stirred at RT for .about.18 hr. The precipitated solid was
collected via suction filtration, washed with Et.sub.2O
(2.times.150 ml), and air-dried to afford 3.036 g of crude
N-(6-chloro-7-methylsulfanyl-9H-.beta.-carbolin-8-yl)-2-methyl-nicotinami-
de as a brown solid. The crude material was purified via HPLC
(yields=.about.40-60%)
[0416] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 11.72 (1H, s)
10.50 (1H, s) 8.97 (1H, s) 8.62 (1H, dd) 8.57 (1H, s) 8.41 (1H, d)
8.33-8.27 (1H, m) 8.21 (1H, d) 2.73 (3H, s) 2.41 (3H, s).
[0417] LCMS (ammonium acetate standard method) retention time=1.89
min.
[0418] MS (M+H.sup.+): 383.
INTERMEDIATE 29:
6-chloro-7-ethylsulfanyl-8-nitro-9H-.beta.-carboline
[0419] A 25 ml round-bottom flask with a magnetic stirrer was
charged with 6-chloro-7-fluoro-8-nitro-9H-.beta.carboline (102 mg,
0.38 mmol) in 5 ml of anhydrous DMF. To the resulting orange
mixture at RT was slowly added sodium thioethoxide (80% pure, 69.7
mg, 0.66 mmol) in powder form. The reaction mixture was stirred at
RT for 45 minutes, then added drop-wise to a 5:1 mixture of
H.sub.2O/saturated sodium bicarbonate (.about.30 ml). The resulting
precipitated solid was collected by suction filtration, washed with
1:1 hexanes/diethyl ether (2.times.20 ml), and air-dried to afford
95.0 mg of 6-chloro-7-ethylsulfanyl-8-nitro-9H-.beta.-carboline as
an orange solid (79%).
[0420] .sup.1H-NMR (300 MHz, CD.sub.3OD, ppm) .delta. 8.91 (1H, s)
8.63 (1H, s) 8.42 (1H, d) 8.18 (1H, d) 3.04 (2H, q) 1.20 (3H,
t).
[0421] Retention Time (LC, formic acid standard method): 1.71
min.
[0422] MS (M+H.sup.+): 308.
INTERMEDIATE 30:
6-chloro-7-ethylsulfanyl-9H-.beta.-carbolin-8-ylamine
[0423] A 50 ml round-bottom flask with magnetic stirrer was charged
with 6-chloro-7-ethylsulfanyl-8-nitro-9H-.beta.-carboline (85.0 mg,
0.28 mmol) in 10 ml anhydrous ethanol. To the resulting orange
mixture at RT was added 0.33 M aqueous ammonium chloride (2.0 ml,
0.66 mmol) and iron powder (680 mg, 12.2 mmol). The reaction
mixture was heated to 60.degree. C. and stirred vigorously for 20
hr. Next, the mixture was cooled to RT, diluted with ethyl acetate
(15 ml), and activated charcoal (.about.180 mg) was added. The
resulting mixture was filtered through a pad of celite and the
filtrate was concentrated to afford 77.8 mg of
6-chloro-7-ethylsulfanyl-9H-.beta.-carbolin-8-ylamine as a yellow
solid (>99%).
[0424] .sup.1H-NMR (300 MHz, CD.sub.3OD, ppm) .delta. 8.94 (1H, s)
8.33-8.29 (1H, m) 8.21-8.18 (1H, m) 7.73 (1H, s) 2.85 (2H, q) 1.21
(3H, t).
[0425] LCMS (ammonium acetate standard method) retention time 2.13
min.
[0426] (M.sup.+=278; M.sup.-=276).
Example 17
N-(6-chloro-7-ethylsulfanyl-9H-.beta.-carbolin-8-yl)-2-methyl-nicotinamide
[0427] A 25 ml round-bottom flask with magnetic stirrer was charged
with 6-chloro-7-ethylsulfanyl-9H-.beta.-carbolin-8-ylamine (37.2
mg, 0.13 mmol) and 2-methylnicotinic acid (36.2 mg, 0.26 mmol) in 3
ml anhydrous pyridine. To the resulting light-orange mixture at RT
was added solid 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (73.2 mg, 0.38 mmol) and the resulting reaction
mixture was heated to 80.degree. C. for 5 days. Next, the reaction
mixture was cooled to RT and concentrated to afford a brown,
viscous syrup. The syrup was dissolved in a minimal amount of MeOH
(.about.2 ml), slowly added to a 5:1 mixture of H.sub.2O/saturated
sodium bicarbonate (.about.20 ml), and stirred at RT for 2.5 hr.
The resulting precipitated solid was collected via suction
filtration, washed with 1:1 hexanes/diethyl ether (2.times.20 ml),
and air-dried to afford 21.0 mg of
N-(6-chloro-7-ethylsulfanyl-9H-.beta.-carbolin-8-yl)-2-methyl-nicotinamid-
e as a tan solid (-38%). LCMS (ammonium acetate standard method)
retention time=2.33 min. (M.sup.+=397; M.sup.-=395).
[0428] .sup.1H-NMR (300 MHz, CD.sub.3OD, ppm) .delta. 8.89 (1H, s)
8.63-8.58 (1H, m) 8.44-8.38 (2H, m) 8.36 (1H, d) 8.15 (1H, d)
7.52-7.44 (1H, m) 2.98 (2H, q) 2.84 (3H, s) 1.20 (3H, t).
INTERMEDIATE 31:
[2-(6-chloro-8-nitro-9H-.beta.-carbolin-7-ylsulfanyl)-ethyl]-dimethyl-ami-
ne
[0429] A 25 ml round-bottom flask with magnetic stirrer was charged
with 6-chloro-7-fluoro-8-nitro-9H-.beta.-carboline (98 mg, 0.37
mmol) in 2 ml of anhydrous DMF. A second 10 ml round-bottom flask
with magnetic stirrer was charged with 2-dimethylamino-ethanethiol
hydrochloride (100 mg, 0.70 mmol) in 2 ml anhydrous DMF. To the
resulting suspension was added n-butyllithium (0.43 ml of 1.6 M
solution in hexanes, 0.69 mmol) via syringe and the mixture was
stirred for 5 min at RT. Next, the thioanion solution was added via
syringe to 6-chloro-7-fluoro-8-nitro-9H-.beta.-carboline, and the
resulting red solution was stirred at RT for 30 min. The reaction
mixture was slowly added to a 5:1 mixture of H.sub.2O/saturated
aqueous sodium bicarbonate (30 ml) and allowed to sit at RT for
several hours. The resulting precipitated solid was collected via
suction filtration and air-dried to afford 109 mg of
[2-(6-chloro-8-nitro-9H-.beta.-carbolin-7-ylsulfanyl)-ethyl]-dimethyl-ami-
ne as an orange solid (83%).
[0430] LCMS (ammonium acetate standard method) retention time=1.35
min.
[0431] (M.sup.+=351; M.sup.-=349).
[0432] .sup.1H-NMR (300 MHz, CD.sub.3OD, ppm) .delta. 8.92 (1H, d,
J=1.0 Hz) 8.66 (1H, s) 8.43 (1H, d) 8.19 (1H, dd) 3.18-3.13 (2H, m)
2.57-2.52 (2H, m) 2.21 (6H, s).
INTERMEDIATE 32:
6-chloro-7-(2-dimethylamino-ethylsulfanyl)-9H-.beta.-carbolin-8-ylamine
[0433] A 50 ml round-bottom flask with a magnetic stirrer was
charged with
[2-(6-chloro-8-nitro-9H-.beta.-carbolin-7-ylsulfanyl)-ethyl]-dimethyl-ami-
ne (106 mg, 0.30 mmol) in 8 ml of anhydrous ethanol. To the
resulting orange mixture at RT was added 0.33 M aqueous ammonium
chloride (1.95 ml, 0.64 mmol) and iron powder (540 mg, 9.67 mmol).
The reaction mixture was heated to 60.degree. C. and stirred
vigorously for 20 hr. Next, the mixture was cooled to RT, diluted
with ethyl acetate (20 ml) and activated charcoal (ca. 150 mg) was
added. The resulting mixture was filtered through a pad of celite
and the resulting filtrate concentrated to afford 103 mg of
6-chloro-7-(2-dimethylamino-ethylsulfanyl)-9H-.beta.-carbolin-8-ylamine
as a yellow solid. The crude product was used directly in the
coupling step.
[0434] LCMS (ammonium acetate standard method) retention time=1.34
min.
[0435] (M.sup.+=321; M.sup.-=319).
[0436] .sup.1H-NMR (300 MHz, CD.sub.3OD, ppm) .delta. 8.87 (1H, s)
8.30 (1H, d) 8.06 (1H, d) 7.75 (1H, s) 3.23-3.13 (4H, m) 2.84 (6H,
s).
Example 18
N-[6-chloro-7-(2-dimethylamino-ethylsulfanyl)-9H-.beta.-carbolin-8-yl]-2-m-
ethyl-nicotinamide
[0437] A 25 ml round-bottom flask with magnetic stirrer was charged
with
6-chloro-7-(2-dimethylamino-ethylsulfanyl)-9H-.beta.-carbolin-8-ylamine
(45.2 mg, 0.14 mmol) and 2-methylnicotinic acid (39.0 mg, 0.28
mmol) in 4.5 ml of anhydrous pyridine. To the resulting
light-orange mixture at RT was added solid
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (95.0
mg, 0.49 mmol). The resulting reaction mixture was heated to
80.degree. C. for 3 days. Next, the reaction was cooled to RT and
concentrated to afford a brown, viscous syrup. The syrup was
dissolved in a minimal amount of MeOH (.about.2 ml), slowly added
to a 5:1 mixture of H.sub.2O/saturated sodium bicarbonate
(.about.25 ml), and extracted with ethyl acetate (3.times.30 ml).
The combined organic layers were washed with brine (1.times.30 ml),
dried over Na.sub.2SO.sub.4, filtered and concentrated to afford a
brown residue (79.6 mg). The residue was redissolved in MeOH
(.about.5 ml), and filtered through a cotton plug. To the resulting
filtrate was added HCl in 1,4-dioxane (1.0 ml, 4.0 mmol), the
resulting solution stirred at RT for 3 hr, and added drop-wise to
diethyl ether (30 ml) with stirring. The resulting precipitated
product was collected via suction filtration, washed with ether and
air-dried to afford 36.3 mg of
N-[6-chloro-7-(2-dimethylamino-ethylsulfanyl)-9H-.beta.-carbolin-8-yl]-2--
methyl-nicotinamide tris-hydrochloride as a yellow powder.
[0438] .sup.1H-NMR (300 MHz, CD.sub.3OD, ppm) .delta. 8.95 (1H, s)
8.62 (1H, dd) 8.43 (1H, s) 8.38-8.34 (2H, m) 8.16 (1H, d) 7.50 (1H,
dd) 3.09 (2H, t) 2.85 (3H, s) 2.30 (2H, t) 1.99 (6H, s).
[0439] LCMS (ammonium acetate standard method) retention time=1.56
min.
[0440] (M.sup.+=440; M.sup.-=438).
INTERMEDIATE 33: morpholine-3(S),4-dicarboxylic acid 4-tert-butyl
ester
[0441] A suspension of morpholine-3(S)-carboxylic acid (2.00 g,
15.3 mmol) in DMF (75 ml) was stirred at RT. Triethylamine (7.47
ml, 53.6 mmol) and di-tert-butyl dicarbonate (BOC.sub.2O, 4.02 g,
18.4 mmol) were added. The suspension was stirred at RT for one
hour, during which time the reaction formed a clear yellow
solution. The solution was concentrated to a reduced volume
(.about.25 ml) and diluted with water (15 ml) and 1N HCl (15 ml).
The mixture was poured into a separatory funnel, diluted further
with water (100 ml) and brine (100 ml), and extracted with
Et.sub.2O (3.times.100 ml). The organic layer was washed with
brine, dried, filtered and concentrated to yield a white solid.
[0442] The solid, which contained excess BOC.sub.2O, was dissolved
in Et.sub.2O (500 ml) and extracted with 1N NaOH (3.times.100 ml).
The aqueous layer was acidified with 6N HCl to approximately a pH
of 2, then extracted quickly with Et.sub.2O (3.times.100 ml). The
Et.sub.2O layer was dried, filtered and concentrated to yield white
solid (3.07 g).
[0443] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 12.95 (br s,
1); 4.34-4.30 (m, 1); 4.18-4.10 (m, 1); 3.83-3.74 (m, 1); 3.59-3.51
(m, 2); 3.39-3.32 (m, 1); 3.21-2.95 (m, 1); 1.41-1.36 (m, 9).
[0444] NH.sub.4OAc standard conditions.
[0445] ELSD R.sub.f=1.08 min.
[0446] M-H=230.
Example 19
4-methyl-morpholine-3(S)-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide
[0447] A slurry of morpholine-3(S)-carboxylic acid (3.00 g, 22.9
mmol) in EtOH (115 ml) was stirred at RT. A solution of aqueous
CH.sub.2O (3.42 ml, 45.8 mmol, 37% w/w in H.sub.2O) was added,
followed by Pd(OH).sub.2 (600 mg, 20% w/w on charcoal). The flask
was charged with hydrogen (1 atm) and the grey slurry was stirred
for 24 hr at RT under a balloon of hydrogen. The flask was purged
with nitrogen and the black slurry was diluted with MeOH, filtered
through filter paper and concentrated to a reduced volume. The pale
grey solution was filtered through a 0.45 .mu.m syringe filter to
remove residual Pd(OH).sub.2 and concentrated to yield a clear
colorless oil. The oil was placed under high vacuum for 24 hr and a
white, solid foam was isolated. The foam was dissolved in pyridine
(200 ml) and 6-chloro-9H-.beta.-carbolin-8-ylamine (3.74 g, 17.2
mmol) was added, followed by EDCI (5.87 g, 30.6 mmol). The clear
pale orange solution was stirred at RT for 24 hr. The solution was
diluted with H.sub.2O (300 ml) and poured into a separatory funnel
containing EtOAc (300 ml). The mixture was shaken and the layers
were separated. The aqueous layer was extracted with EtOAc
(3.times.150 ml) and the combined organic layers were washed with
H.sub.2O and brine. The organic layer was dried, filtered and
concentrated to yield a brown oil which was placed under high
vacuum. The resulting brown foam was triturated with MeOH and the
precipitate which formed was filtered and washed with MeOH. The
resulting pale yellow solid was purified via chiral HPLC to yield a
white solid (3.23 g).
[0448] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 11.36 (s, 1);
10.02 (s, 1); 9.04 (s, 1); 8.38 (d, 1); 8.22-8.21 (m, 1); 8.15 (d,
1); 7.91-7.90 (m, 1); 4.00 (dd, 1); 3.85-3.81 (m, 1); 3.69-3.58 (m,
2); 2.99-2.95 (dd, 1); 2.89-2.85 (m, 1); 2.32 (s, 3); 2.32-2.24 (m,
1). NH.sub.4OAc standard conditions.
[0449] DAD R.sub.f=1.89 min.
[0450] MFH=345.
[0451] Chiral preparative HPLC: 10% v/v EtOH/Hexanes.
[0452] Chiralcel OD column.
[0453] R.sub.f=11.5-14 min.
[0454] Enantiopurity of product.gtoreq.99% ee.
METHOD C: PROCEDURE FOR 4-MORPHOLINE SUBSTITUTED ANALOGS
[0455] AS outlined for Intermediate 34, Intermediate 35 and Example
20:
INTERMEDIATE 34:
3(S)-(6-chloro-9H-.beta.-carbolin-8-ylcarbamoyl)-morpholine-4-carboxylic
acid tert-butyl ester
[0456] A solution of morpholine-3(S),4-dicarboxylic acid
4-tert-butyl ester (2.83 g, 12.7 mmol) in pyridine (106 ml) was
stirred at RT. 6-chloro-9H-.beta.-carbolin-8-ylamine (2.30 g, 10.6
mmol) was added, followed by EDCI (4.06 g, 21.2 mmol). The clear
orange-to-brown solution was stirred at RT for 14 hr. The solution
was diluted with H.sub.2O (120 ml) and poured into a separatory
funnel containing EtOAc (200 ml), H.sub.2O (100 ml) and brine (100
ml). The mixture was shaken and the layers were separated. The
aqueous layer was extracted with EtOAc (2.times.50 ml) and the
combined organic layers were washed with brine. The organic layer
was dried, filtered and concentrated to a reduced volume, then
added drop-wise to a stirring solution of 1:1 Et.sub.2O/hexanes
(500 ml). The precipitate which formed was filtered and washed with
1:1 Et.sub.2O/Hexanes. The filtrate was concentrated to a reduced
volume and a second crop of precipitate was collected. The solid
product was placed under high vacuum for 2 hr to yield
3(S)-(6-chloro-9H-(3-carbolin-8-ylcarbamoyl)-morpholine-4-carboxylic
acid tert-butyl ester as a pale yellow to pale brown solid (4.36
g).
[0457] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 11.30 (s, 1);
10.13 (s, 1); 9.06 (s, 1); 8.40-8.38 (m, 1); 8.19-8.16 (m, 2); 7.98
(s, 1); 4.67-4.47 (m, 2); 3.96-3.60 (m, 2); 3.64-3.39 (m, 3); 1.42
(s, 9).
[0458] NH.sub.4OAc standard conditions.
[0459] DAD R.sub.f=2.31 min.
[0460] M+H=431.
INTERMEDIATE 35:
2(R)-[3(S)-(6-chloro-9H-.beta.-carbolin-8-ylcarbamoyl)-morpholin-4-ylmeth-
yl]-pyrrolidine-1-carboxylic acid tert-butyl ester
[0461] A solution of
3(S)-(6-chloro-9H-.beta.-carbolin-8-ylcarbamoyl)-morpholine-4-carboxylic
acid tert-butyl ester (1.00 g, 2.32 mmol) in CH.sub.2Cl.sub.2 (6
ml) was stirred at RT. Trifluoroacetic acid (6 ml) was added and
the solution was stirred at RT for 45 min, then concentrated to a
residue. The residue was concentrated once more from
CH.sub.2Cl.sub.2 to yield a yellow-brown solid which was dissolved
in THF (13 ml) under argon. Gentle warming was sometimes needed to
ensure complete dissolution. A solution of
N-(tert-butoxycarbonyl)-D-prolinal (693 mg, 3.48 mmol) in THF (2
ml) was added, followed by sodium triacetoxyborohydride (738 mg,
3.48 mmol). The solution was stirred at RT for 30 min, then
quenched via addition of 1N aqueous NaOH (30 ml). The mixture was
poured into a separatory funnel containing EtOAc (100 ml), H.sub.2O
(100 ml), and brine (100 ml). The mixture was shaken and the layers
were separated. The aqueous layer was extracted with EtOAc
(2.times.50 ml) and the combined organic layers were washed with
brine. The organic layer was dried, filtered and concentrated to
yield a light brown solid. Column chromatography (2%-4%
MeOH/CH.sub.2Cl.sub.2) yielded
2(R)-[3(S)-(6-Chloro-9H-.beta.-carbolin-8-ylcarbamoyl)-morpholin-4-ylmeth-
yl]-pyrrolidine-1-carboxylic acid tert-butyl ester as a white solid
(915 mg).
[0462] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 11.30 (s, 1);
9.88 (s, 1); 9.04 (s, 1); 8.39-8.37 (m, 1); 8.20-8.15 (m, 2); 7.95
(s, 1); 3.99-3.82 (m, 3); 3.69-3.63 (m, 2); 3.44-3.32 (m, 1);
3.27-3.11 (m, 3); 2.92-2.80 (m, 1); 2.44-2.32 (m, 1); 1.99-1.67 (m,
5); 1.33 (s, 9).
[0463] HCOOH standard conditions.
[0464] DAD R.sub.f=1.39 min.
[0465] M+H=514.
[0466] Chiral HPLC.
[0467] The enantiopurity of the sample was checked. The samples
were .gtoreq.97% ee.
[0468] Chiralpak AD column.
[0469] 15% v/v EtOH/Hexanes containing 0.1% Et.sub.2NH.
