U.S. patent application number 11/474646 was filed with the patent office on 2006-12-28 for niacin receptor agonists, compositions containing such compounds and methods of treatment.
Invention is credited to Weichun Chen, Steven L. Colletti, Fa-Xiang Ding, Ashley Rouse Lins, Subharekha Raghavan, Darby Rye Schmidt, Hong Shen, Abigail Lee Smenton, James R. Tata, George Scott Tria.
Application Number | 20060293364 11/474646 |
Document ID | / |
Family ID | 37134234 |
Filed Date | 2006-12-28 |
United States Patent
Application |
20060293364 |
Kind Code |
A1 |
Raghavan; Subharekha ; et
al. |
December 28, 2006 |
Niacin receptor agonists, compositions containing such compounds
and methods of treatment
Abstract
The present invention encompasses compounds of Formula I:
##STR1## as well as pharmaceutically acceptable salts and hydrates
thereof, that are useful for treating atherosclerosis,
dyslipidemias and the like. Pharmaceutical compositions and methods
of use are also included.
Inventors: |
Raghavan; Subharekha;
(Teaneck, NJ) ; Colletti; Steven L.; (Princeton
Junction, NJ) ; Ding; Fa-Xiang; (Staten Island,
NY) ; Shen; Hong; (West Windsor, NJ) ; Tata;
James R.; (Westfield, NJ) ; Lins; Ashley Rouse;
(Edison, NJ) ; Smenton; Abigail Lee; (Brooklyn,
NY) ; Chen; Weichun; (Livingston, NJ) ;
Schmidt; Darby Rye; (Clark, NJ) ; Tria; George
Scott; (La Jolla, CA) |
Correspondence
Address: |
MERCK AND CO., INC
P O BOX 2000
RAHWAY
NJ
07065-0907
US
|
Family ID: |
37134234 |
Appl. No.: |
11/474646 |
Filed: |
June 26, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60694711 |
Jun 28, 2005 |
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Current U.S.
Class: |
514/341 ;
514/359; 514/365; 514/366; 514/379; 514/406; 514/459; 514/469;
514/563; 546/269.1; 548/151; 548/202; 548/240; 548/253; 548/375.1;
562/450 |
Current CPC
Class: |
C07C 237/20 20130101;
C07C 2601/16 20170501; C07D 513/04 20130101; A61P 3/10 20180101;
C07C 235/36 20130101; C07D 417/04 20130101; C07D 249/06 20130101;
C07D 231/12 20130101; C07D 307/79 20130101; C07D 271/06 20130101;
C07D 309/14 20130101; C07C 233/55 20130101; A61P 3/00 20180101;
A61P 3/06 20180101; C07D 401/04 20130101; C07C 2601/10 20170501;
C07D 249/08 20130101; C07D 261/20 20130101; C07D 413/04 20130101;
A61P 9/10 20180101; C07D 309/28 20130101; C07D 277/30 20130101;
C07D 257/04 20130101; C07C 233/52 20130101 |
Class at
Publication: |
514/341 ;
514/365; 514/366; 514/379; 514/469; 514/563; 548/151; 548/202;
548/253; 548/240; 562/450; 514/406; 548/375.1; 546/269.1; 514/459;
514/359 |
International
Class: |
A61K 31/4439 20060101
A61K031/4439; A61K 31/43 20060101 A61K031/43; A61K 31/427 20060101
A61K031/427; A61K 31/4245 20060101 A61K031/4245; A61K 31/416
20060101 A61K031/416; A61K 31/4192 20060101 A61K031/4192; A61K
31/35 20060101 A61K031/35; A61K 31/195 20060101 A61K031/195 |
Claims
1. A compound represented by formula I: ##STR133## or a
pharmaceutically acceptable salt or solvate thereof is disclosed
wherein: X represents CH.sub.2, O, S, S(O), SO.sub.2 or NH, such
that when X represents NH, the nitrogen atom may be optionally
substituted with R.sup.6, C(O)R.sup.6, or SO.sub.2R.sup.6, wherein:
R.sup.6 represents C.sub.1-3alkyl optionally substituted with 1-3
groups, 0-3 of which are halo, and 0-1 of which are selected from
the group consisting of: OC.sub.1-3alkyl, OH, NH.sub.2,
NHC.sub.1-3alkyl, N(C.sub.1-3alkyl).sub.2, CN, Hetcy, Aryl and HAR,
said Aryl and HAR being further optionally substituted with 1-3
groups, 1-3 of which are halo, and 0-1 of which are selected from
the group consisting of: OH, NH.sub.2, C.sub.1-3alkyl,
C.sub.1-3alkoxy, haloC.sub.1-3alkyl and haloC.sub.1-3alkoxy groups;
a and b are each integers 1, 2 or 3, such that the sum of a and b
is 2, 3 or 4; ring A represents a 6-10 membered aryl, a 5-13
membered heteroaryl or a partially aromatic heterocyclic group,
said heteroaryl and partially aromatic heterocyclic group
containing at least one heteroatom selected from O, S, S(O),
S(O).sub.2 and N, and optionally containing 1 other heteroatom
selected from O and S, and optionally containing 1-3 additional N
atoms, with up to 5 heteroatoms being present; each R.sup.2 and
R.sup.3 is independently H, C.sub.1-3alkyl, haloC.sub.1-3alkyl,
OC.sub.1-3alkyl, haloC.sub.1-3alkoxy, OH or F; n represents an
integer of from 1 to 5; each R.sup.4 is H or is independently
selected from halo and R.sup.6; R.sup.5 represents --CO.sub.2H,
##STR134## or --C(O)NHSO.sub.2R.sup.e wherein R.sup.e represents
C.sub.1-4alkyl or phenyl, said C.sub.1-4alkyl and phenyl each being
optionally substituted with 1-3 groups, 1-3 of which are selected
from halo and C.sub.1-3alkyl, and 1-2 of which are selected from
the group consisting of: OC.sub.1-3alkyl, haloC.sub.1-3alkyl,
haloC.sub.1-3alkoxy, OH, NH.sub.2 and NHC.sub.1-3alkyl; and each
R.sup.1 is H or is independently selected from the group consisting
of: a) halo, OH, CO.sub.2H, CN, NH.sub.2, S(O).sub.0-2R.sup.e,
C(O)RC, OC(O)R.sup.e and CO.sub.2R.sup.e, wherein R.sup.e is as
previously defined; b) C.sub.1-6 alkyl and OC.sub.1-4alkyl, said
C.sub.1-alkyl and alkyl portion of OC.sub.1-4alkyl being optionally
substituted with 1-3 groups, 1-3 of which are halo and 1-2 of which
are selected from: OH, CO.sub.2H, CO.sub.2C.sub.1-4alkyl,
CO.sub.2C.sub.1-4haloalkyl, OCO.sub.2C.sub.1-4alkyl, NH.sub.2,
NHC.sub.1-4alkyl, N(C.sub.1-4alkyl).sub.2, Hetcy and CN; c)
NHC.sub.1-4alkyl and N(C.sub.1-4alkyl).sub.2, the alkyl portions of
which are optionally substituted as set forth in (b) above; d)
C(O)NH.sub.2, C(O)NHC.sub.1-4alkyl, C(O)N(C.sub.1-4alkyl).sub.2,
C(O)Hetcy, C(O)NHOC.sub.1-4alkyl and
C(O)N(C.sub.1-4alkyl)(OC.sub.1-4alkyl), the alkyl portions of which
are optionally substituted as set forth in (b) above; e)
NR'C(O)R'', NR'SO.sub.2R'', NR'CO.sub.2R'' and NR'C(O)NR''R'''
wherein: R' represents H, C.sub.1-3alkyl or haloC.sub.1-3alkyl, R''
represents (a) C.sub.1-8alkyl optionally substituted with 1-4
groups, 0-4 of which are halo, and 0-1 of which are selected from
the group consisting of: OC.sub.1-6alkyl, OH, CO.sub.2H,
CO.sub.2C.sub.1-4alkyl, CO.sub.2C.sub.1-4haloalkyl, NH.sub.2,
NHC.sub.1-4alkyl, N(C.sub.1-4alkyl).sub.2, CN, Hetcy, Aryl and HAR,
said Hetcy, Aryl and HAR being further optionally substituted with
1-3 halo, C.sub.1-4alkyl, C.sub.1-4alkoxy, haloC.sub.1-4alkyl or
haloC.sub.1-4alkoxy groups; or (b) Hetcy, Aryl or HAR, each being
optionally substituted with 1-3 members selected from the group
consisting of: halo, C.sub.1-4alkyl, C.sub.1-4alkoxy,
haloC.sub.1-4alkyl and haloC.sub.1-4alkoxy groups; and R'''
representing H or R''; f) phenyl or a 5-6 membered heteroaryl or a
Hetcy group attached at any available ring atom and each being
optionally substituted with 1-3 groups, 1-3 of which are selected
from halo, C.sub.1-3alkyl and haloC.sub.1-3alkyl groups, and 1-2 of
which are selected from OC.sub.1-3alkyl and haloOC.sub.1-3alkyl
groups, and 0-1 of which is selected from the group consisting of:
i) OH; CO.sub.2H; CN; NH.sub.2 and S(O).sub.0-2R.sup.e wherein
R.sup.e is as described above; ii) NHC.sub.1-4alkyl and
N(C.sub.1-4alkyl).sub.2, the alkyl portions of which are optionally
substituted with 1-3 groups, 1-3 of which are halo and 1-2 of which
are selected from: OH, CO.sub.2H, CO.sub.2C.sub.1-4alkyl,
CO.sub.2C.sub.1-4haloalkyl, NH.sub.2, NHC.sub.1-4alkyl,
N(C.sub.1-4alkyl).sub.2 and CN; iii) C(O)NH.sub.2,
C(O)NHC.sub.1-4alkyl, C(O)N(C.sub.1-4alkyl).sub.2,
C(O)NHOC.sub.1-4alkyl and C(O)N(C.sub.1-4alkyl)(OC.sub.1-4alkyl),
the alkyl portions of which are optionally substituted as set forth
in b) above; and iv) NR'C(O)R'', NR'SO.sub.2R'', NR'CO.sub.2R'' and
NR'C(O)NR''R''' wherein R', R'' and R''' are as described
above.
2. A compound in accordance with claim 1 wherein up to 4 R.sup.2
and R.sup.3 moieties are selected from the group consisting of:
C.sub.1-3alkyl, haloC.sub.1-3alkyl, OC.sub.1-3alkyl,
haloC.sub.1-3alkoxy, OH and F, and any remaining R.sup.2 and
R.sup.3 moieties represent H.
3. A compound in accordance with claim 1 wherein ring A is a phenyl
or naphthyl group, a 5-6 membered monocyclic heteroaryl group or a
9-13 membered bicyclic or tricyclic heteroaryl group.
4. A compound in accordance with claim 3 wherein ring A is selected
from the group consisting of: phenyl; naphthyl; HAR which
represents a member selected from the group consisting of:
pyrrolyl, isoxazolyl, isothiazolyl, pyrazolyl, pyridyl, oxazolyl,
oxadiazolyl, thiadiazolyl, thiazolyl, imidazolyl, triazolyl,
tetrazolyl, furanyl, triazinyl, thienyl, pyrimidyl, pyridazinyl,
pyrazinyl, benzoxazolyl, benzothiazolyl, benzimidazolyl,
benzofuranyl, benzothiophenyl, benzopyrazolyl, benzotriazolyl,
furo(2,3-b)pyridyl, benzoxazinyl, tetrahydrohydroquinolinyl,
tetrahydroisoquinolinyl, quinolyl, isoquinolyl, indolyl,
dihydroindolyl, quinoxalinyl, quinazolinyl, naphthyridinyl,
pteridinyl, 2,3-dihydrofuro(2,3-b)pyridyl indolinyl,
dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl,
or a member selected from the group consisting of: ##STR135##
5. A compound in accordance with claim 4 wherein ring A is selected
from the group consisting of: phenyl; naphthyl; and HAR which is a
member selected from the group consisting of: isoxazolyl,
pyrazolyl, oxazolyl, oxadiazolyl, thiazolyl, triazolyl, thienyl,
benzothiazolyl, and a member selected from the group consisting of:
##STR136##
6. A compound in accordance with claim 1 wherein each R.sup.1 is H
or is selected from the group consisting of: a) halo, OH, CN,
NH.sub.2 and S(O).sub.0-2R.sup.e wherein R.sup.1 is methyl or
phenyl optionally substituted with 1-3 halo groups; b) C.sub.1-3
alkyl and OC.sub.1-3alkyl, each being optionally substituted with
1-3 groups, 1-3 of which are halo and 1-2 of which are selected
from: OH, NH.sub.2, NHC.sub.1-4alkyl and CN; c) NR'SO.sub.2R'' and
NR'C(O)NR''R''' wherein: R' represents H, C.sub.1-3alkyl or
haloC.sub.1-3alkyl, R'' represents (a) C.sub.1-8alkyl optionally
substituted with 1-4 groups, 0-4 of which are halo, and 0-1 of
which are selected from the group consisting of: OC.sub.1-6alkyl,
OH, CO.sub.2H, CO.sub.2C.sub.1-4alkyl, CO.sub.2C.sub.1-4haloalkyl,
OCO.sub.2C.sub.1-4alkyl, NH.sub.2, NHC.sub.1-4alkyl,
N(C.sub.1-4alkyl).sub.2, CN, Hetcy, Aryl and HAR, said Hetcy, Aryl
and HAR being further optionally substituted with 1-3 groups
selected from: halo, C.sub.1-4alkyl, C.sub.1-4alkoxy,
haloC.sub.1-4alkyl and haloC.sub.1-4alkoxy; (b) Hetcy, Aryl or HAR,
said Aryl and HAR being optionally substituted with 1-3 groups
selected from: halo, C.sub.1-4alkyl, C.sub.1-4alkoxy,
haloC.sub.1-4alkyl and haloC.sub.1-4alkoxy; and R''' representing H
or R''; and d) phenyl or a 5-6 membered heteroaryl or a
heterocyclic group attached at any available point and being
optionally substituted with 1-3 groups, 1-3 of which are halo,
C.sub.1-3alkyl or haloC.sub.1-3alkyl groups, 1-2 of which are
OC.sub.1-3alkyl or haloOC.sub.1-3alkyl groups, and 1 of which is
selected from the group consisting of: i) OH; CO.sub.2H; CN;
NH.sub.2 and S(O).sub.0-2R.sup.e wherein R.sup.e is as described
above; ii) NHC.sub.1-4alkyl, the alkyl portion of which is
optionally substituted with 1-3 groups, 1-3 of which are halo and 1
of which is selected from: OH, CO.sub.2H, CO.sub.2C.sub.1-4alkyl,
CO.sub.2C.sub.1-4haloalkyl, NH.sub.2, NHC.sub.1-4alkyl,
N(C.sub.1-4alkyl).sub.2 and CN; iii) C(O)NH.sub.2,
C(O)NHC.sub.1-4alkyl and C(O)N(C.sub.1-4alkyl).sub.2, the alkyl
portions of which are optionally substituted as set forth in (b)
above; and iv) NR'C(O)R'' and NR'SO.sub.2R'' wherein R' and R'' are
as described above.
7. A compound in accordance with claim 6 wherein each R.sup.1 is H
or is selected from the group consisting of: a) halo, OH, CN and
NH.sub.2 b) C.sub.1-3 alkyl and OC.sub.1-3alkyl, each being
optionally substituted with 1-3 groups, 1-3 of which are halo and
1-2 of which are selected from: OH, NH.sub.2, NHC.sub.1-4alkyl and
CN; c) phenyl or a 5-6 membered heteroaryl or a heterocyclic group
attached at any available point and being optionally substituted
with 1-3 groups, 1-3 of which are halo, C.sub.1-3alkyl or
haloC.sub.1-3alkyl groups, 1-2 of which are OC.sub.1-3alkyl or
haloOC.sub.1-3alkyl groups, and 1 of which is selected from the
group consisting of: i) OH, CN and NH.sub.2.
8. A compound in accordance with claim 1 wherein a and b are 1 or 2
such that the sum of a and b is 2 or 3.
9. A compound in accordance with claim 1 wherein X represents O, S,
N or CH.sub.2.
10. A compound in accordance with claim 9 wherein X represents O or
CH.sub.2.
11. A compound in accordance with claim 1 wherein R.sup.2 and
R.sup.3 are independently H, C.sub.1-3alkyl, OH or
haloC.sub.1-3alkyl.
12. A compound in accordance with claim 11 wherein R.sup.2 and
R.sup.3 are independently H, C.sub.1-3alkyl or
haloC.sub.1-3alkyl.
13. A compound in accordance with claim 12 wherein R.sup.2 and
R.sup.3 are independently H or methyl.
14. A compound in accordance with claim 1 wherein n represents an
integer of from 2 to 4.
15. A compound in accordance with claim 14 wherein n is 2.
16. A compound in accordance with claim 1 wherein each R.sup.4 is H
or is independently selected from the group consisting of: halo,
C.sub.1-3alkyl optionally substituted with 1-3 halo groups or 0-1
OC.sub.1-3alkyl groups.
17. A compound in accordance with claim 16 wherein each R.sup.4 is
H or is independently selected from halo or C.sub.1-3alkyl
optionally substituted with 1-3 halo groups.
18. A compound in accordance with claim 1 wherein R.sup.5
represents --CO.sub.2H.
19. A compound in accordance with claim 1 wherein: ring A is a
phenyl or naphthyl group, a 5-6 membered monocyclic heteroaryl
group or a 9-13 membered bicyclic or tricyclic heteroaryl group;
each R.sup.1 is H or is selected from the group consisting of: a)
halo, OH, CN, NH.sub.2 and S(O).sub.0-2R.sup.e wherein R.sup.e is
methyl or phenyl optionally substituted with 1-3 halo groups; b)
C.sub.1-3 alkyl and OC.sub.1-3alkyl, each being optionally
substituted with 1-3 groups, 1-3 of which are halo and 1-2 of which
are selected from: OH, NH.sub.2, NHC.sub.1-4alkyl and CN; c)
NR'SO.sub.2R'' and NR'C(O)NR''R''' wherein: R' represents H,
C.sub.1-3alkyl or haloC.sub.1-3alkyl, R'' represents (a)
C.sub.1-8alkyl optionally substituted with 1-4 groups, 0-4 of which
are halo, and 0-1 of which are selected from the group consisting
of: OC.sub.1-6alkyl, OH, CO.sub.2H, CO.sub.2C.sub.1-4alkyl,
CO.sub.2C.sub.1-4haloalkyl, OCO.sub.2C.sub.1-4alkyl, NH.sub.2,
NHC.sub.1-4alkyl, N(C.sub.1-4alkyl).sub.2, CN, Hetcy, Aryl and HAR,
said Hetcy, Aryl and HAR being further optionally substituted with
1-3 halo, C.sub.1-4alkyl, C.sub.1-4alkoxy, haloC.sub.1-4alkyl and
haloC.sub.1-4alkoxy groups; (b) Hetcy, Aryl or HAR, said Aryl and
HAR being further optionally substituted with 1-3 halo,
C.sub.1-4alkyl, C.sub.1-4alkoxy, haloC.sub.1-4alkyl and
haloC.sub.1-4alkoxy groups; and R''' representing H or R''; and d)
phenyl or a 5-6 membered heteroaryl or a heterocyclic group
attached at any available point and being optionally substituted
with 1-3 groups, 1-3 of which are halo, C.sub.1-3alkyl or
haloC.sub.1-3alkyl groups, 1-2 of which are OC.sub.1-3alkyl or
haloOC.sub.1-3alkyl groups, and 1 of which is selected from the
group consisting of: i) OH; CO.sub.2H; CN; NH.sub.2;
S(O).sub.0-2R.sup.e wherein R.sup.e is as described above; ii)
NHC.sub.1-4alkyl the alkyl portion of which is optionally
substituted with 1-3 groups, 1-3 of which are halo and 1 of which
is selected from: OH, CO.sub.2H, CO.sub.2C.sub.1-4alkyl,
CO.sub.2C.sub.1-4haloalkyl, NH.sub.2, NHC.sub.1-4alkyl,
N(C.sub.1-4alkyl).sub.2 and CN; iii) C(O)NH.sub.2,
C(O)NHC.sub.1-4alkyl, C(O)N(C.sub.1-4alkyl).sub.2, the alkyl
portions of which are optionally substituted as set forth in (b)
above; and iv) NR'C(O)R'' and NR'SO.sub.2R'' wherein R' and R'' are
as described above; a and b are 1 or 2 such that the sum of a and b
is 2 or 3; X represents O or CH.sub.2; R.sup.2 and R.sup.3 are
independently H, OH, C.sub.1-3alkyl or haloC.sub.1-3alkyl; n
represents 2; R.sup.4 is H or is independently selected from the
group consisting of: halo, C.sub.1-3alkyl optionally substituted
with 1-3 halo groups or 0-1 OC.sub.1-3alkyl groups; and R.sup.5
represents --CO.sub.2H.
20. A compound in accordance with claim 1 wherein: ring A is
selected from the group consisting of: ##STR137## each R.sup.1 is
independently H, CH.sub.3, phenyl, 4-hydroxy-phenyl, OH,
2-hydroxy-phenyl, 3-hydroxy-phenyl, 3-amino-phenyl,
2,3-dihydro-benzofuran-6-yl, 2-chloro-4-hydroxy-phenyl,
1H-pyrazol-4-yl, 5-hydroxy-pyridin-2-yl, 4-hydroxy-pyrazol-1-yl,
1H-[1,2,3]triazol-4-yl, or 5-fluoro-pyridin-2-yl; a and b are 1 or
2 such that the sum of a and b is 2 or 3; X represents CH.sub.2;
each R.sup.2 and R.sup.3 is independently H, OH or CH.sub.3; n
represents 2; R.sup.4 is H, CH.sub.3, CH.sub.2CH.sub.3, CF.sub.3 or
CH.sub.2OCH.sub.3; and R.sup.5 represents --CO.sub.2H.
21. A compound in accordance with claim 1 selected from the
following table: TABLE-US-00004 TABLE 1 Compound 1 ##STR138##
Compound 2 ##STR139## Compound 3 ##STR140## Compound 4 ##STR141##
Compound 5 ##STR142## Compound 6 ##STR143## Compound 7 ##STR144##
Compound 8 ##STR145## Compound 9 ##STR146## Compound 10 ##STR147##
Compound 11 ##STR148## Compound 12 ##STR149## Compound 13
##STR150## Compound 14 ##STR151## Compound 15 ##STR152## Compound
16 ##STR153## Compound 17 ##STR154## Compound 18 ##STR155##
Compound 19 ##STR156## Compound 20 ##STR157## Compound 21
##STR158## Compound 22 ##STR159## Compound 23 ##STR160## Compound
24 ##STR161## Compound 25 ##STR162## Compound 26 ##STR163##
Compound 27 ##STR164## Compound 28 ##STR165## Compound 29
##STR166## Compound 30 ##STR167## Compound 31 ##STR168## Compound
32 ##STR169## Compound 33 ##STR170## Compound 34 ##STR171##
Compound 35 ##STR172## Compound 36 ##STR173## Compound 37
##STR174## Compound 38 ##STR175## Compound 39 ##STR176## Compound
40 ##STR177## Compound 41 ##STR178## or a pharmaceutically
acceptable salt or solvate thereof.
22. A pharmaceutical composition comprising a compound in
accordance with claim 1 in combination with a pharmaceutically
acceptable carrier.
23. A method of treating atherosclerosis in a human patient in need
of such treatment comprising administering to the patient a
compound of claim 1 in an amount that is effective for treating
atherosclerosis.
24. A method of treating dyslipidemia in a human patient in need of
such treatment comprising administering to the patient a compound
of claim 1 in an amount that is effective for treating
dyslipidemias.
25. A method of treating diabetes in a human patient in need of
such treatment comprising administering to the patient a compound
of claim 1 in an amount that is effective for treating
diabetes.
26. A method of treating metabolic syndrome in a human patient in
need of such treatment comprising administering to the patient a
compound of claim 1 in an amount that is effective for treating
metabolic syndrome.
27. A method of treating atherosclerosis, dyslipidemias, diabetes,
metabolic syndrome or a related condition in a human patient in
need of such treatment, comprising administering to the patient a
compound of claim 1 and a DP receptor antagonist, said compounds
being administered in an amount that is effective to treat
atherosclerosis, dyslipidemia, diabetes or a related condition in
the absence of substantial flushing.
28. A method in accordance with claim 27 wherein the DP receptor
antagonist selected from the group consisting of compounds A
through AJ: ##STR179## ##STR180## ##STR181## ##STR182## ##STR183##
##STR184##
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to cycloalkene compounds,
their derivatives, compositions containing such compounds and
methods of treatment or prevention in a mammal relating to
dyslipidemias. Dyslipidemia is a condition wherein serum lipids are
abnormal. Elevated cholesterol and low levels of high density
lipoprotein (HDL) are independent risk factors for atherosclerosis
associated with a greater-than-normal risk of atherosclerosis and
cardiovascular disease. Factors known to affect serum cholesterol
include genetic predisposition, diet, body weight, degree of
physical activity, age and gender. While cholesterol in normal
amounts is a vital building block for cell membranes and essential
organic molecules such as steroids and bile acids, cholesterol in
excess is known to contribute to cardiovascular disease. For
example, cholesterol, through its relationship with foam cells, is
a primary component of plaque which collects in coronary arteries,
resulting in the cardiovascular disease termed atherosclerosis.
[0002] Traditional therapies for reducing cholesterol include
medications such as statins (which reduce production of cholesterol
by the body). More recently, the value of nutrition and nutritional
supplements in reducing blood cholesterol has received significant
attention. For example, dietary compounds such as soluble fiber,
vitamin E, soy, garlic, omega-3 fatty acids, and niacin have all
received significant attention and research funding.
[0003] Niacin or nicotinic acid (pyridine-3-carboxylic acid) is a
drug that reduces coronary events in clinical trials. It is
commonly known for its effect in elevating serum levels of high
density lipoproteins (HDL). Importantly, niacin also has a
beneficial effect on other lipid profiles. Specifically, it reduces
low density lipoproteins (LDL), very low density lipoproteins
(VLDL), and triglycerides (TG). However, the clinical use of
nicotinic acid is limited by a number of adverse side-effects
including cutaneous vasodilation, sometimes called flushing.