Example 20
4-pyrrolidin-2(R)-ylmethyl-morpholine-3(S)-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide, HCl salt)
[0470] To a solution of
2(R)-[3(S)-(6-chloro-9H-.beta.-carbolin-8-ylcarbamoyl)-morpholine-4-ylmet-
hyl]-pyrrolidine-1-carboxylic acid tert-butyl ester (850 mg, 1.65
mmol) in MeOH (16 ml) was added concentrated aqueous HCl (13 ml).
The solution was stirred at RT for 30-45 min, during which time a
fine yellow precipitate formed. The reaction mixture was
concentrated to yield
4-pyrrolidin-2(R)-ylmethyl-morpholine-3(S)-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide, HCl salt) as a pale
yellow solid (755 mg).
[0471] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 13.30 (s, 1);
11.56 (br s, 1); 9.63 (br s, 1); 9.46 (s, 1); 8.87-8.85 (m, 1);
8.66-8.57 (m, 2); 8.25 (s, 1); 4.38-4.26 (m, 1); 4.24-4.08 (m, 1);
4.04-3.86 (m, 2); 3.86-3.68 (m, 2); 3.57-3.39 (m, 1); 3.39-3.02 (m,
4); 2.99-2.76 (m, 1); 2.10-1.84 (m, 3); 1.75-1.56 (m, 1).
[0472] HCOOH standard conditions.
[0473] DAD R.sub.f=0.81 min.
[0474] M+H=414.
INTERMEDIATE 36:
cis-2-(tert-butoxycarbonylamino)-cyclopentanecarboxylic acid
(6-chloro-9H-carbolin-8-yl) amide
[0475] A solution of cis-2-(tert-butoxycarbonylamino)-cyclopentane
carboxylic acid (550 mg, 2.4 mmol) in pyridine (10 ml) was stirred
at RT. 6-chloro-9H-.beta.-carbolin-8-ylamine (436 mg, 2.0 mmol) was
added, followed by EDCI (615 mg, 3.2 mmol), and the orange solution
was stirred at RT for 1.5 hr. The solution was diluted with
H.sub.2O (20 ml) and poured into a separatory funnel containing
H.sub.2O (50 ml) and EtOAc (100 ml). The mixture was shaken and the
layers were separated. The aqueous layer was extracted with EtOAc
(100 ml). The combined organic layers were washed with brine, dried
over MgSO.sub.4, filtered, and concentrated to orange oily solids
which were subsequently triturated with 5% MeOH in Et.sub.2O (20
ml) and captured by filtration to yield
cis-2-(tert-butoxycarbonylamino)-cyclopentanecarboxylic acid
(6-chloro-9H-carbolin-8-yl) amide as a light yellow solid (740
mg).
[0476] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 11.17 (s, 1);
9.89 (s, 1); 9.04 (s, 1); 8.36 (d, 1); 8.18-8.08 (m, 2); 7.95 (s,
1); 6.92 (d, 1); 4.32-4.22 (m, 1); 3.16-3.09 (m, 1); 2.13-2.01 (m,
1); 1.96-1.75 (m, 3); 1.74-1.59 (m, 1); 1.58-1.42 (m, 1); 1.07 (s,
9).
[0477] NH.sub.4OAc standard conditions.
[0478] DAD R.sub.f=2.52 min.
[0479] M+H=429.
Example 21
cis-2-amino-cyclopentanecarboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide
[0480] A solution of
2-(tert-butoxycarbonylamino)-cyclopentanecarboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl) amide (736 mg, 1.72 mmol) in
trifluoroacetic acid (5 ml) was stirred at RT for 20 min, then
concentrated to an orange oil. The oil was dissolved in MeOH (5 ml)
and neutralized with a saturated aqueous sodium bicarbonate
solution (25 ml). The resulting mixture was further diluted with
H.sub.2O (25 ml) and EtOAc (100 ml). The aqueous layer was removed
and extracted with EtOAc (100 ml). The organic layers were
combined, washed with brine, dried over MgSO.sub.4, filtered and
concentrated to yellow solids (507 mg), These solids were dissolved
in MeOH (5 ml) and a solution of HCl in dioxane (4 M, 1.5 ml) was
added. The bright yellow solution was stirred 30 min, then
concentrated to yield cis-2-amino-cyclopentanecarboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide as a yellow powder (600
mg).
[0481] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 9.29 (s, 1);
8.75 (d, 1); 8.53 (d, 1); 8.37 (s, 1); 8.02 (s, 1); 4.05-3.95 (m,
1); 3.42-3.34 (m, 1); 2.46-1.80 (m, 6).
[0482] NH.sub.4OAc standard conditions.
[0483] DAD R.sub.f=1.65 min.
[0484] M+H=329.
Example 22
4-(2-amino-ethyl)-morpholine-3-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide, HCl salt
[0485] Method C was followed using racemic morpholine-3-carboxylic
acid reductively alkylated with 2-aminoacetaldehyde.
[0486] .sup.1H-NMR (300 MHz, MeOH-d.sub.4): .delta. 9.37 (s, 1);
8.76 (d, 1); 8.55 (d, 1); 8.44 (d, 1); 8.06 (d, 1); 4.68-4.55 (m,
2); 4.17-3.99 (m, 3); 3.84-3.73 (m, 2); 3.57-3.39 (m, 4).
[0487] NH.sub.4OAc standard conditions.
[0488] DAD R.sub.f=1.69 min.
[0489] M+H=374.
Example 23
4-(2(S)-amino-propyl)-morpholine-3(S)-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide, HCl salt (first eluting
diastereomer)
[0490] Method C was followed using racemic morpholine-3-carboxylic
acid reductively alkylated with the appropriate alanine aldehyde.
The diastereomers were separated via column chromatography prior to
the deprotection step.
[0491] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 9.32 (s, 1);
8.76 (d); 8.55 (d, 1); 8.41 (s, 1); 8.08 (s, 1); 4.38 (d, 1);
4.32-4.21 (m, 1); 4.16-4.09 (m, 1); 4.04-3.95 (m, 2); 3.79-3.57 (m,
2); 3.47-3.40 (m, 1); 3.22-3.05 (m, 2); 1.44 (d, 3).
[0492] NH.sub.4OAc standard conditions.
[0493] DAD R.sub.f=1.38 min.
[0494] M+H=388.
Example 24
4-(2(S)-amino-propyl)-morpholine-3(R)-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide, HCl salt (second eluting
diastereomer)
[0495] Method C was followed using racemic morpholine-3-carboxylic
acid reductively alkylated with the appropriate alanine aldehyde.
The diastereomers were separated via column chromatography prior to
the deprotection step.
[0496] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 9.34 (s, 1);
8.77 (d, 1); 8.55 (d, 1); 8.42 (s, 1); 8.06 (s, 1); 4.42 (d, 1);
4.30-4.12 (m, 1); 4.07-3.92 (m, 3); 3.89-3.74 (m, 1); 3.65-3.49 (m,
1); 3.25-2.90 (m, 3); 1.36 (d, 3).
[0497] NH.sub.4OAc standard conditions.
[0498] DAD R.sub.f=1.57 min.
[0499] M+H=388.
Example 25
2-amino-cyclohexanecarboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide
[0500] A solution of cis-2-(tert-butoxycarbonylamino)-cyclohexane
carboxylic acid (255 mg, 1.05 mmol) in pyridine (10 ml) was stirred
at RT. 6-chloro-9H-.beta.-carbolin-8-ylamine (218 mg, 1.00 mmol)
was added, followed by EDCI (315 mg, 1.64 mmol) and the slightly
turbid pale orange solution was stirred at RT for 16 hr. The
solution was diluted with H.sub.2O (20 ml) and poured into a
separatory funnel containing H.sub.2O (50 ml) and EtOAc (50 ml).
The mixture was shaken and the layers were separated. The aqueous
layer was extracted with EtOAc (50 ml) and the combined organic
layers were washed with brine. The organic layer was dried,
filtered and concentrated to yield a yellow oil which was placed
under high vacuum for 4 hr. The resulting yellow-brown glass was
slurried in CH.sub.2Cl.sub.2 (10 ml) at RT. Trifluoroacetic acid (5
ml) was added and the slurry instantly dissolved to form a clear
orange solution. The solution was stirred at RT for 45 min, then
concentrated to a brown residue. The residue was azeotroped from
toluene (3.times.10 ml) to yield a yellow solid. A solution of
dilute aqueous Na.sub.2CO.sub.3 was prepared by adding a small
volume of 10% aqueous Na.sub.2CO.sub.3 to H.sub.2O (50 ml) until
the aqueous solution reached a pH of 10. The yellow solid was
dissolved in minimal MeOH and was added drop-wise to the aqueous
solution with stirring. The precipitate which formed was filtered,
washed with H.sub.2O and placed under high vacuum to yield
2-amino-cyclohexanecarboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide as pale yellow solid (147
mg).
[0501] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 9.00 (s, 1);
8.37-8.34 (m, 1); 8.16-8.13 (m, 2); 7.83 (m, 1); 5.66-5.00 (br s,
2); 3.42-3.40 (m, 1); 2.70-2.62 (m, 1); 2.02-1.90 (m, 1); 1.70-1.54
(m, 5); 1.42-1.29 (m, 2). NH.sub.4OAc standard conditions.
[0502] DAD R.sub.f=1.46 min.
[0503] M+H=343.
Example 26
4-(2(R)-amino-propyl)-morpholine-3(S)-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide, HCl salt
[0504] Method C was followed using racemic morpholine-3-carboxylic
acid reductively alkylated with the appropriate alanine aldehyde.
The diastereomers were separated via column chromatography prior to
the deprotection step.
[0505] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 9.35 (s, 1);
8.77 (m, 1); 8.55 (m, 1); 8.43 (s, 1); 8.01 (s, 1); 4.45 (d, 1);
4.26 (m, 1); 4.09-3.91 (m, 3); 3.79 (m, 1); 3.63 (m, 1); 3.28-2.99
(m, 3); 1.37 (d, 3).
[0506] NH.sub.4OAc standard conditions.
[0507] DAD R.sub.f=1.39 min.
[0508] M+H=388.
Example 27
4-(2(R)-amino-3-phenyl-propyl)-morpholine-3(S)-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide, HCl salt
[0509] Method C was followed using racemic morpholine-3-carboxylic
acid reductively alkylated with the appropriate alanine aldehyde.
The diastereomers were separated via column chromatography prior to
the deprotection step.
[0510] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 9.34 (s, 1);
8.77 (d, 1); 8.55 (d, 1); 8.42 (s, 1); 8.04 (s, 1); 7.44-7.23 (m,
5); 4.39 (d, 1); 4.65-4.07 (m, 1); 4.40-3.82 (m, 4); 3.50-3.25 (m,
1); 3.30-3.14 (m, 1); 3.11-2.88 (m, 3); 2.85-2.69 (m, 1).
[0511] NH.sub.4OAc standard conditions.
[0512] DAD R.sub.f=1.90 min.
[0513] M+H=464.
METHOD D: CHROMATOGRAPHY CONDITIONS
LCMS
[0514] Column type: Phenomenex Luna C18(2) columns, 5 um, size
50.times.4.6 mm Run time: 5.00 minute run
[0515] NH.sub.4OAc Conditions:
Solvent A:
10 mM NH.sub.4OAc
99% H.sub.2O
1% MeCN
Solvent B:
10 mM NH.sub.4OAc
5% H2O
95% MeCN
[0516] Standard Gradient:
Initial conditions--95% A, 5% B 3.5 minute gradient from 5%-100% B
3.5-4.3 minutes hold at 100% B 4.3-5 minutes initial conditions
[0517] Polar Gradient:
Initial conditions--70% A, 30% B 3.5 minute gradient from 70%-100%
B 3.5-4.3 minutes hold at 100% B 4.3-5 minutes initial
conditions
[0518] Nonpolar Gradient:
Initial conditions--100% A 3.5 minute gradient from 0%-50% B
3.5-4.3 minutes hold at 100% B 4.3-5 minutes initial conditions
[0519] HCOOH Conditions:
Solvent C,
0.1% HCOOH
99% H.sub.2O
1% MeCN
Solvent D:
0.1% HCOOH
5% H.sub.2O
95% MeCN
[0520] Standard Gradient:
Initial conditions--95% C, 5% D 3.5 minute gradient from 5%-100% D
3.5-4.3 minutes hold at 100% D 4.3-5 minutes initial conditions
[0521] Polar Gradient:
Initial conditions--70% C, 30% D 3.5 minute gradient from 70%-100%
D 3.5-4.3 minutes hold at 100% D 4.3-5 minutes initial
conditions
[0522] Nonpolar Gradient:
Initial conditions--100% C 3.5 minute gradient from 0%-50% D
3.5-4.3 minutes hold at 100% D 4.3-5 minutes initial conditions
INTERMEDIATE 37: 6-chloro-2,3,4,9-tetrahydro-1H-.beta.-carboline,
HCl salt
[0523] 5-chlorotryptamine hydrochloride (5 g, 20 mmol, 1 equiv.)
was dissolved in 40 ml 3 M NaOAc buffer (pH=4.8) and 40 ml water.
Glyoxalic acid (1.84 g, 20 mmol, 1 equiv.) was added in one portion
and the solution was stirred at RT overnight. The resulting thick
slurry was filtered and the light green solids were suspended in
100 ml 6N HCl and heated at 125.degree. C. under a reflux condenser
for 1 hour with intermittent additions of conc HCl (2 ml every 15
min). After cooling to RT, 4.38 g (90%) of
6-chloro-2,3,4,9-tetrahydro-1H-.beta.-carboline, HCl salt as
blue-grey solid was isolated by filtration.
[0524] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 11.33 (br, 2H),
9.62 (br, 2H), 7.53 (d, 1), 7.39 (d, 1), 7.09 (dd, 1), 4.33 (br,
2H), 2.92 (t, 2)
[0525] Formic Acid Standard Conditions.
[0526] DAD RT=1.56 min.
[0527] M+H=207.
INTERMEDIATE 38:
(6-chloro-1,3,4,9-tetrahydro-g-carbolin-2-yl)-phenyl-methanone
[0528] 6-chloro-2,3,4,9-tetrahydro-1H-.beta.-carboline, HCl salt
(10.2 g, 42 mmol, 1 equiv.) was suspended in 100 ml of dry pyridine
under N2 and cooled to 0.degree. C. in an ice water bath. Benzoyl
chloride (7.3 ml, 63 mmol, 1.5 equiv.) was added drop-wise to the
cold solution after which the reaction was removed from the ice
bath and allowed to stir overnight at room temperature. The
reaction was quenched by the addition of water until choked with
solids. These solids were captured by filtration, washed with a
saturated aqueous sodium bicarbonate solution, re-suspended in
water, sonicated, and refiltered to give 1.27 g (94%) of
(6-chloro-1,3,4,9-tetrahydro-.beta.-carbolin-2-yl)-phenyl-methanone
as crystalline orange solids.
[0529] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 10.65-10.59
(br, 1H), 7.0-7.5 (m, 9H), 4.60-4.83 (br, 2H), 3.62-3.99 (br, 2H),
2.75 (br., 2H).
[0530] Formic Acid Standard Conditions.
[0531] DAD RT=2.68 min.
[0532] M+H=311.
INTERMEDIATE 39:
2-benzoyl-6-Chloro-1,2,3,9-tetrahydro-.beta.-carbolin-4-one
[0533]
(6-chloro-1,3,4,9-tetrahydro-.beta.-carbolin-2-yl)-phenyl-methanone
(1.76 g, 5.66 mmol, 1 equiv.) and DDQ (2.31 g, 10.2 mmol, 1.8
equiv.) were mixed as solids and cooled to -78.degree. C. 15 ml of
a 9:1 THF/H.sub.2O solution was cooled to -78.degree. C. and the
resulting slurry was added to the cooled solids followed by an
additional 15 ml of THF (also cooled to -78.degree. C.). The deep
blue solution was stirred at -78.degree. C. for 2 hours and then
gradually warmed to room temperature and stirred an additional two
hours. The reaction was quenched by the addition of 1 N NaOH, and
extracted with 3.times.150 ml EtOAc. The combined organic layers
were washed with 2.times.100 ml 1 N HCl, 1.times.100 ml brine,
dried over MgSO.sub.4, filtered and concentrated to yield 1.38 g
(75%) of
2-benzoyl-6-chloro-1,2,3,9-tetrahydro-.beta.-carbolin-4-one as oily
orange solids.
[0534] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 12.11-12.48
(br, 1H), 7.29-7.88 (m, 8H), 4.93-5.18 (br, 2H), 4.60-4.46 (br,
2H).
[0535] Exact Mass: 324.07.
[0536] Formic Acid Standard Conditions
[0537] DAD RT=2.15 min.
[0538] MPH=325.
INTERMEDIATE 40: 4-amino-6-chloro .beta.-carboline
[0539] Crude
2-benzoyl-6-chloro-1,2,3,9-tetrahydro-.beta.-carbolin-4-one (4 g)
was dissolved in 30 ml of anhydrous hydrazine and stirred at reflux
(130.degree. C. oil bath) under N.sub.2 for 6 hours, after which
the reaction mixture was allowed to cool to room temperature and
sit overnight. The precipitated yellow solids were removed by
filtration and washed with water, 2.times.5 ml, to yield 785 mg
(30%) of 4-amino-6-chloro .beta.-carboline as an off white solid.
Additional water was added to the combined filtrates until no
further precipitation occurred. These solids were also removed by
filtration to give 1.056 g (39%) of 4-amino-6-chloro
.beta.-carboline as yellow solids, (69% total yield).
[0540] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 11.48 (s, 1H),
8.44 (s, 1H), 8.13 (s, 1H), 7.77 (s, 1H), 7.42-7.52 (m, 2H), 5.86
(s, 2H).
[0541] Formic Acid Standard Conditions.
[0542] DAD RT=1.68 min.
[0543] M+H=218.
INTERMEDIATE 41:
N-(6-chloro-9H-.beta.-carbolin-4-yl)-2,2,2-trifluoro-acetamide
[0544] 4-amino-6-chloro .beta.-carboline (1.05 g, 4.82 mmol, 1
equiv.) was dissolved in 4 ml of anhydrous pyridine and 20 ml of
THF and cooled to 0.degree. C. under N.sub.2. Trifluoroacetic
anhydride (3.4 ml, 24 mmol, 5 equiv.) was added drop-wise to the
cooled solution. Upon complete addition, the reaction was removed
from the ice bath and stirred at room temperature for .about.1.5
hours. The reaction was quenched by the slow addition of water (10
ml) and extracted 2.times.150 ml EtOAc, washed 2.times.100 ml
saturated aqueous sodium bicarbonate, 1.times.100 ml brine, dried
over MgSO.sub.4, filtered and concentrated to orange solids. These
solids were titurated by in 10-15 ml Et.sub.2O and captured by
filtration to give 1.23 g (81%) of
N-(6-chloro-9H-.beta.-carbolin-4-yl)-2,2,2-trifluoro-acetamide as a
yellow solid.
[0545] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 12.11 (s, 1H),
11.89 (s, 1H), 8.92 (s, 1H), 8.33 (s, 1H), 7.82 (s, 1H), 7.60-7.70
(m, 2H).
[0546] Formic Acid Standard Conditions.
[0547] DAD RT=2.12 min.
[0548] M+H=314.
INTERMEDIATE 42:
N-(6-chloro-8-nitro-9H-.beta.-carbolin-4-yl)-2,2,2-trifluoro-acetamide
[0549]
N-(6-chloro-9H-.beta.-carbolin-4-yl)-2,2,2-trifluoro-acetamide (125
mg, 0.4 mmol, 1 equiv.) was dissolved in 2 ml TFA and NaNO.sub.2
(541 mg, 7.84 mmol, 2 equiv.) was added in one portion. The
solution was stirred at room temperature for 4 hr. Volatiles were
removed by rotovap, and the resulting oily orange solids were
suspended in water, neutralized with a saturated aqueous sodium
bicarbonate solution and filtered to give 132 mg (92%) of
N-(6-chloro-8-nitro-9H-.beta.-carbolin-4-yl)-2,2,2-trifluoro-ace-
tamide.
[0550] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 12.87 (s, 1H),
12.03 (s, 1H), 9.11 (s, 1H), 8.56 (s, 1H), 8.53 (s, 1H), 8.26 (s,
1H).
[0551] Formic Acid Standard Conditions.
[0552] DAD RT=2.27 min.
[0553] M+H=359.
INTERMEDIATE 43:
N-(8-amino-6-chloro-9H-.beta.-carbolin-4-yl)-2,2,2-trifluoro-acetamide
[0554] The crude
N-(6-chloro-8-nitro-9H-.beta.-carbolin-4-yl)-2,2,2-trifluoro-acetamide
(130 mg, 0.36 mmol) was dissolved in 7 ml of MeOH and the reaction
vessel was vacuum purged 3.times. with N.sub.2. Platinum (20 mg,
20% wt. on activated carbon) was added quickly, and the reaction
vessel was again vacuum purged 3.times. with N.sub.2, followed by 3
additional vacuum purge cycles with H.sub.2. The reaction was
allowed to stir under H.sub.2 at 1 atm overnight. Upon completion,
the reaction vessel was purged of H.sub.2 and filtered over celite.
The celite was washed several times with methanol until the
filtrates were clear. The combined filtrates were concentrated to
give
N-(8-amino-6-chloro-9H-.beta.-carbolin-4-yl)-2,2,2-trifluoro-acetamide
as a yellow solid (112 mg, 95%).
[0555] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 11.83 (s, 1H),
8.98 (s, 1H), 8.30 (s, 1H), 7.10 (s, 1H), 6.82 (s, 1H), 5.87 (br,
2H).
[0556] Formic Acid Standard Conditions.
[0557] DAD RT=1.95 min.
[0558] M+H=329.
INTERMEDIATE 44:
N-[6-chloro-4-(2,2,2-trifluoro-acetylamino)-9H-.beta.-carbolin-8-yl]-2-me-
thyl-nicotinamide
[0559]
N-(8-amino-6-chloro-9H-.beta.-carbolin-4-yl)-2,2,2-trifluoro-acetam-
ide (90 mg, 0.274 mmol, 1 equiv.) and 2-methylnicotinic acid (45
mg, 0.329 mmol, 1.2 equiv.) were dissolved in 1.5 ml of anhydrous
pyridine under N.sub.2. EDCI (84 mg, 0.438 mmol, 1.6 equiv.) was
added in one portion and the reaction mixture was stirred at room
temperature for 2 hours. The reaction was quenched with water and
the resulting dark solids captured by filtration. These solids were
titurated in a 3:1 methanol-DMSO solution to give
N-[6-chloro-4-(2,2,2-trifluoro-acetylamino)-9H-.beta.-carbolin-8-yl]-2-me-
thyl-nicotinamide as light yellow solids (41 mg, 3%
[0560] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 11.94 (br, 1H),
11.78 (s, 1H), 10.61 (s, 1), 9.00 (s, 1), 8.64 (d, 1), 8.38 (s,
1H), 8.13 (d, 1), 8.01 (s, 1H), 7.73 (s, 1H), 7.45 (m, 1H), 2.68
(s, 3H).
[0561] Formic Acid Standard Conditions.
[0562] DAD RT=1.98 min.
[0563] M+H=448.
INTERMEDIATE 45:
N-[6-chloro-4-(2,2,2-trifluoro-acetylamino)-9H-.beta.-carbolin-8-yl]-nico-
tinamide
[0564]
N-(8-amino-6-chloro-9H-.beta.-carbolin-4-yl)-2,2,2-trifluoro-acetam-
ide (90 mg, 0.274 mmol, 1 equiv.) and 2-methylnicotinic acid (40
mg, 0.329 mmol, 1.2 equiv.) were dissolved in 1.5 ml of anhydrous
pyridine under N2. EDCI (84 mg, 0.438 mmol, 1.6 equiv.) was added
in one portion and the reaction mixture was stirred at room
temperature 2 hours. The reaction was quenched and treated
following the preceding protocol to obtain 38 mg (32%) of
N-[6-chloro-4-(2,2,2-trifluoro-acetylamino)-9H-.beta.-carbolin-8-
-yl]-nicotinamide.
[0565] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 11.89 (br, 1H),
10.73 (s, 1), 9.28 (s, 1), 8.97 (s, 1), 8.84 (d, 1), 8.43 (d, 1),
8.37 (s, 1), 7.85 (s, 1), 7.76 (s, 1), 7.67 (m, 1).
[0566] Formic Acid Standard Conditions.
[0567] DAD RT=1.94 min.
[0568] M+H=434.
Example 28
N-(4-amino-6-chloro-9H-.beta.-carbolin-8-yl)-2-methyl-nicotinamide
[0569]
N-[6-chloro-4-(2,2,2-trifluoro-acetylamino)-9H-.beta.-carbolin-8-yl-
]-nicotinamide (41 mg, 0.092 mmol, 1 equiv.) was suspended in 5 ml
of MeOH and a 2 ml aqueous solution of K.sub.2CO.sub.3 (127 mg,
0.92 mmol, 10 equiv.) was added thereto. The resulting clear
solution was heated at 60.degree. C. for 16 hr and then allowed to
cool to RT. Additional water was added producing fine solids that
were captured by filtration, washed once with 10 ml of 5% MeOH in
Et.sub.2O, and dried under high vacuum to give 18 mg of
N-(4-amino-6-chloro-9H-.beta.-carbolin-8-yl)-2-methyl-nicotinamide
as powdery yellow solids (56% yield).
[0570] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 11.18 (s, 1H),
10.41 (s, 1H), 8.62 (d, J=3.6, 1H), 8.32 (s, 1H), 8.20 (s, 1H),
8.11 (d, J=7.5, 1H), 7.91 (s, 1H), 7.79 (s, 1H), 7.43 (m, 1H), 5.91
(br, 2H), 2.66 (s, 3H).
[0571] Formic Acid Standard Conditions.
[0572] DAD RT=1.63 min.
[0573] M+H=352.
Example 29
N-(4-amino-6-chloro-9H-.beta.-carbolin-8-yl)-nicotinamide
[0574]
N-[6-chloro-4-(2,2,2-trifluoro-acetylamino)-9H-.beta.-carbolin-8-yl-
]-nicotinamide (38 mg, 0.088 mmol, 1 equiv.) was suspended in 5 ml
of MeOH and a 2 ml aqueous solution of K.sub.2CO.sub.3 (121 mg,
0.92 mmol, 10 equiv.) was added thereto. The resulting clear
solution was heated at 60.degree. C. for 11 hr. After cooling to
RT, fine solids precipitated that were captured by filtration and
washed with 10 ml of water to give 2.78 mg (10%) of
N-(4-amino-6-chloro-9H-.beta.-carbolin-8-yl)-nicotinamide.
[0575] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 11.32 (s, 1H),
10.56 (s, 1H), 9.26 (s, 1H), 8.82 (d, 1), 8.42 (d, 1), 8.36 (s,
1H), 8.18 (s, 1H), 7.79 (s, 1H), 7.70 (s, 1H), 7.64 (m, 1H), 5.91
(br, 2H).
[0576] Formic Acid Standard Conditions.
[0577] DAD RT=1.55 min.
[0578] M+H=338.
INTERMEDIATE 46: 3-cyanomethyl-indole-1-carboxylic acid tert-butyl
ester
[0579] A solution of 3-indoleacetonitrile (10 g, 64 mmol) in DMF
(160 ml) was stirred at RT. K.sub.2CO.sub.3 (13.3 g, 96 mmol) and
di-tert-butyl dicarbonate (15.35 g, 70 mmol) were added thereto and
the reaction mixture was stirred at RT for 12 hr. H.sub.2O (100 ml)
was added to the reaction mixture and the resulting precipitate was
captured by filtration.
[0580] The solids were dissolved in hot methanol (20 ml) and the
solution was allowed to cool slowly, producing light orange solids
that were isolated by filtration to give
3-cyanomethyl-indole-1-carboxylic acid tert-butyl ester (9.2
g).
[0581] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 8.08, (d, 1);
7.70-7.66 (m, 2); 7.42-7.29 (m, 2); 4.12 (s, 2); 1.63 (3, 9).
[0582] NH.sub.4OAc standard conditions.
[0583] DAD R.sub.f=3.31 min.
[0584] M+H=257.
INTERMEDIATE 47: 3-(cyano-methyl-methyl)-indole-1-carboxylic acid
tert-butyl ester
[0585] A stirred solution of 3-cyanomethyl-indole-1-carboxylic acid
tert-butyl ester (2.15 g, 8.39 mmol) in THF (40 ml) was cooled to
-78.degree. C. under an argon atmosphere. Sodium
bis(trimethylsilyl)amide (1 M in THF, 10 ml, 10 mmol) was added
thereto and the cold solution was stirred for 30 minutes.
Iodomethane (627 uL, 10 mmol) was added thereto and the reaction
mixture was stirred 1.5 hr while gradually warming to 0.degree. C.
H.sub.2O (100 ml) was added thereto and the solution was brought to
RT and diluted with EtOAc (250 ml). The aqueous layer was removed
and extracted with EtOAc (250 ml). The combined organic layers were
washed with aqueous HCl (1N, 3.times.50 ml), followed by brine,
then dried over MgSO.sub.4, filtered, and concentrated to an orange
oil. Purification via column chromatography (hexanes:ethyl acetate)
gave 3-(cyano-methyl-methyl)-indole-1-carboxylic acid tert-butyl
ester (1.8 g).
[0586] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 8.09 (d, 1);
7.73 (d, 1); 7.69 (s, 1); 7.74-7.29 (m, 2); 4.56 (q, 1); 1.66 (d,
J=7.2, 3); 1.63 (s, 9).
INTERMEDIATE 48: 3-(2-amino-1-methyl-methyl)-indole-1-carboxylic
acid tert-butyl ester
[0587] A solution of 3-(cyano-methyl-methyl)-indole-1-carboxylic
acid tert-butyl ester (850 mg, 3.14 mol) in methanol (15 ml) was
stirred at RT. A Raney-Nickel catalyst (50% g/wt suspension in
H.sub.2O, 1 ml) was added thereto and the reaction vessel was
capped and vacuum purged 3 times with argon, followed similarly by
hydrogen. The mixture was stirred 22 hr under 1 atm. of hydrogen,
vacuum purged with argon and filtered through celite. The filtrate
was concentrated to an oil (670 mg) and determined by LCMS to be
composed mainly of the desired compound
3-(2-amino-1-methyl-methyl)-indole-1-carboxylic acid tert-butyl
ester.
[0588] NH.sub.4OAc standard conditions.
[0589] DAD R.sub.f=1.78 min.
[0590] M+H=275.
INTERMEDIATE 49:
4-methyl-2,3,4,9-tetrahydro-1H-.beta.-carboline-1-carboxylic
acid
[0591] Crude 3-(2-amino-1-methyl-methyl)-indole-1-carboxylic acid
tert-butyl ester (670 mg, approx. 2.44 mmol) was dissolved in
trifluoroacetic acid (2 ml) and stirred 30 min at RT, then
concentrated to an oily solid under reduced pressure. The resulting
oil was dissolved in a 3 M of NaOAc:AcOH buffer solution (pH=4.8,
12 ml) and H.sub.2O (6 ml) at RT. Glyoxalic acid (225 mg, 2.44
mmol) was added thereto and the reaction mixture was stirred for 4
hr at RT, then concentrated to dryness. The resulting solids were
determined to be composed mainly of the desired compound
4-methyl-2,3,4,9-tetrahydro-1H-.beta.-carboline-1-carboxylic acid
by LCMS and used subsequently thereafter without purification.
[0592] NH.sub.4OAc standard conditions.
[0593] DAD R.sub.f=1.24 min.
[0594] M+H=231.
INTERMEDIATE 50:
4-methyl-2,3,4,9-tetrahydro-1H-.beta.-carboline
[0595] Crude
4-methyl-2,3,4,9-tetrahydro-1H-.beta.-carboline-1-carboxylic acid
(approx. 2.44 mmol) was suspended in H.sub.2O (5 ml) and HCl (12 N,
5 ml) and the suspension was heated at 120.degree. C. for 1 hr,
then allowed to cool to RT. Dark orange-brown solids were removed
by filtration and then dissolved in methanol (5 ml). A saturated
sodium bicarbonate solution (20 ml) was added thereto, producing a
thick yellow slurry. This reaction mixture was filtered to yield
yellow solids composed mainly of
4-methyl-2,3,4,9-tetrahydro-1H-.beta.-carboline (386 mg) as
determined by LCMS.
[0596] NH.sub.4OAc standard conditions.
[0597] DAD R.sub.f=1.23 min.
[0598] M+H=187.
INTERMEDIATE 51: 4-Methyl-9H-.beta.-carboline
[0599] 4-methyl-2,3,4,9-tetrahydro-1H-.beta.-carboline (214 mg,
1.17 mmol) was suspended in xylenes (10 ml). Pd (10% wt. on carbon,
21 mg) catalyst was added thereto and the reaction mixture was
stirred at 160.degree. C. for 24 hours, then cooled to RT and
filtered through celite. The filtrate was concentrated to dryness
to give 4-methyl-9H-.beta.-carboline (210 mg).
[0600] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 11.73 (br s,
1); 8.78 (br s, 1); 8.19 (d, 1); 8.13 (s, 1); 7.62 (br s, 1); 7.53
(t, 1); 7.25 (t, 1); 2.78 (s, 3),
[0601] NH.sub.4OAc standard conditions.
[0602] DAD R.sub.f=2.24 min.
[0603] M+H=183.
INTERMEDIATE 52: 6-Chloro-4-methyl-9H-.beta.-carboline
[0604] 4-methyl-9H-.beta.-carboline (97 mg, 0.532 mmol) was
dissolved in HCl (1N, 4 ml) and stirred at RT. NCS (85 mg, 0.637
mmol) was added thereto and the reaction mixture was stirred for 5
hr. Saturated sodium bicarbonate solution (20 ml) was added thereto
and the reaction mixture was extracted twice with EtOAc (100 ml).
The combined organic layers were washed with brine, dried over
MgSO.sub.4, filtered, and concentrated to give
6-chloro-4-methyl-9H-.beta.-carboline (108 mg) as an oil.
[0605] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 8.67 (s, 1);
8.19 (s, 1); 8.11 (s, 1); 7.57-7.54 (m, 2); 2.82 (s, 3).
[0606] NH.sub.4OAc standard conditions.
[0607] DAD R.sub.f=2.48 min.
[0608] M+H=217.
INTERMEDIATE 53: 6-chloro-4-methyl-8-nitro-9H-.beta.-carboline
[0609] A solution of 6-chloro-4-methyl-9H-.beta.-carboline (100 mg,
0.462 mmol) in trifluoroacetic acid (10 ml) was stirred at RT.
NaNO.sub.3 (106 mg, 1.25 mmol) was added thereto and the reaction
mixture was stirred 30 min, then concentrated to give an orange
residue. The residue was dissolved in MeOH (5 ml) and neutralized
by an addition of a saturated sodium bicarbonate solution (20 ml)
causing the formation of yellow solids that were captured by
filtration, washed with H.sub.2O (10 ml) and Et.sub.2O (2.times.10
ml) to give 6-chloro-4-methyl-8-nitro-9H-.beta.-carboline (82
mg).
[0610] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 8.90 (s, 1);
8.65 (s, 1); 8.55 (s, 1); 8.29 (s, 1); 2.89 (s, 3). NH.sub.4OAc
standard conditions.
[0611] DAD R.sub.f=3.00 min.
[0612] M+H=262.
INTERMEDIATE 54: 6-chloro-4-methyl-9H-.beta.-carbolin-8-ylamine
[0613] A solution of 6-chloro-4-methyl-8-nitro-9H-.beta.-carboline
(80 mg, 0.31 mmol) in MeOH (10 ml) was stirred at RT. Platinum (10%
wt. on carbon, 24 mg) catalyst was added thereto and the reaction
vessel was capped and vacuum purged 3 times with argon, followed
similarly by hydrogen. The reaction mixture was stirred 1.5 hr
under 1 atm hydrogen, then vacuum purged with argon, diluted with
DCM (10 ml) and filtered through a 0.2 uM syringe filter. The
filtrate was concentrated to yield
6-chloro-4-methyl-9H-.beta.-carbolin-8-ylamine (67 mg) as a light
brown oil.
[0614] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 9.04 (s, 1);
8.29 (s, 1); 7.74 (d, 1); 7.10 (d, 1); 2.98 (s, 3).
[0615] NH.sub.4OAc standard conditions.
[0616] DAD R.sub.f=1.89 min,
[0617] M+H=231.
Example 30
N-(6-chloro-4-methyl-9H-.beta.-carbolin-8-yl)-nicotinamide
[0618] A solution of 6-chloro-4-methyl-9H-.beta.-carbolin-8-ylamine
(43 mg, 0.19 mmol) in pyridine (4 ml) was stirred at RT under an
argon atmosphere. Nicotinoyl chloride hydrochloride (40 mg, 0.22
mmol) was added thereto and the reaction mixture was stirred for 12
hr. The solution was diluted with H.sub.2O (5 ml) and poured into a
separatory funnel containing H.sub.2O (5 ml) and EtOAc (25 ml). The
reaction mixture was shaken and the layers were separated. The
aqueous layer was extracted with EtOAc (2.times.25 ml). The
combined organic layers were washed with a saturated sodium
bicarbonate solution (15 ml), followed by brine, then dried over
MgSO.sub.4, filtered, and concentrated to yield
N-(6-chloro-4-methyl-9H-.beta.-carbolin-8-yl)-nicotinamide (5.2 mg)
as an orange viscous oil.
[0619] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 11.67 (s, 1);
10.70 (s, 1); 9.27 (s, 1); 8.83 (s, 2); 8.45 (s, 1); 8.20-8.12 (m,
2); 7.83 (s, 1); 7.66 (s, 1); 2.80 (s, 3).
[0620] NH.sub.4OAc standard conditions.
[0621] DAD R.sub.f=1.92 min.
[0622] M+H=337.
INTERMEDIATE 55:
1,1-Dioxo-1.lamda..sup.6-thiomorpholine-3,4-dicarboxylic acid
4-tert-butyl ester
[0623] NOM Thiomorpholine-3,4-dicarboxylic acid 4-tert-butyl ester
(120 mg, 0.485 mmol) was dissolved in Et.sub.2O (8 ml). To the
solution was added mCPBA (172 mg, 0.994 mmol), followed later by a
second portion of mCPBA (84 mg, 0.485 mmol). The precipitate which
formed was filtered, washed with Et.sub.2O and dried to yield a
white solid of
1,1-dioxo-1.lamda..sup.6-thiomorpholine-3,4-dicarboxylic acid
4-tert-butyl ester (74 mg).
Example 31
1,1-Dioxo-1.lamda..sup.6-thiomorpholine-3-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide
[0624] A slurry of 6-chloro-7-fluoro-9H-.beta.-carbolin-8-ylamine
(43 mg, 0.198 mmol),
1,1-dioxo-1.lamda..sup.6-thiomorpholine-3,4-dicarboxylic acid
4-tert-butyl ester (72 mg, 0.257 mmol), and EDCI (76 mg, 0.396
mmol) in pyridine (2 ml) was heated to 70.degree. C. After 1 hr,
the solvent was removed under reduced pressure and the resulting
dark oil was dissolved in MeOH (1 ml). The MeOH solution was added
drop-wise to a stirring solution of aqueous NaHCO.sub.3 and a
yellow precipitate was formed. The solid was filtered, dried, and
dissolved in 2 M HCl in Et.sub.2O. After stirring overnight the
resulting yellow solid was filtered and dried to yield a yellow
solid, the di-HCl salt of
1,1-Dioxo-1.lamda..sup.6-thiomorpholine-3-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide (78 mg).
[0625] .sup.1H-NMR (300 MHz, MeOH-d.sub.4): .delta. 9.26 (s, 1);
8.74 (d, 1); 8.54 (d, 1); 8.37 (d, 1); 7.94 (d, 1); 5.01 (dd, 1);
4.15-3.75 (m, 4); 3.64-3.58 (m, 2).
[0626] NH.sub.4OAc standard conditions.
[0627] DAD R.sub.f=1.57 min.
[0628] M+H=379.