[0004] Despite the attention focused on traditional and alternative
means for controlling serum cholesterol, serum triglycerides, and
the like, a significant portion of the population has total
cholesterol levels greater than about 200 mg/dL, and are thus
candidates for dyslipidemia therapy. There thus remains a need in
the art for compounds, compositions and alternative methods of
reducing total cholesterol, serum triglycerides, and the like, and
raising HDL.
[0005] The present invention relates to compounds that have been
discovered to have effects in modifying serum lipid levels.
[0006] The invention thus provides compositions for effecting
reduction in total cholesterol and triglyceride concentrations and
raising HDL, in accordance with the methods described.
[0007] Consequently one object of the present invention is to
provide a nicotinic acid receptor agonist that can be used to treat
dyslipidemias, atherosclerosis, diabetes, metabolic syndrome and
related conditions while minimizing the adverse effects that are
associated with niacin treatment.
[0008] Yet another object is to provide a pharmaceutical
composition for oral use.
[0009] These and other objects will be apparent from the
description provided herein.
SUMMARY OF THE INVENTION
[0010] A compound represented by formula I: ##STR2## or a
pharmaceutically acceptable salt or solvate thereof is disclosed
wherein:
[0011] X represents CH.sub.2, O, S, S(O), SO.sub.2 or NH, such that
when X represents NH, the nitrogen atom may be optionally
substituted with R.sup.6, C(O)R.sup.6, or SO.sub.2R.sup.6,
wherein:
[0012] R.sup.6 represents C.sub.1-3alkyl optionally substituted
with 1-3 groups, 0-3 of which are halo, and 0-1 of which are
selected from the group consisting of: OC.sub.1-3alkyl, OH,
NH.sub.2, NHC.sub.1-3alkyl, N(C.sub.1-3alkyl).sub.2, CN, Hetcy,
Aryl and HAR,
[0013] said Aryl and HAR being further optionally substituted with
1-3 groups, 1-3 of which are halo, and 0-1 of which are selected
from the group consisting of: OH, NH.sub.2, C.sub.1-3alkyl,
C.sub.1-3alkoxy, haloC.sub.1-3alkyl and haloC.sub.1-3alkoxy
groups;
[0014] a and b are each integers 1, 2 or 3, such that the sum of a
and b is 2, 3 or 4;
[0015] ring A represents a 6-10 membered aryl, a 5-13 membered
heteroaryl or a partially aromatic heterocyclic group, said
heteroaryl and partially aromatic heterocyclic group containing at
least one heteroatom selected from O, S. S(O), S(O).sub.2 and N,
and optionally containing 1 other heteroatom selected from O and S,
and optionally containing 1-3 additional N atoms, with up to 5
heteroatoms being present;
[0016] each R.sup.2 and R.sup.3 is independently H, C.sub.1-3alkyl,
haloC.sub.1-3alkyl, OC.sub.1-3alkyl, haloC.sub.1-3alkoxy, OH or
F;
[0017] n represents an integer of from 1 to 5;
[0018] each R.sup.4 is H or is independently selected from halo and
R.sup.6;
[0019] R.sup.5 represents --CO.sub.2H, ##STR3## or
--C(O)NHSO.sub.2R.sup.e wherein R.sup.e represents C.sub.1-4alkyl
or phenyl, said C.sub.1-4alkyl and phenyl each being optionally
substituted with 1-3 groups, 1-3 of which are selected from halo
and C.sub.1-3alkyl, and 1-2 of which are selected from the group
consisting of: OC.sub.1-3alkyl, haloC.sub.1-3alkyl,
haloC.sub.1-3alkoxy, OH, NH.sub.2 and NHC.sub.1-3alkyl;
[0020] and each R.sup.1 is H or is independently selected from the
group consisting of:
[0021] a) halo, OH, CO.sub.2H, CN, NH.sub.2, S(O).sub.0-2R.sup.e,
C(O)R.sup.e, OC(O)R.sup.e and CO.sub.2R.sup.e, wherein R.sup.e is
as previously defined;
[0022] b) C.sub.1-6 alkyl and OC.sub.1-alkyl, said C.sub.1-6alkyl
and alkyl portion of OC.sub.1-6alkyl being optionally substituted
with 1-3 groups, 1-3 of which are halo and 1-2 of which are
selected from: OH, CO.sub.2H, CO.sub.2C.sub.1-4alkyl,
CO.sub.2C.sub.1-4haloalkyl, OCO.sub.2C.sub.1-4alkyl, NH.sub.2,
NHC.sub.1-4alkyl, N(C.sub.1-4alkyl).sub.2, Hetcy and CN;
[0023] c) NHC.sub.1-4alkyl and N(C.sub.1-4alkyl).sub.2, the alkyl
portions of which are optionally substituted as set forth in (b)
above;
[0024] d) C(O)NH.sub.2, C(O)NHC.sub.1-4alkyl,
C(O)N(C.sub.1-4alkyl).sub.2, C(O)Hetcy, C(O)NHOC.sub.1-4alkyl and
C(O)N(C.sub.1-4alkyl)(OC.sub.1-4alkyl), the alkyl portions of which
are optionally substituted as set forth in (b) above;
[0025] e) NR'C(O)R'', NR'SO.sub.2R'', NR'CO.sub.2R'' and
NR'C(O)NR''R''' wherein:
[0026] R' represents H, C.sub.1-3alkyl or haloC.sub.1-3alkyl,
[0027] R'' represents (a) C.sub.1-8alkyl optionally substituted
with 1-4 groups, 0-4 of which are halo, and 0-1 of which are
selected from the group consisting of: OC.sub.1-6alkyl, OH,
CO.sub.2H, CO.sub.2C.sub.1-4alkyl, CO.sub.2C.sub.1-4haloalkyl,
NH.sub.2, NHC.sub.1-4alkyl, N(C.sub.1-4alkyl).sub.2, CN, Hetcy,
Aryl and HAR, [0028] said Hetcy, Aryl and HAR being further
optionally substituted with 1-3 halo, C.sub.1-4alkyl,
C.sub.1-4alkoxy, haloC.sub.1-4alkyl or haloC.sub.1-4alkoxy groups;
[0029] (b) Hetcy, Aryl or HAR, each being optionally substituted
with 1-3 members selected from the group consisting of: halo,
C.sub.1-4alkyl, C.sub.1-4alkoxy, haloC.sub.1-4alkyl and
haloC.sub.1-4alkoxy groups;
[0030] and R''' representing H or R'';
[0031] f) phenyl or a 5-6 membered heteroaryl or a Hetcy group
attached at any available ring atom and each being optionally
substituted with 1-3 groups, 1-3 of which are selected from halo,
C.sub.1-3alkyl and haloC.sub.1-3alkyl groups, and 1-2 of which are
selected from OC.sub.1-3alkyl and haloOC.sub.1-3alkyl groups, and
0-1 of which is selected from the group consisting of: [0032] i)
OH; CO.sub.2H; CN; NH.sub.2 and S(O).sub.0-2R.sup.e wherein R.sup.e
is as described above; [0033] ii) NHC.sub.1-4alkyl and
N(C.sub.1-4alkyl).sub.2, the alkyl portions of which are optionally
substituted with 1-3 groups, 1-3 of which are halo and 1-2 of which
are selected from: OH, CO.sub.2H, CO.sub.2C.sub.1-4alkyl,
CO.sub.2C.sub.1-4haloalkyl, NH.sub.2, NHC.sub.1-4alkyl,
N(C.sub.1-4alkyl).sub.2 and CN; [0034] iii) C(O)NH.sub.2,
C(O)NHC.sub.1-4alkyl, C(O)N(C.sub.1-4alkyl).sub.2,
C(O)NHOC.sub.1-4alkyl and C(O)N(C.sub.1-4alkyl)(OC.sub.1-4alkyl),
the alkyl portions of which are optionally substituted as set forth
in b) above; and [0035] iv) NR'C(O)R'', NR'SO.sub.2R'',
NR'CO.sub.2R'' and NR'C(O)NR''R''' wherein R', R'' and R''' are as
described above.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The invention is described herein in detail using the terms
defined below unless otherwise specified.
[0037] "Alkyl", as well as other groups having the prefix "alk",
such as alkoxy, alkanoyl and the like, means carbon chains which
may be linear, branched, or cyclic, or combinations thereof,
containing the indicated number of carbon atoms. If no number is
specified, 1-6 carbon atoms are intended for linear and 3-7 carbon
atoms for branched alkyl groups. Examples of alkyl groups include
methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl,
pentyl, hexyl, heptyl, octyl, nonyl and the like. Cycloalkyl is a
subset of alkyl; if no number of atoms is specified, 3-7 carbon
atoms are intended, forming 1-3 carbocyclic rings that are fused.
"Cycloalkyl" also includes monocyclic rings fused to an aryl group
in which the point of attachment is on the non-aromatic portion.
Examples of cycloalkyl include cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, tetrahydronaphthyl,
decahydronaphthyl, indanyl and the like. Haloalkoxy and haloalkyl
are used interchangeably and refer to halo substituted alkyl groups
linked through the oxygen atom. Haloalkyl and haloalkoxy include
mono-substituted as well as multiple substituted alkyl and alkoxy
groups, up to perhalo substituted alkyl and alkoxy. For example,
trifluoromethyl and trifluoromethoxy are included.
[0038] "Alkenyl" means carbon chains which contain at least one
carbon-carbon double bond, and which may be linear or branched or
combinations thereof. Examples of alkenyl include vinyl, allyl,
isopropenyl, pentenyl, hexenyl, heptenyl, 1-propenyl, 2-butenyl,
2-methyl-2-butenyl, and the like.
[0039] "Alkynyl" means carbon chains which contain at least one
carbon-carbon triple bond, and which may be linear or branched or
combinations thereof. Examples of alkynyl include ethynyl,
propargyl, 3-methyl-1-pentynyl, 2-heptynyl and the like.
[0040] "Aryl" (Ar) means mono- and bicyclic aromatic rings
containing 6-10 carbon atoms. Examples of aryl include phenyl,
naphthyl, indenyl and the like.
[0041] "Heteroaryl" (HAR) unless otherwise specified, means mono-,
bicyclic and tricyclic aromatic ring systems containing at least
one heteroatom selected from O, S, S(O), SO.sub.2 and N, with each
ring containing 5 to 6 atoms. HAR groups may contain from 5-14,
preferably 5-13 atoms. Examples include, but are not limited to,
pyrrolyl, isoxazolyl, isothiazolyl, pyrazolyl, pyridyl, oxazolyl,
oxadiazolyl, thiadiazolyl, thiazolyl, imidazolyl, triazolyl,
tetrazolyl, furanyl, triazinyl, thienyl, pyrimidyl, pyridazinyl,
pyrazinyl, benzoxazolyl, benzothiazolyl, benzimidazolyl,
benzofuranyl, benzothiophenyl, benzopyrazolyl, benzotriazolyl,
furo(2,3-b)pyridyl, benzoxazinyl, tetrahydrohydroquinolinyl,
tetrahydroisoquinolinyl, quinolyl, isoquinolyl, indolyl,
dihydroindolyl, quinoxalinyl, quinazolinyl, naphthyridinyl,
pteridinyl, 2,3-dihydrofuro(2,3-b)pyridyl and the like. Heteroaryl
also includes aromatic carbocyclic or heterocyclic groups fused to
heterocycles that are non-aromatic or partially aromatic, and
optionally containing a carbonyl. Examples of additional heteroaryl
groups include indolinyl, dihydrobenzofuranyl,
dihydrobenzothiophenyl, dihydrobenzoxazolyl, and aromatic
heterocyclic groups fused to cycloalkyl rings. Examples also
include the following: ##STR4## Heteroaryl also includes such
groups in charged form, e.g., pyridinium.
[0042] "Heterocyclyl" (Hetcy) unless otherwise specified, means
mono- and bicyclic saturated and partially saturated rings and ring
systems containing at least one heteroatom selected from N, S and
O, each of said ring having from 3 to 10 atoms in which the point
of attachment may be carbon or nitrogen. Examples of "heterocyclyl"
include, but are not limited to, azetidinyl, pyrrolidinyl,
piperidinyl, piperazinyl, imidazolidinyl, tetrahydrofuranyl,
1,4-dioxanyl, morpholinyl, thiomorpholinyl, tetrahydrothienyl and
the like. Heterocycles can also exist in tautomeric forms, e.g., 2-
and 4-pyridones. Heterocycles moreover includes such moieties in
charged form, e.g., piperidinium.
[0043] "Halogen" (Halo) includes fluorine, chlorine, bromine and
iodine.
[0044] The phrase "in the absence of substantial flushing" refers
to the side effect that is often seen when nicotinic acid is
administered in therapeutic amounts. The flushing effect of
nicotinic acid usually becomes less frequent and less severe as the
patient develops tolerance to the drug at therapeutic doses, but
the flushing effect still occurs to some extent and can be
transient. Thus, "in the absence of substantial flushing" refers to
the reduced severity of flushing when it occurs, or fewer flushing
events than would otherwise occur. Preferably, the incidence of
flushing (relative to niacin) is reduced by at least about a third,
more preferably the incidence is reduced by half, and most
preferably, the flushing incidence is reduced by about two thirds
or more. Likewise, the severity (relative to niacin) is preferably
reduced by at least about a third, more preferably by at least
half, and most preferably by at least about two thirds. Clearly a
one hundred percent reduction in flushing incidence and severity is
most preferable, but is not required.
[0045] One aspect of the invention relates to a compound
represented by formula I: ##STR5## or a pharmaceutically acceptable
salt or solvate thereof is disclosed wherein:
[0046] X represents CH.sub.2, O, S, S(O), SO.sub.2 or NH, such that
when X represents NH, the nitrogen atom may be optionally
substituted with R.sup.6, C(O)R.sup.6, or SO.sub.2R.sup.6,
wherein:
[0047] R.sup.6 represents C.sub.1-3alkyl optionally substituted
with 1-3 groups, 0-3 of which are halo, and 0-1 of which are
selected from the group consisting of: OC.sub.1-3alkyl, OH,
NH.sub.2, NHC.sub.1-3alkyl, N(C.sub.1-3alkyl).sub.2, CN, Hetcy,
Aryl and HAR,
[0048] said Aryl and HAR being further optionally substituted with
1-3 groups, 1-3 of which are halo, and 0-1 of which are selected
from the group consisting of: OH, NH.sub.2, C.sub.1-3alkyl,
C.sub.1-3alkoxy, haloC.sub.1-3alkyl and haloC.sub.1-3alkoxy
groups;
[0049] a and b are each integers 1, 2 or 3, such that the sum of a
and b is 2, 3 or 4;
[0050] ring A represents a 6-10 membered aryl, a 5-13 membered
heteroaryl or a partially aromatic heterocyclic group, said
heteroaryl and partially aromatic heterocyclic group containing at
least one heteroatom selected from O, S, S(O), S(O).sub.2 and N,
and optionally containing 1 other heteroatom selected from O and S,
and optionally containing 1-3 additional N atoms, with up to 5
heteroatoms being present;
[0051] each R.sup.2 and R.sup.3 is independently H, C.sub.1-3alkyl,
haloC.sub.1-3alkyl, OC.sub.1-3alkyl, haloC.sub.1-3alkoxy, OH or
F;
[0052] n represents an integer of from 1 to 5;
[0053] each R.sup.4 is H or is independently selected from halo and
R.sup.6;
[0054] R.sup.5 represents --CO.sub.2H, ##STR6## or
--C(O)NHSO.sub.2R.sup.e wherein R.sup.e represents C.sub.1-4alkyl
or phenyl, said C.sub.1-4alkyl and phenyl each being optionally
substituted with 1-3 groups, 1-3 of which are selected from halo
and C.sub.1-3alkyl, and 1-2 of which are selected from the group
consisting of: OC.sub.1-3alkyl, haloC.sub.1-3alkyl,
haloC.sub.1-3alkoxy, OH, NH.sub.2 and NHC.sub.1-3alkyl;
[0055] and each R.sup.1 is H or is independently selected from the
group consisting of:
[0056] a) halo, OH, CO.sub.2H, CN, NH.sub.2, S(O).sub.0-2R.sup.e,
C(O)R.sup.e, OC(O)R.sup.e and CO.sub.2R.sup.e, wherein R.sup.e is
as previously defined;
[0057] b) C.sub.1-6 alkyl and OC.sub.1-6alkyl, said C.sub.1-6alkyl
and alkyl portion of OC.sub.1-6alkyl being optionally substituted
with 1-3 groups, 1-3 of which are halo and 1-2 of which are
selected from: OH, CO.sub.2H, CO.sub.2C.sub.1-4alkyl,
CO.sub.2C.sub.1-4haloalkyl, OCO.sub.2C.sub.1-4alkyl, NH.sub.2,
NHC.sub.1-4alkyl, N(C.sub.1-4alkyl).sub.2, Hetcy and CN;
[0058] c) NHC.sub.1-4alkyl and N(C.sub.1-4alkyl).sub.2, the alkyl
portions of which are optionally substituted as set forth in (b)
above;
[0059] d) C(O)NH.sub.2, C(O)NHC.sub.1-4alkyl,
C(O)N(C.sub.1-4alkyl).sub.2, C(O)Hetcy, C(O)NHOC.sub.1-4alkyl and
C(O)N(C.sub.1-4alkyl)(OC.sub.1-4alkyl), the alkyl portions of which
are optionally substituted as set forth in (b) above;
[0060] e) NR'C(O)R'', NR'SO.sub.2R'', NR'CO.sub.2R'' and
NR'C(O)NR''R''' wherein:
[0061] R' represents H, C.sub.1-3alkyl or haloC.sub.1-3alkyl,
[0062] R'' represents (a) C.sub.1-8alkyl optionally substituted
with 1-4 groups, 0-4 of which are halo, and 0-1 of which are
selected from the group consisting of: OC.sub.1-6alkyl, OH,
CO.sub.2H, CO.sub.2C.sub.1-4alkyl, CO.sub.2C.sub.1-4haloalkyl,
NH.sub.2, NHC.sub.1-4alkyl, N(C.sub.1-4alkyl).sub.2, CN, Hetcy,
Aryl and HAR, [0063] said Hetcy, Aryl and HAR being further
optionally substituted with 1-3 halo, C.sub.1-4alkyl,
C.sub.1-4alkoxy, haloC.sub.1-4alkyl or haloC.sub.1-4alkoxy groups;
or [0064] (b) Hetcy, Aryl or HAR, each being optionally substituted
with 1-3 members selected from the group consisting of: halo,
C.sub.1-4alkyl, C.sub.1-4alkoxy, haloC.sub.1-4alkyl and
haloC.sub.1-4alkoxy groups;
[0065] and R''' representing H or R'';
[0066] f) phenyl or a 5-6 membered heteroaryl or a Hetcy group
attached at any available ring atom and each being optionally
substituted with 1-3 groups, 1-3 of which are selected from halo,
C.sub.1-3alkyl and haloC.sub.1-3alkyl groups, and 1-2 of which are
selected from OC.sub.1-3alkyl and haloOC.sub.1-3alkyl groups, and
0-1 of which is selected from the group consisting of: [0067] i)
OH; CO.sub.2H; CN; NH.sub.2 and S(O).sub.0-2R.sup.e wherein R.sup.e
is as described above; [0068] ii) NHC.sub.1-4alkyl and
N(C.sub.1-4alkyl).sub.2, the alkyl portions of which are optionally
substituted with 1-3 groups, 1-3 of which are halo and 1-2 of which
are selected from: OH, CO.sub.2H, CO.sub.2C.sub.1-4alkyl,
CO.sub.2C.sub.1-4haloalkyl, NH.sub.2, NHC.sub.1-4alkyl,
N(C.sub.1-4alkyl).sub.2 and CN; [0069] iii) C(O)NH.sub.2,
C(O)NHC.sub.1-4alkyl, C(O)N(C.sub.1-4alkyl).sub.2,
C(O)NHOC.sub.1-4alkyl and C(O)N(C.sub.1-4alkyl)(OC.sub.1-4alkyl),
the alkyl portions of which are optionally substituted as set forth
in b) above; and [0070] iv) NR'C(O)R'', NR'SO.sub.2R'',
NR'CO.sub.2R'' and NR'C(O)NR''R''' wherein R', R'' and R''' are as
described above.
[0071] One aspect of the invention that is of interest relates to a
compound of formula I wherein up to 4 R.sup.2 and R.sup.3 moieties
are selected from the group consisting of: C.sub.1-3alkyl,
haloC.sub.1-3alkyl, OC.sub.1-3alkyl, haloC.sub.1-3alkoxy, OH and F,
and any remaining R.sup.2 and R.sup.3 moieties represent H.
[0072] Another subset of compounds that is of interest relates to
compounds of formula I wherein ring A is a phenyl or naphthyl
group, a 5-6 membered monocyclic heteroaryl group or a 9-13
membered bicyclic or tricyclic heteroaryl group. Within this subset
of compounds, all other variables are as defined with respect to
formula I.
[0073] More particularly, a subset of compounds that is of interest
relates to compounds of formula I wherein ring A is selected from
the group consisting of: phenyl; naphthyl;
[0074] HAR which is a member selected from the group consisting of:
pyrrolyl, isoxazolyl, isothiazolyl, pyrazolyl, pyridyl, oxazolyl,
oxadiazolyl, thiadiazolyl, thiazolyl, imidazolyl, triazolyl,
tetrazolyl, furanyl, triazinyl, thienyl, pyrimidyl, pyridazinyl,
pyrazinyl, benzoxazolyl, benzothiazolyl, benzimidazolyl,
benzofuranyl, benzothiophenyl, benzopyrazolyl, benzotriazolyl,
furo(2,3-b)pyridyl, benzoxazinyl, tetrahydrohydroquinolinyl,
tetrahydroisoquinolinyl, quinolyl, isoquinolyl, indolyl,
dihydroindolyl, quinoxalinyl, quinazolinyl, naphthyridinyl,
pteridinyl, 2,3-dihydrofuro(2,3-b)pyridyl indolinyl,
dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl,
or a member selected from the group consisting of: ##STR7##
[0075] Even more particularly, an aspect of the invention that is
of interest relates to a compound of formula I wherein ring A is
selected from the group consisting of: phenyl; naphthyl;
[0076] HAR which is a member selected from the group consisting of:
isoxazolyl, pyrazolyl, oxazolyl, oxadiazolyl, thiazolyl, triazolyl,
thienyl, benzothiazolyl, or a member selected from the group
consisting of: ##STR8## Within this subset of compounds, all other
variables are as defined with respect to formula I.
[0077] Another subset of compounds that is of interest relates to
compounds of formula I wherein each R.sup.1 is H or is selected
from the group consisting of:
[0078] a) halo, OH, CN, NH.sub.2 and S(O).sub.0-2R.sup.e wherein
R.sup.e is methyl or phenyl optionally substituted with 1-3 halo
groups;
[0079] b) C.sub.1-3 alkyl and OC.sub.1-3alkyl, each being
optionally substituted with 1-3 groups, 1-3 of which are halo and
1-2 of which are selected from: OH, NH.sub.2, NHC.sub.1-4alkyl and
CN;
[0080] c) NR'SO.sub.2R'' and NR'C(O)NR''R''' wherein:
[0081] R' represents H, C.sub.1-3alkyl or haloC.sub.1-3alkyl,
[0082] R'' represents (a) C.sub.1-8alkyl optionally substituted
with 1-4 groups, 0-4 of which are halo, and 0-1 of which are
selected from the group consisting of: OC.sub.1-6alkyl, OH,
CO.sub.2H, CO.sub.2C.sub.1-4alkyl, CO.sub.2C.sub.1-4haloalkyl,
OCO.sub.2C.sub.1-4alkyl, NH.sub.2, NHC.sub.1-4alkyl,
N(C.sub.1-4alkyl).sub.2, CN, Hetcy, Aryl and HAR, [0083] said
Hetcy, Aryl and HAR being further optionally substituted with 1-3
groups selected from: halo, C.sub.1-4alkyl, C.sub.1-4alkoxy,
haloC.sub.1-4alkyl and haloC.sub.1-4alkoxy; [0084] (b) Hetcy, Aryl
or HAR, said Aryl and HAR being optionally substituted with 1-3
groups selected from: halo, C.sub.1-4alkyl, C.sub.1-4alkoxy,
haloC.sub.1-4alkyl and haloC.sub.1-4alkoxy;
[0085] and R''' representing H or R''; and
[0086] d) phenyl or a 5-6 membered heteroaryl or a heterocyclic
group attached at any available point and being optionally
substituted with 1-3 groups, 1-3 of which are halo, C.sub.1-3alkyl
or haloC.sub.1-3alkyl groups, 1-2 of which are OC.sub.1-3alkyl or
haloOC.sub.1-3alkyl groups, and 1 of which is selected from the
group consisting of: [0087] i) OH; CO.sub.2H; CN; NH.sub.2 and
S(O).sub.2R.sup.e wherein R.sup.e is as described above; [0088] ii)
NHC.sub.1-4alkyl, the alkyl portion of which is optionally
substituted with 1-3 groups, 1-3 of which are halo and 1 of which
is selected from: OH, CO.sub.2H, CO.sub.2C.sub.1-4alkyl,
CO.sub.2C.sub.1-4haloalkyl, NH.sub.2, NHC.sub.1-4alkyl,
N(C.sub.1-4alkyl).sub.2 and CN; [0089] iii) C(O)NH.sub.2,
C(O)NHC.sub.1-4alkyl and C(O)N(C.sub.1-4alkyl).sub.2, the alkyl
portions of which are optionally substituted as set forth in (b)
above; and [0090] iv) NR'C(O)R'' and NR'SO.sub.2R'' wherein R' and
R'' are as described above. Within this subset of compounds, all
other variables are as defined with respect to formula I.
[0091] In particular, another subset of compounds that is of
interest relates to compounds of formula I wherein each R.sup.1 is
H or is selected from the group consisting of:
[0092] a) halo, OH, CN and NH.sub.2;
[0093] b) C.sub.1-3alkyl and OC.sub.1-3alkyl, each being optionally
substituted with 1-3 groups, 1-3 of which are halo and 1-2 of which
are selected from: OH, NH.sub.2, NHC.sub.1-4alkyl and CN;
[0094] c) phenyl or a 5-6 membered heteroaryl or a heterocyclic
group attached at any available point and being optionally
substituted with 1-3 groups, 1-3 of which are halo, C.sub.1-3alkyl
or haloC.sub.1-3alkyl groups, 1-2 of which are OC.sub.1-3alkyl or
haloOC.sub.1-3alkyl groups, and 1 of which is selected from the
group consisting of: [0095] i) OH, CN and NH.sub.2. Within this
subset of compounds, all other variables are as defined with
respect to formula I.