INTERMEDIATE 56: 6,6-Dimethyl-morpholine-3,4-dicarboxylic acid
4-tert-butyl ester
[0629] To a suspension of 6,6-dimethyl-morpholine-3-carboxylic acid
(5.56 g, 34.9 mmol) in dioxane (58 mL) was added aqueous potassium
carbonate (1M, 58 mL). To the resulting clear colorless solution
was added di-tert-butyl dicarbonate (9.14 g, 41.9 mmol). The
solution was stirred at room temperature overnight. The reaction
mixture was diluted with water (200 mL) and the pH of the solution
was confirmed to be approximately 7. The reaction mixture was
poured into a separatory funnel and extracted with Et.sub.2O
(2.times.100 mL) to remove excess di-tert-butyl dicarbonate. The
aqueous layer was acidified by addition of 6N aqueous HCl with
stirring until a pH of 3 was reached. The mixture was quickly
extracted with Et.sub.2O (2.times.200 mL) and the organic layers
were combined, dried over magnesium sulfate, filtered, and
concentrated to yield a clear colorless oil. The oil was dissolved
in Et.sub.2O (50 mL), triturated with hexanes (150 mL), and
concentrated to yield a white solid. The solid product was placed
on the high-vacuum pump for several hours, after which 8.85 g of
6,6-dimethyl-morpholine-3,4-dicarboxylic acid 4-tert-butyl ester
was obtained (97% yield).
[0630] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 12.95 (s, 1);
4.35 ("dd", 1); 3.98-3.83 (m, 2); 3.48 ("dd", 1); 2.81 ("dd", 1);
1.39 ("dd", 9); 1.15 (s, 3); 1.08 ("dd", 3).
[0631] NH.sub.4OAc standard conditions.
[0632] DAD R.sub.f=0.98 min.
[0633] M-H=258.
INTERMEDIATE 57:
(S)-5-(6-Chloro-9H-.beta.-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholine-
-4-carboxylic acid tert-butyl ester
[0634] The desired compound was prepared according to Method C from
6-chloro-9H-.beta.-carboline-8-ylamine and
6,6-dimethyl-morpholine-3,4-dicarboxylic acid 4-tert-butyl ester in
87% yield.
[0635] .sup.1H-NMR (DMSO-d.sub.6, 300 MHz) .delta. 11.33 (s, 1);
10.14 (s, 1); 9.05 (s, 1); 8.38 (d, 1); 8.21 (d, 1); 8.16 (d, 1);
7.94 (s, 1); 4.70-4.56 (m, 1); 4.25-4.14 (m, 1); 4.07 (dd, 1);
3.64-3.56 (m, 1); 3.30-3.14 (m, 1); 1.41 ("dd", 9); 1.21 (s, 3);
1.15 (s, 3).
[0636] NH.sub.4OAc standard conditions.
[0637] DAD R.sub.f=1.84 min.
[0638] M+H=459.
[0639] Chiral HPLC: 95% ee.
[0640] Chiralpak AD column.
[0641] 15% v/v EtOH/Hexanes containing 0.1% Et.sub.2NH.
INTERMEDIATE 58:
[(S)-4-((S)-2-Amino-propyl)-6,6-dimethyl-morpholine-3-carboxylic
acid (6-chloro-9H-beta-carbolin-8-yl)-amide trifluoroacetate
salt
[0642] The desired compound was prepared using the same procedure
as for Intermediate 35 starting from
(S)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholine-4-
-carboxylic acid tert-butyl ester and using Boc-alaninal in the
reductive alkylation step in 60% yield.
[0643] .sup.1H-NMR (300 MHz, D.sub.2O): .delta. 1.16 (d, 3H), 1.25
(s, 3H), 1.28 (s, 3H), 2.41 (d, 1H), 2.59 (dd, 1H), 2.89 (dd, 1H),
2.95 (d, 1H), 3.35-3.50 (m, 2H), 3.95-4.15 (m, 2H), 7.59 (d, 1H),
7.97 (d, 1H), 8.11 (d, 1H), 8.30 (d, 1H), 8.40 (d, 1H), 8.94 (s,
1H).
[0644] Retention Time (LC, method: ammonium acetate standard): 1.57
min.
[0645] MS (M+H.sup.+): 416.2.
METHOD E: Coupling procedure using
[(S)-4-((S)-2-Amino-propyl)-6,6-dimethyl-morpholine-3-carboxylic
acid (6-chloro-9H-beta-carbolin-8-yl)-amide trifluoroacetate
salt
[0646]
[(S)-4-((S)-2-Amino-propyl)-6,6-dimethyl-morpholine-3-carboxylic
acid (6-chloro-9H-beta-carbolin-8-yl)-amide trifluoroacetate salt
(1.0 mmole), TBTU (1.2 mmoles), the acid (1.25 mmoles) to be
coupled and Et.sub.3N (4-6 mmoles, basic pH) were taken into
acetonitrile (10 ml). The resulting mixture was stirred at ambient
temperature for 4-15 hrs. The reaction mixture was then partitioned
into EtOAc and 10% aqueous Na.sub.2CO.sub.3 solution. The separated
aqueous phase was further extracted with EtOAc. The combined
extracts were successively washed with 10% aqueous Na.sub.2CO.sub.3
solution and brine, dried over Na.sub.2SO.sub.4 and concentrated
completely. The residue was purified on silica (2-7%
MeOH/CH.sub.2Cl.sub.2) to give the corresponding product.
Example 39
6,6-Dimethyl-4-[2-(2,2,2-trifluoro-acetylamino)-propyl]-morpholine-3-carbo-
xylic acid (6-chloro-9H-.beta.-carbolin-8-yl)-amide
[0647] 4-(2-Amino-propyl)-6,6-dimethyl-morpholine-3-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide (3 CF.sub.3COOH salt) (1.5
g) was suspended in dichloromethane (80 mL) along with 5
equivalents of triethylamine. Trifluoroacetic anhydride (56 .mu.L,
2 equivalents) was added and the mixture stirred at room
temperature for an hour. The solvent was removed by rotary
evaporation. The product was purified by silica gel flash
chromatography (5% methanol/dichloromethane, product Rf 0.3) to
afford 1 g.
[0648] .sup.1H-NMR (300 MHz, relative to CDCl.sub.3 peak at 7.3
ppm) .delta. 10 (s, 1H), 9.7 (d, 1H), 8.7 (s, 1H), 8.6 (s, 1H), 8.2
(d, 1H), 7.6 (s, 2H), 6.6 (s, 1H), 4.3 (m, 1H), 3.9 (m, 1H), 3.8
(t, 1H), 3.2 (m, 1H), 2.7-2.9 (m, 2H), 2.5 (m, 1H), 2.2 (d, 1H),
1.4 (d, 3H), 1.3 (s, 3H), 1.2 (s, 3H).
[0649] LCMS (ammonium acetate standard method) retention time=1.84
min.
[0650] (M.sup.+=512; M.sup.-=510).
Example 40
4-((S)-2-Acetylamino-propyl)-6,6-dimethyl-morpholine-3-(S)-carboxylic
acid (6-chloro-9H-.beta.-carbolin-8-yl)-amide
[0651] The desired compound was prepared according to the previous
example from
4-(2-Amino-propyl)-6,6-dimethyl-morpholine-3-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide (3 CF.sub.3COOH salt) and
acetic anhydride.
[0652] .sup.1H-NMR (300 MHz, methyl-d.sub.3 alcohol-d): .delta.
1.15 (d, 3H), 1.23 (s, 3H), 1.39 (s, 3H), 1.98 (s, 3H), 2.24 (d,
1H), 2.38 (m, 1H), 2.68 (m, 1H), 2.92 (d, 1H), 3.24 (m, 1H), 3.98
(m, 2H), 4.22 (m, H), 7.78 (d, 1H), 7.98 (m, 2H), 8.27 (d, 1H),
8.84 (s, 1H).
[0653] Retention Time (LC, method: ammonium acetate standard): 1.54
min.
[0654] MS (M+H.sup.+): 458.
Example 41
4-((8)-2-Methanesulfonylamino-propyl)-6,6-dimethyl-morpholine-3-(S)-carbox-
ylic acid (6-chloro-9H-.beta.-carbolin-8-yl)-amide
[0655] The desired compound was prepared according to Method E.
[0656] .sup.1H-NMR (300 MHz, methyl-d.sub.3 alcohol-d): .delta.
1.28 (s, 3H), 1.29 (d, 3H), 1.43 (s, 3H), 2.28 (d, 1H), 2.57 (m,
1H), 2.66 (m, 1H), 2.98 (s, 3H), 3.03 (d, 1H), 3.34 (m, 1H), 3.66
(m, 1H), 4.05 (m, 2H), 7.67 (d, 1H), 8.10 (m, 2H), 8.32 (d, 1H),
8.89 (s, 1H).
[0657] Retention Time (LC, method: ammonium acetate standard): 1.54
min.
[0658] MS (M+H.sup.+): 494.
Example 42
4-{2-[(-4,6-Dimethyl-pyrimidine-5-carbonyl)-amino]-propyl}-6,6-dimethyl-mo-
rpholine-3-carboxylic acid
(6-chloro-9H-beta-carbolin-8-yl)-amide
[0659] The desired compound was prepared using
[4-(2-Amino-propyl)-6,6-dimethyl-morpholin-3-ylmethyl]-(6-chloro-9H-beta--
carbolin-8-yl)-amine and 4,6-dimethylpyrimidine-5-carboxylic acid
following Method E in 51% yield.
[0660] .sup.1H-NMR (300 MHz, DMSO): .delta. 11.27 (1H, s), 10.02
(1H, s), 9.0 (1H, s), 8.86 (1H, s), 8.5 (1H, d), 8.3 (1H, d), 8.22
(2H, m), 7.88 (1H, s), 4.1 (1H, m), 3.9 (2H, m), 2.99 (2H, m), 2.36
(6H, s), 2.1 (2H, m), 1.3 (3H, s), 1.24 (6H, m).
[0661] Retention time (LC, method: ammonium acetate standard): 1.50
min.
[0662] MS (M+H.sup.+): 551
Example 43
(S)-6,6-Dimethyl-4-((S)-2-[(2-methyl-pyridine-3-carbonyl)amino]propyl)-mor-
pholine-3-carboxylic acid
(6-chloro-9H-beta-carbolin-8-yl)-amide
[0663] The desired compound was prepared according to Method E from
(S)-4-((S)-2-Amino-propyl)-6,6-dimethyl-morpholine-3-carboxylic
acid (6-chloro-9H-beta-carbolin-8-yl)-amide trifluoroacetate salt
and 2-methyl-nicotinic acid in 75% yield.
[0664] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 1.21 (s, 3H),
1.22 (d, 3H), 1.36 (s, 3H), 2.10 (d, 1H), 2.42 (m, 1H), 2.60 (m,
1H), 2.99 (d, 1H), 3.20 (m, 1H), 3.92 (m, 2H), 4.22 (m, 1H), 7.22
(dd, 1H), 7.65 (d, 1H), 7.90 (s, 1H), 8.16 (d, 1H), 8.23 (s, 1H),
8.31 (d, 1H), 8.38 (d, 1H), 8.45 (d, 1H), 9.02 (s, 1H), 10.04 (s,
1H), 11.26 (s, 1H).
[0665] Retention Time (LC, method: ammonium acetate standard): 2.16
min.
[0666] MS (M+H.sup.+): 535.5.
Example 44
6,6-Dimethyl-4-{2-[(tetrahydro-pyran-4-carbonyl)-amino]-propyl}-morpholine-
-3-carboxylic acid (6-chloro-9H-.beta.-carbolin-8-yl)-amide
[0667] 4-(2-Amino-propyl)-6,6-dimethyl-morpholine-3-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide (3 CF.sub.3COOH salt) (300
mg) was suspended in methylene chloride (12 mL) along with 3
equivalents of triethylamine. Morpholine-4-carbonyl chloride (70
mg, 1.3 equivalents) was added and the mixture stirred at room
temperature overnight. The solvent was removed by rotary
evaporation. The product was separated by preparative TLC on silica
plates (10/90 methanol/ethyl acetate as eluent, product Rf 0.4)
Yield: 83 mg.
[0668] .sup.1H-NMR (300 MHz, relative to CD.sub.3OD peak at 3.3
ppm) .delta. 8.8 (s, 1H), 8.27 (d, 1H), 7.9-7.99 (m, 3H), 3.95-4.15
(m, 2H), 3.85-3.95 (m, 2H), 3.5-3.6 (m, 5H), 3.3-3.45 (m, 2H),
3.15-3.3 (m, 2H), 2.85-2.95 (d, 1H), 2.6-2.72 m, 1H), 2.3-2.43 (m,
1H), 2.2-2.28 (d, 1H), 2.0 (s, 2H), 1.35-1.45 (d, 3H), 1.05-1.25
(m, 6H).
[0669] LCMS (ammonium acetate standard method) retention time=2.39
min. (M+=529; M-=527).
Example 45
4-{(S)-2-[(1-Acetyl-pyrrolidine-2-(S)-carbonyl)-amino]-propyl}-6,6-dimethy-
l-morpholine-3-(S)-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide
[0670] The desired compound was prepared according to Method E.
[0671] .sup.1H-NMR (300 MHz, methyl-d.sub.3 alcohol-d): .delta.
1.21 (d, 3H), 1.28 (s, 3H), 1.39 (s, 3H), 1.95 (m, 3H), 2.05 (s,
3H), 2.17 (m, 1H), 2.28 (d, 1H), 2.51 (m, 1H), 2.74 (m, 1H), 3.05
(d, 1H), 3.30 (m, 1H), 3.55 (m, 1H), 3.61 (m, 1H), 4.05 (m, 2H),
4.18 (m, 1H), 4.34 (m 1H), 7.78 (d, 1H), 8.10 (m, 2H), 8.33 (d,
1H), 8.90 (s, 1H).
[0672] Retention Time (LC, method: ammonium acetate standard): 2.07
min.
[0673] MS (M+H.sup.+): 555.
Example 46
6,6-Dimethyl-4-{-2-[(5-methyl-isoxazole-3-carbonyl)-amino]-propyl}-morphol-
ine-3-carboxylic acid (6-chloro-9H-beta-carbolin-8-yl)-amide
[0674] The desired compound was prepared using
[4-(2-Amino-propyl)-6,6-dimethyl-morpholin-3-ylmethyl]-(6-chloro-9H-beta--
carbolin-8-yl)-amine and 5-methylisoxazole carbonyl chloride
following Method E in 61% yield.
[0675] .sup.1H-NMR (300 MHz, DMSO): .delta. 11.2 (1H, s), 9.98 (1H,
s), 9.0 (1H, s), 8.7 (1H, d), 8.6 (1H, d), 8.2 (2H, m), 7.9 (1H,
s). 6.47 (1H, s), 3.87 (2H, m), 3.17 (2H, m), 2.9 (1H, d), 2.7 (1H,
m), 2.3 (4H), 2.1 (1H, d), 1.29 (3H, s), 1.15 (6H, m).
[0676] Retention time (LC, method: ammonium acetate standard): 1.81
min.
[0677] MS (M+H+): 526
Example 47
6,6-Dimethyl-4-[2-(3-methyl-ureido)-prop1yl]-morpholine-3-carboxylic
acid (6-chloro-9H-.beta.-carbolin-8-yl)-amide
[0678] 4-(2-Amino-propyl)-6,6-dimethyl-morpholine-3-carboxylic acid
(6-chloro-9H-.quadrature.-carbolin-8yl)-amide (3 HCl salt) (300 mg)
was suspended in dichloromethane (10 mL) triethyl amine (4
equivalents) and methyl isocyanate (2 equivalents) were added
simultaneously. After one hour at room temperature solvent was
removed by rotary evaporation. The product was purified by silica
gel flash chromatography (5% methanol/dichloromethane, product
Rf=0.3) to afford 200 mg.
[0679] .sup.1H-NMR (300 MHz, relative to CDCl.sub.3 peak at 7.3
ppm) .delta. 12.3 (s, 1H), 10.2 (s, 1H), 8.9 (s, 1H), 8.6 (s, 1H),
8.4 (d, 1H), 7.9 (d, 1H), 7.8 (s, 1H), 5.4 (s, 1H), 5.2 (d, 1H),
4.2 (s, 1H), 3.8 (m, 2H), 3.2 (m, 1H), 2.6-3 (m, 7H), 2.3 (d, 1H),
2.2 (t, 1H), 1.4 (s, 3H), 1.2 (s, 3H), 1.1 (d, 3H)
[0680] LCMS (ammonium acetate standard method) retention time=1.62
min. (M.sup.+=473; M.sup.-=471).
Example 49
{(S)-2-[(S)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morph-
olin-4-yl]-1-methyl-ethyl}-carbamic acid methyl ester
[0681] To a solution of
(S)-4-((S)-2-Amino-propyl)-6,6-dimethyl-morpholine-3 carboxylic
acid (6-chloro-9H-beta-carbolin-8-yl)-amide hydrochloride salt
(3.45 g, 6.59 mole) in 68 ml of dry pyridine, was added in three
portions over 1.5 hr, a 3M DCM solution of methyl chloroformate
(9.2 ml, 27.6 mole, 4.2 eq). After 2 h, 10 ml of water were added
and the mixture was concentrated to dryness. The residue was
partitioned into 150 ml 0f EtOAc and 100 ml of an aqueous 0.5M
solution of K.sub.2CO.sub.3. The separated aqueous phase was
extracted with 50 ml of EtOAc. The combined organic extracts were
successively washed with water (2.times.50 ml) and brine (50 ml),
dried over Na.sub.2SO.sub.4 and concentrated to dryness. The
residue was purified on silica (5% MeOH/CH.sub.2Cl.sub.2) to give
2.48 g (thick oil, 77% yield) of the desired product.
[0682] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. 1.15 (d, 3H),
1.28 (s, 3H), 1.42 (s, 3H), 2.33 (dd, 1H), 2.42 (d, 1H), 2.78 (dd,
1H), 2.86 (d, 1H), 3.32 (dd, 1H), 3.86 (s, 3H), 3.92 (t, 1H), 4.01
(dd, 1H), 4.18 (m, 1H), 4.78 (d, 1H), 7.95 (d, 1H), 7.97 (s, 1H),
8.29 (s, 1H), 8.50 (d, 1H), 8.98 (s, 1H), 9.88 (s, 1H), 10.94 (s,
1H).
[0683] Retention Time (LC, method: ammonium acetate standard): 1.70
min.
[0684] MS (M+H.sup.+): 474.1
Example 50
4-{2-[(2,4-Dimethyl-pyridine-3-carbonyl)-amino]-propyl}-6,6-dimethyl-morph-
oline-3-carboxylic acid (6-chloro-9H-beta-carbolin-8-yl)-amide
[0685] The desired compound was prepared from
[4-(2-Amino-propyl)-6,6-dimethyl-morpholin-3-ylmethyl]-(6-chloro-9H-beta--
carbolin-8-yl)-amine and 2,4-dimethyl nicotinic acid following
Method E in 50% yield.
[0686] .sup.1H-NMR (300 MHz, DMSO): .delta. 11.27 (1H, s), 10 (1H,
s), 8.9 (1H, s), 8.37 (2H, d), 8.24 (3H, m), 7.8 (1H, s), 7.04 (1H,
d), 3.91 (2H, m), 3.1 (2H, m), 2.36 (4H, m), 2.1 (3H, m), 2.05 (1H,
d), 1.3 (3H, s), 1.2 (6H, m).
[0687] Retention time (LC, method: ammonium acetate standard): 1.53
min.
[0688] MS (M+H.sup.+): 550
Example 51
6,6-dimethyl-4-(2-[(pyrazine-2-carbonyl)-amino]-propyl)-morpholine-3-carbo-
xylic acid (6-chloro-9H-beta-carbolin-8-yl)-amide
[0689] The desired compound was prepared from
[4-(2-Amino-propyl)-6,6-dimethyl-morpholin-3-ylmethyl]-(6-chloro-9H-beta--
carbolin-8-yl)-amine and 2-pyrazine carboxylic acid following
Method E in 62% yield.
[0690] .sup.1H-NMR (300 MHz, DMSO): .delta. 12.9 (1H, s), 10.89
(1H, s), 9.42 (1H, s), 9.0 (1H, s), 8.8 (1H, d), 8.6 (1H, d), 8.4
(1H, s), 8.2 (1H, m), 4.5 (1H, m), 4.1 (2H, m), 3.1 (1H, m), 2.1
(4H, m), 1.23 (9H, m).
[0691] Retention time (LC, method: ammonium acetate standard): 1.69
min.