[0096] Another subset of compounds that is of interest relates to
compounds of formula I wherein a and b are 1 or 2 such that the sum
of a and b is 2 or 3. Within this subset of compounds, all other
variables are as defined with respect to formula I.
[0097] Another subset of compounds that is of interest relates to
compounds of formula I wherein X represents O, S, N or CH.sub.2.
Within this subset of compounds, all other variables are as defined
with respect to formula I.
[0098] More particularly, another subset of compounds that is of
interest relates to compounds of formula I wherein X represents O
or CH.sub.2. Within this subset of compounds, all other variables
are as defined with respect to formula I.
[0099] Another subset of compounds that is of interest relates to
compounds of formula I wherein R.sup.2 and R.sup.3 are
independently H, C.sub.1-3alkyl, OH or haloC.sub.1-3alkyl. Within
this subset of compounds, all other variables are as defined with
respect to formula I.
[0100] More particularly, another subset of compounds that is of
interest relates to compounds of formula I wherein R.sup.2 and
R.sup.3 are independently H, C.sub.1-3alkyl or haloC.sub.1-3alkyl.
Within this subset of compounds, all other variables are as defined
with respect to formula I.
[0101] More particularly, a subset of compounds that is of interest
relates to compounds of formula I wherein R.sup.2 and R.sup.3 are
independently H or methyl. Within this subset of compounds, all
other variables are as defined with respect to formula I.
[0102] Another subset of compounds that is of interest relates to
compounds of formula I wherein n represents an integer of from 2 to
4. Within this subset of compounds, all other variables are as
defined with respect to formula I.
[0103] More particularly, a subset of compounds that is of interest
relates to compounds of formula I wherein n is 2. Within this
subset of compounds, all other variables are as defined with
respect to formula I.
[0104] Another subset of compounds that is of interest relates to
compounds of formula I wherein each R.sup.4 is H or is
independently selected from the group consisting of: halo,
C.sub.1-3alkyl optionally substituted with 1-3 halo groups and 0-1
OC.sub.1-3alkyl groups. Within this subset of compounds, all other
variables are as defined with respect to formula I.
[0105] Another subset of compounds that is of interest relates to
compounds of formula I wherein each R.sup.4 is H or is
independently selected from halo or C.sub.1-3alkyl optionally
substituted with 1-3 halo groups. Within this subset of compounds,
all other variables are as defined with respect to formula I.
[0106] Another subset of compounds that is of interest relates to
compounds of formula I wherein R.sup.5 represents --CO.sub.2H.
Within this subset of compounds, all other variables are as defined
with respect to formula I.
[0107] A particular subset of compounds that is of interest relates
to compounds of formula I or a pharmaceutically acceptable salt or
solvate thereof wherein:
[0108] ring A is a phenyl or naphthyl group, a 5-6 membered
monocyclic heteroaryl group or a 9-13 membered bicyclic or
tricyclic heteroaryl group;
[0109] each R.sup.1 is H or is selected from the group consisting
of:
[0110] a) halo, OH, CN, NH.sub.2 and S(O).sub.0-2R.sup.e wherein
R.sup.e is methyl or phenyl optionally substituted with 1-3 halo
groups;
[0111] b) C.sub.1-3 alkyl and OC.sub.1-3alkyl, each being
optionally substituted with 1-3 groups, 1-3 of which are halo and
1-2 of which are selected from: OH, NH.sub.2, NHC.sub.1-4alkyl and
CN;
[0112] c) NR'SO.sub.2R'' and NR'C(O)NR''R''' wherein:
[0113] R' represents H, C.sub.1-3alkyl or haloC.sub.1-3alkyl,
[0114] R'' represents (a) C.sub.1-8alkyl optionally substituted
with 1-4 groups, 0-4 of which are halo, and 0-1 of which are
selected from the group consisting of: OC.sub.1-6alkyl, OH,
CO.sub.2H, CO.sub.2C.sub.1-4alkyl, CO.sub.2C.sub.1-4haloalkyl,
OCO.sub.2C.sub.1-4alkyl, NH.sub.2, NHC.sub.1-4alkyl,
N(C.sub.1-4alkyl).sub.2, CN, Hetcy, Aryl and HAR, [0115] said
Hetcy, Aryl and HAR being further optionally substituted with 1-3
halo, C.sub.1-4alkyl, C.sub.1-4alkoxy, haloC.sub.1-4alkyl and
haloC.sub.1-4alkoxy groups; [0116] (b) Hetcy, Aryl or HAR, said
Aryl and HAR being further optionally substituted with 1-3 halo,
C.sub.1-4alkyl, C.sub.1-4alkoxy, haloC.sub.1-4alkyl and
haloC.sub.1-4alkoxy groups;
[0117] and R''' representing H or R''; and
[0118] d) phenyl or a 5-6 membered heteroaryl or a heterocyclic
group attached at any available point and being optionally
substituted with 1-3 groups, 1-3 of which are halo, C.sub.1-3alkyl
or haloC.sub.1-3alkyl groups, 1-2 of which are OC.sub.1-3alkyl or
haloOC.sub.1-3alkyl groups, and 1 of which is selected from the
group consisting of: [0119] i) OH; CO.sub.2H; CN; NH.sub.2;
S(O).sub.2R.sup.e wherein R.sup.e is as described above; [0120] ii)
NHC.sub.1-4alkyl the alkyl portion of which is optionally
substituted with 1-3 groups, 1-3 of which are halo and 1 of which
is selected from: OH, CO.sub.2H, CO.sub.2C.sub.1-4alkyl,
CO.sub.2C.sub.1-4haloalkyl, NH.sub.2, NHC.sub.1-4alkyl,
N(C.sub.1-4alkyl).sub.2 and CN; [0121] iii) C(O)NH.sub.2,
C(O)NHC.sub.1-4alkyl, C(O)N(C.sub.1-4alkyl).sub.2, the alkyl
portions of which are optionally substituted as set forth in (b)
above; and [0122] iv) NR'C(O)R'' and NR'SO.sub.2R'' wherein R' and
R'' are as described above;
[0123] a and b are 1 or 2 such that the sum of a and b is 2 or
3;
[0124] X represents O or CH.sub.2;
[0125] R.sup.2 and R.sup.3 are independently H, OH, C.sub.1-3alkyl
or haloC.sub.1-3alkyl;
[0126] n represents 2;
[0127] R.sup.4 is H or is independently selected from the group
consisting of: halo, C.sub.1-3alkyl optionally substituted with 1-3
halo groups or 0-1 OC.sub.1-3alkyl groups; and
[0128] R.sup.5 represents --CO.sub.2H.
[0129] A more particular subset of compounds that is of interest
relates to compounds of formula I or a pharmaceutically acceptable
salt of solvate thereof wherein:
[0130] ring A is selected from the group consisting of:
##STR9##
[0131] each R.sup.1 is independently H, CH.sub.3, phenyl,
4-hydroxy-phenyl, OH, 2-hydroxy-phenyl, 3-hydroxy-phenyl,
3-amino-phenyl, 2,3-dihydro-benzofuran-6-yl,
2-chloro-4-hydroxy-phenyl, 1H-pyrazol-4-yl, 5-hydroxy-pyridin-2-yl,
4-hydroxy-pyrazol-1-yl, 1H-[1,2,3]triazol-4-yl, or
5-fluoro-pyridin-2-yl;
[0132] a and b are 1 or 2 such that the sum of a and b is 2 or
3;
[0133] X represents CH.sub.2;
[0134] each R.sup.2 and R.sup.3 is independently H, OH or
CH.sub.3;
[0135] n represents 2;
[0136] R.sup.4 is H, CH.sub.3, CH.sub.2CH.sub.3, CF.sub.3 or
CH.sub.2OCH.sub.3; and
[0137] R.sup.5 represents --CO.sub.2H.
[0138] Representative examples of species that are of interest are
shown below in Table I. Within this subset of compounds, all other
variables are as originally defined with respect to formula I.
TABLE-US-00001 TABLE 1 Compound 1 ##STR10## Compound 2 ##STR11##
Compound 3 ##STR12## Compound 4 ##STR13## Compound 5 ##STR14##
Compound 6 ##STR15## Compound 7 ##STR16## Compound 8 ##STR17##
Compound 9 ##STR18## Compound 10 ##STR19## Compound 11 ##STR20##
Compound 12 ##STR21## Compound 13 ##STR22## Compound 14 ##STR23##
Compound 15 ##STR24## Compound 16 ##STR25## Compound 17 ##STR26##
Compound 18 ##STR27## Compound 19 ##STR28## Compound 20 ##STR29##
Compound 21 ##STR30## Compound 22 ##STR31## Compound 23 ##STR32##
Compound 24 ##STR33## Compound 25 ##STR34## Compound 26 ##STR35##
Compound 27 ##STR36## Compound 28 ##STR37## Compound 29 ##STR38##
Compound 30 ##STR39## Compound 31 ##STR40## Compound 32 ##STR41##
Compound 33 ##STR42## Compound 34 ##STR43## Compound 35 ##STR44##
Compound 36 ##STR45## Compound 37 ##STR46## Compound 38 ##STR47##
Compound 39 ##STR48## Compound 40 ##STR49## Compound 41
##STR50##
[0139] Pharmaceutically acceptable salts and solvates thereof are
included as well.
[0140] Many of the compounds of formula I contain asymmetric
centers and can thus occur as racemates and racemic mixtures,
single enantiomers, diastereomeric mixtures and individual
diastereomers. All such isomeric forms are included.
[0141] Moreover, chiral compounds possessing one stereocenter of
general formula I, may be resolved into their enantiomers in the
presence of a chiral environment using methods known to those
skilled in the art. Chiral compounds possessing more than one
stereocenter may be separated into their diastereomers in an
achiral environment on the basis of their physical properties using
methods known to those skilled in the art. Single diastereomers
that are obtained in racemic form may be resolved into their
enantiomers as described above.
[0142] If desired, racemic mixtures of compounds may be separated
so that individual enantiomers are isolated. The separation can be
carried out by methods well known in the art, such as the coupling
of a racemic mixture of compounds of Formula I to an
enantiomerically pure compound to form a diastereomeric mixture,
which is then separated into individual diastereomers by standard
methods, such as fractional crystallization or chromatography. The
coupling reaction is often the formation of salts using an
enantiomerically pure acid or base. The diasteromeric derivatives
may then be converted to substantially pure enantiomers by cleaving
the added chiral residue from the diastereomeric compound.
[0143] The racemic mixture of the compounds of Formula I can also
be separated directly by chromatographic methods utilizing chiral
stationary phases, which methods are well known in the art.
[0144] Alternatively, enantiomers of compounds of the general
Formula I may be obtained by stereoselective synthesis using
optically pure starting materials or reagents.
[0145] Some of the compounds described herein exist as tautomers,
which have different points of attachment for hydrogen accompanied
by one or more double bond shifts. For example, a ketone and its
enol form are keto-enol tautomers. Or for example, a
2-hydroxyquinoline can reside in the tautomeric 2-quinolone form.
The individual tautomers as well as mixtures thereof are
included.
Dosing Information
[0146] The dosages of compounds of formula I or a pharmaceutically
acceptable salt or solvate thereof vary within wide limits. The
specific dosage regimen and levels for any particular patient will
depend upon a variety of factors including the age, body weight,
general health, sex, diet, time of administration, route of
administration, rate of excretion, drug combination and the
severity of the patient's condition. Consideration of these factors
is well within the purview of the ordinarily skilled clinician for
the purpose of determining the therapeutically effective or
prophylactically effective dosage amount needed to prevent,
counter, or arrest the progress of the condition. Generally, the
compounds will be administered in amounts ranging from as low as
about 0.01 mg/day to as high as about 2000 mg/day, in single or
divided doses. A representative dosage is about 0.1 mg/day to about
1 g/day. Lower dosages can be used initially, and dosages increased
to further minimize any untoward effects. It is expected that the
compounds described herein will be administered on a daily basis
for a length of time appropriate to treat or prevent the medical
condition relevant to the patient, including a course of therapy
lasting months, years or the life of the patient.
Combination Therapy
[0147] One or more additional active agents may be administered
with the compounds described herein. The additional active agent or
agents can be lipid modifying compounds or agents having other
pharmaceutical activities, or agents that have both lipid-modifying
effects and other pharmaceutical activities. Examples of additional
active agents which may be employed include but are not limited to
HMG-CoA reductase inhibitors, which include statins in their
lactonized or dihydroxy open acid forms and pharmaceutically
acceptable salts and esters thereof, including but not limited to
lovastatin (see U.S. Pat. No. 4,342,767), simvastatin (see U.S.
Pat. No. 4,444,784), dihydroxy open-acid simvastatin, particularly
the ammonium or calcium salts thereof, pravastatin, particularly
the sodium salt thereof (see U.S. Pat. No. 4,346,227), fluvastatin
particularly the sodium salt thereof (see U.S. Pat. No. 5,354,772),
atorvastatin, particularly the calcium salt thereof (see U.S. Pat.
No. 5,273,995), pitavastatin also referred to as NK-104 (see PCT
international publication number WO 97/23200) and rosuvastatin,
also known as CRESTOR.RTM.; see U.S. Pat. No. 5,260,440); HMG-CoA
synthase inhibitors; squalene epoxidase inhibitors; squalene
synthetase inhibitors (also known as squalene synthase inhibitors),
acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitors
including selective inhibitors of ACAT-1 or ACAT-2 as well as dual
inhibitors of ACAT-1 and -2; microsomal triglyceride transfer
protein (MTP) inhibitors; endothelial lipase inhibitors; bile acid
sequestrants; LDL receptor inducers; platelet aggregation
inhibitors, for example glycoprotein IIb/IIIa fibrinogen receptor
antagonists and aspirin; human peroxisome proliferator activated
receptor gamma (PPAR-gamma) agonists including the compounds
commonly referred to as glitazones for example pioglitazone and
rosiglitazone and, including those compounds included within the
structural class known as thiazolidine diones as well as those
PPAR-gamma agonists outside the thiazolidine dione structural
class; PPAR-alpha agonists such as clofibrate, fenofibrate
including micronized fenofibrate, and gemfibrozil; PPAR dual
alpha/gamma agonists; vitamin B.sub.6 (also known as pyridoxine)
and the pharmaceutically acceptable salts thereof such as the HCl
salt; vitamin B.sub.12 (also known as cyanocobalamin); folic acid
or a pharmaceutically acceptable salt or ester thereof such as the
sodium salt and the methylglucamine salt; anti-oxidant vitamins
such as vitamin C and E and beta carotene; beta-blockers;
angiotensin II antagonists such as losartan; angiotensin converting
enzyme inhibitors such as enalapril and captopril; renin
inhibitors, calcium channel blockers such as nifedipine and
diltiazem; endothelin antagonists; agents that enhance ABCA1 gene
expression; cholesteryl ester transfer protein (CETP) inhibiting
compounds, 5-lipoxygenase activating protein (FLAP) inhibiting
compounds, 5-lipoxygenase (5-LO) inhibiting compounds, farnesoid X
receptor (FXR) ligands including both antagonists and agonists;
Liver X Receptor (LXR)-alpha ligands, LXR-beta ligands,
bisphosphonate compounds such as alendronate sodium;
cyclooxygenase-2 inhibitors such as rofecoxib and celecoxib; and
compounds that attenuate vascular inflammation.
[0148] Cholesterol absorption inhibitors can also be used in the
present invention. Such compounds block the movement of cholesterol
from the intestinal lumen into enterocytes of the small intestinal
wall, thus reducing serum cholesterol levels. Examples of
cholesterol absorption inhibitors are described in U.S. Pat. Nos.
5,846,966, 5,631,365, 5,767,115, 6,133,001, 5,886,171, 5,856,473,
5,756,470, 5,739,321, 5,919,672, and in PCT application Nos. WO
00/63703, WO 00/60107, WO 00/38725, WO 00/34240, WO 00/20623, WO
97/45406, WO 97/16424, WO 97/16455, and WO 95/08532. The most
notable cholesterol absorption inhibitor is ezetimibe, also known
as
1-(4-fluorophenyl)-3(R)-[3(S)-(4-fluorophenyl)-3-hydroxypropyl)]-4(S)-(4--
hydroxyphenyl)-2-azetidinone, described in U.S. Pat. Nos. 5,767,115
and 5,846,966.
[0149] Therapeutically effective amounts of cholesterol absorption
inhibitors include dosages of from about 0.01 mg/kg to about 30
mg/kg of body weight per day, preferably about 0.1 mg/kg to about
15 mg/kg.
[0150] For diabetic patients, the compounds used in the present
invention can be administered with conventional diabetic
medications. For example, a diabetic patient receiving treatment as
described herein may also be taking insulin or an oral antidiabetic
medication. One example of an oral antidiabetic medication useful
herein is metformin.
[0151] In the event that these niacin receptor agonists induce some
degree of vasodilation, it is understood that the compounds of
formula I may be co-dosed with a vasodilation suppressing agent.
Consequently, one aspect of the methods described herein relates to
the use of a compound of formula I or a pharmaceutically acceptable
salt or solvate thereof in combination with a compound that reduces
flushing. Conventional compounds such as aspirin, ibuprofen,
naproxen, indomethacin, other NSAIDs, COX-2 selective inhibitors
and the like are useful in this regard, at conventional doses.
Alternatively, DP antagonists are useful as well. Doses of the DP
receptor antagonist and selectivity are such that the DP antagonist
selectively modulates the DP receptor without substantially
modulating the CRTH2 receptor. In particular, the DP receptor
antagonist ideally has an affinity at the DP receptor (i.e.,
K.sub.i) that is at least about 10 times higher (a numerically
lower K.sub.i value) than the affinity at the CRTH2 receptor. Any
compound that selectively interacts with DP according to these
guidelines is deemed "DP selective". This is in accordance with US
Published Application No. 2004/0229844A1 published on Nov. 18,
2004, incorporated herein by reference.
[0152] Dosages for DP antagonists as described herein, that are
useful for reducing or preventing the flushing effect in mammalian
patients, particularly humans, include dosages ranging from as low
as about 0.01 mg/day to as high as about 100 mg/day, administered
in single or divided daily doses. Preferably the dosages are from
about 0.1 mg/day to as high as about 1.0 g/day, in single or
divided daily doses.
[0153] Examples of compounds that are particularly useful for
selectively antagonizing DP receptors and suppressing the flushing
effect include the following: ##STR51## ##STR52## ##STR53##
##STR54## ##STR55## ##STR56## as well as the pharmaceutically
acceptable salts and solvates thereof.
[0154] The compound of formula I or a pharmaceutically acceptable
salt or solvate thereof and the DP antagonist can be administered
together or sequentially in single or multiple daily doses, e.g.,
bid, tid or qid, without departing from the invention. If sustained
release is desired, such as a sustained release product showing a
release profile that extends beyond 24 hours, dosages may be
administered every other day. However, single daily doses are
preferred. Likewise, morning or evening dosages can be
utilized.
Salts and Solvates
[0155] Salts and solvates of the compounds of formula I are also
included in the present invention, and numerous pharmaceutically
acceptable salts and solvates of nicotinic acid are useful in this
regard. Alkali metal salts, in particular, sodium and potassium,
form salts that are useful as described herein. Likewise alkaline
earth metals, in particular, calcium and magnesium, form salts that
are useful as described herein. Various salts of amines, such as
ammonium and substituted ammonium compounds also form salts that
are useful as described herein. Similarly, solvated forms of the
compounds of formula I are useful within the present invention.
Examples include the hemihydrate, mono-, di-, tri- and
sesquihydrate.
[0156] The compounds of the invention also include esters that are
pharmaceutically acceptable, as well as those that are
metabolically labile. Metabolically labile esters include C.sub.1-4
alkyl esters, preferably the ethyl ester. Many prodrug strategies
are known to those skilled in the art. One such strategy involves
engineered amino acid anhydrides possessing pendant nucleophiles,
such as lysine, which can cyclize upon themselves, liberating the
free acid. Similarly, acetone-ketal diesters, which can break down
to acetone, an acid and the active acid, can be used.
[0157] The compounds used in the present invention can be
administered via any conventional route of administration. The
preferred route of administration is oral.
Pharmaceutical Compositions
[0158] The pharmaceutical compositions described herein are
generally comprised of a compound of formula I or a
pharmaceutically acceptable salt or solvate thereof, in combination
with a pharmaceutically acceptable carrier.
[0159] Examples of suitable oral compositions include tablets,
capsules, troches, lozenges, suspensions, dispersible powders or
granules, emulsions, syrups and elixirs. Examples of carrier
ingredients include diluents, binders, disintegrants, lubricants,
sweeteners, flavors, colorants, preservatives, and the like.
Examples of diluents include, for example, calcium carbonate,
sodium carbonate, lactose, calcium phosphate and sodium phosphate.
Examples of granulating and disintegrants include corn starch and
alginic acid. Examples of binding agents include starch, gelatin
and acacia. Examples of lubricants include magnesium stearate,
calcium stearate, stearic acid and talc. The tablets may be
uncoated or coated by known techniques. Such coatings may delay
disintegration and thus, absorption in the gastrointestinal tract
and thereby provide a sustained action over a longer period.
[0160] One embodiment of the invention that is of interest is a
tablet or capsule that is comprised of a compound of formula I or a
pharmaceutically acceptable salt or solvate thereof in an amount
ranging from about 0.1 mg to about 1000 mg, in combination with a
pharmaceutically acceptable carrier.
[0161] In another embodiment of the invention, a compound of
formula I or a pharmaceutically acceptable salt or solvate thereof
is combined with another therapeutic agent and the carrier to form
a fixed combination product. This fixed combination product may be
a tablet or capsule for oral use.
[0162] More particularly, in another embodiment of the invention, a
compound of formula I or a pharmaceutically acceptable salt or
solvate thereof (about 0.1 to about 1000 mg) and the second
therapeutic agent (about 0.1 to about 500 mg) are combined with the
pharmaceutically acceptable carrier, providing a tablet or capsule
for oral use.
[0163] Sustained release over a longer period of time may be
particularly important in the formulation. A time delay material
such as glyceryl monostearate or glyceryl distearate may be
employed. The dosage form may also be coated by the techniques
described in the U.S. Pat. Nos. 4,256,108; 4,166,452 and 4,265,874
to form osmotic therapeutic tablets for controlled release.
[0164] Other controlled release technologies are also available and
are included herein. Typical ingredients that are useful to slow
the release of nicotinic acid in sustained release tablets include
various cellulosic compounds, such as methylcellulose,
ethylcellulose, propylcellulose, hydroxypropylcellulose,
hydroxyethylcellulose, hydroxypropylmethylcellulose,
microcrystalline cellulose, starch and the like. Various natural
and synthetic materials are also of use in sustained release
formulations. Examples include alginic acid and various alginates,
polyvinyl pyrrolidone, tragacanth, locust bean gum, guar gum,
gelatin, various long chain alcohols, such as cetyl alcohol and
beeswax.
[0165] Optionally and of even more interest is a tablet as
described above, comprised of a compound of formula I or a
pharmaceutically acceptable salt or solvate thereof, and further
containing an HMG Co-A reductase inhibitor, such as simvastatin or
atorvastatin. This particular embodiment optionally contains the DP
antagonist as well.
[0166] Typical release time frames for sustained release tablets in
accordance with the present invention range from about 1 to as long
as about 48 hours, preferably about 4 to about 24 hours, and more
preferably about 8 to about 16 hours.
[0167] Hard gelatin capsules constitute another solid dosage form
for oral use. Such capsules similarly include the active
ingredients mixed with carrier materials as described above. Soft
gelatin capsules include the active ingredients mixed with
water-miscible solvents such as propylene glycol, PEG and ethanol,
or an oil such as peanut oil, liquid paraffin or olive oil.
[0168] Aqueous suspensions are also contemplated as containing the
active material in admixture with excipients suitable for the
manufacture of aqueous suspensions. Such excipients include
suspending agents, for example sodium carboxymethylcellulose,
methylcellulose, hydroxypropylmethylcellulose, sodium alginate,
polyvinylpyrrolidone, tragacanth and acacia; dispersing or wetting
agents, e.g., lecithin; preservatives, e.g., ethyl, or n-propyl
para-hydroxybenzoate, colorants, flavors, sweeteners and the
like.
[0169] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredients in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those
already mentioned above.
[0170] Syrups and elixirs may also be formulated.
[0171] More particularly, a pharmaceutical composition that is of
interest is a sustained release tablet that is comprised of a
compound of formula I or a pharmaceutically acceptable salt or
solvate thereof, and a DP receptor antagonist that is selected from
the group consisting of compounds A through AJ in combination with
a pharmaceutically acceptable carrier.
[0172] Yet another pharmaceutical composition that is of more
interest is comprised of a compound of formula I or a
pharmaceutically acceptable salt or solvate thereof and a DP
antagonist compound selected from the group consisting of compounds
A, B, D, E, X, AA, AF, AG, AH, AI and AJ, in combination with a
pharmaceutically acceptable carrier.
[0173] Yet another pharmaceutical composition that is of more
particular interest relates to a sustained release tablet that is
comprised of a compound of formula I or a pharmaceutically
acceptable salt or solvate thereof, a DP receptor antagonist
selected from the group consisting of compounds A, B, D, E, X, AA,
AF, AG, AH, AI and AJ, and simvastatin or atorvastatin in
combination with a pharmaceutically acceptable carrier.
[0174] The term "composition", in addition to encompassing the
pharmaceutical compositions described above, also encompasses any
product which results, directly or indirectly, from the
combination, complexation or aggregation of any two or more of the
ingredients, active or excipient, or from dissociation of one or
more of the ingredients, or from other types of reactions or
interactions of one or more of the ingredients. Accordingly, the
pharmaceutical composition of the present invention encompasses any
composition made by admixing or otherwise combining the compounds,
any additional active ingredient(s), and the pharmaceutically
acceptable excipients.
[0175] Another aspect of the invention relates to the use of a
compound of formula I or a pharmaceutically acceptable salt or
solvate thereof and a DP antagonist in the manufacture of a
medicament. This medicament has the uses described herein.
[0176] More particularly, another aspect of the invention relates
to the use of a compound of formula I or a pharmaceutically
acceptable salt or solvate thereof, a DP antagonist and an HMG Co-A
reductase inhibitor, such as simvastatin, in the manufacture of a
medicament. This medicament has the uses described herein.