[0692] MS (M+): 522.2
Example 52
Pyridine-3,4-dicarboxylic acid
4-({2-[5-(6-chloro-9H-.beta.-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morphol-
in-4-yl)-1-methyl-ethyl]-amide}3-methylamide
[0693] To a solution of
[4-(2-Amino-propyl)-6,6-dimethyl-morpholin-3-ylmethyl]-(6-chloro-9H-beta--
carbolin-8-yl)-amine (100 mg, 0.132 mmole) in 0.6 ml of dry
acetonitrile, was added 3,4-pyridinedicarboxylic anhydride (21 mg,
0.15 mmole) and triethylamine (102 ml, 0.8 mmole). The reaction
mixture was stirred at ambient temperature for 1 h. The solvent was
then removed under reduced pressure and the residue was taken up
into pyridine (0.6 ml). To the resulting mixture was added 2M
methylamine solution in THF (0.2 ml, 0.4 mmole) and EDCI (40 mg,
0.21 mmole). The reaction mixture was stirred for 4 hrs, the
solvent was removed under reduce pressure and the residue was
partitioned into EtOAc and 1M aqueous K.sub.2CO.sub.3. The
separated aqueous phase was extracted twice with EtOAc. The
combined organic phases were successively washed with water and
brine, dried over MgSO.sub.4, and concentrated completely. The
residue was purified on silica gel (10% MeOH--CH.sub.2Cl.sub.2) to
give the title compound as a white solid in 36% yield.
[0694] .sup.1H-NMR (300 MHz, DMSO): .delta. 9.24 (1H, s), 8.17 (1H,
s), 7.81 (2H, m), 7.5 (3H, m), 7.3 (1H, s), 7.0 (1H, s) 6.5 (1H,
d), 3.08 (2H, d), 3.35 (1H, m), 2.4 (3H, m), 1.86 (4H, m), 0.5 (3H,
s), 0.37 (6H, m).
[0695] Retention time (LC, method: ammonium acetate standard): 1.46
min.
[0696] MS (M+H.sup.+): 579
Example 53
6.6-Dimethyl-4-{(4-methyl-pyrimidine-5-carbonyl)-amino)-propyl}-morpholine-
-3-carboxylic acid (6-chloro-9H-beta-carbolin-8-yl)-amide
[0697] The desired compound was prepared according to Method E from
[4-(2-Amino-propyl)-6,6-dimethyl-morpholin-3-ylmethyl]-(6-chloro-9H-beta--
carbolin-8-yl)-amine and 4-methyl-pyrimidine-5-carboxylic acid in
55% yield.
[0698] .sup.1H-NMR (300 MHz, DMSO): .delta. 11.26 (1H, s), 10.04
(1H, s) 9.04 (2H, m), 8.66 (1H, d) 8.3 (2H, m) 8.22-8.17 (2H, m),
7.9 (1H, s), 4.2 (1H, m), 3.93 (2H, m), 3.2 (1H, m), 2.98 (2H, m),
2.6 (3H, m), 2.1 (2H, m), 1.36 (3H, s), 1.23 (6H, m).
[0699] Retention time (LC, method: ammonium acetate standard): 1.55
min.
[0700] MS (M+H+): 537
Example 54
(S)-6,6-Dimethyl-4-{(S)-2-[(4-methyl-pyridine-3-carbonyl)-amino]-propyl}-m-
orpholine-3-carboxylic acid
(6-chloro-9H-beta-carbolin-8-yl)-amide
[0701] The desired compound was prepared according to Method E from
(8)-4-((S)-2-Amino-propyl)-6,6-dimethyl-morpholine-3-carboxylic
acid (6-chloro-9H-beta-carbolin-8-yl)-amide trifluoroacetate salt
and 4-methyl-nicotinic acid in 79% yield.
[0702] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 1.21 (s, 3H),
1.22 (d, 3H), 1.36 (s, 3H), 2.10 (d, 1H), 2.40 (m, 1H), 2.62 (m,
1H), 2.99 (d, 1H), 3.22 (m, 1H), 3.94 (m, 2H), 4.23 (m, 1H), 7.26
(d, 1H), 7.90 (s, 1H), 8.16 (d, 1H), 8.23 (s, 1H), 8.34-8.46 (m,
3H), 9.02 (s, 1H), 10.04 (s, 1H), 11.27 (s, 1H).
[0703] Retention Time (LC, method: ammonium acetate standard): 2.22
min.
[0704] MS (M+H.sup.+): 535.5.
Example 47
4-[2-(2-Amino-2-methyl-propionyl)]-6,6-dimethyl-morpholine-3-carboxylic
acid (6-chloro-9H-.beta.-carbolin-8-yl)-amide
[0705] A solution of
{2-[5-(6-Chloro-9H-.beta.-carbolin-8-ylcarbamoyl)
2,2-dimethyl-morpholin-4-yl]-1-methyl-ethyl}-carbamic acid
tert-butyl ester (70.2 mg, 0.14 mmol) in TFA (2 mL) was stirred at
room temperature. After 15 min, the reaction was concentrated and
the crude product was azeotroped with CH.sub.2Cl.sub.2 (2.times.5
mL). A mixture of the crude intermediate, TBTU (54.0 mg, 0.17
mmol), triethylamine (0.2 mL, 1.43 mmol) and
2-tert-butoxycarbonylamino-2-methyl-propionic acid (45.0 mg, 0.22
mmol) in MeCN (1 mL) was stirred at room temperature for 18 h. The
solution was diluted with H.sub.2O (20 mL) and poured into a
separatory funnel containing EtOAc (50 mL), and brine (50 mL). The
mixture was shaken and the layers were separated. The aqueous layer
was extracted with EtOAc (2.times.50 mL). The combined organic
layers were dried, filtered and concentrated. The crude product was
purified by flash chromatography to yield a yellow solid (51.0 mg,
62%) which was shown by NMR and LCMS to be
4-[2-(2-amino-2-methyl-propionyl]-6,6-dimethyl-morpholine-3-carboxylic
acid (6-chloro-9H-.beta.-carbolin-8-yl)-amide.
[0706] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 12.94 (br s,
1); 11.36 (br s, 1); 10.16 (s, 1); 9.06 (s, 1); 8.39 (d, 1); 8.22
(d, 1); 8.19 (d, 1); 7.95 (s, 1); 4.77-4.52 (m, 1); 4.28-4.13 (m,
1); 4.13-4.00 (m, 1); 3.68-3.52 (m, 1); 3.22-3.12 (m, 1); 1.44 (s,
3); 1.41-1.38 (m, 6); 1.28-1.24 (m, 2); 1.22 (s, 3); 1.25 (s, 3);
1.11-1.07 (m, 1).
[0707] NH.sub.4OAc standard conditions.
[0708] DAD R.sub.f=1.31 min.
[0709] M+H=501.
Example 55
6,6
Dimethyl-4-(1,2,3,4-tetrahydroisoquinolin-3-ylmethyl)-morpholine-3-car-
boxylic acid (6-chloro-9H-beta-carbolin-8-yl)amide
[0710] The desired compound was made following the procedure
outlined in Method C using 6,6-dimethyl morpholine-3-carboxylic
acid and (s)-tetrahydroisoquinoline aldehyde.
[0711] .sup.1H-NMR (300 MHz, D.sub.2O): .delta. 9.1 (1H, s), 8.68
(1H, d), 8.52 (1H, d), 8.41 (1H, d), 7.68 (1H, d), 7.27 (1H, d),
7.06 (1H, m), 6.97 (1H, d), 6.84 (1H, m), 4.31 (2H, m), 4.09 (2H,
m), 3.68 (1H, m), 3.56 (1H, t), 3.2 (2H, m), 3.06 (2H, m), 2.7 (2H,
m) 1.48 (3H, s), 1.32 (3H, s)
[0712] Retention time (LC, method: ammonium acetate standard): 2.43
min.
[0713] MS (M+H.sup.+): 505
INTERMEDIATE 59: 6,6-Dimethyl-morpholine-3-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide, HCl salt
[0714] To a clear brown solution of
5-(6-chloro-9H-(3-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholine-4-carbo-
xylic acid tert-butyl ester (10.4 g, 22.7 mmol) in methanol (41 mL)
was added HCl in dioxane (4M, 91 mL). The reaction was stirred for
30 minutes at room temperature, during which time a pale brown
precipitate began to form. The mixture was poured into a 250-mL
volume of vigorously stirring Et.sub.2O. The resulting slurry was
stirred at room temperature for 15 minutes, then filtered to yield
a pale orange solid. The solid was placed on the high-vacuum pump
overnight, after which 9.71 g of
6,6-dimethyl-morpholine-3-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide was obtained (99%
yield).
[0715] .sup.1H-NMR (DMSO-d.sub.6, 300 MHz) .delta. 13.47 (s, 1);
11.77 (s, 1); 9.42 (s, 1); 8.86 (d, 1); 8.66 (d, 1); 8.58 (d, 1);
8.25 (d, 1); 4.41-4.37 (m, 2); 4.05 (dd, 1); 3.32-3.28 (m, 1);
3.04-3.00 (m, 1); 1.34 (s, 3); 1.30 (s, 3).
[0716] NH.sub.4OAc standard conditions.
[0717] DAD R.sub.f=1.48 min.
[0718] M+H=359.
Example 56
4-(2-Amino-butyl)-6,6-dimethyl-morpholine-3-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide (also INTERMEDIATE 60)
[0719] Method C was followed, using
5-(6-chloro-9H-.beta.-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholine-4-c-
arboxylic acid tert-butyl ester and the appropriate aldehyde,
(1-formyl-propyl)-carbamic acid tert-butyl ester.
[0720] .sup.1H-NMR (DMSO-d.sub.6, 300 MHz) .delta. 9.06 (s, 1);
8.37 (d, 1); 8.19 (d, 1); 8.15 (d, 1); 7.85 (d, 1); 6.75 (br s, 2);
3.96-3.85 (m, 2); 3.17-3.13 (m, 1); 2.89 (d, 1); 2.78-2.74 (m, 1);
2.67-2.59 (m, 1); 2.26-2.20 (m, 1); 2.14 (d, 1); 1.58-1.50 (m, 1);
1.32 (s, 3); 1.32-1.23 (m, 1); 1.18 (s, 3); 0.87 (t, 3).
[0721] NH.sub.4OAc standard conditions.
[0722] DAD R.sub.f=1.27 min.
[0723] M+H=430.
Example 57
6,6-Dimethyl-4-(2-[(2-methyl-pyridine-3-carbonyl)-amino]-butyl)-morpholine-
-3-carboxylic acid (6-chloro-9H-.beta.-carbolin-8-yl)-amide, HCl
salt
[0724] To a solution of
4-(2-amino-butyl)-6,6-dimethyl-morpholine-3-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide (100 mg, 0.233 mmol) in
pyridine (4 mL) was added 2-methyl-nicotinic acid (38.4 mg, 0.280
mmol) and EDCI (71.5 mg, 0.373 mmol). The solution was stirred
overnight at room temperature, then diluted with water (5 mL). The
mixture was poured into a separatory funnel and diluted further
with water (20 mL). The mixture was extracted with EtOAc
(2.times.20 mL), then the combined organic layers were washed with
brine. The organic layer was dried over magnesium sulfate,
filtered, and concentrated to yield a yellow-brown oil which was
purified via column chromatography. The resulting yellow solid was
dissolved in methanol (2 mL) and HCl in Et.sub.2O (2M, 2 mL) was
added. The mixture was stirred for 5 minutes, then concentrated to
yield 103 mg of
6,6-dimethyl-4-(2-[(2-methyl-pyridine-3-carbonyl)-amino]-butyl)-morpho-
line-3-carboxylic acid (6-chloro-9H-.beta.-carbolin-8-yl)-amide
(71% yield).
[0725] .sup.1H-NMR (DMSO-d.sub.6, 300 MHz) .delta. 13.31 (br s, 1);
11.41 (br s, 1); 11.14 (br s, 1); 9.44 (s, 1); 8.85 (d, 1);
8.73-8.64 (m, 3); 8.52 (s, 1); 8.32 (s, 1); 7.72 (s, 1); 4.64-3.55
(m, 6); 3.24-3.06 (m, 1); 2.95-2.81 (m, 1); 2.71 (s, 3); 1.89-1.74
(m, 1); 1.55-1.41 (m, 1); 1.32 (s, 3); 1.23 (s, 3); 0.93 (t,
3).
[0726] NH.sub.4OAc standard conditions.
[0727] DAD R.sub.f=1.66 min.
[0728] M+H=549.
INTERMEDIATE 61:
4-(2-Amino-3-methyl-butyl)-6,6-dimethyl-morpholine-3-carboxylic
acid (6-chloro-9H-.beta.-carbolin-8-yl)-amide
[0729] Method C was followed, using
5-(6-chloro-9H-.beta.-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholine-4-c-
arboxylic acid tert-butyl ester and the appropriate aldehyde,
(1-formyl-2-methyl-propyl)-carbamic acid tert-butyl ester.
[0730] .sup.1H-NMR (DMSO-d.sub.6, 300 MHz) .delta. 9.05 (s, 1);
8.37 (d, 1); 8.21 (d, 1); 8.16 (dd, 1); 7.90 (d, 1); 6.71 (br s,
2); 3.93-3.86 (m, 2); 3.18-3.14 (m, 1); 2.91 (d, 1); 2.70-2.65 (m,
2); 2.21 (dd, 1); 2.15 (d, 1); 1.74-1.66 (m, 1); 1.31 (s, 3); 1.19
(s, 3); 0.85 (d, 3); 0.80 (d, 3).
[0731] NH.sub.4OAc standard conditions.
[0732] DAD R.sub.f=1.27 min.
[0733] M+H=444.
Example 58
6,6-Dimethyl-4-{3-methyl-2-[(2-methyl-pyridine-3-carbonyl)-amino]-butyl}-m-
orpholine-3-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide
[0734] To a solution of
4-(2-amino-3-methyl-butyl)-6,6-dimethyl-morpholine-3-carboxylic
acid (6-chloro-9H-.beta.-carbolin-8-yl)-amide (1.47 g, 3.31 mmol)
in pyridine (35 mL) was added 2-methyl-nicotinic acid (544 mg, 3.97
mmol) and EDCI (1.02 g, 5.30 mmol). The solution was stirred 6.5
hours at room temperature, then diluted with water (100 mL). The
mixture was poured into a separatory funnel and diluted further
with water (50 mL) and EtOAc (150 mL). The layers were shaken and
separated. The aqueous layer was extracted with EtOAc (3.times.50
mL), then the combined organic layers were washed with brine. The
organic layer was dried over magnesium sulfate, filtered, and
concentrated to yield an orange semi-solid residue which was
purified via column chromatography. The resulting yellow solid was
placed on the high-vacuum pump overnight, after which 1.43 g of
6,6-dimethyl-4-{3-methyl-2-[(2-methyl-pyridine-3-carbonyl)-amino]-butyl}--
morpholine-3-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide was obtained (77%
yield).
[0735] .sup.1H-NMR (DMSO-d.sub.6, 300 MHz) .delta. 11.32 (s, 1);
10.08 (s, 1); 9.02 (s, 1); 8.46 (dd, 1); 8.38 (d, 1); 8.21-8.14 (m,
3); 7.97 (d, 1); 7.64 (dd, 1); 7.23 (dd, 1); 4.23-4.14 (m, 1);
3.99-3.87 (m, 2); 3.22-3.19 (m, 1): 3.02 (d, 1); 2.85 (dd, 1); 2.52
(s, 3); 2.30 (dd, 1); 2.11 (d, 1); 2.05-1.95 (m, 1); 1.32 (s, 3);
1.21 (s, 3); 0.93 (d, 3); 0.86 (d, 3).
[0736] NH.sub.4OAc standard conditions.
[0737] DAD R.sub.f=1.67 min.
[0738] M+H=563.
Example 59
6,6-Dimethyl-4-{3-methyl-2-(S)-[(tetrahydro-furan-3-carbonyl)-amino]-butyl-
}-morpholine-3-(S)-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide
[0739] The desired compound was prepared following Method E from
Intermediate 61 and the appropriate acid.
[0740] .sup.1H-NMR (300 MHz, methyl-d.sub.3 alcohol-d): .delta.
0.87 (m, 6H), 1.25 (d, 3H), 1.37 (d, 3H), 1.81 (m, 1H), 2.10-2.47
(m, 4H), 2.93 (m, 2H), 3.10 (m, 1H), 3.26 (m, 1H), 3.80 (m, 1H),
3.86-4.07 (m, 6H), 7.86 (d, 1H), 8.10 (m, 2H), 8.32 (d, 1H), 8.89
(s, 1H).
[0741] Retention Time (LC, method: ammonium acetate standard): 1.73
min. MS (M+H.sup.+): 542.
INTERMEDIATE 62:
((S)-2-[(S)-5-(6-Chloro-4-methyl-9H-.beta.-carbolin-8-ylcarbamoyl)-2,2-di-
methyl-morpholin-4-yl]-1-methyl-ethyl)-carbamic acid tert-butyl
ester
[0742] A solution of
(S)-4-((S)-2-tert-butoxycarbonylamino-propyl)-6,6-dimethyl-morpholine-3-c-
arboxylic acid (3.316 g, 10.5 mmol) (prepared by reductively
alkylating (S)-6,6-dimethyl-morpholine-3-carboxylic acid with
N-(tert-butoxycarbonyl)-L-alanal) in anhydrous pyridine (75 mL) was
stirred at room temperature.
6-chloro-4-methyl-9H-.beta.-carbolin-8-ylamine (1.869 g, 8.09 mmol)
was added, followed by EDCI (2.894 g, 15.1 mmol). The reaction was
stirred at room temperature for 14-18 hours under argon. The
reaction was partially concentrated, diluted with H.sub.2O (20 mL)
and transferred to a reparatory funnel. The mixture was diluted
with brine (50 mL) and extracted with EtOAc (3.times.100 mL). The
combined organic layers were washed with brine, dried, filtered and
concentrated to afford a dark residue. Column chromatography (0-8%
MeOH/CH.sub.2Cl.sub.2) yielded
f(S)-2-[(S)-5-(6-Chloro-4-methyl-9H-.beta.-carbolin-8-ylcarbamoyl)-2,2-di-
methyl-morpholin-4-yl]-1-methyl-ethyl)-carbamic acid tert-butyl
ester as a light tan solid (2.688 g).
[0743] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 11.25 (s, 1)
9.94 (s, 1) 8.88 (s, 1) 8.18 (s, 1) 8.02 (s, 1) 7.92 (s, 1) 6.73
(d, 1) 3.95-3.85 (m, 2) 3.66 (br s, 1) 3.16-3.08 (m, 1) 2.88 (d, 1)
2.76 (s, 3) 2.51-2.40 (m, 1) 2.23 (dd, 1) 1.99 (d, 1) 1.34 (br s,
12) 1.17 (s, 3) 1.08 (d, 3).
[0744] NH.sub.4OAc standard conditions.
[0745] ELSD R.sub.f=2.07 min.
[0746] M+H=530.
Example 60
(S)-6,6-Dimethyl-4-{(S)-2-[(2-methyl-pyridine-3-carbonyl)-amino]-propyl}-m-
orpholine-3-carboxylic acid
(6-chloro-4-methyl-9H-.beta.-carbolin-8-yl)-amide
[0747] A solution of
{(S)-2-[(S)-5-(6-Chloro-4-methyl-9H-.beta.-carbolin-8-ylcarbamoyl)-2,2-di-
methyl-morpholin-4-yl]-1-methyl-ethyl}-carbamic acid tert-butyl
ester (2.688 g, 5.08 mmol) in EtOH (60 mL) was stirred at room
temperature. Concentrated HCl (10 mL) was added and the reaction
stirred for 14 hours at room temperature under argon. The reaction
was concentrated to afford a yellow solid (2.84 g). The solid was
dissolved in anhydrous pyridine (40 mL) and stirred at room
temperature under argon. Triethylamine (2.20 mL, 15.7 mmol) and
EDCI (1.39 g, 7.28 mmol) were added. The reaction mixture was
stirred at room temperature for 10 minutes and 2-methyl-nicotinic
acid (0.868 g, 6.33 mmol) was added. The reaction was stirred at
room temperature for 14-18 hours and diluted with H.sub.2O (40 mL).
The mixture was poured into a separatory funnel containing H.sub.2O
(40 mL), brine (40 mL), and EtOAc (40 mL). The mixture was shaken
and the layers were separated. The aqueous layer was extracted with
EtOAc (2.times.40 mL) and the combined organic layers washed with
brine. The organic layer was dried, filtered, and concentrated. The
resulting residue was dissolved in EtOAc (10-20 mL) and added
dropwise to a stirring solution of 4:1 hexanes/Et.sub.2O (300 mL).
The precipitate which formed was collected via filtration and air
dried to yield
(S)-6,6-Dimethyl-4-{(S)-2-[(2-methyl-pyridine-3-carbonyl)-amino]-propyl}--
morpholine-3-carboxylic acid
(6-chloro-4-methyl-9H-.beta.-carbolin-8-yl)-amide as a tan solid
(3.151 g).
[0748] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 11.28 (s, 1)
9.98 (s, 1) 8.81 (s, 1) 8.45-8.39 (m, 1) 8.26 (d, 1) 8.13 (s, 1)
7.97 (s, 1) 7.89 (s, 1) 7.64-7.56 (m, 1) 7.24-7.12 (m, 1) 4.25-4.10
(m, 1) 3.91-3.82 (m, 2) 3.20-3.10 (m, 1) 2.94 (d, 1) 2.71 (s, 3)
2.61-2.49 (m, 1) 2.37 (s, 3) 2.06-2.02 (m, 2) 1.30 (s, 3) 1.16 (d,
3) 1.15 (s, 3).
[0749] NH.sub.4OAc standard conditions.
[0750] ELSD R.sub.f=1.57 min. M+H=549.
INTERMEDIATE 63: 3,5-Difluoro-4-tributylstannanyl-pyridine
[0751] n-Butyl lithium (1.0 eq, 76 mmol, 47.6 mL, 1.6 M in hexanes)
was added via dropping funnel to a solution of diisopropylamine
(1.05 eq, 80 mmol, 11.2 mL) in THF (300 mL) at -78.degree. C. under
nitrogen (N.sub.2). The solution was stirred for 30 min at
-78.degree. C., then a solution of 3,5-difluoropyridine (1.05 eq,
80 mmol, 9.2 g) in THF (20 mL) was added dropwise via syringe. A
beige precipitate was observed to form. The reaction stirred at
-78.degree. C. for 90 min then tributyltin chloride (1.0 eq, 76
mmol, 20.7 mL) was added dropwise via syringe and the resulting
solution allowed to warm to RT over 2 h. Water (5 mL) was added,
then roughly 250 mL of THF was removed on a rotary evaporator. The
resulting material was diluted with diethyl ether (350 mL) and
washed successively with water (2.times.200 mL), saturated sodium
chloride solution (1.times.150 mL), dried over magnesium sulfate,
filtered and concentrated in vacuo to afford the
3,5-Difluoro-4-tributylstannanyl-pyridine as a colourless oil (27.5
g, 88%). This material was used crude without further
purification.
[0752] Retention Time (LC, method: ammonium acetate standard): 3.35
min. MS (M+H.sup.+): 406.
INTERMEDIATE 64:
4-Chloro-2-(3,5-difluoro-pyridin-4-yl)-phenylamine
[0753] Stille coupling: A dimethyl formamide (256 mL) solution of
crude Intermediate 63 (1.1 eq, 70 mmol, 27.5 g) and
2-iodo-4-chloro-phenylamine (1.0 eq, 64 mmol, 16.2 g) was degassed
with N.sub.2 for 15 min. Dichlorobis(triphenylphosphine)palladium
(II) (0.05 eq, 3.2 mmol, 2.2 g) and copper (I) iodide (0.1 eq, 6.4
mmol, 1.2 g) were added and the suspension heated at reflux for 15
h under N.sub.2. The mixture was cooled to RT, filtered through a
short plug of Celite.RTM. and the dimethyl formamide removed on a
rotary evaporator. The crude material was dissolved in acetonitrile
(300 mL), washed with hexanes (2.times.200 mL) then concentrated in
vacuo. The material was then dissolved in ethyl acetate (400 mL)
and washed successively with water (2.times.200 mL), saturated
sodium bicarbonate solution (1.times.200 mL), saturated sodium
chloride solution (200 mL), dried over magnesium sulfate, filtered
and concentrated in vacuo. The resulting solid was triturated with
diethyl ether (50 mL) to remove the dark colour, then dissolved in
the minimum volume of methanol, filtered to remove an insoluble
impurity and concentrated in vacuo to afford the
4-Chloro-2-(3,5-difluoro-pyridin-4-yl)-phenylamine as a tan solid
(12.3 g, .about.80%,) which was used in the subsequent step without
further purification.
[0754] .sup.1H-NMR (300 MHz, dmso-d.sub.6): .delta. 5.28 (s, 2H),
6.77 (d, 1H), 7.08 (d, 1H), 7.19 (dd, 1H) and 8.58 (s, 2H).
[0755] Retention Time (LC, method: ammonium acetate standard): 1.70
min. MS (M+H.sup.+): not observed.
INTERMEDIATE 65: 6-Chloro-4-fluoro-9H-.beta.-carboline
[0756] Sodium bis(trimethylsilyl)amide (3.0 eq, 130 mmol, 130 mL,
1.0M in THF) was added via dropping funnel to a solution of crude
Intermediate 64 (1.0 eq, 43 mmol, 10.4 g) in THF at RT under
N.sub.2. After stirring for 15 h the excess base was quenched by
the cautious addition of saturated ammonium chloride solution (100
mL) and the majority of the THF removed on a rotary evaporator. The
resulting slurry was extracted with ethyl acetate (400 mL then
2.times.200 mL), then the combined organics were washed
successively with saturated sodium bicarbonate solution (300 mL),
saturated sodium chloride solution (300 mL), dried over sodium
sulfate and filtered. Silica gel was added and the slurry
concentrated on a rotary evaporator. The material was purified
using a Biotage Flash 75 purification system (short column) eluting
with 96:4 dichloromethane/methanol to afford the
6-Chloro-4-fluoro-9H-.beta.-carboline as an off-white solid (7.8 g,
82%).
[0757] .sup.1H-NMR (300 MHz, dmso-d.sub.6): .delta. 7.71-7.61 (m,
3H), 8.11 (d, 1H) and 12.16 (s, 1H).
[0758] Retention Time (LC, method: ammonium acetate standard): 1.70
min. MS (M+H.sup.+): 221.
INTERMEDIATE 66: 6-chloro-4-fluoro-9H-.beta.-carbolin-8-ylamine
[0759] Sodium nitrate (1.5 eq, 53 mmol, 4.5 g) was added
portionwise to a solution of Intermediate 65 (1.0 eq, 35 mmol, 7.8
g) in trifluoroacetic acid (200 mL) and the resulting mixture
heated at 70.degree. C. for 3 h. After cooling to RT the
trifluoroacetic acid was removed on a rotary evaporator to afford a
crude solid which was suspended in a small volume of methanol and
added dropwise to a vigorously stirred mixture of saturated sodium
bicarbonate solution (500 mL). The resulting slurry was stirred for
15 min then the precipitated solids were collected by suction
filtration, washed with water (300 mL) and then dried in vacuo to
afford 6-chloro-4-fluoro-8-nitro-9H-.beta.-carboline (about 9.5 g)
which was used in the subsequent step without further
purification.
[0760] Retention Time (LC, method: ammonium acetate standard): 1.79
min. MS (M+H.sup.+): 266
[0761] Sulfated platinum (.about.0.1 eq, 1 g) was added to a
suspension of 6-chloro-4-fluoro-8-nitro-9H-.beta.-carboline (1.0
eq, 35 mmol, 9.3 g) and ammonium formate (3.0 eq, 105 mmol, 6.6 g)
in ethanol (175 mL) and the resulting mixture heated at 75.degree.
C. for 4 h. After cooling to RT the mixture was filtered through a
short plug of Celite.RTM. washing with copious amounts of methanol,
and then the filtrate concentrated in vacuo to afford a beige
solid. The solid was suspended in the minimum volume of methanol
and added dropwise to a vigorously stirred mixture of saturated
sodium bicarbonate solution and saturated sodium chloride solution.
After stirring for 15 min the precipitated solids were collected by
suction filtration, washed with water (200 mL) and dried in vacuo
to afford 6-chloro-4-fluoro-9H-.beta.-carbolin-8-ylamine (5.8 g,
70% 2 steps) as a beige powder.
[0762] .sup.1H-NMR (300 MHz, dmso-d.sub.6): .delta. 5.76 (s, 2H),
6.81 (d, 1H), 7.29 (d, 1H), 8.24 (d, 1H), 8.82 (d, 1H), and 11.71
(s, 1H).
[0763] Retention Time (LC, method: ammonium acetate standard): 1.59
min. MS (M+H.sup.+): 236.
Example 61
4-(2-Acetylamino-propyl)-6,6-dimethyl-morpholine-3-carboxylic acid
(6-chloro-4-fluoro-9H-.beta.-carbolin-8-yl)-amide
[0764] The desired compound was prepared from Intermediate 66 and
acetic anhydride.
[0765] .sup.1H-NMR (300 MHz, dmso-d.sub.6): .delta. 11.70 (s, 1H),
10.17 (s, 1H), 8.92 (s, 1H), 8.34 (d, 1H), 7.95 (s, 2H), 7.81 (d,
1H), 4.05-3.95 (m, 1H), 3.92-3.83 (m, 2H), 3.18-3.12 (m, 1H), 2.87
(d, 1H), 2.55-2.47 (m, 1H), 2.40-2.31 (m, 1H), 2.03 (d, 1H), 1.78
(s, 3H), 1.32 (s, 3H), 1.63 (s, 3H) and 1.08 (d, 3H).
[0766] Retention Time (LC, method: ammonium acetate standard): 1.73
min. MS (M+H.sup.+): 474.
Example 62
6,6-Dimethyl-4-{2-[(2-methyl-pyridine-3-carbonyl)-amino]-propyl}-morpholin-
e-3-carboxylic acid
(6-chloro-4-fluoro-9H-.beta.-carbolin-8-yl)-amide
[0767] The desired compound was prepared according to Method E from
Intermediate 66 and methylnicotinic acid.
[0768] .sup.1H-NMR (300 MHz, dmso-d.sub.6): .delta. 11.63 (s, 1H),
.delta. 10.10 (s, 1H), 5, 8.90 (s, 1H), 6, 8.44 (d, 1H), 5, 8.34
(s, 1H), .delta. 8.29 (d, 1H), 7.94 (s, 2H), 7.65 (d, 1H), 7.21
(dd, 1H), 4.25-4.15 (m, 1H), 3.97-3.88 (m, 2H), 3.24-3.15 (m, 1H),
2.99 (d, 1H), 2.59 (t, 1H), 2.49 (s, 3H), 2.40 (dd, 1H), 2.09 (d,
1H), 1.35 (s, 3H), 1.21 (d, 3H) and 1.20 (s, 3H).
[0769] Retention Time (LC, method: ammonium acetate standard): 1.67
min. MS (M+H.sup.+): 553.
Example 63
[(S)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4--
yl]-acetic acid
[0770] To a suspension of (S)-6,6-Dimethyl-morpholine-3-carboxylic
acid (6-chloro-9H-beta-carbolin-8-yl)-amide (2.6 g, 6.0 mmoles) in
60 ml of methanol was added 1.7 ml of triethylamine (2.0 eq.),
sodium cyanoborohydride (575 mg, 9.1 mmoles) and glyoxylic acid
(780 mg, 8.5 mmoles). The reaction mixture was stirred at ambient
temperature for 1.5 hrs. Water was added (5 ml) and the mixture was
concentrated to a thick yellow slurry. More water was then added
(30 ml) and the resulting slurry was stirred at ambient temperature
for 10 min. and was filtered. The collected yellow solid was washed
with water and dried under high vacuum to give 1.80 g (71%) of the
desired product.
[0771] .sup.1H-NMR (300 MHz, DMSO-d.sub.5): .delta. 1.17 (s, 3H),
1.30 (s, 3H), 2.81 (d, 1H), 3.34 (d, 1H), 3.46 (d, 1H), 3.56 (dd,
1H), 3.84-3.90 (m, 2H), 7.92 (s, 1H), 8.15 (d, 1H), 8.21 (s, 1H),
8.36 (d, 1H), 9.01 (s, 1H), 10.28 (s, 1H), 11.40 (s, 1H).
[0772] Retention Time (LC, method: ammonium acetate standard): 1.26
min.
[0773] MS (M+H.sup.+): 417.1.
METHOD F: Coupling procedure for reverse amides from
[(8)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-
-yl]-acetic acid
[0774]
[(S)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morph-
olin-4-yl]-acetic acid (1.0 mmol), EDCI (1.6 mmol) and the amine
(1.2 mmol) to be coupled were taken in a round-bottom flask and
suspended in pyridine (5 ml). The resulting mixture was stirred
overnight. The pyridine was then removed under reduced pressure and
the residue was partitioned in EtOAc and 5% aqueous
Na.sub.2CO.sub.3 solution. The separated aqueous phase was further
extracted with EtOAc. The combined extracts were successively
washed with water and brine, dried over Na.sub.2SO.sub.4 and
concentrated completely. The residue was purified on silica to give
the desired product.
Example 64
(S)-6,6-Dimethyl-4-(2-oxo-2-pyrrolidin-1-yl-ethyl)-morpholine-3-carboxylic
acid (6-chloro-9H-beta-carbolin-8-yl)-amide
[0775] The desired compound was prepared according to Method F from
[(S)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-
-yl]-acetic acid and pyrrolidine in 82% yield.
[0776] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 1.21 (s, 3H),
1.29 (s, 3H), 1.75-1.92 (m, 4H), 2.46 (d, 1H), 2.77 (d, 1H),
3.35-3.68 (m, 7H), 3.94 (m, 2H), 8.08 (s, 1H), 8.19 (d, 1H), 8.23
(s, 1H), 8.41 (d, 1H), 9.05 (s, 1H), 10.71 (s, 1H), 11.51 (s,
1H).
[0777] Retention Time (LC, method: ammonium acetate standard): 1.75
min.
[0778] MS (M+H.sup.+): 470.3.
Example 65
(S)-6,6-Dimethyl-4-(2-oxo-2-piperidin-1-yl-ethyl)morpholine-3-carboxylic
acid (6-chloro-9H-beta-carbolin-8-yl)-amide
[0779] The desired compound was prepared according to Method F from
[(S)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-
-yl]-acetic acid and piperidine in 90% yield.
[0780] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 1.17 (s, 3H),
1.29 (s, 3H), 1.35-1.60 (m, 6H), 2.25 (d, 1H), 2.71 (d, 1H), 3.15
(d, 1H), 3.26 (dd, 1H), 3.37 (dd, 1H), 3.45-3.65 (m, 2H), 3.65 (d,
1H), 3.70 (m, 1H), 3.89 (m, 2H) 7.95 (d, 1H), 8.15 (d, 1H), 8.20
(d, 1H), 8.37 (d, 1H), 9.01 (s, 1H), 10.43 (s, 1H), 11.32 (s,
1H)
[0781] Retention Time (LC, method: ammonium acetate standard): 1.86
min.
[0782] MS (M+H.sup.+): 484.3.
Example 66
(S)-6,6-Dimethyl-4-(2-morpholin-4-yl-2-oxo-ethyl)-morpholine-3-carboxylic
acid (6-chloro-9H-beta-carbolin-8-yl)-amide
[0783] The desired compound was prepared according to Method F from
[(S)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-
-yl]-acetic acid and morpholine in 86% yield.
[0784] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta. 1.17 (s, 3H),
1.30 (s, 3H), 2.19 (d, 1H), 2.71 (d, 1H), 3.03 (d, 1H), 3.16 (d,
1H), 3.22 (dd, 1H), 3.45-3.72 (m, 7H), 3.80-3.98 (m, 3H), 7.94 (d,
1H), 8.15 (d, 1H), 8.21 (d, 1H), 8.37 (d, 1H), 9.03 (s, 1H), 10.35
(s, 1H), 11.28 (s, 1H)
[0785] Retention Time (LC, method: ammonium acetate standard): 1.56
min.
[0786] MS (M+H.sup.+): 486.3.
Example 67
(8)-4-{[(2-Hydroxy-ethyl)-methyl-carbamoyl]-methyl}-6,6-dimethyl-morpholin-
e-3-carboxylic acid (6-chloro-9H-beta-carbolin-8-yl)-amide
[0787] The desired compound was prepared according to Method F from
[(S)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-
-yl]-acetic acid and 2-methylamino-ethanol in 55% yield.
[0788] .sup.1HNMR (300 MHz, DMSO-d.sub.6): .delta. 1.21 (s, 3H),
1.30 (s, 3H), 2.39 (m, 1H), 2.75 (bd, 1H), 2.96 (s, 1.5H), 3.17 (s,
1.5H), 3.50-3.65 (m, 2.5H), 3.70-3.85 (m, 1.5H), 3.93 (m, 2H), 4.69
(m, 0.5H), 4.94 (m, 0.5H), 8.05 (d, 1H), 8.18-8.25 (m, 2H), 8.42
(d, 1H), 9.06 (s, 1H), 10.68 (s, 1H), 11.38 (s, 1H).
[0789] Retention Time (LC, method: ammonium acetate standard): 1.47
min.
[0790] MS (M+H.sup.+): 474.
Example 68
(8)-6,6-Dimethyl-4-(pyridin-3-ylcarbamoylmethyl)-morpholine-3-carboxylic
acid (6-chloro-9H-beta-carbolin-8-yl)-amide
[0791] The desired compound was prepared according to Method F from
[(S)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-
-yl]-acetic acid and 3-aminopyridine. The product was isolated as
hydrochloride salt in 55% yield.
[0792] .sup.1HNMR (300 MHz, D.sub.2O): .delta. 1.34 (s, 3H), 1.54
(s, 3H), 2.60 (d, 1H), 3.4 (d, 1H), 3.50 (d, 1H), 3.70 (t, 1H),
3.81 (d, 1H) 4.23 (d, 2H), 7.69 (d, 1H), 7.98 (d, 1H), 8.01 (d,
1H), 8.17 (d, 1H), 8.42 (d, 1H), 8.48-8.55 (m, 3H), 9.09 (s, 1H),
9.30 (d, 1H).
[0793] Retention Time (LC, method: ammonium acetate standard): 1.56
min.
[0794] MS (M+H.sup.+): 493.2.
Example 69
(S)-6,6-Dimethyl-4-{[(pyridin-4-ylmethyl)-carbamoyl]-methyl}-morpholine-3--
carboxylic acid (6-chloro-9H-beta-carbolin-8-yl)-amide
[0795] The desired compound was prepared according to Method F from
[(S)-5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-
-yl]-acetic acid and 4-(aminomethyl)pyridine. The product was
isolated as hydrochloride salt in 53% yield.
[0796] .sup.1HNMR (300 MHz, D.sub.2O): .delta. 1.34 (s, 3H), 1.49
(s, 3H), 2.64 (d, 1H), 3.03 (d, 1H), 3.48 (d, 1H), 3.72 (t, 1H),
3.74 (d, 1H), 4.15-4.25 (m, 2H), 7.66 (d, 1H), 7.88 (s, 1H), 7.91
(s, 1H), 8.21 (d, 1H), 8.44 (d, 1H), 8.53 (d, 1H), 8.56 (s, 1H),
8.58 (s, 1H), 9.08 (s, 1H).
[0797] Retention Time (LC, method: ammonium acetate standard): 1.47
min.
[0798] MS (M+H.sup.+): 507.3.
Example 70
4-[2-(4-Hydroxymethyl-piperidin-1-yl)-2-oxo-ethyl]-6,6-dimethyl-morpholine-
-3-carboxylic acid (6-chloro-9H-beta-carbolin-8-yl)-amide
[0799]
[5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-
-4-yl]-acetic acid (200 mg, 0.48 mmol) and piperidin-4-yl-methanol
(111 mg, 0.96 mmol) were dissolved in pyridine (4 mL). The
resulting yellow solution was stirred at room temperature for 10
min and then EDC (184 mg, 0.96 mmol) was added in a single portion.