[0177] Compounds of the present invention have anti-hyperlipidemic
activity, causing reductions in LDL-C, triglycerides, lipoprotein
(a), free fatty acids and total cholesterol, and increases in
HDL-C. Consequently, the compounds of the present invention are
useful in treating dyslipidemias. The present invention thus
relates to the treatment, prevention or reversal of atherosclerosis
and the other diseases and conditions described herein, by
administering a compound of formula I or a pharmaceutically
acceptable salt or solvate in an amount that is effective for
treating, preventing or reversing said condition. This is achieved
in humans by administering a compound of formula I or a
pharmaceutically acceptable salt or solvate thereof in an amount
that is effective to treat or prevent said condition, while
preventing, reducing or minimizing flushing effects in terms of
frequency and/or severity.
[0178] One aspect of the invention that is of interest relates to a
compound in accordance with formula I or a pharmaceutically
acceptable salt or solvate thereof for use in a method of treatment
of the human or animal body by therapy.
[0179] Another aspect of the invention that is of interest relates
to a compound in accordance with formula I or a pharmaceutically
acceptable salt or solvate thereof for use in a method for the
treatment of atherosclerosis, dyslipidemia, diabetes, metabolic
syndrome or a related condition in the human or animal body by
therapy.
[0180] More particularly, an aspect of the invention that is of
interest is a method of treating atherosclerosis in a human patient
in need of such treatment comprising administering to the patient a
compound of formula I or a pharmaceutically acceptable salt or
solvate thereof in an amount that is effective for treating
atherosclerosis in the absence of substantial flushing.
[0181] Another aspect of the invention that is of interest relates
to a method of raising serum HDL levels in a human patient in need
of such treatment, comprising administering to the patient a
compound of formula I or a pharmaceutically acceptable salt or
solvate thereof in an amount that is effective for raising serum
HDL levels.
[0182] Another aspect of the invention that is of interest relates
to a method of treating dyslipidemia in a human patient in need of
such treatment comprising administering to the patient a compound
of formula I or a pharmaceutically acceptable salt or solvate
thereof in an amount that is effective for treating
dyslipidemia.
[0183] Another aspect of the invention that is of interest relates
to a method of reducing serum VLDL or LDL levels in a human patient
in need of such treatment, comprising administering to the patient
a compound of formula I or a pharmaceutically acceptable salt or
solvate thereof in an amount that is effective for reducing serum
VLDL or LDL levels in the patient in the absence of substantial
flushing.
[0184] Another aspect of the invention that is of interest relates
to a method of reducing serum triglyceride levels in a human
patient in need of such treatment, comprising administering to the
patient a compound of formula I or a pharmaceutically acceptable
salt or solvate thereof in an amount that is effective for reducing
serum triglyceride levels.
[0185] Another aspect of the invention that is of interest relates
to a method of reducing serum Lp(a) levels in a human patient in
need of such treatment, comprising administering to the patient a
compound of formula I or a pharmaceutically acceptable salt or
solvate thereof in an amount that is effective for reducing serum
Lp(a) levels. As used herein Lp(a) refers to lipoprotein (a).
[0186] Another aspect of the invention that is of interest relates
to a method of treating diabetes, and in particular, type 2
diabetes, in a human patient in need of such treatment comprising
administering to the patient a compound of formula I or a
pharmaceutically acceptable salt or solvate thereof in an amount
that is effective for treating diabetes.
[0187] Another aspect of the invention that is of interest relates
to a method of treating metabolic syndrome in a human patient in
need of such treatment comprising administering to the patient a
compound of formula I or a pharmaceutically acceptable salt or
solvate thereof in an amount that is effective for treating
metabolic syndrome.
[0188] Another aspect of the invention that is of particular
interest relates to a method of treating atherosclerosis,
dyslipidemias, diabetes, metabolic syndrome or a related condition
in a human patient in need of such treatment, comprising
administering to the patient a compound of formula I or a
pharmaceutically acceptable salt or solvate thereof and a DP
receptor antagonist, said combination being administered in an
amount that is effective to treat atherosclerosis, dyslipidemia,
diabetes or a related condition in the absence of substantial
flushing.
[0189] Another aspect of the invention that is of particular
interest relates to the methods described above wherein the DP
receptor antagonist is selected from the group consisting of
compounds A through AJ and the pharmaceutically acceptable salts
and solvates thereof.
Methods of Synthesis for Compounds of Formula I
[0190] Compounds of Formula I have been prepared by the following
representative reaction schemes. It is understood that similar
reagents, conditions or other synthetic approaches to these
structure classes are conceivable to one skilled in the art of
organic synthesis. Therefore these reaction schemes should not be
construed as limiting the scope of the invention. All substituents
are as defined above unless indicated otherwise. ##STR57##
[0191] Compounds of Formula I, where X represents CH.sub.2, a and b
equal 1 and RCOOH represents: ##STR58## can be prepared as
illustrated in Scheme 1 by treatment of commercially available
methyl 2-oxocyclopentane-1-carboxylate 1 with ammonium acetate in a
polar solvent such a methanol or ethanol to give the methyl 2-amino
cyclopent-1-ene-1-carboxylate 2. The amine 2 can be coupled with
the appropriate acid in the presence of methanesulfonyl chloride
(MsCl) and DMAP to give the desired amide 3. Finally, the ester can
be saponified by one skilled in the art using such methods as NaOH
or LiOH-dioxane to give compounds with the structure 4.
##STR59##
[0192] Compounds of Formula I, where X represents CH.sub.2, a
represents 2, and b represents 1 such that the sum of a and b is 3,
can be prepared as illustrated in Scheme 2 by coupling commercially
available methyl or ethyl 2-amino-cyclo-hex-1-ene-1-carboxylate 5
or 6 with the appropriate acid in the presence of methanesulfonyl
chloride and DMAP to give the desired amide 7. The ester can be
saponified to the desired compound 8 by methods known to those
skilled in the art. ##STR60##
[0193] Shown in Scheme 3 is the preparation of acid of the
structure 10 from commercially available material 9 by methods
known to one skilled in the art, such as hydrogenation in a polar
solvent, such as methanol or ethanol, using Pd/C as a catalyst.
##STR61##
[0194] Compounds with the structure 19 can be prepared by the
chemistry outlined in Scheme 4. Thus, 6-methoxy-2-naphthaldehyde 11
can be treated with a suitable ylide such as
(tert-Butoxycarbonyl-methylene)triphenyl-phospharane in a non-polar
solvent such as toluene or xylenes under refluxing conditions to
give the desired olefin 12. Hydrogenation of the double bond can be
accomplished using standard conditions such as H.sub.2(g), Pd/C in
a suitable polar solvent like methanol or ethanol to give 13.
Removal of the methyl group in the methoxynaphthyl moiety can be
accomplished with boron tribromide at low temperature, followed by
a careful quenching of the reaction with methanol to give the
trans-esterified product 14. Saponification of the ester was
accomplished using conditions described earlier. The naphthol can
be protected as the TBS ether using TBSOTf or TBS-Cl in the
presence of a suitable base such as triethylamine or imidazole in
dichloromethane. The TBS ester can be hydrolyzed with a mild acid
such as acetic acid in THF--H.sub.2O to give the desired acid 17.
This acid can be coupled to the methyl or ethyl
2-aminocyclo-hex-1-ene-1-carboxylate in the presence of
methanesulfonyl chloride and DMAP to give the desired amide 18.
Finally, TBS ether removal and methyl ester saponification can be
achieved using NaOH/THF--H.sub.2O providing compounds of the
structure 19. ##STR62## ##STR63##
[0195] Compounds with the structure 28 can be prepared by the
chemistry outlined in Scheme 5. Thus, treating a suitable tetralone
such as 20 with LDA at low temperature followed by the addition of
a suitable acylating agent such as 4-chloro-4-oxobutyrate provides
the desired diketo-ester 21. The ester can be saponified using
standard conditions known to one skilled in the art to give the
acid 22. The di-ketone 22 can be converted to the fused isoxazole
of the structure 23 by refluxing with hydroxylamine hydrochloride
in the presence of a base such as triethylamine in an alcoholic
solvent such as methanol or ethanol. De-protection of the methyl
ether can be done with boron tribromide in a suitable solvent such
as dichloromethane to give the desired alcohol 24. Treatment of the
intermediate 24 with a silylating agent such as TBS-Cl in the
presence of a base such as imidazole or triethylamine in a
chlorinated solvent like DCM gives the bis-silyl protected ester
25. The silyl ester 25 can be treated with oxalyl chloride in a
solvent such as DCM under anhydrous conditions followed by coupling
with methyl 2-aminocyclo-pent-1-ene-1-carboxylate to give the
desired amide 26. The TBS group can be removed using aqueous TBAF.
Finally, the methyl ester can be saponified using standard
conditions to give compounds of the structure 28. ##STR64##
[0196] Scheme 6 outlines the strategy used to synthesize compounds
of the structure 32. Coupling commercially available methyl or
ethyl 2-aminocyclo-hex-1-ene-1-carboxylate 5 or 6 with
3-(3-bromophenyl) propionic acid 29 in the presence of
methanesulfonyl chloride and DMAP gives the desired amide 30. The
bromide 30 can be converted to 31 via a Suzuki reaction with a
suitable boronic acid such as 4-hydroxy phenyl boronic acid in the
presence of a catalyst such as Bis-tert-butyl-ferrocene palladium
dichloride. The ester can be saponified by methods known to those
skilled in the art providing compounds of the structure 32.
##STR65##
[0197] Scheme 7 outlines the strategy used to synthesize compounds
of the structure 37. Homologating aldehyde 33, followed by
reduction provides 34 which may be resolved into its enantiomers
via chiral HPLC. One enantiomer is shown in Scheme 7 for
illustrative purposes. Hydrolysis of the ethyl esters provides the
acid 35, followed by demethylation and silylation to give 36. This
intermediate can be acylated with the cyclopentene fragment, and
saponified to provide biaryl products such as 37. ##STR66##
##STR67##
[0198] Scheme 8 outlines the strategy used to synthesize compounds
of the structure 43. The aminobenzothiazole 38 may be N-alkylated
and cyclized to form intermediate 39. The ester can be reduced to
the aldehyde, and homologated to the enoate 40. This intermediate
can then be reduced and saponified to provide the acid 41.
Demethylation and silylation affords intermediate 42, which can be
acylated and saponified once again as in Scheme 7 above, to provide
products such as 43. ##STR68##
[0199] Scheme 9 outlines the strategy used to synthesize compounds
of the structure 50. Adiponitrile can be converted to the
amino-nitrile 45 via a Thorpe-Ziegler reaction using a suitable
base such as LDA in a solvent such as THF. Coupling the amino
nitrile 45 to 3-(4-bromophenyl) propionic acid 46 in the presence
of methanesulfonyl chloride and DMAP gives the desired amide 47.
The bromide 47 can be converted to 49 via a Suzuki reaction with a
suitable boronic acid such as phenyl boronic acid in the presence
of a catalyst such as 1,1 bis(di-tert-butylphosphino)-ferrocene
palladium dichloride. Finally, treatment of the nitrile 49 with
NaN.sub.3 in the presence of a Lewis acid such as zinc bromide, in
a suitable solvent mixture such as dioxane-water gives tetrazoles
such as 50. ##STR69##
[0200] Scheme 10 outlines the strategy used to synthesize compounds
of the structure 54. The heterocyclic bromo aldehyde 51 can be
homologated to the intermediate 52 via several transformations
including the displacement of an activated bromide with a malonate
anion. The bromide 52 can be arylated and demethylated to provide
acid 53, which in turn may be acylated and deprotected to provide
compounds such as 54. ##STR70##
[0201] Scheme 11 displays a method for generating compounds of the
structure 55. The acid intermediate 52 from Scheme 10 above may be
acylated, the bromide coupled with a heterocyclic boronate ester,
and this intermediate deprotected to provide compounds such as 55.
##STR71##
[0202] Scheme 12 demonstrates a synthetic strategy to access
compounds of the structure 59. Starting from a pyridyl bromo
nitrile such as 56, the bromide may be displaced, the nitrile
transformed to an N-hydroxy amidine, this intermediate acylated and
then cyclized to provide the intermediate 57. Saponification of 57
can give acid 58, which may be acylated, and after deprotection
provide compounds such as 59. ##STR72##
[0203] Scheme 13 outlines the strategy used to synthesize compounds
of the structure 63. The pyrazole 60 may be N-arylated, this
intermediate homologated to nitro 61, which in turn may be
transformed to hydroxy acid 62. Upon acylation and deprotection,
compounds such as 63 may be obtained. ##STR73##
[0204] Scheme 14 displays a method to access compounds of the
structure 66. The intermediate 64 generated in Scheme 12, may be
elaborated into 65 via an intermediate oxime cycloaddition with an
alkyne. The hydroxy acid 65 can be protected, acylated and
deprotected to provide compounds such as 66. ##STR74##
[0205] Scheme 15 shows a synthetic route used to generate compounds
of the structure 68. The intermediate 67 may be accessed from an
oxidative cleavage of the requisite olefin. This alpha-methyl acid
67 may then be condensed with an N-hydroxy amidine, and this
intermediate elaborated into compounds such as 68 using methods
illustrated in the Schemes above. ##STR75##
[0206] Scheme 16 outlines the strategy used to synthesize compounds
of the structure 70. This methodology follows closely to that
illustrated in Scheme 15 above, where intermediate 69 can now be
doubly deprotected in one step to provide compounds such as 70,
containing a methyl group alpha to the amide moiety. ##STR76##
##STR77##
[0207] Scheme 17 displays methodology to synthesize compounds of
the structure 74. An amino heterocycle such as 71 can be converted
to its halide and displaced with a nitrogen anionic heterocycle,
followed by hydroxyl introduction to provide intermediate 72. This
ester 72 may be homologated to 73, and upon acylation and
protecting group manipulation, converted to compounds such as 74.
##STR78##
[0208] Scheme 18 displays a method for generating compounds of the
structure 77. The intermediate 75 can be accessed from
4-methoxyaniline via dipolar cycloaddition, and then homologated
into 76. Following some of the methods illustrated in the Schemes
above, 76 can be converted into compounds such as 77. ##STR79##
[0209] Scheme 19 displays a method to access compounds of the
structure 80. Alkyne 78 may be homologated and undergo a
cycloaddition reaction to generate intermediate 79. The enoate 79
may then be converted into compounds such as 80 using methods
illustrated in the Schemes above. ##STR80##
[0210] Scheme 20 illustrates a strategy used to synthesize
compounds of the structure 83. Malic acid 81 can be orthogonally
protected, condensed with an N-hydroxy amidine, and deprotected to
generate 82. The bis-hydroxyacid 82 may be globally silylated, and
then acylated and deprotected to provide alpha-hydroxy compounds
such as 83. ##STR81##
[0211] Scheme 21 displays a strategy used to generate compounds of
the structure 86. Orthogonally protected acid ester intermediate 84
can be obtained via oxidative degradation of the requisite olefinic
starting material. The acid 84 may then be condensed with an
N-hydroxy amidine, and manipulated to provide a primary carboxamide
85. A primary carboxamide intermediate such as 85 may undergo a
coupling reaction with an enol triflate, and upon further
deprotection reactions, provide geminal dimethyl compounds such as
86. ##STR82##
[0212] Scheme 22 outlines a methodology to access compounds of the
structure 88. Commercially available (S)-pulegone can be converted
into intermediate 87 via reverse aldol, acylation with Mander's
reagent, and enamine formation. Using a similar alpha-methyl ester
intermediate illustrated in Scheme 16, chiral amine 87 may be
elaborated into compounds such as 88. ##STR83##
[0213] Scheme 23 displays a strategy to access compounds of the
structure 89. Commercially available symmetrical ketones, such as
4-ethylcyclohexanone, can be acylated with Mander's reagent,
followed by enol triflate formation with Comins' reagent. Using
similar metal catalyzed coupling methodology illustrated in Scheme
21, different regioisomerically substituted cyclohexene compounds
such as 89 may be accessed. ##STR84##
[0214] Scheme 24 illustrates a methodology to access compounds of
the structure 90. Commercially available 3-(trifluoromethyl)phenol
can be reduced to a hydroxy cyclohexane, oxidized with Dess-Martin
reagent to the ketone, acylated with Mander's reagent, and followed
by enamine formation. Using similar methodologies illustrated
above, compounds such as 90 may be obtained that possess a
trifluoromethyl substituted cyclohexene. ##STR85##
[0215] Scheme 25 displays a strategy to access compounds of the
structure 91. Commercially available 3-methyl-2-cyclohexen-1-one
can be substituted to generate a 3-geminal-dialkyl cyclohexanone.
Upon acylation with Mander's reagent, enamine formation, and
following similar methodologies illustrated above, compounds such
as 91 may be obtained that possess a geminal dialkyl substituted
cyclohexene. ##STR86##
[0216] Scheme 26 shows a method to access compounds of the
structure 94. Commercially available pyridine 92 can be fluorinated
and incorporated into a fluoro biaryl intermediate such as 93.
Subsequent hydrolysis, acylation, and saponification following
similar methodologies illustrated above, may provide fluoropyridyl
compounds such as 94. ##STR87##
[0217] Scheme 27 illustrates a method to generate compounds of the
structure 97. Commercially available 3-hydroxybenzaldehyde can be
converted into the ether substituted cyclohexane intermediate 95
via the key reduction of the phenyl ring. Upon ketone formation and
similar methodologies described above, the ether substituted
cyclohexene aminoester 96 can be obtained. This enamine
intermediate may be acylated and deprotected as described above, to
generate compounds such as 97 that possess an ether substituted
cyclohexene. ##STR88##
[0218] Scheme 28 displays methodology to access compounds of the
structure 100. Commercially available tetrahydro-4-H-pyran-4-one
can be converted into the dihydropyran triflate intermediate 98 via
similar methodologies described above. In parallel, commercially
available 6-methoxy-2-naphthaldehyde can be converted into the
primary carboxamide intermediate 99, also via similar methodologies
described above. Intermediates 98 and 99 may be coupled under
similar metal catalyzed methodology illustrated in Scheme 21, to
generate compounds such as 100 that possess a dihydropyran
carboxylic acid moiety. ##STR89##
[0219] Scheme 29 illustrates a methodology to access compounds of
the structure 105. The ketone 101 (for preparation see Danishefsky,
et al J. Am. Chem. Soc. 2004, 126, 14358) can be converted to the
olefin 102, followed by reduction of the double bond using standard
hydrogenation conditions, and acid catalyzed removal of the ketal
protecting group to provide the ketone 103. This material can be
acylated using Mander's reagent to give the desired ketoester that
is converted to the enol trifate 104 with Comins' reagent.
Intermediates 104 and 99 may be coupled using similar metal
catalyzed methodology illustrated in Scheme 21. Saponification of
the methyl ester using standard conditions can generate vicinal
disubstituted cyclohexene compounds such as 105.
[0220] The various organic group transformations and protecting
groups utilized herein can be performed by a number of procedures
other than those described above. References for other synthetic
procedures that can be utililized for the preparation of
intermediates or compounds disclosed herein can be found in, for
example, M. B. Smith, J. March Advanced Organic Chemistry, 5.sup.th
Edition, Wiley-Interscience (2001); R. C. Larock Comprehensive
Organic Transformations, A Guide to Functional Group Preparations,
2.sup.nd Edition, VCH Publishers, Inc. (1999); T. L. Gilchrist
Heterocyclic Chemistry, 3.sup.rd Edition, Addison Wesley Longman
Ltd. (1997); J. A. Joule, K. Mills, G. F. Smith Heterocyclic
Chemistry, 3.sup.rd Edition, Stanley Thornes Ltd. (1998); G. R.
Newkome, W. W. Paudler Contempory Heterocyclic Chemistry, John
Wiley and Sons (1982); or Wuts, P. G. M.; Greene, T. W.; Protective
Groups in Organic Synthesis, 3.sup.rd Edition, John Wiley and Sons,
(1999), all six incorporated herein by reference in their
entirety.
REPRESENTATIVE EXAMPLES
[0221] The following examples are provided to more fully illustrate
the present invention, and shall not be construed as limiting the
scope in any manner. Unless stated otherwise:
[0222] (i) all operations were carried out at room or ambient
temperature (RT), that is, at a temperature in the range
18-25.degree. C.;
[0223] (ii) evaporation of solvent was carried out using a rotary
evaporator under reduced pressure (4.5-30 mmHg) with a bath
temperature of up to 50.degree. C.;
[0224] (iii) the course of reactions was followed by thin layer
chromatography (TLC) and/or tandem high performance liquid
chromatography (HPLC) followed by mass spectroscopy (MS), herein
termed LCMS, and any reaction times are given for illustration
only;
[0225] (iv) yields, if given, are for illustration only;
[0226] (v) the structure of all final compounds was assured by at
least one of the following techniques: MS or proton nuclear
magnetic resonance (.sup.1H NMR) spectrometry, and the purity was
assured by at least one of the following techniques: TLC or
HPLC;
[0227] (vi) .sup.1H NMR spectra were recorded on either a Varian
Unity or a Varian Inova instrument at 500 or 600 MHz using the
indicated solvent; when line-listed, NMR data is in the form of
delta values for major diagnostic protons, given in parts per
million (ppm) relative to residual solvent peaks (multiplicity and
number of hydrogens); conventional abbreviations used for signal
shape are: s. singlet; d. doublet (apparent); t. triplet
(apparent); m. multiplet; br. broad; etc.;
[0228] (vii) MS data were recorded on a Waters Micromass unit,
interfaced with a Hewlett-Packard (Agilent 1100) HPLC instrument,
and operating on MassLynx/OpenLynx software; electrospray
ionization was used with positive (ES+) or negative ion (ES-)
detection; the method for LCMS ES+ was 1-2 mL/min, 10-95% B linear
gradient over 5.5 min (B=0.05% TFA-acetonitrile, A=0.05%
TFA-water), and the method for LCMS ES- was 1-2 mL/min, 10-95% B
linear gradient over 5.5 min (B=0.1% formic acid-acetonitrile,
A=0.1% formic acid-water), Waters XTerra C18-3.5 um-50.times.3.0
mmID and diode array detection;
[0229] (viii) automated purification of compounds by preparative
reverse phase HPLC was performed on a Gilson system using a
YMC-Pack Pro C18 column (150.times.20 mm i.d.) eluting at 20 mL/min
with 0-50% acetonitrile in water (0.1% TFA);
[0230] (ix) the manual purification of compounds by preparative
reverse phase HPLC (RPHPLC) was conducted on either a Waters
Symmetry Prep C18-5 um-30.times.100 mmID, or a Waters Atlantis Prep
dC18-5 um-20.times.100 mmID; 20 mL/min, 10-100% B linear gradient
over 15 min (B=0.05% TFA-acetonitrile, A=0.05% TFA-water), and
diode array detection;
[0231] (x) the purification of compounds by preparative thin layer
chromatography (PTLC) was conducted on 20.times.20 cm glass prep
plates coated with silica gel, commercially available from
Analtech;
[0232] (xi) flash column chromatography was carried out on a glass
silica gel column using Kieselgel 60, 0.063-0.200 mm (SiO.sub.2),
or a Biotage SiO.sub.2 cartridge system including the Biotage
Horizon and Biotage SP-1 systems;
[0233] (xii) chemical symbols have their usual meanings, and the
following abbreviations have also been used: h (hours), min
(minutes), v (volume), w (weight), b.p. (boiling point), m.p.
(melting point), L (litre(s)), mL (millilitres), g (gram(s)), mg
(milligrams(s)), mol (moles), mmol (millimoles), eq or equiv
(equivalent(s)), IC50 (molar concentration which results in 50% of
maximum possible inhibition), EC50 (molar concentration which
results in 50% of maximum possible efficacy), uM (micromolar), nM
(nanomolar);
[0234] (xiii) definitions of acronyms are as follows:
TABLE-US-00002 BBr.sub.3 is boron tribromide B(OMe).sub.3 is
trimethyl borate Comins' reagent is 2-[N,N- CDI is 1,1'-carbonyl
diimidazole Bis(trifluromethylsulfonyl)amino]-5- chloropyridine DCM
is dichloromethane (methylene chloride) DIBALH is diisobutyl
aluminum hydride DMF is dimethylformamide DMAP is 4-dimethyl amino
pyridine DMSO is dimethyl sulfoxide iPrMgCl is isopropyl magenisium
chloride KHMDS is potassium bis(trimethylsilyl) amide LDA is
lithium diisopropyl amide LiHMDS is lithium bis(trimethylsilyl)
amide Mander's reagent is methyl cyanoformate NBS is
N-bromo-succinimide NaOCl is sodium hypochlorite NMO is
4-methylmorpholine N-oxide OTf is triflate Pd(PPh.sub.3).sub.4 is
tetrakis triphenylphosphine palladium (0) Pd.sub.2(dba).sub.3 is
Tris(dibenzylideneacetone) TBAF is tetrabutylammonium fluoride;
dipalladium (0); TBS Chloride is t-bulyl dimethyl silyl chloride
TBSOTF is t-butyl dimethyl silyl trifluoromethane sulfonate TFA is
trifluoroacetic acid THF is tetrahydrofuran XANTPHOS is
9,9-Dimethyl-4,5-bis(diphenyl- phosphino)xanthene
Example 1
[0235] ##STR90##
[0236] To a solution of methyl-2-oxocyclopentane-1-carboxylate (1.5
g, 10.55 mmol) in methanol was added ammonium acetate (4.07 g,
52.76 mmol). After stirring the reaction at room temperature for 18
h, it was concentrated in vacuo. The residue was dissolved in DCM,
washed with water, brine, dried over anhydrous Na.sub.2SO.sub.4,
filtered and concentrated in vacuo. This cyclopentene aminoester
intermediate was used without any further purification.
[0237] To a solution of 3-(2-naphthyl)acrylic acid (1.5 g, 7.56
mmol) in 1:1 ethanol-ethyl acetate (50 mL) was added Pd/C and the
resulting mixture stirred under a H.sub.2 balloon for 18 h. The
reaction mixture was filtered through celite, and concentrated in
vacuo to give the desired saturated naphthyl acid as a white
solid.
[0238] To a solution of this saturated naphthyl acid intermediate
(150 mg, 0.75 mmol) in DCM (6 mL) cooled to 0.degree. C. was added
DMAP (201 mg, 1.65 mmol) followed by methanesulfonyl chloride
(0.059 mL, 0.75 mmol). After 5 min, the cyclopentene aminoester
intermediate (95 mg, 0.67 mmol) was added as a solid. The mixture
was stirred at RT for 18 h, and quenched with saturated NH.sub.4Cl
solution. The resulting mixture was extracted with DCM. The organic
layer was dried over anhydrous Na.sub.2SO.sub.4 filtered and
concentrated in vacuo. The residue was purified by flash
chromatography using 10% ethyl acetate-hexanes as the eluant to
give the desired amide product as a methyl ester.
[0239] To a solution of this ester intermediate (15 mg, 0.046 mmol)
in THF (2 mL), was added methanol (1 mL) followed by 1N NaOH (1
mL). The resulting reaction mixture was stirred at 23.degree. C.
for 6 h. It was neutralized to pH=7 by the addition of 1N HCl and
extracted with ethyl acetate. The organic layer was dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The
residue was purified by reverse phase HPLC (Gilson) to provide
Example 1. .sup.1H NMR (500 MHz, CD.sub.3OD) .delta. 1.85 (m, 2H),
2.49 (m, 2H), 2.8 (t, 2H), 3.1 (m, 4H), 7.4 (m, 3H), 7.7 (bs, 1H),
7.8 (m, 3H); LCMS m/z 308 (M-1).
Preparation of Cyclohexene Aminoester and Enol Triflate Ester
Intermediates
[0240] ##STR91##
[0241] To a slurry of NaH (5.6 g, 60%) in 200 mL of THF was slowly
added methyl cyclohexanone 2-carboxylate (19.9 g, 90%) at 0.degree.
C. After 30 min, the mixture was warmed to 23.degree. C. and
stirred for 15 min. The resulting mixture was cooled to 0.degree.
C., and to it was added Commin's reagent (50 g) in portions. The
resulting mixture was warmed to RT and stirred for 2.5 h. The
solution was then concentrated, and the residue was partitioned
between ethyl acetate and water. The organic layer was dried with
sodium sulfate and concentrated. The residue was purified by
Biotage (5-10% ethyl acetate in hexane) to give the cyclohexene
enol triflate ester.
[0242] To a solution of methyl cyclohexanone 2-carboxylate (10.3 g,
90%) in 100 ml of methanol was added ammonium acetate (8.5 g). The
resulting mixture was stirred at room temperature overnight. The
mixture was then concentrated, and the residue was dissolved in
ethyl acetate. The solid was filtered, and the filtrate was washed
with water, brine and dried over sodium sulfate. The resulting
solution was concentrated to give the cyclohexene aminoester as an
oil, which crystallized from hexane as a white solid.
Example 2
[0243] ##STR92##
[0244] To a solution of the saturated naphthyl acid intermediate
from Example 1 (194 mg, 0.97 mmol) in DCM (6 mL), was added DMAP
(236 mg, 1.93 mmol) followed by methanesulfonyl chloride (0.05 mL,
0.64 mmol). After 5 min, a solution of methyl
2-aminocyclohex-1-ene-1-carboxylate (100 mg, 0.64 mmol) in DCM (1
mL) was added. The reaction mixture was stirred at RT for 18 h, and
quenched with saturated NH.sub.4Cl solution. The resulting mixture
was extracted with DCM dried over anhydrous Na.sub.2SO.sub.4,
filtered and concentrated in vacuo. The residue was purified by
flash chromatography using 7% ethyl acetate-hexanes to provide the
amide as a methyl ester.
[0245] To a solution of this ester intermediate in THF (2 mL) was
added MeOH (1 mL) and 1N NaOH (1 mL). The resulting reaction
mixture was stirred at room temperature for 18 h, then neutralized
to pH=7 by the addition of 1N HCl, and extracted with ethyl
acetate. The organic layer was dried over anhydrous
Na.sub.2SO.sub.4, filtered, concentrated in vacuo and purified by
reverse phase HPLC (Gilson) to provide Example 2. .sup.1H NMR (500
MHz, CD.sub.3OD) .delta. 1.55 (m, 4H), 2.3 (m, 2H), 2.7 (t, 2H),
2.85 (m, 2H), 3.1 (t, 2H), 7.45 (m, 3H), 7.66 (s, 1H), 7.77 (m,
3H); LCMS m/z 324 (M+1).
Example 3
[0246] ##STR93##
[0247] To a solution of 6-methoxy-2-naphthaldehyde (3.6 g, 19.38
mmol) in toluene (100 mL) placed in a pressure vessel, was added
(tert-butoxycarbonyl-methylene)triphenyl-phospharane (8.76 g, 23.25
mmol). The resulting mixture was refluxed at 120.degree. C. for 18
h. The reaction mixture was concentrated in vacuo and purified
using a Biotage flash 40M column with 15% ethyl acetate-hexanes as
the eluant to give the enoate intermediate.
[0248] To a solution of this
tert-butyl-3-(6-methoxy-2-naphthyl)acrylate (4.88 g, 17.16 mmol) in
ethanol (100 mL) was added Pd/C. The resulting mixture was stirred
under a H.sub.2 balloon for 18 h. The reaction mixture was filtered
through celite and concentrated in vacuo to give the saturated
ester as a white solid.
[0249] To a solution of this ether ester intermediate (150 mg, 0.52
mmol) in DCM cooled to 0.degree. C., was added BBr.sub.3 (5.23 mL,
1.0M in DCM). After 30 min, the reaction mixture was quenched by
the addition of methanol (2 mL). The reaction mixture was
concentrated in vacuo, and the residue was dissolved in ethyl
acetate and washed with water. The organic layer was dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The
residue was purified by flash chromatography using 20% ethyl
acetate-hexanes as eluant. This transesterified methyl ester (97
mg, 0.42 mmol) was dissolved in THF (3 mL) and MeOH was added (2
mL) followed by 1N NaOH (2 mL). After stirring for 6 h, the mixture
was neutralized to pH=7 by the addition of 1N HCl. The resulting
solution was extracted with ethyl acetate, dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated in vacuo. This
naphtholic acid was used in the next step without any further
purification.
[0250] To a solution of this naphtholic acid (106 mg, 0.49 mmol) in
DCM (5 mL) cooled to 0.degree. C., was added TBSOTf (0.17 mL, 0.73
mmol) followed by triethylamine (0.14 mL, 0.98 mmol). After warming
the mixture to 23.degree. C. and stirring for 2 h, it was quenched
by the addition of water. The resulting mixture was extracted with
DCM, dried over anhydrous Na.sub.2SO.sub.4, filtered and
concentrated in vacuo. This bis-silylated material was dissolved in
1:1 THF/H.sub.2O (2 mL) and AcOH (3 mL) was added. After stirring
the mixture at RT for 1 h, it was diluted with water and extracted
with ethyl acetate. The organic layer was dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated in vacuo to give the
desired acid intermediate.
[0251] To a solution of this acid (5 mg, 0.154 mmol) in DCM (2 mL),
was added DMAP (48 mg, 0.39 mmol) followed by methanesulfonyl
chloride (0.012 mL, 0.154 mmol). After 5 min, methyl
2-aminocyclohex-1-ene-1-carboxylate (20 mg, 0.128 mmol) was added
as a solid. The reaction mixture was heated to 50.degree. C. for 18
h, and then cooled to RT and quenched by the addition of saturated
ammonium chloride. The resulting mixture was extracted with DCM,
dried over anhydrous Na.sub.2SO.sub.4 filtered and concentrated in
vacuo. The residue was purified by flash chromatography using 10%
ethyl acetate-hexanes as the eluant to provide the amide
product.
[0252] To a solution of this intermediate ester (109 mg, 0.23 mmol)
in THF (3 mL), was added 1N NaOH (1 mL) followed by MeOH (1.5 mL).
After the reaction was complete, it was neutralized to pH=7 by the
addition of 1N HCl. The resulting mixture was extracted with ethyl
acetate, dried over anhydrous Na.sub.2SO.sub.4, filtered and
concentrated in vacuo. The residue was purified by reverse phase
HPLC (Gilson) to provide Example 3. .sup.1H NMR (500 MHz,
DMSO-d.sub.6) .delta. 1.5 (m, 4H), 2.2 (bt, 2H), 2.63 (t, 2H), 2.8
(bt, 2H), 2.92 (t, 2H), 7.1 (m, 2H), 7.27 (d, 1H), 7.52 (m, 2H),
7.65 (d, 1H), 9.6 (bs, 1H), 11.6 (bs, 1H), 12.5 (bs, 1H); LCMS m/z
338 (M-1).
Example 4
[0253] ##STR94##
[0254] To a solution of methyl 3-(3-bromophenyl)propionate (100 mg,
0.411 mmol) in toluene (2 mL) was added phenyl boronic acid (100
mg, 0.82 mmol), 1M Na.sub.2CO.sub.3 solution (1 mL) followed by
Pd(PPh.sub.3).sub.4. The resulting reaction mixture was refluxed in
a pressure tube. After 2 h the mixture was cooled to 23.degree. C.,
diluted with ethyl acetate, washed with water, brine, dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The
residue was purified by flash chromatography using 10% ethyl
acetate-hexanes to give the biaryl product.
[0255] To a solution of this ester intermediate (85 mg, 0.35 mmol)
in THF (1 mL) was added MeOH (1 mL) and 5N NaOH (1 mL). After
stirring for 1 h, the reaction mixture was neutralized to pH=7 by
the addition of 1N HCl. The resulting mixture was extracted with
ethyl acetate, and the organic phase was dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The acid was
used in the next step without any further purification. This acid
intermediate was coupled to methyl-2-amino-cyclohexene using the
similar procedures as described in the Examples above. Example 4
was prepared by saponification of the penultimate ester using
similar procedures as described in the Examples above. .sup.1H NMR
(500 MHz, CD.sub.3OD) .delta. 1.6 (m, 4H), 2.3 (m, 2H), 2.68 (t,
2H), 2.85 (m, 2H), 3.01 (t, 2H), 7.2 (d, 1H), 7.35 (m, 2H), 7.45
(m, 4H), 7.58 (d, 2H); LCMS m/z 348 (M-1).
Example 5
[0256] ##STR95##
[0257] The coupling of 3-(3-bromophenyl) propionic acid with methyl
2-aminocyclohex-1-ene-1-carboxylate followed the similar procedures
as described in the Examples above. To a solution of this aryl
bromide intermediate (50 mg, 0.136 mmol) and 4-hydoxy-phenyl
boronic acid (28 mg, 0.2 mmol) in THF (0.5 mL), was added
K.sub.2CO.sub.3 (0.5 mL, 1.0M solution), followed by
1,1(bis-tert-butyl-phosphino) ferrocene palladium dichloride
ligand. The reaction vessel was flushed with N.sub.2 and heated to
85.degree. C. After 30 min, the reaction mixture was cooled to RT
and diluted with ethyl acetate. The resulting mixture was washed
with water, brine, dried over anhydrous Na.sub.2SO.sub.4, filtered
and concentrated in vacuo. The residue was purified by flash
chromatography using 15% ethyl acetate-hexanes as the eluant to
obtain the hydroxy biaryl methyl ester.
[0258] This intermediate ester was saponified following similar
procedures described in the Examples above. .sup.1H NMR (500 MHz,
DMSO-d.sub.6) .delta. 1.55 (m, 4H), 2.24 (m, 2H), 2.63 (t, 2H),
2.80 (bt, 2H), 2.9 (t, 2H), 6.83 (d, 2H), 7.13 (d, 1H), 7.33 (t,
1H), 7.38 (d, 1H), 7.42 (s, 1H), 7.45 (d, 2H), 9.52 (s, 1H); LCMS
m/z 364 (M-1).
Example 6
[0259] ##STR96##
[0260] To a solution of diisopropylamine (5.3 g, 52 mmol) in 200 mL
of THF was added n-butyllithium (22.4 mL, 56 mmol, 2.5 M in hexane)
at -78.degree. C. The resulting solution was stirred at -78.degree.
C. for 30 min, and then at RT for an additional 30 min. The
solution was re-cooled to -78.degree. C., and to this solution, was
added dropwise a solution of tetralone 20 (7.03 g, 39.9 mmol) in 80
mL of THF. After 1 h at -78.degree. C., to the above solution was
added 4-chloro-4-oxobutyrate (8.43 g, 6.84 mL, 56 mmol) in one
portion. The resulting solution was warmed to 23.degree. C. over 2
h. The solvent was then evaporated, and the residue was diluted
with 200 mL of THF/MeOH/water (v:v:v=3:1:1). To this mixture was
added 100 mL of lithium hydroxide (1 M in water), and the resulting
solution was stirred overnight. After removing some solvent in
vacuo, the remaining aqueous layer was extracted with ethyl
acetate. The aqueous phase was acidified with HCl until pH=3. The
mixture was extracted with ethyl acetate, and the combined organic
fractions were dried with sodium sulfate and concentrated in vacuo
to give the ketoacid as a grey solid.
[0261] To a solution of this ketoacid intermediate (0.72 g, 2.6
mmol) in 15 mL of ethanol were added hydroxylamine hydrochloride
(0.22 g, 3.1 mmol.) and triethylamine (320 mg, 0.44 mL, 3.1 mmol).
The resulting mixture was heated at reflux for 5 h. After removing
ethanol in vacuo, the residue was diluted with ethyl acetate (100
mL) and 1N HCl (20 mL). The aqueous layer was further extracted
with 30% of isopropanol in chloroform (2.times.30 mL). The organic
fractions were combined, dried with sodium sulfate and concentrated
in vacuo to give the tricycle as a pale yellow solid. This
intermediate was dissolved in dichloromethane (20 mL) and
borontribromide (10 mL, 1 M in dichloromethane) was added at
0.degree. C. The resulting dark solution was stirred at room
temperature for 4 h before it was quenched with 100 mL of water at
0.degree. C. The mixture was extracted with 30% isopropanol in
chloroform. The aqueous layer contained a lot of product as a
yellow solid, which was collected by filtration. The aqueous layer
was further extracted with 30% isopropanol in chloroform. The
organic phase was dried with sodium sulfate and concentrated in
vacuo to give the hydroxy product as a yellow solid after reverse
phase-HPLC purification.
[0262] To a solution of this hydroxy acid intermediate (110 mg,
0.42 mmol) in 15 mL of dichloromethane were added imidazole (87 mg,
1.3 mmol) and tert-butyldimethylsilyl chloride (192 mg, 1.3 mmoL)
at RT. The resulting mixture was stirred for 4 h. The mixture was
then purified by Biotage to give the product as a colorless
oil.
[0263] To a solution of this bis-silyl intermediate (50 mg, 0.10
mmol) in 3 mL of dichloromethane were added 1 drop of DMF and
oxalyl chloride (0.13 mL, 0.25 mmol, 2 M in dichloromethane) at
0.degree. C. After 2 h at 0.degree. C., the mixture was warmed to
room temperature and stirred for 30 min. The volatiles were removed
in vacuo, and to the residue was added 2 mL of dichloromethane
followed by methyl 2-aminocyclopent-1-ene-1-carboxylate (35 mg,
0.25 mmol). The mixture was stirred overnight and then DMAP (10 mg)
was added. The resulting mixture was stirred for an additional 2 h.
The crude mixture was directly purified by Biotage (2-10% ethyl
acetate/hexane) to give the amide product as a colorless oil.
[0264] To a solution of this silyl ether methyl ester intermediate
(14 mg, 0.023 mmol, 23%) in 5 mL of THF/MeOH/water (v:v:v=3:1:1),
was added 1 N sodium hydroxide (1 mL) and 3 drops of TBAF (1 M in
THF). After 5 min at 23.degree. C., the mixture was concentrated in
vacuo and the residue was dissolved in DMSO, which was purified by
reverse phase HPLC (Gilson) to give a colorless oil. This material
was then dissolved in 3 mL of THF/MeOH/water (v:v:v=3:1:1). To this
solution was added lithium hydroxide (2 mL, 1 M in water). The
resulting mixture was stirred at room temperature for 3 h. The
mixture was concentrated and dissolved in DMSO. The mixture was
purified by reverse phase HPLC (Gilson) to provide Example 6 as a
light brown solid. .sup.1H NMR (acetone-d.sub.6, 500 MHz) .delta.
10.4 (1H, s), 7.44 (1H, d), 6.85 (s, 1H), 6.80 (1H, dd), 3.13 (2H,
t), 2.98 (4H, q), 2.83 (2H, t), 2.73 (2H, t), 2.49 (2H, t), 1.87
(2H, t); LCMS m/z 369 (M+1).
Examples 7-15
[0265] The following compounds were prepared under conditions
similar to those described in Examples 1-6 above and illustrated in
Schemes 1-6. Example 15 utilized triethylamine as base instead of
imidazole/DMAP described for the TBSCl silylation step in Example
6. TABLE-US-00003 EXAMPLE LCMS 7 ##STR97## 322 (M - 1) 8 ##STR98##
336 (M - 1) 9 ##STR99## 348 (M - 1) 10 ##STR100## 364 (M - 1) 11
##STR101## 364 (M - 1) 12 ##STR102## 363 (M - 1) 13 ##STR103## 390
(M - 1) 14 ##STR104## 334 (M - 1) 15 ##STR105## 383 (M + 1) NMR
data for selected Examples: EXAMPLE 7 .sup.1H NMR (500 MHz,
DMSO-d.sub.6) .delta. 1.5 (m, 4H), 2.2 (bt, 2H), 2.65 (t, 2H), 2.8
(bt, 2H), 3.34 (t, 2H), 7.4 (m, 2H), 7.54 (m, 2H), 7.78 (d, 1H),
7.92 (d, 1H), 8.08 (d, 1H). EXAMPLE 8 .sup.1H NMR (500
MHz,CD.sub.3OD) .delta. 0.9 (d, 3H), 1.3 (m, 1H), 1.57 (m, 1H), 1.7
(m, 1H), 2.24 (m, 1H), 2.35 (m, 1H), 2.45 (m, 1H), 2.7 (t, 2H),
3.05 (dd, 1H), 3.1 (t, 2H), 7.34-7.43 (m, 3H), 7.65 (s, 1H), 7.72
(m, 3H). EXAMPLE 9 .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 1.55
(m, 4H), 2.2 (bt, 2H), 2.6 (t, 2H), 2.8 (bt, 2H), 2.9 (t, 2H), 7.3
(m, 3H), 7.44 (t, 2H), 7.56 (d, 2H), 7.62 (d, 2H), 11.6 (bs, 1H).
EXAMPLE 10 .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 1.55 (m,
4H), 2.4 (t, 2H), 2.62 (t, 2H), 2.84 (bt, 2H), 2.9 (t, 2H), 6.85
(t, 1H), 6.95 (d, 1H), 7.15 (m, 2H), 7.22 (d, 1H), 7.3 (t, 1H),
7.38 (m, 2H), 9.4 (s, 1H), 11.6 (s, 1H), 12.55 (bs, 1H). EXAMPLE 11
.sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 1.55 (m, 4H), 2.24 (bt,
2H), 2.66 (t, 2H), 2.82 (bt, 2H), 2.94 (t, 2H), 6.75 (d, 1H), 6.9
(m, 1H), 7.05 (d, 1H), 7.25 (m, 2H), 7.35 (t, 1H), 7.4 (d, 1H),
7.45 (s, 1H), 9.49 (s, 1H). EXAMPLE 12 .sup.1H NMR (500 MHz,
DMSO-d.sub.6) .delta. 1.55 (m, 4H), 2.22 (bt, 2H), 2.62 (t, 2H),
2.8 (bt, 2H), 2.9 (t, 2H), 6.9 (d, 1H), 7.2 (m, 3H), 7.3-7.4 (m,
3H), 7.44 (s, 1H), 11.6 (s, 1H). EXAMPLE 13 .sup.1H NMR (500 MHz,
DMSO-d.sub.6) .delta. 1.55 (m, 4H), 2.2 (m, 2H), 2.6 (t, 2H), 2.8
(m, 2H), 2.9 (t, 2H), 3.2 (t, 2H), 4.55 (t, 2H), 6.82 (d, 1H), 7.14
(d, 1H), 7.3 (t, 1H), 7.39 (m, 2H), 7.42 (s, 1H), 7.49 (s, 1H),
11.62 (s, 1H), 12.5 (bs, 1H). EXAMPLE 14 .sup.1H NMR (500 MHz,
DMSO-d.sub.6) .delta. 1.75 (m, 2H), 2.35 (bt, 2H), 2.7 (t, 2H),
2.85 (t, 2H), 3.02 (t, 2H), 7.35 (m, 3H), 7.44 (t, 2H), 7.56 (d,
2H), 7.62 (d, 2H). EXAMPLE 15 .sup.1H NMR (acetone-d.sub.6, 500
Mhz) .delta. 11.8 (1H, s), 7.43 (1H, d), 6.81 (1H, d), 6.80 (1H,
dd), 2.96 (8H, m), 2.72 (4H, q), 1.61 (4H, m). EXAMPLE 16
##STR106##
[0266] A solution of the aldehyde intermediate (1.45 g, 6.7 mmol),
ethyl triphenylphosphonium methyl acetate (3.1 g, 8.1 mmol) in 15
mL of toluene was heated at 130.degree. C. for 16 h. The mixture
was directly purified by Biotage (5-20% ethyl acetate in hexane) to
give the enoate as a light yellow solid.
[0267] This enoate intermediate (1.74 g, 5.8 mmol) and Pd/C (10%,
170 mg) in 200 mL of methanol was stirred under 1 atm of hydrogen
gas (balloon) for 12 h. The slurry was filtered and concentrated in
vacuo. The residue was dissolved in ethanol/methanol (1:1) and
purified by chiral OJ-H (9 mL/min, 28% isopropanol/heptane,
isocratic, 40 min/run) to give the enantiomers as white solids.
Elution times of these enantiomeric intermediates were 18 min and
22 min using analytical Chiralcel-OJ, (25% isopropanol in heptane,
isocratic).
[0268] The ethyl ester enantiomer (400 mg, 1.32 mmol.) was combined
with concentrated HCl (2 mL) and 4 mL of acetic acid, and was
heated at 80.degree. C. for 3 h. The mixture was concentrated in
vacuo, and to it was added 15 mL of water. The mixture was
extracted with 30% isopropanol/chloroform. The organic layer was
dried with sodium sulfate and concentrated in vacuo to give the
acid product as a white solid.
[0269] To a solution of the methyl ether (410 mg, 1.50 mmol) in 20
mL of dichloromethane was added borontribromide (7.5 mL, 1 M in
dichloromethane) at 0.degree. C. The mixture was warmed to RT and
stirred for 18 h. The mixture was quenched with water at 0.degree.
C. and concentrated in vacuo without further purification.
[0270] To a solution of the phenol in 60 mL of dichloromethane were
added TBSCl (0.57 g, 3.8 mmol), imidazole (0.26 g, 3.8 mmol) and
DMAP (37 mg, 0.3 mmol). The mixture was stirred at 23.degree. C.
for 5 h. The mixture was concentrated and purified by RP-HPLC to
give monosilyl ether (0.37 g), which was resubmitted to a solution
of TBSCl (225 mg), triethylamine (0.21 mL) and DMAP (20 mg) in 15
mL of dichloromethane. The reaction mixture was stirred for 3 h and
washed with brine. The mixture was then dried with sodium sulfate
and concentrated in vacuo to give the bis-silylated intermediate as
a crude brown oil. Following the previously described amide
formation and hydrolysis procedures using oxalyl chloride and
lithium hydroxide respectively, the enantiomers of Example 16 were
obtained. .sup.1H NMR (methanol-d.sub.4, 500 MHz) .delta. 7.41 (1H,
s), 7.16 (2H, d), 6.88 (2H, d), 3.07 (2H, m), 2.73 (3H, m), 2.46
(2H, m), 2.15 (3H, s), 1.87 (2H, m), 1.23 (3H, d); LCMS m/z 370
(M+1).
Example 17
[0271] ##STR107##
[0272] The enantiomers of Example 17 were prepared under similar
conditions as described in the Examples above. .sup.1H NMR
(methanol-d.sub.4, 500 MHz) .delta. 7.43 (1H, s), 7.18 (2H, dd),
6.89 (2H, dd), 2.86 (2H, m), 2.76 (1H, dd), 2.63 (1H, dd), 2.58
(1H, m), 2.30 (2H, m), 2.16 (3H, s), 1.60 (4H, m), 1.23 (3H, d);
LCMS m/z 384 (M+1).
Example 18
[0273] ##STR108##
[0274] A mixture of the methoxy aminobenzothiazole (8.5 g, 47
mmol.) and ethyl .alpha.-bromopyruvate (12.9 g, 59 mmol) was heated
in 120 mL of DME under reflux for 2 h. After cooling to RT, the
precipitate was collected by filtration to afford the product as a
yellow solid, which was then heated in a solution of ethanol (200
mL) under reflux for 4 h. The partitioning of the resulting residue
after concentration using ethyl acetate and saturated aqueous
sodium carbonate solution gave an organic fraction, which was dried
with sodium sulfate. The concentration in vacuo led to the
tricyclic intermediate as a solid.
[0275] To a solution of this ester (2.67 g, 9.65 mmol) in 100 mL of
dichloromethane was added DIBALH (14.5 mL, 1 M in hexane, 14.5
mmol) at -78.degree. C. After 1 h at -78.degree. C., the mixture
was quenched with water and slowly warmed to 23.degree. C. A
saturated aqueous Rochelle's salt solution was added, and the
mixture turned clear overnight. The organic phase was washed with
water and concentrated. The resulting residue was filtered to give
the aldehyde as a yellow solid.
[0276] To a solution of trimethyl phosphonoacetate (0.71 mL, 4.33
mmol) in 40 mL of THF was added nBuLi (2.9 mL, 4.6 mmol., 1.6 M in
hexane) at 0.degree. C. After 30 min, to the solution was added the
aldehyde (0.67 g, 2.88 mmol). After 10 min, the mixture was
quenched with water and diluted with ethyl acetate. The organic
phase was concentrated and purified by Biotage (20-30% ethyl
acetate/hexane) to give the enoate as a white solid.
[0277] To a solution of this enoate intermediate (0.43 g, 1.49
mmol) in 200 mL of methanol was added tosylhydrazide (2.77 g, 14.9
mmol). The mixture was heated under reflux for overnight. The
resulting clear solution was concentrated and purified by Gilson to
give the product as a white solid.
[0278] To a solution of this ester (230 mg) in 50 mL of
THF/MeOH/water (v:v:v=3:1:1) was added 10 mL of 1 N aqueous LiOH
solution. After 1.5 h, the mixture was acidified using HCl to pH=4.
The mixture was extracted with 30% of isopropanol in chloroform.