The reaction mixture was allowed to stir over night (16 h). Water
(4 mL) was added and the mixture was concentrated under reduced
pressure, The resulting residue was partitioned between ethyl
acetate (50 mL) and 1 M aqueous potassium carbonate. The aqueous
layer was back-extracted with ethyl acetate (3.times.50 mL) and the
combined extracts were washed with water and brine, dried over
sodium sulfate, filtered, and concentrated. The crude residue was
purified by silica gel chromatography (methylene chloride and
methanol gradient) to afford pure
4-[2-(4-hydroxymethyl-piperidin-1-yl)-2-oxo-ethyl]-6,6-dimethyl-morpholin-
e-3-carboxylic acid (6-chloro-9H-beta-carbolin-8-yl)-amide as a
yellow foam (152 mg, 62%). The bis-HCl salt was prepared by adding
2 equivalents of conc. HCl to an ethanolic solution of the
freebase. Concentration, followed by ether trituration afforded the
salt as a free-flowing, yellow powder.
[0800] .sup.1H-NMR (freebase, 300 MHz, CDCl.sub.3) .delta.: 11.03
(d, 1H), 10.51 (d, 1H), 8.97-8.80 (m, 1H), 8.47-8.27 (m, 2H),
7.94-7.76 (m, 2H), 4.84 (d, 1H), 4.16-3.77 (m, 3H), 3.68-3.26 (m,
5H), 3.08 (ddd, 1H), 2.89-2.64 (m, 2H), 2.50-2.37 (m, 2H),
1.98-1.70 (m, 3H), 1.38 (s, 3H), 1.25 (s, 3H), 1.19-0.97 (m,
1).
[0801] MS (NH.sub.4OAc standard conditions, ES+) e/z=514
(M+H).sup.+
[0802] DAD R.sub.f=1.51 min
Example 71
4-[2-(4-Hydroxy-piperidin-1-yl)-2-oxo-ethyl]-6,6-dimethyl-morpholine-3-car-
boxylic acid (6-chloro-9H-beta-carbolin-8-yl)-amide
[0803] The desired compound was synthesized using
[5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-
-acetic acid and 4-hydroxypiperazine following Method F in 31%
yield.
[0804] .sup.1H-NMR (300 MHz, D.sub.2O): .delta. 8.85 (1H, s), 8.19
(2H, s), 7.82 (1H, s), 7.44 (1H, s), 4.15 (2H, m), 3.9-3.75 (4H,
m), 3.6 (2H, m), 3.56 (2H, m), 3.12 (1H, m), 2.9 (3H, m), 2.8 (1H,
m), 1.7 (2H, m) 1.34 (3H, s), 1.19 (3H, s)
[0805] Retention time (LC, method: ammonium acetate standard): 1.5
min
[0806] MS (M+H.sup.+): 501
Example 72
4-Diethylcarbamoylmethyl-6,6-dimethyl-morpholine-3-carboxylic acid
(6-chloro-9H-beta-carbolin-8-yl)-amide
[0807] The desired compound was made using of
[5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-
-acetic acid and diethylamine following Method F in 60% yield.
[0808] .sup.1H-NMR (300 MHz, D.sub.2O): .delta. 8.87 (1H, s), 8.26
(2H, m), 7.8 (1H, s), 7.52 (1H, s), 4.1 (3H, m), 3.8 (2H, m), 3.22
(2H, m), 3.0 (1H, d), 2.7 (1H, d), 1.3 (3H, s), 1.19 (3H, s), 0.09
(6H, m).
[0809] Retention time (LC, method: ammonium acetate standard): 1.96
min
[0810] MS (M+H.sup.+): 472
Example 73
6,6-dimethyl-4-[-2-(4-methyl-piperazin-1-yl)-2-oxo-ethyl]-morpholine-3-car-
boxylic acid (6-chloro-9H-beta-carbolin-8-yl)-amide
[0811] The desired compound was prepared using
[5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-
-acetic acid and 1-methyl piperazine following Method F in 12%
yield.
[0812] .sup.1H-NMR (DMSO) .delta. 11.28 (1H, s), 10.35 (1H, s),
9.01 (1H, s), 8.37 (1H, s), 8.21-8.15 (2H, m), 7.9 (1H, s), 3.89
(2H, m), 3.7 (5H, m), 3.1 (2H, m), 2.85 (2H, m), 2.49 (3H, s), 2.4
(3H, m), 1.2 (3H, s), 1.16 (3H, s)
[0813] Retention time (LC, method: ammonium acetate standard): 1.34
min.
[0814] MS (M+H.sup.+): 500
Example 74
4-[2-(2,6-Dimethyl-morpholin-4-yl)-2-oxo-ethyl]-6,6-dimethyl-morpholine-3--
carboxylic acid (6-chloro-9H-b-carbolin-8-yl)-amide
[0815] The desired compound was prepared using
[5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-yl]-
-acetic acid and 2,6-dimethyl morpholine following Method F.
Chromatographic purification gave the desired product in 70-80%
yield.
[0816] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. 9.31 (s, 1H),
8.75 (d, 1H), 8.55 (d, 1H), 8.37 (d, 1H), 8.14 (m, 1H), 4.73 (m,
2H), 4.96 (m, 1H), 4.37 (m, 2H), 3.69 (m, 2H), 3.57 (m, 4H), 2.82
(t, 1H), 2.47 (t, 1H), 1.50 (d, 3H), 1.42 (d, 3H), 1.18 (m, 6H)
[0817] Retention Time (LC, method: formic acid standard): 1.14 min
(Diode Array).
[0818] MS (M+H.sup.+): 514, (M-H.sup.+): 512
Example 75
1-{2-[5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-4-
-yl]-acetyl}-piperidine-4-carboxylic acid methyl ester
[0819]
[5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-
-4-yl]-acetic acid (200 mg, 0.48 mmol) and methyl isonipecotate
(137 mg, 130 uL, 0.96 mmol) were dissolved in pyridine (4 mL) and
stirred at room temperature 10 min. EDC was added and the reaction
mixture was allowed to stir at room temperature over night (16 h).
Additional methyl isonipecotate (137 uL, 0.96 mmol) was added and
the mixture was stirred an additional 24 h. Water (4 mL) was added
and the mixture concentrated. The crude residue was partitioned
between ethyl acetate (75 mL) and 1 M aqueous potassium carbonate
(50 mL). The aqueous phase was back-extracted with additional ethyl
acetate (75+50 mL). The combined extracts were washed with water
and brine, dried over sodium sulfate, filtered, concentrated and
purified by silica gel chromatography (methylene chloride/methanol
gradient) to afford the freebase as a yellow foam (150 mg, 57%).
The bis-HCl salt was prepared by adding 2 equivalents of conc. HCl
to an ethanolic solution of the freebase. Concentration, followed
by ether trituration afforded the salt as a free-flowing, yellow
powder.
[0820] .sup.1H-NMR (freebase, 300 MHz, CDCl.sub.3) .delta. 11.04
(d, 1H), 10.48 (d, 1H), 8.95 (s, 1H), 8.48-8.31 (m, 2H), 7.96-7.79
(m, 2H), 4.48 (dd, 1H), 4.13-3.92 (m, 2H), 3.88-2.97 (m, 9H),
2.84-2.35 (m, 3H), 2.09-1.90 (m, 2H), 1.85-1.52 (m, 2H), 1.38 (s,
3H), 1.23 (s, 3H).
[0821] MS (NH.sub.4OAc standard conditions, ES+) e/z=, 542
(M+H).sup.+
[0822] DAD R.sub.f=1.72 min
Example 76
(S)-4-[2-(3,3-Dimethyl-morpholin-4-yl)-2-oxo-ethyl]-6,6-dimethyl-morpholin-
e-3-carboxylic acid
(6-chloro-9H-beta-carbolin-8-yl)-amide-bis-hydrochloride salt
[0823] 2,2-Dimethylmorpholine (3.0 g, 26.1 mmol) (prepared
according to the procedure of Cottle, D. L., et al. J. Org. Chem.
1946, 11, 286-291) was dissolved in dichloromethane (60 mL).
Triethylamine (3.6 mL, 2.66 g, 26.1 mmol) was added and the
reaction was cooled to -10.degree. C. Bromoacetyl chloride (2.2 mL,
4.08 g, 26.1 mmol) was added dropwise and the solution was warmed
slowly to room temperature. The reaction was concentrated to
dryness in vacuo, redissolved in ethyl acetate and passed through a
plug of silica gel. The eluant was concentrated to a yellow oil
(3.45 g, 56%) that was carried to the next step.
[0824] Retention Time (LC, method: ammonium acetate standard): 1.20
min.
[0825] MS (M+H.sup.+): 237.
[0826] Intermediate 59 (60 mg, 0.14 mmol) was suspended in
dichloromethane (3 mL). A solution of 1M potassium carbonate (0.5
mL) was added. The organic layer was separated, dried over
MgSO.sub.4 and concentrated to dryness. The free base was dissolved
in DMF (1 mL) and stirred at room temperature.
2-Bromo-1-(3,3-dimethyl-morpholin-4-yl)-ethanone (30 mg, 0.13 mmol)
was dissolved in DMF (1 mL) and added dropwise. The reaction was
stirred for 3 hours at room temperature and concentrated to dryness
in vacuo. Flash column chromatography (93:7
dichloromethane:methanol) afforded a yellow oil, which was
dissolved in 4N HCl/dioxane (2 mL). Concentration in vacuo afforded
the title compound as a yellow solid (22 mg, 29%). .sup.1HNMR (300
MHz, MeOH-d.sub.4): .delta. 1.35 (s, 3H).sub., 1.41 (s, 3H), 1.43
(s, 6H), 2.98 (m, 1H), 3.45 (m, 4H), 3.56 (m, 1H), 3.76 (m, 3H),
4.30 (m, 4H), 8.04 (s, 1H), 8.39 (s, 1H), 8.52 (d, 1H), 8.73 (d,
1H), 9.24 (s, 1H)
[0827] Retention Time (LC, method: ammonium acetate standard): 1.16
min.
[0828] MS (M+H.sup.+): 514
Example 77
4-[(Trans-4-hydroxy-cyclohexylcarbamoyl)-methyl]-6,6-dimethyl-morpholine-3-
-carboxylic acid (6-chloro-9H-beta-carbolin-8-yl)-amide
[0829]
[5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-
-4-yl]-acetic acid (200 mg, 0.48 mmol), trans-4-aminocyclohexanol
hydrochloride (145 mg, 0.96 mmol) and diisopropylethylamine (124
mg, 167 uL, 0.96 mmol) were dissolved in pyridine (4 mL) and
stirred at room temperature 10 min. EDC (184 mg) was added and the
reaction was stirred at room temperature over night (16 h). Water
(2 mL) was added and the resulting mixture was concentrated under
reduced pressure, The resulting residue was diluted with 50 mL 1 M
aqueous potassium carbonate and extracted with ethyl acetate
(75+2.times.50 mL). The combined extracts were washed with water
and brine, dried over sodium sulfate, filtered and concentrated.
The crude residue was purified by silica gel chromatography
(methanol/methylene chloride gradient) to afford pure
4-[(trans-4-hydroxy-cyclohexylcarbamoyl)-methyl]-6,6-dimethyl-morpholine--
3-carboxylic acid (6-chloro-9H-beta-carbolin-8-yl)-amide (148 mg,
59%) as a yellow foam. The bis-HCl salt was prepared by adding 2
equivalents of conc. HCl to an ethanolic solution of the freebase.
Concentration, followed by ether trituration afforded the salt as a
free-flowing, yellow powder.
[0830] .sup.1H-NMR (freebase, 300 MHz, CDCl.sub.3) .delta. 11.71
(br s, 1H), 10.21 (s, 1H), 9.06 (s, 1H), 8.45-8.27 (m, 2H),
8.08-7.84 (m, 2H), 6.36 (br s, 1H), 4.13-3.81 (m, 3H), 3.72-3.56
(m, 1H), 3.51-3.37 (m, 2H), 3.03 (d, 1H), 2.79-2.26 (m, 5H),
2.16-1.94 (m, 4H), 1.54-1.30 (m, 5H), 1.22 (s, 3H).
[0831] MS (NH.sub.4OAc standard conditions, ES+) e/z=514
(M+H).sup.+
[0832] DAD R.sub.f=1.39 min
Example 78
(S)-4-[2-((2R,5R)-2,5-Dimethyl-pyrrolidin-1-yl)-2-oxo-ethyl]-6,6-dimethyl--
morpholine-3-carboxylic acid
(6-chloro-9H-beta-carbolin-8-yl)-amide-bis-hydrochloride salt
[0833] (2R,5R)-2,5-Dimethyl-pyrrolidine hydrochloride (270 mg, 2.0
mmol) (prepared using the procedure of Masamune, S., et al. J. Org.
Chem. 1989, 54, 1756) was dissolved in dichloromethane (3 mL) and
cooled to 0.degree. C. Triethylamine (405 mg, 0.56 mL, 4.0 mmol)
was added. Chloroacetyl chloride (226 mg, 0.16 mL, 2.0 mmol) was
dissolved in dichloromethane (1 mL) and added dropwise. The mixture
was warmed to room temperature and stirred an additional 30
minutes. The reaction was diluted with dichloromethane (5 mL),
extracted with 1N HCl and brine, then dried over MgSO.sub.4. The
organic layer was concentrated to a brown oil. Wt.: 242 mg
(82%).
[0834] Retention Time (LC, method: ammonium acetate standard): 1.24
min.
[0835] MS (M+H.sup.+): 176.5. The material was carried to the next
step without further purification.
[0836] Intermediate 59 (495 mg, 1.15 mmol) was dissolved in a
mixture of acetonitrile (8 mL) and water (2 mL). Potassium
carbonate (477 mg, 3.45 mmol) was added and the reaction was warmed
to 40.degree. C.
2-Chloro-1-((2R,5R)-2,5-dimethyl-pyrrolidin-1-yl)-ethanone (242 mg,
1.38 mmol) (prepared as described above) was dissolved in
acetonitrile (1 mL) and added dropwise. The reaction was warmed to
80.degree. C. and stirred overnight. The reaction was cooled to
40.degree. C. Sodium iodide (207 mg, 1.38 mmol) was dissolved in
acetone (1 mL) and added in one portion. The reaction was stirred
overnight at 40.degree. C. The reaction was concentrated in vacuo
and diluted with ethyl acetate (40 mL). The organic layer was
extracted twice with water, brine then dried over MgSO.sub.4. The
organic layer was filtered, and concentrated to an orange foam in
vacuo. Flash column chromatography (95:5 dichloromethane:methanol)
afforded a yellow solid, which was dissolved in 4N HCl/dioxane (1
mL) and concentrated to dryness. Trituration with ether afforded
the title compound as a yellow solid (18 mg, 3%).
[0837] .sup.1H-NMR (300 MHz, MeOH-d.sub.4): .delta. 1.23 (dd, 6H),
1.38 (s, 3H), 1.48 (s, 3H), 1.65 (m, 2H), 2.23 (m, 2H), 3.18 (m,
1H), 3.50 (m, 1H), 3.66 (m, 1H), 4.18 (m, 2H), 4.35 (m, 4H), 8.15
(s, 1H), 8.42 (s, 1H), 8.55 (d, 1H), 8.78 (d, 1H), 9.29 (s,
1H).
[0838] Retention Time (LC, method: ammonium acetate standard): 2.02
min.
[0839] MS (M+H.sup.+): 498.
Example 79
4-{2-[4-(1-Hydroxy-1-methyl-ethyl)-piperidin-1-yl]-2-oxo-ethyl}-6,6-dimeth-
yl-morpholine-3-carboxylic acid
(6-chloro-9H-.beta.-carbolin-8-yl)-amide
[0840] A solution of
1-(2-[5-(6-chloro-9H-.beta.-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholi-
n-4-yl]-acetyl)-piperidine-4-carboxylic acid methyl ester (62 mg,
0.11 mmol) in a mixture of anhydrous ether and toluene (1 ml: 1 ml)
was stirred at 0.degree. C. under N.sub.2. To this solution was
slowly added methylmagnesium bromide (3.0 M in ether, 306 .mu.L,
0.917 mmol). The reaction mixture was stirred at room temperature
overnight, and then quenched by adding saturated aqueous sodium
bicarbonate. The resulting mixture was further diluted with water
(10 ml) and ethyl acetate (30 ml). The aqueous layer was removed
and extracted with ethyl acetate (30.times.2 ml). The organic
layers were combined, washed with brine, dried over magnesium
sulfate, filtered and concentrated to afford a yellow solid (85
mg). The residue was purified by HPLC, to afford the pure product
(13 mg, 20%).
[0841] .sup.1H-NMR (300 MHz, HCDCl.sub.3): .delta. 10.94 (d, 1H),
10.47 (d, 1H), 8.94 (s, 1H), 8.41 (d, 1H), 8.32 (d, 1H), 7.89-7.81
(m, 2H), 4.91 (d, 1H), 4.04-3.91 (m, 2H); 3.64-3.57 (m, 1H), 3.43
(s, 1H), 3.37-3.31 (m, 1H), 3.15-2.90 (m, 1H), 2.77-2.58 (m, 2H),
2.47-2.41 (m, 1H), 2.00-1.86 (m, 2H), 1.53-1.03 (m, 17H).
[0842] NH.sub.4OAc standard conditions.
[0843] DAD R.sub.f=1.85 min
[0844] M+H=542
Example 80
4-[2-(3,3-Dimethyl-4-oxo-piperidin-1-yl)-2-oxo-ethyl]-6,6-dimethyl-morphol-
ine-3-carboxylic acid (6-chloro-9H-beta-carbolin-8-yl)-amide
[0845] 4-Oxo-piperidine-1-carboxylic acid tert-butyl ester (5 g, 25
mmol) was dissolved in tetrahydrofuran (100 mL) and the resulting
solution was cooled to 0.degree. C. Sodium hydride (60% in mineral
oil, 2.10 g, 53 mmol) was added to the cooled solution in a single
portion, and the resulting cloudy mixture was allowed to stir 10
min. Methyl iodide was subsequently added and the mixture was
allowed to warm to room temperature over several hours. Stirring
continued over night (12 h). The light orange mixture was
concentrated under reduced pressure. The residue was partitioned
between ether and water. The aqueous phase was back-extracted with
additional ether. The combined extracts were washed with water and
brine, dried over sodium sulfate, filtered and concentrated to a
pale yellow solid. The solid was triturated with 4% ethyl acetate
in hexanes (50 mL) to afford
3,3-dimethyl-4-oxo-piperidine-1-carboxylic acid tert-butyl ester as
a cream-colored solid (1.8 g, 32%).
[0846] .sup.1H-NMR (CDCl.sub.3, 300 MHz) .delta. 3.73 (t, 2H), 3.43
(br s, 2H), 2.49 (t, 2H), 1.49 (s, 9H), 1.13 (s, 6H).
[0847] The 3,3-dimethyl-4-oxo-piperidine-1-carboxylic acid
tert-butyl ester thus prepared (450 mg, 1.97 mmol) was dissolved in
methylene chloride (10 mL). Trifluoroacetic acid was added (305 uL)
and the resulting solution stirred at room temperature 2 h.
Additional trifluoroacetic acid was added (300 uL) and the reaction
stirred at room temperature 3 days. The pale yellow solution was
concentrated to afford an oily residue, which was triturated with
ether. The solids were collected by suction filtration and dried in
vacuo. 3,3-Dimethyl-piperidin-4-one was isolated and used as its
trifluoroacetic acid salt (381 mg, 80%).
[0848] .sup.1H-NMR (d.sub.6-DMSO, 300 MHz) .delta. 3.44-3.33 (m,
4H), 2.63-2.57 (m, 2H), 1.11 (s, 6H).
[0849]
[5-(6-Chloro-9H-beta-carbolin-8-ylcarbamoyl)-2,2-dimethyl-morpholin-
-4-yl]-acetic acid (100 mg, 0.24 mmol),
3,3-dimethyl-piperidin-4-one trifluoroacetatic acid salt (116 mg,
0.48 mmol) and diisopropylethylamine (62 mg, 85 HL) were dissolved
in pyridine (3 mL) and stirred 10 min. EDC (92 mg, 0.48 mmol) was
added and the mixture was stirred at room temperature 4 days. Water
was added (3 mL) and the quenched reaction was concentrated. The
residue was partitioned between ethyl acetate (50 mL) and 1 M
aqueous sodium carbonate (50 mL). The aqueous phase was extracted
with additional ethyl acetate (50 mL), and the extracts were
combined. The extracts were then washed with water and brine, dried
over sodium sulfate, filtered, dried and concentrated under reduced
pressure. The resulting residue was purified by silica gel
chromatography (chloroform, ethyl acetate, methanol gradient),
affording
4-[2-(3,3-dimethyl-4-oxo-piperidin-1-yl)-2-oxo-ethyl]-6,6-dimethyl-morpho-
line-3-carboxylic acid (6-chloro-9H-beta-carbolin-8-yl)-amide as a
yellow foam (91 mg, 73%). The bis-HCl salt was prepared by adding 2
equivalents of conc. HCl to an ethanolic solution of the
freebase.