The organic phase was concentrated to dryness to give the acid as a
white solid.
[0279] To a solution of the methyl ether (220 mg, 0.80 mmol) in 40
mL of dichloromethane was added borontribromide (6.4 mL, 1 M in
dichloromethane) at 0.degree. C. The mixture was warmed to RT and
stirred for 12 h. The mixture was quenched with water at 0.degree.
C. and washed with 30% of isopropanol in chloroform. After
concentration of the organic solvent, the product was obtained as a
solid.
[0280] To a solution of this phenol in 60 mL of dichloromethane
were added TBSCl (380 mg) and triethylamine (3 mL). The reaction
mixture was stirred for 3 h and washed with water. After
concentration of the organic fraction, the residue was purified by
RP-HPLC to give the mono-TBS product (0.26 g), which was
resubmitted to a solution of TBSCl (156 mg), triethylamine (0.24
mL) and DMAP (13 mg) in 60 mL of dichloromethane. The reaction
mixture was stirred at 0.degree. C. for 1 hr, and to the mixture
was added additional 1 equivalent of triethylamine and TBSCl. The
solution was stirred overnight at RT before it washed with water.
The organic phase was then dried with sodium sulfate and
concentrated in vacuo to give the bis-silylated product as a crude
brown oil. Following the previously described amide formation and
hydrolysis procedures using oxalyl chloride and lithium hydroxide
respectively, Example 18 was obtained. .sup.1H NMR
(methanol-d.sub.4, 500 MHz) .delta. 8.13 (1H, s), 7.88 (1H, d),
7.41 (1H, d), 7.11 (1H, dd), 3.16 (4H, m), 2.89 (2H, t), 2.50 (2H,
t), 1.91 (2H, m); LCMS m/z 372 (M+1).
Example 19
[0281] ##STR109##
[0282] Example 19 was prepared under similar conditions as
described in the Examples above. .sup.1H NMR (methanol-d.sub.4, 500
MHz) .delta. 8.04 (1H, s), 7.83 (1H, d), 7.37 (1H, d), 7.08 (1H,
dd), 3.12 (2H, t), 2.93 (2H, m), 2.81 (2H, t), 2.34 (2H, m), 1.65
(4H, m); LCMS m/z 386 (M+1).
Example 20
[0283] ##STR110##
[0284] To a solution of adiponitrile (0.569 mL, 5.0 mmol) in
anhydrous THF cooled to -78.degree. C. under a nitrogen atmosphere,
was added LDA (2.62 mL, 5.25 mmol, 2.0 M solution in THF). The
reaction was warmed to -20.degree. C. over 10 min, and then
quenched with saturated NH.sub.4Cl solution. The resulting mixture
was extracted with ethyl acetate. The organic layer was washed with
brine, dried over anhydrous Na.sub.2SO.sub.4, filtered and
concentrated in vacuo. This material was purified by flash
chromatography using 15% ethyl acetate-hexanes as the eluant to
give the cyclopentene aminonitrile as an off white solid. This
intermediate cyclopentene aminonitrile was coupled with
3-(4-bromophenyl) propionic acid, using similar procedures as
described in the Examples above, to provide the amide bromide.
[0285] To a solution of this arylbromide intermediate (88 mg, 0.28
mmol) in THF (0.5 mL) was added phenyl boronic acid (48 mg, 0.41
mmol) followed by 1M K.sub.2CO.sub.3 (0.5 mL) and 1,
lbis(di-tert-butylphosphino)ferrocene palladium dichloride ligand
(18 mg, 0.03 mmol). After stirring the reaction in a sealed tube at
85.degree. C. for 18 h, it was diluted with ethyl acetate, washed
with H.sub.2O and saturated NaCl. The organic layer was dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The
residue was purifed by flash chromatography using 10% ethyl
acetate-hexanes to give the biaryl intermediate as a colorless
oil.
[0286] To a solution of this intermediate nitrile (34 mg, 0.11
mmol) in a 2:1 mixture of dioxane-H.sub.2O (0.9 mL) was added
sodium azide (21 mg, 0.32 mmol) and zinc bromide (29 mg, 0.13
mmol). The reaction mixture was stirred at 120.degree. C. in a
sealed tube for 18 h. The mixture was then cooled to room
temperature and 1N HCl was added until the the pH=7. The reaction
mixture was concentrated in vacuo and purified by reverse phase
HPLC (Gilson) to provide Example 20. .sup.1H NMR (500 MHz,
CD.sub.3OD) .delta. 2.02 (m, 2H), 2.46 (m, 2H), 2.85 (m, 2H), 3.15
(m, 4H), 7.18 (d, 2H), 7.31 (t, 1H), 7.41 (t, 2H), 7.52 (d, 2H),
7.56 (t, 2H); LCMS m/z 360 (M+1).
Example 21
[0287] ##STR111##
[0288] To the commercially available thiazole bromo aldehyde (4.97
g) shown in Scheme 10, in 200 mL of methanol was added NaBH.sub.4
(0.98 g) in portions at 0.degree. C. The mixture was stirred for 2
h and concentrated. The residue was suspended in saturated
NH.sub.4Cl solution (200 mL) to adjust the pH to 6. The mixture was
then basified by NaOH (aq) to pH 11 before the extraction with
ethyl acetate (4.times.200 mL). The combined organic fractions were
dried with sodium sulfate and concentrated to give the alcohol.
[0289] To a solution of this hydroxy intermediate (4.91 g) in 120
mL of dichloromethane at 0.degree. C. was added triphenylphosphine
(9.96 g). To this solution was then added dropwise, carbon
tetrabromide (12.6 g) in 30 mL of dichloromethane. After 2.5 h at
23.degree. C., the mixture was stirred at -20.degree. C. overnight.
The solvent was then removed, and the residue was purified by
Biotage (5-10% ethyl acetate in hexane) to give the bromide as a
white solid.
[0290] To a solution of diethyl methylmalonate (5.19 g) in 100 mL
of THF was added NaH (1.2 g, 60%) at 0.degree. C. in portions. The
mixture was stirred at 0.degree. C. for 10 min, and to this
solution was then added the bromide intermediate (3.84 g) in one
portion. The mixture was warmed to 23.degree. C. and stirred for 1
h before the addition of water. The resulting mixture was extracted
with ethyl acetate, concentrated and purified by Biotage (5-10%
ethyl acetate in hexane) to give the diester containing some
diethyl methyl malonate contaminant as a crude oil.
[0291] To this diester (5 g) was added THF/MeOH/water (.about.100
mL, 3:1:1), LiOH (.about.50 mL, 1 N) at 23.degree. C. The resulting
solution was stirred overnight. After the removal of the organic
solvent, to the residue was added concentrated HCl until pH=4. The
mixture was extracted with ethyl acetate (5.times.100 mL). The
combined organic layers were dried with sodium sulfate and
concentrated to give the diacid as a white solid containing some
.alpha.-methyl malonate acid contaminant.
[0292] The solution of this diacid intermediate (4.9 g) in 20 mL of
DMF was heated at 150.degree. C. for 7 min and then cooled to
0.degree. C. The solution was diluted with ethyl acetate, washed
with brine, dried with sodium sulfate and concentrated to give the
monoacid containing some propanoic acid. The mixture was further
purified by RP-HPLC to give pure alpha-methylacid as a colorless
oil.
[0293] The mixture of this bromoacid intermediate (0.71 g), the
aryl boronic acid (0.67 g), Pd(PPh.sub.3).sub.4 (323 mg),
NaHCO.sub.3 solution (11.2 mL, 1 N) and dioxane (40 mL) was heated
at 100.degree. C. under nitrogen overnight in a sealed tube. The
mixture was then partitioned between ethyl acetate and 1 N NaOH
solution. The organic layer was washed with 1 N NaOH solution. The
combined aqueous layers were acidified with concentrated HCl until
pH=4-5. The resulting mixture was extracted with ethyl acetate
three times. The combined organic layers were washed with brine and
dried with sodium sulfate. The removal of solvent afforded the
biaryl product.
[0294] To a solution of the methoxyaryl intermediate (0.88 g) in 60
mL of dichloromethane was added BBr.sub.3 (22.7 mL, 1 M in
dichloromethane) at 0.degree. C. The mixture was warmed to room
temperature and stirred overnight. To the mixture was then added
water at 0.degree. C., and the mixture was extracted with 30%
isopropanol in chloroform. After concentrating the organic layer,
the residue was hydrolyzed with LiOH (1 N) in 3:1:1 THF/MeOH/water
for 2 h. After the removal of the organic solvent, the residue was
washed with ethyl acetate. The alkaline aqueous phase was acidified
with HCl until pH=4-5, and the mixture extracted with ethyl acetate
(2.times.). The combined organic layers were washed with brine,
dried with sodium sulfate and concentrated to give the phenol as a
brown solid.
[0295] To a mixture of this phenolic acid intermediate (0.51 g) in
10 mL of dichloromethane was added triethylamine (0.84 mL). To this
solution at 0.degree. C. was added tert-butyldimethylsilyl chloride
(0.91 g) and DMAP (42 mg). After 6 h at 23.degree. C., the mixture
was washed with water, brine and dried with sodium sulfate. The
resulting organic fraction was concentrated in vacuo. To the
resulting residue in dichloromethane (40 mL) was added one drop of
DMF, and then a solution of oxalyl chloride (4 mL, 2 N in
dichloromethane). The mixture was warmed to 23.degree. C. and
stirred for additional 4 h. The resulting mixture was concentrated
in vacuo and then dissolved in dichloromethane (28 mL). To the
resulting solution was then added the enamine fragment (717 mg) as
in the examples above. The resulting mixture was stirred for
overnight. The crude material was purified by RP-HPLC to give 430
mg of amide. To the resulting amide was added 6 mL of
THF:methanol:water (3:1:1) and a solution of lithium hydroxide (10
mL, 1N). After 5 h, most of the low boiling solvent was removed in
vacuo. To the residue was added concentrated HCl until pH=3. The
mixture was extracted with 30% isopropanol in chloroform. The
organic layer was concentrated and purified by RP-HPLC to give the
desired Example 21. .sup.1H NMR (DMSO-d.sub.6, 500 MHz) .delta.
12.6 (1H, s), 11.6 (1H, s), 10.4 (1H, s), 7.95 (1H, d), 7.62 (1H,
s), 6.94 (1H, d), 6.85 (1H, dd), 3.10 (1H, dd), 2.89 (1H, dd), 2.79
(2H, m), 2.62 (1H, m), 2.20 (2H, m), 1.52 (4H, m), 1.15 (3H, d);
LCMS m/z 421 (M+1).
Example 22
[0296] ##STR112##
[0297] To the intermediate alpha-methylacid bromothiazole above
(197 mg, Compound 52 in Scheme 11) in dichloromethane (10 mL) was
added one drop of DMF, and then a solution of oxalyl chloride (1.6
mL, 2 N in dichloromethane). The mixture was warmed to room
temperature and stirred for 1 h. The resulting mixture was
concentrated in vacuo and then dissolved in dichloromethane (10
mL). To the resulting solution was then added the common
2-aminocyclohex-1-ene-1-carboxylate ester (400 mg). The resulting
mixture was stirred overnight. The crude material was purified by
Biotage (5-10% ethyl acetate in hexane) to give the amide as a
colorless oil.
[0298] The mixture of this bromo intermediate (55 mg), phosphine
ligand (14 mg), K.sub.2CO.sub.3 (440 mg, in 3.2 mL of water) in THF
(4 mL) was degassed with argon followed by the addition of boronate
ester (35 mg). The mixture was heated at 55.degree. C. for 1 h and
then 65.degree. C. overnight. The resulting mixture was partitioned
between ethyl acetate and brine. The organic layer was dried with
sodium sulfate and purified by RP-HPLC to give the biaryl product.
The similar hydrolysis procedure as described in the Examples above
gave Example 22 as a white solid. .sup.1H NMR (DMSO-d.sub.6, 500
MHz) .delta. 11.6 (1H, s), 8.05 (1H, s), 7.43 (1H, d), 3.04 (1H,
dd), 2.90 (1H, dd), 2.76 (2H, m), 2.58 (1H, m), 2.18 (2H, m), 1.50
(4H, m), 1.13 (3H, d); LCMS m/z 361 (M+1).
Example 23
[0299] ##STR113##
[0300] To NaH (7.2 g, 60%) was added DMF (100 mL) followed by
4-methoxybenzyl alcohol (18.7 mL) at 0.degree. C. After 25 min at
0.degree. C., the mixture was warmed to 23.degree. C. and stirred
for additional 30 min. To the resulting solution was added the
pyridyl cyanobromide (22.9 g) in one portion. The reaction was
exothermic and stirred for 10 min before it was cooled to room
temperature. The mixture was diluted with 500 mL of ethyl acetate,
washed with water (500 mL.times.3). The first two aqueous phases
were extracted with dichloromethane (500 mL.times.2). The combined
dichloromethane phase was washed with water (500 mL.times.3). The
combined organic phases were dried over sodium sulfate and
concentrated to give the PMB ether as a white solid.
[0301] To the suspension of this intermediate (24.6 g) and
hydroxylamine hydrochloride (8.55 g) in ethanol (500 mL) was added
NaOH (4.92 g in 50 mL of water) dropwise. The mixture was stirred
at RT overnight. The solid was collected by filtration to give the
N-hydroxy amidine as a white solid.
[0302] To this amidine intermediate (15.4 g) was added pyridine (40
mL) and the acid chloride shown in Scheme 12 (8.3 mL). The mixture
was heated at 120.degree. C. for 2 h and then 130.degree. C. for 1
h. After removing most pyridine, the residue was partitioned
between water and dichloromethane. The organic phase was washed
with water four times and then dried with sodium sulfate. After
removing the solvent, to the residue was added some methanol. The
resulting slurry was filtered. The solid collected by the
filtration was washed with methanol and dried in vacuo to give the
methyl ester intermediate as a pale pink solid.
[0303] To this ester (30 g) suspended in 3:1:1 THF/MeOH/water (700
mL) was added LiOH (300 mL, 1 N). The mixture was stirred at RT for
1 h. After removing most of the solvent, the aqueous layer was
acidified to pH=3. Filtration of the resulting slurry gave a white
solid, which was washed with water, diethyl ether and azeotroped
with toluene to give the acid as a white solid.
[0304] To a mixture of this acid (26.9 g) in 300 mL of
dichloromethane were added 0.1 mL of DMF, and then a solution of
oxalyl chloride (76 mL, 2 N in dichloromethane) at 0.degree. C.
After 0.5 h, the mixture was warmed to RT and stirred for
additional 0.5 h. The resulting mixture was concentrated in vacuo
and then dissolved in dichloromethane (250 mL). To the resulting
solution was then added the methyl
2-aminocyclohex-1-ene-1-carboxylate (29 g). The resulting mixture
was stirred for overnight. The solution was then washed with water
(200 mL), saturated sodium bicarbonate solution (200 mL) and dried
with sodium sulfate to give the amide as a crude material.
[0305] To a solution of the PMB ether intermediate (10.2 g) in 50
mL of dichloromethane was added triisopropylsilane (12.3 mL) and
trifluoroacetic acid (20 mL) dropwise. The mixture was stirred at
RT for 10 min, and the solvent was removed in vacuo. To the residue
containing this hydroxy product was added 300 mL of
THF:methanol:water (3:1:1) followed by a solution of lithium
hydroxide (200 mL, 1N). After 12 h, most of the low-boiling solvent
was removed in vacuo. To the residue was added ethyl acetate (200
mL.times.2), then the aqueous layer was neutralized to pH=5. The
precipitate was collected by filtration to give the desired Example
23 as a light brown solid. .sup.1H NMR (DMSO-d.sub.6, 500 MHz)
.delta. 12.6 (1H, s), 11.7 (1H, s), 10.6 (1H, s), 8.25 (1H, d),
7.88 (1H, d), 7.29 (1H, dd), 3.18 (2H, t), 2.89 (2H, t), 2.48 (2H,
m), 2.21 (2H, m), 1.51 (4H, m); LCMS m/z 359 (M+1).
Example 24
[0306] ##STR114##
[0307] To a flask containing 20 mL diglyme and KH (1.33 g, 30%) at
room temperature was added methylpyrazole (820 mg) in one portion.
After 2 h, to this mixture was added the pyridyl nitrobromide (1.83
g). The mixture was then heated at 130.degree. C. overnight. To the
resulting mixture was added 100 mL of water and 100 mL of ethyl
acetate. The aqueous layer was extracted with 100 mL of
dichloromethane. The combined organic layers were dried with sodium
sulfate and concentrated in vacuo to give a green slurry. To the
slurry was added hexane to remove the mineral oil. The mixture was
then filtered to give the pyridylpyrazole as a green solid.
[0308] The mixture of this methylated intermediate (204 mg), NBS
(330 mg) and 5 mL of CCl.sub.4 under light was refluxed for 3.5 h.
The mixture was filtered and the filtrate was washed with saturated
aqueous sodium sulfite (100 mL). The mixture was extracted with 30%
of isopropanol in chloroform. The organic layer was washed with
water, dried with sodium sulfate and concentrated. The residue was
purified by Biotage eluting with 5-30% ethyl acetate in
hexane/dichloromethane to give the bromide as a light yellow
solid.
[0309] To NaH (350 mg, 60%) in 20 mL of THF was added dimethyl
malonate (1.14 g) at 0.degree. C. After 20 min, to the clear
solution was added the bromomethylene intermediate (490 mg) in 10
mL of THF dropwise. The mixture was warmed to RT and stirred for
additional 1 h. To the mixture was then added water (50 mL). The
mixture was extracted with ethyl acetate (200 mL). The combined
organic layers were concentrated, and the residue was submitted to
10 mL of LiOH (1N) and 50 mL of THF/MeOH/water (3:1:1). After 3 h,
the mixture was acidified to pH=4 using concentrated HCl. The
mixture was concentrated to remove organic solvents, and the
residue was extracted with 30% isopropanol in chloroform. The
organic layer was concentrated and purified by RP-HPLC to give the
acidester. The mixture of this alpha-carboxyacid (1 g) in 10 mL of
DMF was heated at 150.degree. C. for 10 min. The mixture was then
purified by RP-HPLC to give the monoester (880 mg) as a yellow
solid. To this nitro intermediate (100 mg) in 6 mL of acetic acid
was added Zn (234 mg). The slurry was heated at 60.degree. C. for
30 min and filtered through celite. The filtrated was purified by
RP-HPLC to give the amino methyl ester as a reddish oil.
[0310] To the mixture of this aminopyridine (580 mg), sodium
nitrite (200 mg) was added in 2.5 mL of 10% sulfuric acid. The
mixture was heated at 80.degree. C. for 1 h. The mixture was
purified by RP-HPLC to give the hydroxypyridine. To this
hydroxyacid (86 mg) was added 5 mL of dichloromethane, 0.18 mL of
triethyl amine and 139 mg of TBSCl. After 3 h, to the mixture was
added water. The mixture was extracted with dichloromethane and 30%
isopropanol in chloroform. The combined organic fractions were
dried with sodium sulfate and concentrated in vacuo. The resulting
residue was dissolved in 5 mL of dichloromethane. To the solution
was added 1 drop of DMF, and 1 mL of oxalyl chloride (2 M in
dichloromethane) at 0.degree. C. The resulting mixture was warmed
to 23.degree. C. and stirred for 30 min before the mixture was
concentrated in vacuo. The residue was diluted into 5 mL of
dichloromethane, and to the solution was added 100 mg of methyl
2-aminocyclohex-1-ene-1-carboxylate. The mixture was stirred
overnight. The reaction mixture was concentrated, and to the
residue was added 20 mL of THF/MeOH/water (3:1:1) and 8 mL of LiOH
(1 N). The mixture was stirred at RT for 8 h and concentrated to a
smaller volume. To the residue was added concentrated HCl dropwise
until pH<3. The mixture was extracted with 30% isopropanol in
chloroform. The organic fraction was concentrated and the residue
was purified by RP-HPLC to give Example 24. .sup.1H NMR
(Acetone-d.sub.6, 500 MHz) .delta. 11.7 (1H, s), 8.32 (1H, s), 8.01
(1H, s), 7.78 (1H, d), 7.56 (1H, s), 7.40 (1H, d), 2.93 (2H, m),
2.87 (2H, t), 2.63 (2H, t), 2.32 (2H, m), 1.60 (4H, m); LCMS m/z
357 (M+1).
Example 25
[0311] ##STR115##
[0312] The cyanopyridine prepared above, was reduced with DIBAL-H
under standard conditions, and the aldehyde (173 mg), in 5 mL of
THF and 2 mL of water was combined with hydroxylamine hydrochloride
(99 mg). The mixture was stirred for 5 h and concentrated in vacuo.
The residue was purified by Biotage eluting with 5%-20% of ethyl
acetate in 1:1 mixture of dichloromethane and hexane to give the
oxime.
[0313] To this oxime (50 mg) and 4-pentynoic acid (76 mg) in 10 mL
of dichloromethane at 0.degree. C. was added 0.4 mL of NaOCl
(>=4% in water). After 12 h, the solvent was removed, and to the
residue was added 6 mL of DMF and 3 mL of NaOCl (>=4% in water).
The mixture was stirred at RT for 2 days. The mixture was filtered,
and the filtrate was purified with RP-HPLC to give the
isoxazole.
[0314] To this PMB ether intermediate (42 mg), was added 1 mL of
dichloromethane and 1 mL of TFA. After 30 min, the mixture was
concentrated, and to the residue was added 10 mL of
dichloromethane, 73 uL of triethyl amine and 48 mg of TBSCl. After
3 h, to the mixture was added water. The mixture was then extracted
with dichloromethane and 30% isopropanol in chloroform. The
combined organic fractions were dried with sodium sulfate and
concentrated in vacuo. Following similar procedures as described in
the Examples above, acylation and deprotection provided Example 25.
.sup.1H NMR (Acetone-d.sub.6, 500 MHz) .delta. 11.8 (1H, s), 8.31
(1H, s), 7.94 (1H, s), 7.39 (1H, d), 6.73 (1H, s), 2.93 (2H, t),
2.82 (2H, t), 2.68 (2H, m), 2.33 (2H, m), 1.60 (4H, m); LCMS m/z
358 (M+1).
Example 26
[0315] ##STR116##
[0316] To the commercially available olefin (5 g) in 20 mL of
propanol was added 0.5 mL of concentrated sulfuric acid. The
mixture was heated at reflux for 2 days. The reaction mixture was
purified by Biotage (5% ethyl acetate in hexane) to give the propyl
ester as a colorless oil.
[0317] To a solution of this ester (4.8 g) and NMO (9.9 g) in 30 mL
of dichloromethane was added OSO.sub.4 (4.2 mL, 4% in water). The
mixture was stirred for 12 h at RT. To the resulting solution was
added water (150 mL) and dichloromethane (300 mL). The organic
layer was concentrated. To the residue was added acetone (300 mL)
and sodium periodate (14.4 g) in water (80 mL). A white slurry was
formed. After 30 min, the slurry was filtered, and the filtrate was
concentrated in vacuo. The residue was purified by Biotage (5%
ethyl acetate in hexane) to give the corresponding aldehyde as a
colorless oil. To this aldehyde was added t-butanol (25 mL),
2-methyl-2-butene (15 mL), a mixture of sodium chlorite (14.5 g,
80%) and sodium dihydrophosphate (18 g) in water (75 mL) at
0.degree. C. The resulting brown solution was slowly warmed to
23.degree. C. and stirred for 1.5 h. To this mixture was added NaOH
(1 N) until pH=8. The organic layer was removed. To the aqueous
layer was added concentrated HCl until pH=3. The mixture was then
extracted with ethyl acetate (200 mL.times.3). The combined organic
layers were dried to give the monoacid as a colorless oil.
[0318] To this acid intermediate (1 g) in 10 mL of toluene was
added thionyl chloride (2 mL) at room temperature. The mixture was
heated at 80.degree. C. for 1 h, and the volatiles were removed and
azetroped with toluene. The residue was then dissolved in pyridine
(10 mL), and to the mixture was added the N-hydroxy amidine (1.0 g)
described in the above Examples. The resulting mixture was heated
at 120.degree. C. for 2 h and then purified by Biotage (540% ethyl
acetate in hexane) to give the oxadiazole as a brown oil. Following
similar procedures described in the Examples above, acylation and
deprotection gave the desired Example 26 as a white solid. .sup.1H
NMR (DMSO-d.sub.6, 500 MHz) .delta. 11.6 (1H, s), 10.7 (1H, bs),
8.27 (1H, d), 7.90 (1H, d), 7.31 (1H, dd), 2.86 (1H, dd), 2.80 (1H,
dd), 2.74 (3H, m), 2.22 (2H, m), 1.52 (4H, m), 1.37 (3H, d); LCMS
m/z 373 (M+1).
Example 27
[0319] ##STR117##
[0320] The similar procedures as described for the preparation of
Example 26 above, gave the racemic oxadiazole ester intermediate
shown below. ##STR118##
[0321] This oxadiazole intermediate (5 g) was purified by chrial
AD-H to give two enantiomers. To each enantiomer (1.5 g) in 100 mL
of THF/MeOH/water was added LiOH (15 mL, 1 N) at 0.degree. C. After
30 min at 0.degree. C., the mixture was acidified with HCl to
pH=2-3. After the removal of the organic solvent in vacuo, the
residue was extracted with 30% isopropanol in chloroform. The
organic layer was dried with sodium sulfate. The removal of solvent
in vacuo gave the acid containing some inorganic salt.
[0322] This material was submitted to amide formation following the
same procedures as described in the Examples above, which was
subsequently treated with dichloromethane (20 mL),
triisopropylsilane (2 mL) and treated dropwise with TFA (10 mL).
The resulting mixture was stirred at 0.degree. C. for 25 min, and
the mixture was concentrated in vacuo. The residue was dissolved in
DMSO and purified by RP-HPLC to give an enriched single enantiomer
of Example 27 (83% ee determined by chiral OJ-R). The same
procedure starting with the opposite enantiomer gave
enantiomerically enriched Example 27 (71% ee determined by chrial
OJ-R).
[0323] These enantiomers of Example 27 were subsequently repurified
and resolved by preparative SFC chiral chromatography (ChiralPak
AD, 35% methanol(TFA)-CO.sub.2) to obtain each enantiomer at 98-99%
ee. Enantiomer A: .sup.1H NMR (DMSO-d.sub.6, 500 MHz) .delta. 11.7
(1H, s), 10.7 (1H, bs), 8.27 (1H, d), 7.88 (1H, d), 7.31 (1H, dd),
3.24 (1H, dd), 3.07 (1H, dd), 3.02 (1H, m), 2.75 (2H, m), 2.21 (2H,
m), 1.51 (4H, m), 1.27 (3H, d); LCMS m/z 373 (M+1). Enantiomer B:
.sup.1H NMR (CD.sub.3OD-d.sub.6, 500 MHz) .delta. 8.25 (1H, d),
8.05 (1H, d), 7.42 (1H, dd), 3.34 (1H, dd), 3.12 (2H, m), 2.87 (2H,
m), 2.34 (2H, m), 1.62 (4H, m), 1.37 (3H, d); LCMS m/z 373
(M+1).
Example 28
[0324] ##STR119##
[0325] To a preheated (50.degree. C.) slurry of copper (II)
chloride (932 mg), 10 mL of acetonitrile was added, along with the
thiazole aminoester (1 g) and amyl nitrite (737 mg). The mixture
was heated at 50.degree. C. for 2 h. The resulting mixture was
concentrated and purified by Biotage (5-10% ethyl acetate in
hexane) to give the chloride as a brown solid.
[0326] To 4-iodopyrazole (715 mg) in 15 mL of THF was added NaH
(161 mg, 60%) at 0.degree. C. After 30 min, to this mixture was
added the chloride intermediate (595 mg). After 30 min at 0.degree.
C., the mixture was warmed to RT and stirred for 8 h. The mixture
was quenched with water and extracted with ethyl acetate. The
organic layer contained some white solid which is pure biaryl
product, and was collected by filtration. The filtrate was
concentrated and further purified by Biotage (20-100% ethyl acetate
in hexane) to give additional biaryl product as a white solid.
[0327] To this iodo intermediate (750 mg) in 30 mL of THF was
dropwise added iPrMgCl (1.4 mL, 2 M in diethyl ether) at
-78.degree. C. under nitrogen to give a light brown solution. After
1 h at -78.degree. C., to the resulting solution was added
B(OMe).sub.3 (0.29 mL). The mixture was slowly warmed to 23.degree.
C. and stirred for 12 h. The mixture was partitioned between ethyl
acetate and water. The organic layer was concentrated and treated
with 10 mL of 30% hydrogen peroxide and 50 mL of THF. The mixture
was heated at 50.degree. C. for overnight. The mixture was then
concentrated and purified by RP-HPLC to give the
hydroxypyrazole.
[0328] To this alcohol intermediate (200 mg) was added 15 mL of
dichloromethane, 0.15 mL of triethylamine and 148 mg of TBSCl. The
crude mixture was concentrated and purified by Biotage (5-10% ethyl
acetate in hexane) to give the silyl ether as an off-white
solid.
[0329] To this methyl ester (265 mg) in 20 mL of dichloromethane
was added DIBAL-H (5 mL, 1 M in hexane) at -78.degree. C. The
mixture was warmed to RT and stirred for 5 h before it was quenched
with a saturated solution of Rochelle's salt. The slurry was
stirred vigorously, and the aqueous layer was extracted with
dichloromethane. The combined organic layers were dried with sodium
sulfate and concentrated to give the hydroxymethylene product as a
crude oil, which was directly used for the next step.
[0330] To this crude alcohol (300 mg) in 10 mL of dichloromethane
was added sodium bicarbonate (121 mg) and Dess-Martin periodinane
(490 mg). After 2 h, the crude mixture was purified by Biotage (10%
ethyl acetate in hexane) to give the aldehyde.
[0331] To a solution of trimethyl phosphonate acetate (182 mg) in
20 mL of THF was added n-butyllithium (0.75 mL, 1.6 M in hexane) at
0.degree. C. The resulting solution was stirred at this temperature
for 30 min. To this solution was added a THF solution (5 mL) of the
aldehyde intermediate (270 mg). The mixture was slowly warmed to RT
and stirred for 2 hours. After quenching the mixture with water,
the mixture was extracted with ethyl acetate, concentrated and
purified by Biotage to give the methyl enoate as a white solid.
[0332] A mixture of methyl enoate (159 mg) and p-toluenesulfonyl
hydrazide (2 g) in 30 mL of methanol was heated at 65.degree. C.
for 2.5 days. The solvent was removed, and the residue was purified
by RP-HPLC to give the saturated methyl ester as a white solid.
[0333] To this methyl ester (40 mg) in 5 mL of THF:MeOH:water
(3:1:1) was added LiOH (1.5 mL, 1 M). The mixture was stirred for 2
hours. After being acidified with concentrated HCl until pH=3, the
slurry was extracted with 30% isopropanol in chloroform, dried with
sodium sulfate and concentrated in vacuo to give the acid as an
oily solid.
[0334] Following the similar amide formation and hydrolysis
procedures described in the Examples above, Example 28 was obtained
as a white solid. .sup.1H NMR (Acetone-d.sub.6, 500 MHz) .delta.
11.8 (1H, s), 7.84 (1H, s), 7.42 (1H, s), 7.27 (1H, s), 3.13 (2H,
t), 2.95 (2H, t), 2.71 (2H, t), 2.34 (2H, m), 1.62 (4H, m); LCMS
m/z 363 (M+1).
Example 29
[0335] ##STR120##
[0336] To a solution of 4-methoxyaniline (1.57 g) in 10% HCl (18
mL) was added sodium nitrite (0.87 g) in 4 mL of water at 0.degree.
C. After being stirred at 0.degree. C. for 30 min, to this mixture
was added dropwise, a solution of methyl isocyanoacetate (1.05 g)
and sodium acetate (6.63 g) in methanol (40 mL) and water (12 mL)
at 0.degree. C. The mixture was stirred at 0.degree. C. for 1.5 h.
The solvent was removed in vacuo and the residue was extracted with
ethyl acetate, washed with 5% HCl, saturated sodium bicarbonate
solution and brine. The solution was then dried with sodium sulfate
and purified by Biotage (40-80% ethyl acetate in hexane) to give
the triazole intermediate.
[0337] To a solution of this triazole ester (0.7 g) in THF (40 mL)
was added LiBH.sub.4 (79 mg). The mixture was heated under reflux
for 1 h and cooled to 23.degree. C. The mixture was then quenched
with 1 N HCl. After removing the solvent, the residue was dissolved
in ethyl acetate, washed with saturated sodium bicarbonate
solution, brine and dried with sodium sulfate. The concentration of
this mixture gave the hydroxymethylene intermediate.
[0338] To this alcohol (0.46 g) was added 50 mL of dichloromethane
and Dess-Martin reagent (227 mg) at 0.degree. C. The mixture was
warmed to RT and stirred for an additional 3 h. The mixture was
purified by Biotage (40-80% ethyl acetate in hexane) to give the
aldehyde.
[0339] To a solution of trimethyl phosphosphonoacetate (301 mg) in
20 mL of THF was added n-BuLi (0.73 mL, 2.5 M in hexane) at
0.degree. C. After 30 min, to this solution was added the aldehyde
intermediate (0.30 g). The resulting solution was stirred at RT for
1 h. To the solution was then added dichloromethane/water. The
mixture was extracted with 30% isopropanol in chloroform. The
organic layer was concentrated and purified by Biotage (40-80%
ethyl acetate in hexane) to give the methyl enoate.
[0340] A mixture of the methyl enoate intermediate (186 mg), ca. 60
mg Pd/C (10%) and 200 mL of methanol/dichloromethane (1:1) was
subjected to hydrogenation under a hydrogen balloon. After 20 min,
the reaction mixture was filtered and the filtrate was concentrated
to give the saturated methyl ester as a white solid.
[0341] Following the similar amide formation and hydrolysis
procedures described in the Examples above, Example 29 was obtained
as a white solid. .sup.1H NMR (DMSO-d.sub.6, 500 MHz) a 12.5 (1H,
bs), 11.7 (1H, s), 9.77 (1H, s), 7.57 (2H, d), 6.88 (2H, dd), 2.95
(2H, t), 2.82 (2H, m), 2.72 (2H, t), 2.23 (2H, m), 1.53 (4H, m);
LCMS m/z 357 (M+1).
Example 30
[0342] ##STR121##
[0343] BuLi (10 mmol, 1.3 eq, 2.5M/THF, 4 mL) was added to a THF (8
mL) solution of trimethyl phosphonoacetate (9.23 mmol, 1.2 eq, 1.68
g) at -78.degree. C. and stirred for 30 min. The solution was
warmed to 0.degree. C. for 10 min and re-cooled to -78.degree. C.
Then a THF (5 mL) solution of 4-ethynylbenzaldehyle (7.69 mmol, 1
eq, 1.00 g) was added dropwise and stirred for 2 h at RT. The
reaction was partitioned between AcOEt and H.sub.2O. The organic
layer was dried, and the residue was recrystalized with
CH.sub.2Cl.sub.2/MeOH to obtain a light yellow solid product.
[0344] A mixture of this methyl enoate acetylide (460 mg, 1 eq,
2.47 mmol), CuI (0.1 eq. 24 mg) and azidotrimethylsilane (427 mg,
1.5 eq, 3.71 mmol) were mixed in DMF/MeOH (5 mL, 9/1) in a sealed
tube and heated to 100.degree. C. for 15 h. The reaction solution
was cooled to RT and diluted with AcOEt (10 mL). The solution was
filtered through celite and dried under reduced pressure. The
residue was recrystalized with CH.sub.2Cl.sub.2/MeOH to obtain a
light yellow solid product triazole.
[0345] Then LiOH (0.5 M, 8 mL) was added to this methyl ester (450
mg, 1.97 mmol) in MeOH/THF (10 mL, 1/9) and stirred until all
solids were dissolved (about 2 h). Then 20 mL of MeOH was added to
this solution, followed by Pd/C (10 mg), and the mixture was
subjected to hydrogenation under balloon pressure for 15 h. The
reaction solution was filtered and acidified to pH=7. The product
white solid was obtained by filtration of the precipitate.
[0346] Following the similar amide formation and hydrolysis
procedures described in the Examples above, Example 30 was
obtained. .sup.1H NMR (CD.sub.3OD, 500 MHz) .delta. 8.12 (s, 1H),
7.75 (d, 2H), 7.33 (d, 2H), 3.00 (t, 2H), 2.89 (t, 2H), 2.65 (t,
2H), 2.32 (t, 2H), 1.62 (m, 4H); LCMS m/z 339 (M-1).
Example 31
[0347] ##STR122##
[0348] To racemic malic acid (1.03 g) was added
2,2-dimethoxypropane (25 mL) and p-TsOH hydrate (30 mg). The
mixture was stirred overnight before the addition of sodium
acetate. The mixture was stirred for additional 3 h and filtered.
The filtrate was concentrated, and the mono-protected acid was
crystallized from chloroform/hexane as a white solid.
[0349] To a solution of this acid intermediate (281 mg) in 10 mL of
dichloromethane was added CDI (524 mg). The resulting mixture was
stirred for 1 h, and to this mixture was added the N-hydroxy
amidine (1.32 g) and dichloromethane (10 mL). The mixture was
stirred over 2 days and then filtered. The filtrate was
concentrated, and the residue was suspended in toluene (40 mL) and
heated to 120.degree. C. for 6 h and 130.degree. C. for 2 h. After
removing the solvent, the residue was purified by Biotage (20-40%
ethyl acetate in hexane) to give the oxadiazole intermediate, which
was dissolved in chloroform (5 mL) and treated with TFA (2.5 mL)
for 20 min. The mixture was concentrated, and to the residue was
added 5% KOH in ethanol (50 ml). The resulting mixture was stirred
for 6 h and acidified with HCl until pH=4. The solution was
extracted with 30% isopropanol in chloroform. The extracts were
concentrated and purified by RP-HPLC to give the hydroxypyridyl
alpha-hydroxy acid intermediate. Following similar procedures as
described for the Examples above, Example 31 was obtained after
silylation, amide formation and hydrolysis. .sup.1H NMR
(CD.sub.3OD, 500 MHz) .delta. 8.25 (1H, s), 8.06 (1H, dd), 7.42
(1H, dd), 4.63 (1H, dd), 3.62 (1H, m), 3.52 (1H, m), 2.92 (1H, m),
2.35 (1H, m), 1.63 (6H, m); LCMS m/z 375 (M+1).
Example 32
[0350] ##STR123##
[0351] A solution of the commercially available olefinic acid shown
in Scheme 21 (20 g) in ethanol (150 mL) in the presence of 0.5 mL
of concentrated sulfueric acid, was heated under reflux for 1 day.
A pad of 3A molecular sieves above the reaction flask was used to
absorb the water generated from the reaction. The mixture
containing the volatile ester was cooled to 0.degree. C., and to
the mixture was added NMO (21.9 g) and 4% of OSO.sub.4 (1 mL). The
solution was stirred at 0.degree. C. for 1 h and then warmed to
23.degree. C. and stirred overnight. Most of the solvent in this
mixture was removed in vacuo, the residue was partitioned between
water and ethyl acetate. The organic layer was concentrated to give
the diol intermediate as a brown oil.
[0352] To a solution of this diol in 300 mL of acetone at 0.degree.
C. was added a slurry of sodium periodate (87 g) in 400 mL of
water. The resulting white slurry was slowly warmed to RT and
stirred for 1.5 h. The slurry was filtered and washed with acetone,
and the filtrate was extracted with dichloromethane. The combined
organic layers were carefully concentrated to provide the volatile
aldehyde as a brown oil. At 0.degree. C., to the solution of this
aldehyde and tert-butanol (150 mL) was added 2-methyl-2-butene (20
mL) and a solution of sodium dihydrophosphate (15 g) and sodium
chlorite (41 g, .about.80%). The resulting brown mixture was slowly
warmed to RT, and the mixture was stirred for 5 h. To the mixture
was added 10% sodium hydroxide until pH>11. The mixture was then
washed with ethyl acetate, and the aqueous layer was acidified with
concentrated HCl until pH=4. The resulting aqueous fraction was
extracted with ethyl acetate. The combined organic fractions were
dried with sodium sulfate and concentrated in vacuo to give the
mono-acid mono-ester as a colorless oil.
[0353] To a solution of this acid intermediate (13.5 g) in 120 mL
of dichloromethane were added 50 .mu.L of DMF and 97 mL of oxalyl
chloride (2 M in dichloromethane) at 0.degree. C. The mixture was
stirred at 0.degree. C. for 30 min, warmed to 23.degree. C., and
the resulting solution was stirred for an additional 2 h. After
removing the volatiles, to the residue was added the N-hydroxy
amidine (21.2 g) and 100 mL of pyridine. The mixture was then
heated at 130.degree. C. for overnight. The pyridine was removed in
vacuo, and the residue was partitioned between water and
dichloromethane. The organic phase was concentrated and purified by
Biotage (eluting with 10-40% ethyl acetate in hexane) to give the
bi-heterocyclic intermediate as a white solid.
[0354] To this ester (15.1 g) in 400 mL of THF/MeOH/water (3:1:1),
was added LiOH (200 mL, 1 N) dropwise. The mixture was stirred at
room temperature for 2 h. After removing most organic solvent in
vacuo, the aqueous layer was acidified with 1 N HCl to pH=3. The
precipitate was extracted with dichloromethane thrice. The combined
organic phase was dried with sodium sulfate and concentrated to
give the acid as a white solid.
[0355] To a solution of this acid intermediate (14.3 g) in 300 mL
of dichloromethane, were added N-hydroxysuccinimide (4.52 g) and
EDCI (7.53 g). The mixture was stirred for 2.5 h and then diluted
to 1 L of dichloromethane. The resulting solution was washed with
brine and dried with sodium sulfate. The solution was concentrated,
and the resulting residue was dissolved in 700 mL of dioxane. To
this solution was added ammonia in water (60 mL, 28-30%) at
0.degree. C. The resulting mixture was stirred at RT for 15 min.
The volatiles were removed in vacuo, and the residue was
partitioned between water and dichloromethane. The organic phase
was washed with water, saturated sodium bicarbonate, and then dried
with sodium sulfate. The resulting solution was concentrated to
give the primary carboxamide as a white solid.
[0356] A mixture of this carboxamide (6.83 g), the enol triflate
(12.9 g), Pd.sub.2(dba).sub.3 (1.31 g), Cs.sub.2CO.sub.3 (9.9 g,
anhydrous), Xantphos (2.48 g) and 200 mL of dioxane was heated
under argon at 80.degree. C. for overnight. The mixture was cooled
and filtered through celite, concentrated, and purified by Biotage
(20-40% ethyl acetate in hexane) to give the cyclohexenylamide
ester as an oil.
[0357] To this ester (8.6 g) in 84 mL of dichloromethane was added
triisopropylsilane (8.4 mL) and TFA (40 mL) at 0.degree. C. The
mixture was stirred at room temperature for 15 min, the solvents
removed, the residue dissolved in 200 mL of THF/MeOH/water (3:1:1),
and the mixture treated dropwise with excess of LiOH (1 N). The
mixture was stirred at RT overnight, and after removing most
organic solvents in vacuo, the aqueous layer was washed with ethyl
acetate. The aqueous layer was then acidified with 1N HCl to pH=5.
The precipitate was collected by filtration, washed with water and
diethyl ether to give the product as a crude material. This
material was dissolved in 30% isopropanol in chloroform and
filtered. The filtrate was concentrated to a small volume and the
homogeneous mixture was kept at 0.degree. C. overnight. The
precipitate was collected, washed with methanol, then diethyl ether
and dried under vacuum to provide Example 32 as a white solid.
.sup.1H NMR (Acetone-d.sub.6, 500 MHz) .delta. 12.1 (1H, s), 8.36
(1H, s), 7.98 (1H, d), 7.41 (1H, d), 3.27 (2H, s), 2.95 (2H, m),
2.37 (2H, m), 1.63 (4H, m), 1.46 (6H, s); LCMS m/z 387 (M+1).
Example 33
[0358] ##STR124##
[0359] To (S)-pulegone (4 g) was added concentrated HCl (3.8 mL)
and 12 mL of water. The mixture was heated at reflux for 20 h under
vigorous stirring. The mixture was distilled to remove acetone and
the heating oil was heated at 180.degree. C. to distill off both
organic layer and HCl (aq). The organic layer was separated from
the aqueous layer and then was diluted with diethyl ether and
washed with sodium bicarbonate solution. The organic layer was then
dried with sodium sulfate and concentrated carefully to remove most
of the diethyl ether to give the (S)-3-methylcyclohexanone.
[0360] To this (S)-3-methylcyclohexanone (2 g) in 50 mL of THF, was
added LiHMDS (20 mL, 1 M in THF) at -78.degree. C. The mixture was
slowly warmed to 0.degree. C. and stirred for 30 min before the
solution was re-cooled to -78.degree. C. To this mixture was added
methyl cyanoformate (1.55 mL), and the mixture was stirred at
-20.degree. C. for 2 h before the addition of 1 N HCl solution. The
aqueous layer was extracted with diethyl ether and purified by
Biotage (10-20% diethyl ether in hexane) to give the ketoester as a
colorless oil.
[0361] To this ketoester intermediate (1 g) in 20 mL of methanol,
was added ammonium acetate (4.6 g), and the mixture was stirred
overnight. After the removal of the solvent, the residue was
diluted with ethyl acetate. The solid was filtered off, and the
filtrate was washed with water, brine and dried with sodium
sulfate. The liquid was then concentrated to give the
(S)-cyclohexene aminoester as an oily solid.
[0362] The enantiomeric ester intermediates from Example 27 above
(200 mg) were each heated at 70.degree. C. in 1 mL of concentrated
HCl/HOAc (v:v=1:2) for 30 min. The resulting mixture was
concentrated under high vacuum overnight. The enantiomeric acids
were then submitted directly for the next amidation step with the
(S)-cyclohexene aminoester as described in the Examples above.
[0363] Following the similar hydrolysis procedures as described in
the Examples above, provided two of the four possible diasteromers
of Example 33 containing minor epimerization. (S)-Diastereomer A:
.sup.1H NMR (DMSO-d.sub.6, 500 MHz) a 12.6 (1H, s), 11.7 (1H, s),
10.6 (1H, s), 8.26 (1H, s), 7.88 (1H, d), 7.30 (1H, d), 3.02 (4H,
m), 2.33 (2H, m), 2.16 (1H, m), 1.60 (2H, m), 1.27 (3H, s), 1.09
(1H, m), 0.91 (3H, d); LCMS m/z 387 (M+1). (S)-Diastereomer B:
.sup.1H NMR (DMSO-d.sub.6, 500 MHz) b 12.6 (1H, s), 11.69 (1H, s),
10.6 (1H, s), 8.26 (1H, s), 7.88 (1H, d), 7.30 (1H, d), 3.02 (4H,
m), 2.33 (2H, m), 2.16 (1H, m), 1.60 (2H, m), 1.27 (3H, s), 1.09
(1H, m), 0.91 (3H, d); LCMS m/z 387 (M+1).
[0364] Utilizing the commercially available
(R)-3-methylcyclohexanone, provided access to the additional other
two diastereomers of Example 33. Thus to a suspension of sodium
hydride (3.57 g, 89.15 mmol, 60% dispersion in oil) in anhydrous
dioxane (25 mL) was added dimethyl carbonate (30 mL, 356.6 mmol).
The resulting mixture was heated to 85.degree. C., and a solution
of (R)-3-methylcyclohexanone (5.0 g, 44.64 mmol) in dioxane (50 mL)
was added dropwise via an addition funnel. After stirring at
80.degree. C. for 2 hours, the reaction mixture was cooled to room
temperature and quenched with 1N HCl. The resulting mixture was
concentrated, the residue was extracted with ether, and the organic
layer was washed with brine, dried over magnesium sulfate, filtered
and concentrated. The residue was purified by Biotage using a
gradient of 0-5% ethyl acetate-hexanes to give the desired product
as a white crystalline solid.
[0365] To a solution of this methyl ketoester intermediate (2.5 g,
14.7 mmol) in methanol (50 mL) was added ammonium acetate (5.66 g,
73.52 mmol). The reaction mixture was left stirring at RT for 16 h.
It was then concentrated, and the residue was diluted with ethyl
acetate and washed with water. The organic layer was washed with
brine, dried over anhydrous sodium sulfate, filtered and
concentrated. A white solid was obtained for this methyl
(R)-cyclohexene aminoester intermediate.
[0366] As before, the enantiomeric ester intermediates from Example
27 above were each converted to their respective acids. One
enantiomeric acid was then submitted directly for the next
amidation step with the methyl (R)-cyclohexene aminoester as
described in the Examples above. Following the similar hydrolysis
procedures as described in the Examples above, and subsequent
purification by reverse phase HPLC, provided the third diastereomer
of Example 33 containing minor epimerization. (R)-Diastereomer A:
.sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.73 (s, 1H), 8.26 (s,
1H), 7.9 (d, 1H), 7.37 (dd, 1H), 3.26 (m, 1H), 3.0 (m, 3H), 2.4 (m,
2H), 2.1 (m, 1H), 1.6 (m, 2H), 1.28 (d, 3H), 1.08 (m, 1H), 0.93 (d,
3H); LCMS m/z 387 (M+1).
[0367] To obtain the fourth diastereomer of Example 33, a solution
of the (R)-3-methyl cyclohexanone (2.18 g, 19.46 mmol) in anhydrous
THF (50 mL), cooled to -78.degree. C., was treated with LiHMDS
(23.35 mL, 1.0 M in THF). After 15 minutes, benzyl cyanoformate was
added. The reaction mixture was slowly warmed to 0.degree. C. over
an hour, and quenched by the addition of 1N HCl. The resulting
mixture was extracted with ethyl acetate. The organic layer was
washed with brine, dried over anhydrous sodium sulfate, filtered
and concentrated. The residue was purified by Biotage SP-I using a
gradient of 0-15% ethyl acetate-hexanes to give the desired product
as a colorless oil.
[0368] To a solution of this benzyl ketoester intermediate (1.0 g,
4.06 mmol) in methanol (20 mL) was added ammonium acetate (1.57 g,
20.32 mmol). After stirring the reaction at 23.degree. C. for 16
hours it was concentrated. The residue was diluted with ethyl
acetate and washed with water. The organic layer was washed with
brine, dried over anhydrous sodium sulfate, filtered and
concentrated. A white solid was obtained for this benzyl
(R)-cyclohexene aminoester intermediate.
[0369] As before, the enantiomeric ester intermediates from Example
27 above were each converted to their respective acids. One
enantiomeric acid was then submitted directly for the next
amidation step with the benzyl (R)-cyclohexene aminoester as
described in the Examples above. Following the similar PMB-ether
deprotection procedures as described in the Examples above,
provided the benzyl ester penultimate intermediate.
[0370] To a solution of this benzyl ester intermediate (27 mg,
0.056 mmol) in methanol (2 mL) was added Pd/C (10 mg). The
resulting solution was stirred under a hydrogen balloon for 15
minutes. The reaction mixture was filtered through celite. The
filtrate was concentrated and purified by reverse phase HPLC to
provide the fourth diastereomer of Example 33 containing minor
epimerization. (R)-Diastereomer B: .sup.1H NMR (500 MHz,
DMSO-d.sub.6) .delta. 11.69 (s, 1H), 8.26 (s, 1H), 7.88 (d, 1H),
7.3 (dd, 1H), 3.24 (m, 1H), 3.0 (m, 3H), 2.4 (m, 2H), 2.1 (m, 1H),
1.6 (m, 2H), 1.26 (d, 3H), 1.1 (m, 1H), 0.92 (d, 3H); LCMS m/z 387
(M+1).
Example 34
[0371] ##STR125##
[0372] The enantiomers of Example 34 were generated from the
respective methyl (R)-cyclohexene aminoester and methyl
(S)-cyclohexene aminoester intermediates prepared in Example 33
above. These enantiomeric methyl cyclohexene aminoesters were
acylated with the requisite carboxylic acid intermediate from
Example 23, and the amides converted to the desired products
following the procedures described in the Examples above.
(S)-Enantiomer: .sup.1H NMR (CD.sub.3OD, 500 MHz) .delta. 8.23 (1H,
s), 8.01 (1H, d), 7.34 (1H, d), 3.29 (2H, m), 3.08 (1H, bd), 2.99
(2H, bs), 2.50 (1H, bd), 2.41 (1H, m), 2.28 (1H, m), 1.70 (1H, m),
1.66 (1H, m), 1.18 (1H, m), 1.01 (3H, d); LCMS m/z 373 (M+1).