[0850] Concentration, followed by ether trituration afforded the
salt as a free-flowing, yellow powder.
[0851] .sup.1H-NMR (CDCl.sub.3, 300 MHz) .delta. 10.67 (s, 1H),
10.37 (d, 1H), 8.93 (s, 1H), 8.41 (d, 1H), 8.15 (dd, 1H), 7.82 (d,
1H), 7.71 (d, 1H), 4.07-3.92 (m, 3H), 3.83-3.89 (m, 6H), 2.79-2.68
(m, 1H), 2.62-2.40 (m, 3H), 1.41-1.36 (m, 3H), 1.27-1.22 (m, 3H),
1.18-1.13 (m, 3H), 1.07-0.99 (m, 3H).
[0852] MS (NH.sub.4OAc standard conditions, ES+) e/z=526
(M+H).sup.+
[0853] DAD R.sub.f=1.79 min
Example 81
6,6-Dimethyl-4-(2-oxo-2-pyrrolidin-1-yl-ethyl)-morpholine-3-carboxylic
acid (6-chloro-4-methyl-9H-.beta.-carbolin-8-yl)-amide
[0854] The desired compound was prepared according to Methods C, E
and F from 6-chloro-4-methyl-9H-.beta.-carbolin-8-ylamine
(Intermediate 54) and pyrrolidine.
[0855] .sup.1H-NMR (300 MHz, MeOD-d.sub.4) .delta. 9.17 (s, 1) 8.39
(s, 1) 8.26 (d, 1) 8.17 (d, 1) 4.74-4.52 (m, 3) 4.47-4.30 (m, 1)
3.80-3.52 (m, 3) 3.48-3.34 (m, 4) 3.01 (s, 3) 1.76-1.55 (m, 4) 1.50
(s, 3) 1.43 (s, 3).
[0856] NH.sub.4OAc standard conditions ELSD R.sub.f=2.01 min.
[0857] M+H=498
Biological Testing
[0858] Compounds of this invention are effective inhibitors of
I.kappa.B kinase (IKK), and therefore, are useful for treating
conditions caused or aggravated by the activity of this kinase. The
in vitro and in vivo I.kappa.B kinase inhibitory activities of the
compounds of formula I may be determined by various procedures
known in the art. The potent affinities for I.kappa.B kinase
exhibited by the inventive compounds can be measured as an
IC.sub.50 value (in nM), which is the concentration (in nM) of
compound required to provide 50% inhibition of I.kappa.B
kinase.
[0859] Following are examples of assays that can be useful for
evaluating and selecting a compound that modulates IKK.
Assay for Measuring I.kappa.B Kinase Enzyme Inhibition
[0860] An in vitro assay for detecting and measuring inhibition
activity against I.kappa.B kinase complex by candidate
pharmacological agents can employ a polypeptide spanning both
Ser.sup.32 and Ser.sup.36 of I.kappa.B (SwissProt Accession No.
P25963, Swiss Institute of Bioinformatics, Geneva, Switzerland) and
an agent for detection of the phosphorylated product, e.g. a
specific antibody binding only to the phosphorylated form of the
polypeptide, being either monoclonal or polyclonal (e.g.,
commercially-available anti-phospho-serine.sup.32 I.kappa.B
antibodies). In the example of detecting the phosphorylated product
by an anti-phosphoserine.sup.32 I.kappa.B antibody, once the
antibody-phospho-polypeptide complex is formed, the complex can be
detected by a variety of analytical methods (e.g., radioactivity,
luminescence, fluorescence, or optical absorbance). For the use of
the DELFIA (Dissociation Enhancement Lanthanide Fluorescence
Immunoassay) method (time-resolved fluorometry, Perkin Elmer Life
and Analytical Sciences Inc., Boston, Mass.), the complex can be
immobilized either onto a biotin-binding plate (e.g., Neutravidin
coated plate) and detected with a secondary antibody conjugated to
Europium, or onto an antibody-binding plate (e.g., Protein-A coated
plate) and detected with biotin-binding protein conjugated to
Europium (e.g., Streptavidin-Europium). The level of activity can
be correlated with a standard curve using synthetic phosphopeptides
corresponding to the substrate polypeptide. How to prepare
materials for and conduct this assay are described in more detail
below.
Isolation of the I.kappa.B Kinase Complex
[0861] An I.kappa.B-.alpha. kinase complex was prepared by first
diluting 10 ml of HeLa S3 cell-extracts 5100 fraction (Lee et al.
(1997) Cell 88:213-222) with 40 ml of 50 mM HEPES pH 7.5. Then, 40%
ammonium sulfate was added and incubated on ice for 30 minutes. The
resulting precipitated pellet was redissolved with 5 ml of SEC
buffer (50 mM HEPES pH 7.5, 1 mM DTT, 0.5 mM EDTA, 10 mM
2-glycerophosphate), clarified by centrifugation at 20,000.times.g
for 15 min., and filtrated through a 0.22 .mu.m filter unit. The
sample was loaded onto a 320 ml SUPEROSE-6 gel filtration FPLC
column (Amersham Biosciences AB, Uppsala, Sweden) equilibrated with
a SEC buffer operated at 2 ml/min flow rate at 4.degree. C.
Fractions spanning the 670-kDa molecular-weight marker were pooled
for activation. A kinase-containing pool was then activated by
incubation with 100 nM MEKK1.DELTA. (Lee et al. (1997) Cell
88:213-222), 250 .mu.M MgATP, 10 mM MgCl.sub.2, 5 mM DTT, 10 mM
2-glycerophosphate, 2.5 .mu.M Microcystin-LR, for 45 minutes at
37.degree. C. The activated enzyme was stored at -80.degree. C.
until further use.
Measurement of I.kappa.B Kinase Phosphotransferase Activity
[0862] At the per well of a 96 well plate, compounds of various
concentrations in 5 .mu.L of 20% DMSO were preincubated for 30
minutes at 25.degree. C. with 40 .mu.L of activated enzyme diluted
1:25 with assay buffer (50 mM Hepes pH 7.5, 5 mM DTT, 10 mM
MgCl.sub.2, 10 mM 2-glycerophosphate, 2 .mu.M Microcystin-LR, 0.1%
Bovine Serum Albumin). 5 .mu.L of peptide substrate
(biotin-(CH.sub.2)-6-DRHDSGLD(phosphoS)MKD-CONH.sub.2) at 200
.mu.M+500 .mu.M ATP were added to each well and incubated for 1
hour before quenching with 50 .mu.L of 50 mM Hepes pH 7.5, 0.1%
BSA, 100 mM EDTA. 5 .mu.L of quenched kinase reaction were
transferred to a Protein A plate (Pierce Biotechnology, Inc.,
Rockford, Ill., USA) containing 90 .mu.L of anti-phospho I.kappa.B
S32/S36 antibody (Cell Signaling Technologies Beverly, Mass., USA)
at 2 .mu.g/ml. Samples were incubated for 2 hours with shaking.
Following 3 washes with PBS+0.05% Tween20, 90 .mu.L of streptavidin
linked europium chelate (Perkin Elmer Life and Analytical Sciences,
Boston, Mass., USA) at 0.1 .mu.g/ml were added to each well and
incubated for 1 hour with shaking. Following 3 washes with PBS
0.05% Tween20, 100 .mu.L of DELFIA Enhancement Solution (Perkin
Elmer Life and Analytical Sciences, Boston, Mass., USA) were added
to each well. An europium signal was read with an excitation of 330
nM and emission of 615 nM on a Wallac Victor plate reader (Perkin
Elmer Life and Analytical Sciences, Boston, Mass.). As the assay
was previously shown to be linear with respect to enzyme
concentration and time for the enzyme dilution tested, levels of
europium signal were used to determine the inhibition activity of
candidate pharmacological agents.
[0863] The compounds of the invention were active inhibitors of the
IKK complex. It will be appreciated that compounds of this
invention can exhibit I.kappa.B kinase inhibitor activities of
varying degrees. Following assay procedures such as the in vitro
and cell-based assays described herein, the I.kappa.B kinase
inhibition average IC.sub.50 values for the inventive compounds
were generally below about 10 micromolar, preferably, below about
1.0 micromolar and more preferably below about 100 nanomolar. The
inventive compounds were also selective for inhibiting IKK-2 as
opposed to IKK-1.
Cellular Assays
Multiple Myeloma (MM) Cell Lines and Patient-Derived MM Cells
Isolation
[0864] RPMI 8226 and U266 human MM cells were obtained from
American Type Culture Collection (Manassas, Va.). All MM cell lines
were cultured in RPMI-1640 containing 10% fetal bovine serum (FBS,
Sigma-Aldrich Co., St. Louis, Mo.), 2 mM L-glutamine, 100 U/mL
penicillin (Pen) and 100 .mu.g/mL streptomycin (Strep) (GIBCO brand
cell culture products available from Invitrogen Life Technologies,
Carlsbad, Calif.). Patient-derived MM cells were purified from
patient bone marrow (BM) aspirates using ROSETTESEP (B cell
enrichment kit) separation system (StemCell Technologies,
Vancouver, Canada). The purity of MM cells was confirmed by flow
cytometry using PE-conjugated anti-CD138 antibody (BD Biosciences,
Bedford, Mass.).
Bone Marrow Stroma Cell Cultures
[0865] Bone marrow (BM) specimens were obtained from patients with
MM. Mononuclear cells (MNCs) separated by Ficoll-Hipaque density
sedimentation were used to established long-term BM cultures as
previously described (Uchiyama et al., Blood 1993, 82:3712-3720).
Cells were harvested in Hank's Buffered Saline Solution (HESS)
containing 0.25% trypsin and 0.02% EDTA, washed, and collected by
centrifugation.
Cell Proliferation Via Measurement of DNA-Synthesis Rate
[0866] Proliferation was measured as previously described
(Hideshima et al., Blood 96:2943 (2000)). MM cells
(3.times.10.sup.4 cells/well) were incubated in 96-well culture
plates (Corning Life Sciences, Corning, N.Y.) in the presence of
media or an IKK inhibitor of this invention for 48 h at 37.degree.
C. DNA synthesis was measured by [.sup.3H]-thymidine
([.sup.3H]-TdR, New England Nuclear division of Perkin Elmer Life
and Analytical Sciences, Boston, Mass.) incorporation into dividing
cells. Cells were pulsed with [.sup.3H]TdR (0.5 .mu.Ci/well) during
the last 8 h of 48 h cultures. All experiments were performed in
triplicate.
MTT Cell Viability Assay
[0867] The inhibitory effect of the present compounds on MM growth
was assessed by measuring the reduction of yellow tetrazolium MTT
(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) by
metabolically active cells (J. Immunol. Methods 174: 311-320,
1994). Cells from 48 h cultures were pulsed with 10 .mu.L of 5
mg/mL MTT to each well for the last 4 h of the 48 h cultures,
followed by 100 .mu.L isopropanol containing 0.04N HCl. Absorbance
was measured at 570 nm using a spectrophotometer (Molecular Devices
Corp., Sunnyvale Calif.).
NF-.kappa.B Activation Via Electrophoretic Mobility Shift Assay
[0868] Electrophoretic mobility shift analyses (BMSA) were carried
out as previously described (Hideshima et al., Oncogene 2001,
20:4519). Briefly, MM cells were pre-incubated with an IKK
inhibitor of this invention (10 .mu.M for 90 min) before
stimulation with TNF-.alpha. (5 ng/mL) for 10 to 20 min. Cells were
then pelleted, resuspended in 400 .mu.L of hypotonic lysis buffer
(20 mM HEPES, pH 7.9, 10 mM KCl, 1 mM EDTA, 0.2% Triton X-100, 1 mM
Na.sub.3VO.sub.4, 5 mM NaF, 1 mM PMSF, 5 .mu.g/mL leupeptin, 5
.mu.g/mL aprotinin), and kept on ice for 20 min. After
centrifugation (14000 g for 5 min) at 4.degree. C., the nuclear
pellet was extracted with 100 .mu.L hypertonic lysis buffer (20 mM
HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM Na.sub.3VO.sub.4, 5 mM
NaF, 1 mM PMSF, 5 .mu.g/mL leupeptin, 5 .mu.g/mL aprotinin) on ice
for 20 min. After centrifugation (14000 g for 5 min) at 4.degree.
C., the supernatant was collected as nuclear extract.
Double-stranded NF-.kappa.B consensus oligonucleotide probe
(5'-GGGGACTTTCCC-3', Santa Cruz Biotechnology Inc., Santa Cruz
Calif.) was end-labeled with [(.sup.32P]ATP (50 .mu.Ci at 222
TBq/mM; New England Nuclear division of Perkin Elmer Life and
Analytical Sciences, Boston, Mass.). Binding reactions containing 1
ng of oligonucleotide and 5 .mu.g of nuclear protein were conducted
at room temperature for 20 min in a total volume of 10 .mu.L of
binding buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM
MgCl.sub.2, 0.5 mM EDTA, 0.5 mM DTT, 4% glycerol (v/v), and 0.5
.mu.g poly(dI-dC) (Amersham Biosciences AB, Uppsala, Sweden). For
supershift analysis, 1 .mu.g of anti-p65 NF-.kappa.B Ab was added 5
min before the reaction mixtures, immediately after addition of
radiolabeled probe. The samples were loaded onto a 4%
polyacrylamide gel, transferred to Whatman paper (Whatman
International, Maidstone, U.K.), and visualized by
autoradiography.
Diffuse Large B-Cell Lymphoma (DLBCL) Cell Proliferation Assay
[0869] ABC-like (LY3 and Ly10) and GCB-like (Ly7 and Ly19) DLBCL
cell lines (Alizadeh et al (2000) Nature 403:503-511; Davis et al.
(2001) J. Exp. Med. 194:1861-1874) were maintained in growth medium
(GM, Iscove's DMEM+10% FBS) by passaging cells twice per week.
Cells were starved overnight in Iscove's DMEM medium+0.5% FBS
overnight before plated in proliferation assay. On the day of the
assay, cells were counted and viability was checked using Trypan
Blue staining. For the Ly3 and Ly10 cells, 5000 cell were plated in
GM per well in a 96-well plate. The Ly7 and Ly19 cells were plated
at 10,000 cells per well. IKK inhibitors were first dissolved in
DMSO and then diluted in GM to reach the final concentrations of 80
.mu.M-0.01 .mu.M. Each concentration was plated in triplicate. Cell
viability was determined using a standard WST-1 cell viability
assay (Roche Applied Science, Indianapolis, Ind.).
Human Peripheral Blood Monocyte (PBMC) Cytokine Release Assay
[0870] Human PBMC was purified from normal donor whole blood by
Ficoll gradient method. After a PBS wash, PBMC were re-suspended in
AIM-V medium. Serially diluted IKK inhibitors of this invention in
100% DMSO was added at 1 .mu.l to the bottom of a 96-well plate and
mixed with 180 .mu.l 4.5.times.10.sup.5 PBMC in AIM-V media per
well. After preincubating PBMC with inhibitor at 37.degree. C. for
40 min, cells were stimulated with 20 .mu.l of either with LPS (100
ng/ml) or with anti-CD3 (0.25 .mu.g/ml) and anti-CD28 (0.25
.mu.g/ml) (Pharmingen division of BD Biosciences, Bedford, Mass.)
at 37.degree. C. for 5 hours. The supernatants were collected and
assessed for IL-1.beta. or TNF-.alpha. release using standard
commercially available ELISA kits.
Human Chondrocyte Matrix Metalloproteases (MMPs) Release Assay
[0871] Human chondrocyte cell line SW1353 (ATCC, Manassas, Va.) was
cultured containing 10% fetal bovine serum (Hyclone, Logan, Utah),
2 mM L-glutamine (GIBCO brand cell culture products available from
Invitrogen Life Technologies, Carlsbad, Calif.) and 1% Pen/Strep
(GIBCO). Cells were seeded in 96-well Poly-D-Lysine plate (BD
BIOCOAT, Black/Clear bottom, BD Biosciences, Bedford, Mass.).
Serially diluted IKK inhibitors at 1 .mu.l were added to each well
of 96-well plates and mixed with 180 .mu.l 4.5.times.10.sup.5
chondrocytes per well. After pre-incubating cells with compounds
for 1 hr at 37.degree. C., cells were stimulated with 20 .mu.l
IL-1.beta. (10 ng/mL, R&D Systems Inc.) at 37.degree. C. for 24
hrs. The supernatants were then collected and assessed for
production of matrix metalloproteinases (MMPs) using commercially
available ELISA kits.
Human Fibroblast Like Synoviocyte (HFLS) Assay
[0872] HFLS isolated from RA synovial tissues obtained at joint
replacement surgery was provided by Cell Applications Inc. (San
Diego, Calif.). IKK inhibitors of the invention were tested for
their ability to block the TNF- or IL-1.beta.-induced release of
IL-6 or IL-8 from these cells using commercially available ELISA
kits. Cell culture conditions and assay methods were described in
Aupperle et al., Journal of Immunology, 163:427-433 (1999).
Human Cord Blood Derived Mast Cell Assay
[0873] Human cord blood was obtained from Cambrex (Walkersville,
Md.). Mast cells were differentiated and cultured in a manner
similar to that described by Hsieh et al., J. Exp. Med.,
193:123-133 (2001). IKK inhibitors of the invention were tested for
their ability to block the IgE- or LPS-induced TNF.alpha. release
using commercially available ELISA kits.
Osteoclast Differentiation and Functional Assays
[0874] Human osteoclast precursors were obtained as cryopreserved
form from Cambrex (Walkersville, Md.). The cells were
differentiated in culture based on instructions from the
manufacturer. IKK inhibitors of the invention were tested for their
ability to block the differentiation, bone resorption and collagen
degradation as described previously (see Khapli, S. M., Journal of
Immunol, 171:142-151 (2003); Karsdal, M. A., J Biol Chem,
278:44975-44987 (2003); and Takami, M., Journal of Immunol,
169:1516-1523 (2002)).
Rat Models for Rheumatoid Arthritis
[0875] Certain compounds of this invention were found to be active
in one or more rat models for rheumatoid arthritis. Such testing is
known in the literature and include a standard rat LPS model as
described in Conway et al., "Inhibition of Tumor Necrosis
Factor-.alpha. (TNF-.alpha.) Production and Arthritis in the Rat by
GW3333, a Dual Inhibitor of TNF-Converting Enzyme and Matrix
Metalloproteinases", J. Pharmacol. Exp. Ther. 298(3), 900-908
(2001); a rat adjuvant induced arthritis model as described in
Pharmacological Methods in the Control of Inflammation (1989) p
363-380 "Rat Adjuvant Arthritis: A Model of Chronic Inflammation"
Barry M. Weichman author of book chapter {Alan R. Liss Inc
Publisher}; and a rat collagen induced arthritis model as described
in Pharmacological Methods in the Control of Inflammation (1989) p
395-413 "Type II Collagen Induced Arthritis in the Rat" DE Trentham
and RA Dynesuis-Trentham authors of book chapter {Alan R. Liss Inc
Publisher}, See also, "Animal Models of Arthritis: Relevance to
Human Disease" (1999) by A. Bendele, J. McComb, T. Gould, T.
McAbee, G. Sennello, E. Chlipala and M. Guy. Toxicologic Pathology
Vol 27 (1) 134-142.
[0876] Based on the results of one or more rat models such as the
ones described above, compounds of formula II-C were found to be
surprisingly superior compared to other compounds where Ring A is a
pyridine ring. Also in the rat models, compounds of formula
III-A-a, especially compounds of formula III-A-aa, were found to be
surprisingly superior compared to other compounds where Ring A is a
morpholine ring.
[0877] While we have described a number of embodiments of this
invention, it is apparent that our basic examples may be altered to
provide other embodiments, which utilize the compounds and methods
of this invention. Therefore, it will be appreciated that the scope
of this invention is to be defined by the appended claims rather
than by the specific embodiments, which have been represented by
way of example.
* * * * *