(R)-Enantiomer: .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 11.66
(s, 1H), 8.26 (s, 1H), 7.91 (d, 1H), 7.32 (dd, 1H), 3.2 (t, 2H),
3.0 (dd, 1H), 2.9 (m, 2H), 2.4 (m, 2H), 2.1 (m, 1H), 1.6 (m, 2H),
1.15 (m, 1H), 0.95 (d, 3H); LCMS m/z 373 (M+1).
Example 35
[0373] ##STR126##
[0374] Commercially available 4-ethylcyclohexanone was converted to
its methyl ketoester via Mander's reagent under the conditions
described in the Examples above. This material as an orange oil was
used in the next step without any further purification.
[0375] To a solution of this methyl ketoester intermediate (475 mg,
2.6 mmol) in anhydrous THF (25 mL) cooled to 0.degree. C., was
added NaH (113 mg, 2.84 mmol, 60%). After 30 min,
2-[N,N-bis(trifluromethylsulfonyl)amino]-5-chloropyridine (1.13 g,
2.84 mmol) was added, and the resulting reaction stirred at room
temperature for 2 h. The reaction mixture was quenched with 1N HCl,
then extracted with EtOAc. The organic layers were washed with
brine, dried over Na.sub.2SO.sub.4, filtered and concentrated to
give a brown oil. This material was purified by Biotage using 50%
EtOAc/hexanes as eluant to give the desired enol triflate
intermediate.
[0376] As shown in Scheme 23, the methyl ester intermediate from
Example 23 can be directly converted to its primary carboxamide.
Thus to a solution of this methyl ester in dioxane in a pressure
tube was added 7N ammonia in methanol. The resulting solution was
heated at 70.degree. C. for 16 hours. The reaction mixture was
cooled to room temperature and concentrated to give the desired
primary carboxamide intermediate as a white solid.
[0377] To a solution of the enol triflate intermediate (150 mg,
0.37 9 mmol) in anhydrous dioxane (3 mL) was added the primary
carboxamide intermediate (112 mg, 0.316 mmol), XANTPHOS (37 mg,
0.06 mmol), cesium carbonate (46 mg, 0.36 mmol) and
Pd.sub.2(dba).sub.3 (20 mg, 0.019 mmol). The resulting mixture was
de-gassed for 2 minutes by bubbling N.sub.2. The reaction was
heated at 60.degree. C. under a N.sub.2 atmosphere for 18 h. The
reaction mixture was cooled to RT, and filtered through celite. The
filtrate was concentrated, and the residue was purified by Prep-TLC
(30% ethyl acetate-hexanes) to give the desired amide product.
[0378] To a solution of this intermediate (89 mg, 0.171 mmol) in
DCM (2 mL) was added triisopropyl silane (0.3 mL) followed by TFA
(1 mL). After stirring the mixture for 20 min, it was cooled to
0.degree. C. and carefully quenched by the addition of saturated
sodium bicarbonate solution. The resulting mixture was extracted
with 30% isopropanol/chloroform. The organic layer was dried over
anhydrous sodium sulfate, filtered and concentrated. This material
was dissolved in THF (5 mL), 1N NaOH (2 mL) was added followed by
enough MeOH to obtain a homogenous solution. After stirring the
reaction at room temperature for 18 h, it was neutralized with 1N
HCl. The resulting mixture was extracted with 20%
isopropanol/chloroform. The organic layer was washed with brine and
dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue
was purified by reverse phase HPLC (10-100% acetonitrile/H.sub.2O
(1% TFA)) to provide Example 35. .sup.1H NMR (CD.sub.3OD, 500 MHz),
.delta. 8.25 (d, 1H), 7.90-7.88 (d, 1H) 7.31-7.29 (dd, 1H), 3.18
(t, 2H), 2.99-2.88 (m, 3H), 2.69-2.63 (m, 1H), 2.44-2.40 (m, 1H),
1.78-1.73 (m, 2H), 1.30-1.23 (m, 3H), 1.10-1.06 (m, 1H), 0.866 (t,
3H); LCMS m/z 387 (M+1).
Example 36
[0379] ##STR127##
[0380] To a solution of 3-(trifluromethyl)-phenol (3.0 g, 18.51
mmol) in methanol (20 mL) was added Rh/Al.sub.2O.sub.3 (100 mg).
The resulting mixture was stirred under hydrogen atmosphere (50
psi) for 16 h. The reaction was filtered through celite and
concentrated to give the cyclohexanol as a colorless oil.
[0381] To a solution of this cyclohexanol intermediate (3.0 g,
17.85 mmol) in dichloromethane (100 mL) was added Dess-Martin
reagent (9.0 g, 21.42 mmol). After stirring at room temperature for
4 h, the mixture was quenched with saturated sodium bicarbonate
solution. The resulting mixture was extracted with dichloromethane.
The organic layer was dried over anhydrous sodium sulfate, filtered
and concentrated to give the ketone as a colorless oil.
[0382] To a solution of this cyclohexanone intermediate (2.0 g,
12.04 mmol) in anhydrous THF cooled to -78.degree. C. was added
LiHMDS (14.5 mL, 14.5 mmol, 1.0 M in THF). The resulting mixture
was warmed to 0.degree. C. and stirred for 20 min. The reaction
mixture was cooled back to -78.degree. C. and methyl cyanoformate
(1.15 mL, 14.46 mmol) was added. The reaction mixture was warmed to
-20.degree. C. and quenched with 1N HCl. The resulting mixture was
extracted with ethyl acetate. The organic layer was washed with
brine, dried over anhydrous sodium sulfate, filtered and
concentrated. The residue was purified by flash chromatography
using 10% ethyl acetate-hexanes to give the ketoester as a
colorless oil.
[0383] To this ketoester intermediate (860 mg, 3.83 mmol) in
methanol was added ammonium acetate (1.48 g, 19.2 mmol). After
stirring the mixture at room temperature for 16 h, it was
concentrated. The residue was diluted with ethyl acetate and washed
with water. The organic layer was washed with brine, dried over
anhydrous sodium sulfate, filtered and concentrated. A white solid
of the trifluorocyclohexene aminoester was obtained.
[0384] As shown in Scheme 24, the carboxylic acid intermediate from
Example 23 can be used to acylate this trifluorocyclohexene
aminoester. Thus to a solution of the carboxylic acid (100 mg,
0.281 mmol) in anhydrous dichloromethane (5 mL) cooled to 0.degree.
C. under nitrogen atmosphere, was added DMF (10 .mu.L) followed by
oxalyl chloride (0.562 mL, 2.0 M solution in DCM). The reaction
mixture was warmed to 23.degree. C. and stirred for 30 min. The
reaction mixture was concentrated, and the residue was dissolved in
anhydrous dichloromethane. A solution of the trifluorocyclohexene
aminoester (140 mg, 0.627 mmol) in dichloromethane (2 mL) was
added. After stirring the mixture at RT for 16 h, it was quenched
by the addition of saturated sodium bicarbonate solution. The
resulting mixture was extracted with dichloromethane. The organic
layer was dried over anhydrous sodium sulfate, filtered and
concentrated. The residue was purified by flash chromatography
using 40% ethyl acetate-hexanes to give the desired amide methyl
ester as a white solid.
[0385] To a solution of this ester intermediate (44 mg) in THF (2
mL) was added 0.5 N NaOH (2 mL), followed by MeOH (1 mL). After
stirring the mixture for 1 h, it was quenched by neutralizing with
1N HCl (1 mL). The resulting mixture was concentrated, and the
residue was diluted with water (5 mL) and extracted with ethyl
acetate. The organic layer was dried over anhydrous sodium sulfate,
filtered and concentrated. The residue was purified by reverse
phase HPLC to provide Example 36. .sup.1H NMR (500 MHz,
DMSO-d.sub.6) .delta. 11.64 (s, 1H), 10.62 (bs, 1H), 8.26 (bs, 1H),
7.9 (d, 1H), 7.32 (dd, 1H), 3.2 (m, 3H), 2.9 (m, 2H), 2.75 (m, 1H),
2.6 (m, 2H), 2.2 (m, 1H), 1.9 (m, 1H), 1.4 (m, 1H); LCMS m/z 427
(M+1).
Example 37
[0386] ##STR128##
[0387] To a suspension of copper(I) iodide (3.8 g, 20 mmol) in
anhydrous ether (20 mL) cooled to 0.degree. C. under a nitrogen
atmosphere was added dropwise a solution of methyl lithium (25 mL,
40 mmol, 1.6 M in diethylether). After stirring the mixture at
0.degree. C. for 20 min, it was cooled to -78.degree. C. and
3-methyl 2-cyclohexen-1-one was added. The reaction mixture was
slowly warmed to -20.degree. C. over 1 h and quenched by the
addition of conc. ammonium hydroxide solution (10 mL). The
resulting biphasic solution was stirred for 20 min. The aqueous
layer was extracted with ether, and the organic layer was washed
with brine, dried over anhydrous sodium sulfate, filtered and
concentrated. The residue was purified by flash chromatography
using 10% ethyl acetate-hexanes to give the desired geminal
dimethyl cyclohexanone as a yellow oil.
[0388] To a solution of this cyclohexanone intermediate (740 mg,
5.86 mmol) in anhydrous THF (20 mL) cooled to -78.degree. C. under
a nitrogen atmosphere, was added LiHMDS (7 mL, 7 mmol, 1.0 M
solution). After 15 min, methyl cyanoformate (0.558 mL, 7.03 mmol)
was added. After stirring the mixture at -78.degree. C. for 20 min,
it was quenched with 1N HCl. The biphasic mixture was extracted
with ethyl acetate, washed with brine and dried over anhydrous
sodium sulfate. The organic layer was filtered, concentrated and
purified by flash chromatography using 10% ethyl acetate hexanes to
give the ketoester as a colorless oil.
[0389] To a solution of this ketoester intermediate (500 mg, 2.72
mmol) in methanol (30 mL) was added ammonium acetate (1.05 g, 13.6
mmol). After stirring the reaction mixture at room temperature for
48 h, it was concentrated, and the residue was diluted with ethyl
acetate and washed with water. The organic layer was washed with
brine, dried over anhydrous sodium sulfate, filtered and
concentrated. A white solid was obtained for the geminal dimethyl
cyclohexene aminoester intermediate.
[0390] As shown in Scheme 25, the carboxylic acid intermediate from
Example 23 can be used to acylate this cyclohexene aminoester. Thus
to a solution of the carboxylic acid (100 mg, 0.281 mmol) in
methylene chloride (4 mL) and DMF (20 .mu.L), was added oxalyl
chloride (0.281 mL, 0.563 mmol) at 0.degree. C. The solution was
stirred at RT for 30 min, then concentrated. To the resulting
residue was added methylene chloride (3 mL) followed by a solution
of the cyclohexene aminoester intermediate (129 mg, 0.703 mmol) in
methylene chloride (2 mL). The reaction was stirred at 23.degree.
C. under N.sub.2 for 3 h, then quenched with saturated NaHCO.sub.3
solution and extracted with methylene chloride. The combined
organic layers were dried over anhydrous sodium sulfate, filtered
and concentrated. The residue was purified on silica, eluting with
a gradient of 20%-60% EtOAc/hexanes over 10 column volumes, then
60%-100% EtOAc/hexanes over 4 column volumes, affording the desired
amide as a white solid.
[0391] To a solution of this PMB ether intermediate (84 mg, 0.161
mmol) in methylene chloride (4 mL) was added triisopropylsilane
(500 .mu.L, 2.441 mmol) then TFA (2 mL, 26.0 mmol). The reaction
was stirred for 5 min, then slowly quenched at 0.degree. C. with
saturated aqueous NaHCO.sub.3 in an ice bath. This mixture was
extracted with methylene chloride then 30% IPA/CHCl.sub.3. The
combined organic layers were dried over anhydrous sodium sulfate,
filtered and concentrated. The solid residue was used in the next
step without further purification.
[0392] To this ester intermediate was added THF (2 mL), NaOH (2 mL,
1.0 mmol) and methanol (1 mL). The mixture was stirred overnight,
then quenched with 1N HCl (1 mL) and concentrated. The mixture was
diluted with water and extracted with EtOAc. The combined organic
layers were dried over anhydrous sodium sulfate, filtered and
concentrated to an oily residue. The residue was purified by
reverse phase HPLC to provide Example 37. .sup.1H NMR (500 MHz,
DMSO-d.sub.6) .delta. 11.67 (s, 1H), 8.26 (d, 1H), 7.89 (d, 1H),
7.30 (dd, 1H), 3.18 (t, 2H), 2.90 (t, 2H), 2.59 (s, 2H), 2.25 (t,
2H), 1.29 (t, 2H), 0.87 (s, 6H); LCMS m/z 369 (M-17).
Example 38
[0393] ##STR129##
[0394] To a suspension of 5-amino-2-cyano pyridine (20.0 g, 0.168
mol) in HF-pyridine (100 g) in an Erlenmeyer flask cooled to
0.degree. C. was added sodium nitrite (17.4 g, 0.251 mol) in four
portions. After 45 min at 0.degree. C. the reaction mixture was
stirred at room temperature for 30 min and then heated to
80.degree. C. for 90 min. The reaction mixture was quenched by
pouring into ice/water mixture. The resulting mixture was extracted
with DCM. The organic layer was dried over anhydrous sodium
sulfate, filtered and concentrated to give the fluoropyridine as an
orange solid.
[0395] To a suspension of this fluoropyridine nitrile intermediate
(16.0 g, 0.131 mol) in methanol (200 mL) was added hydroxylamine
(9.63 mL, 0.157 mmol, 50% by wt). After stirring the reaction at
room temperature for 48 h, it was filtered through a fritted
funnel. The precipitate was washed with ether and dried under
vacuum to give the N-hydroxy amidine as a yellow solid.
[0396] To a suspension of this amidine intermediate (5.32 g, 34.32
mmol) in anhydrous pyridine (10 mL) was added 4-chloro-4-oxo-methyl
butyrate (5 mL, 41.18 mmol). The resulting reaction mixture was
heated at 120.degree. C. for 2 h. The mixture was cooled to RT and
concentrated. The residue was dissolved in ethyl acetate and washed
with 1N HCl, water and brine. The organic layer was dried over
anhydrous sodium sulfate, filtered and concentrated to give a dark
brown solid. This material was purified by Biotage using 25%-60%
ethyl acetate-hexanes gradient to give the heterobiaryl
intermediate as a light yellow solid.
[0397] To a solution of this ester intermediate (900 mg, 3.58 mmol)
in THF (4 mL) was added methanol (2 mL) followed by 5N NaOH (1 mL).
After 30 min, the reaction mixture was neutralized by the addition
of 1N HCl (5 mL). The reaction mixture was concentrated. The
residue was extracted with ethyl acetate, and the organic layer was
washed with brine, dried over anhydrous sodium sulfate, filtered
and concentrated to give a light yellow solid of the carboxylic
acid.
[0398] To a solution of this acid intermediate (50 mg, 0.21 mmol)
in anhydrous dichloromethane (4 mL) cooled to 0.degree. C. under
nitrogen atmosphere was added DMF (10 .mu.L) followed by oxalyl
chloride (0.21 mL, 2.0 M solution in DCM). The reaction was warmed
to 23.degree. C. and stirred for 30 min. The reaction mixture was
concentrated, and the residue was dissolved in anhydrous
dichloromethane (2 mL) and cooled to 0.degree. C. A dichloromethane
(2 mL) solution of the methyl (R)-cyclohexene aminoester described
in the Examples above (90 mg, 0.525 mmol) was then added. The
ice-bath was removed, and the resulting solution was stirred at RT
for 16 h. The reaction mixture was quenched by the addition of
saturated sodium bicarbonate solution. The resulting mixture was
extracted with dichloromethane. The organic layer was dried over
anhydrous sodium sulfate, filtered and concentrated. The residue
was purified by flash chromatography using 25% then 35% ethyl
acetate-hexanes to give the amide as a white solid.
[0399] To a solution of this amide ester intermediate (41 mg) in
THF was added 1N LiOH (1 mL). The resulting mixture was stirred for
16 h. The reaction mixture was neutralized by the addition of 1N
HCl (1 mL). The resulting biphasic mixture was extracted with ethyl
acetate. The organic layer was dried over anhydrous sodium sulfate,
filtered and concentrated. The residue was purified by reverse
phase HPLC to provide Example 38 as the (R)-enantiomer. .sup.1H NMR
(500 MHz, DMSO d.sub.6) .delta. 12.6 (bs, 1H), 11.64 (s, 1H), 8.76
(d, 1H), 8.14 (dd, 1H), 7.93 (dt, 1H), 3.2 (t, 2H), 2.9 (m, 3H),
2.3 (m, 2H), 2.15 (m, 1H), 1.6 (m, 2H), 1.1 (m, 1H), 0.92 (d, 3H);
LCMS m/z 397 (M+Na).
Example 39
[0400] ##STR130##
[0401] To a solution of 3-hydroxy benzaldehyde (1.2 g, 10 mmol) in
anhydrous DCM (50 mL) was added imidazole (1.02 g, 15 mmol)
followed by TBSCl (1.65 g, 11 mmol). After stirring the reaction at
room temperature for 1 h, it was quenched by pouring into saturated
sodium bicarbonate solution. The resulting mixture was extracted
with DCM, and the organic layer was dried over anhydrous sodium
sulfate, filtered and concentrated to give the silyl ether
aldehyde.
[0402] To a solution of this aldehyde intermediate (2.24 g, 9.5
mmol) in ethanol (50 mL) was added sodium borohydride (0.5 g, 14.5
mmol). After stirring the mixture at RT for 1 h, it was
concentrated, and the residue was dissolved in ethyl acetate and
washed with water and brine. The organic layer was dried over
anhydrous sodium sulfate, filtered and concentrated to give the
hydroxy methylene intermediate.
[0403] To a solution of this alcohol (2.25 g, 9.5 mmol) in
anhydrous THF (50 mL) cooled to 0.degree. C. under a nitrogen
atmosphere was added sodium hydride (0.57 g, 14.25 mmol). After 15
min, methyl iodide (0.89 mL, 14.25 mmol) was added. After stirring
the mixture at room temperature for 1 h, it was quenched with
saturated ammonium chloride solution. The resulting mixture was
extracted with ethyl acetate, washed with brine, dried over
anhydrous sodium sulfate, filtered and concentrated. The crude
material was dissolved in THF (20 mL) and TBAF (2 mL) was added.
After 1 h, the reaction mixture was concentrated and the residue
purified by flash chromatography using 30% ethyl acetate hexanes to
give the methyl ether as a colorless oil.
[0404] To a solution of this phenol intermediate (0.9 g, 6.52 mmol)
in methanol (20 mL) was added Rh/Al.sub.2O.sub.3 (50 mg). The
resulting mixture was stirred under a hydrogen balloon for 18 h.
The mixture was filtered through celite and concentrated to give
the desired cyclohexanol as a colorless oil.
[0405] To a solution of this cyclohexanol (870 mg, 6 mmol) in DCM
cooled to -78.degree. C. was added DMSO (0.85 mL, 12 mmol) followed
by oxalyl chloride (4.5 mL, 2M in DCM). After 10 min, triethylamine
(1.67 mL, 12 mmol) was added and the reaction mixture slowly warmed
to 0.degree. C. over 1 h. The mixture was quenched by pouring into
saturated sodium bicarbonate solution. The resulting mixture was
extracted with DCM. The organic layer was dried over anhydrous
sodium sulfate, filtered and concentrated. The residue was purified
by flash chromatography using 10% ethyl acetate-hexanes to give the
cyclohexanone intermediate.
[0406] To a suspension of sodium hydride (0.225 g, 5.62 mmol, 60%
dispersion in oil) in anhydrous dioxane (5 mL) was added dimethyl
carbonate (1 mL, 11.87 mmol). The resulting mixture was heated to
85.degree. C., and a solution the cyclohexanone intermediate (400
mg, 2.81 mmol) in dioxane (5 mL) was added dropwise via an addition
funnel. After stirring at 80.degree. C. for 2 h, the reaction
mixture was cooled to room temperature and quenched with 1N HCl.
The resulting mixture was concentrated. The residue was extracted
with ether. The organic layer was washed with brine, dried over
magnesium sulfate, filtered and concentrated. The residue was
purified by flash chromatography 30% ethyl acetate-hexanes to give
the ketoester.
[0407] To a solution of this ketoester (199 mg, 0.994 mmol) in
methanol (12 mL), was added ammonium acetate (383 mg, 4.97 mmol).
The reaction was left stirring at room temperature, and
concentrated. The residue was dissolved in EtOAc and washed with
water and brine. The organic phase was dried over anhydrous sodium
sulfate, filtered and concentrated to give the methoxymethyl ether
substituted cyclohexene aminoester.
[0408] Following similar procedures as described for the Examples
above, Example 39 was obtained after amide formation and
hydrolysis. The residue was purified by reverse phase HPLC to
provide Example 39. .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta.
11.66 (s, 1H), 8.76 (d, 1H), 8.14 (dd, 1H), 7.94 (m, 1), 3.24-3.20
(m, 7H), 3.01-2.89 (m, 3H), 2.43 (t, 1H), 2.37 (d, 1H), 2.16 (m,
1H), 1.75 (m, 1H), 1.68 (br d, 1H), 1.14 (m, 1H); LCMS m/z 403
(M-1).
Example 40
[0409] ##STR131##
[0410] To a solution of tetrahydro-4-H-pyran-4-one (1 mL, 10.82
mmol) in anhydrous THF (50 mL) cooled to -78.degree. C. under a
nitrogen atmosphere, was added lithium diisopropylamide (6.5 mL,
13.02 mmol, 2.0 M solution). After 20 min, methyl cyanoformate
(1.03 mL, 13.03 mmol) was added. The resulting mixture was slowly
warmed to -20.degree. C., and quenched with saturated ammonium
chloride solution. The biphasic mixture was extracted with ethyl
acetate, washed with brine and dried over anhydrous sodium sulfate.
The organic layer was filtered, concentrated and purified by flash
chromatography using 30% ethyl acetate hexanes to give the
ketoester as a colorless oil.
[0411] To a solution of this ketoester intermediate (0.450 g, 2.85
mmol) in anhydrous THF (20 mL) cooled to 0.degree. C., was added
sodium hydride (0.171 g, 4.27 mmol, 60% by weight). After 30 min,
2-[N,N-Bis(trifluromethylsulfonyl)amino]-5-chloropyridine (1.34 g,
3.42 mmol) was added. After stirring the reaction mixture at room
temperature for 2 h, it was quenched with saturated ammonium
chloride solution. The resulting mixture was extracted with ethyl
acetate, and the combined organic layers were washed with brine,
dried over anhydrous sodium sulfate, filtered and concentrated. The
residue was purified by flash chromatography using 30% ethyl
acetate-hexanes to give the enol triflate as colorless oil.
[0412] To a solution of 6-methoxy-2-naphthaldehyde (3.72 g, 20.0
mmol) in toluene (40 mL) placed in a pressure vessel, was added
methyl(triphenylphosphoranylidene)acetate (6.7 g, 20 mmol). The
resulting mixture was refluxed at 120.degree. C. for 18 h. The
reaction mixture was concentrated and purified using a Biotage
flash 40M column with 15% ethyl acetate-hexanes as the eluant, to
provide the enoate.
[0413] To a solution of this enoate (4.64 g, 19.14 mmol) in 1:1
dichloromethane-methanol (100 mL) was added Pd/C. The resulting
mixture was stirred under a H.sub.2 balloon for 18 h. The reaction
mixture was filtered through celite and concentrated to give the
methoxy ester as a white solid.
[0414] To a solution of this methyl ether intermediate (3.0 g, 12.3
mmol) in DCM (80 mL) cooled to 0.degree. C., was added BBr.sub.3
(61.5 mL, 11.0M in DCM). After 30 min, the mixture was quenched
with methanol (50 mL) followed by cold water. The resulting mixture
was concentrated, and the residue diluted with water and extracted
with dichloromethane. The organic layer was dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated. This naphtholic ester
was used in the next step without any further purification.
[0415] To a solution of this ester intermediate (3.0 g, 12.3 mmol)
in 1,4-dioxane (50 mL) placed in a pressure tube, was added
concentrated NH.sub.4OH solution. The resulting mixture was stirred
at RT for 18 h. The reaction mixture was concentrated and the
residue was suspended in ethyl acetate, washed with water, dried
over anhydrous sodium sulfate filtered and concentrated. The
residue was purified by flash chromatography using 50% ethyl
acetate-hexanes then 100% ethyl acetate as the eluant to give the
naphtholic primary carboxamide as an off-white solid.
[0416] To a solution of the enol triflate intermediate (100 mg,
0.344 mmol) in anhydrous dioxane (3 mL) was added the primary
carboxamide intermediate (61 mg, 0.287 mmol), XANTPHOS (40 mg,
0.068 mmol), cesium carbonate (157 mg, 0.481 mmol) and
Pd.sub.2(dba).sub.3 (19 mg, 0.02 mmol). The resulting mixture was
degassed for 2 min by bubbling N.sub.2 gas. The reaction mixture
was heated at 50.degree. C. under a N.sub.2 atmosphere for 2 h. The
reaction mixture was cooled to room temperature, and filtered
through celite. The filtrate was concentrated and purified by flash
chromatography using 40% ethyl acetate-hexanes to give the amide
product.
[0417] To a solution of this ester penultimate intermediate (44 mg)
in THF (2 mL) was added 1N NaOH (1 mL) followed by MeOH (1 mL). The
resulting mixture was stirred at 23.degree. C. for 5 h. The
reaction mixture was quenched by the addition of 1N HCl (1 mL). The
resulting mixture was concentrated, and the residue was extracted
with ethyl acetate. The combined organic layers were washed with
brine, dried over anhydrous sodium sulfate, filtered and
concentrated. The residue was purified by reverse phase HPLC
(Gilson) to provide Example 40. .sup.1H NMR (500 MHz, DMSO-d.sub.6)
.delta. 11.38 (s, 1H), 9.59 (bs, 1H), 7.65 (d, 1H), 7.56 (m, 2H),
7.28 (d, 1H), 7.05 (m, 2H), 4.16 (s, 2H), 3.65 (t, 2H), 2.9 (t,
2H), 2.86 (bt, 2H), 2.65 (t, 2H); LCMS m/z 342 (M+1).
Example 41
[0418] ##STR132##
[0419] Potassium hexamethyldisilazide (35 mL of a 0.5 M solution in
THF, 17.5 mmol) was added to propyl triphenylphosphonium bromide
(7.1 g, 18.5 mmol) in toluene (75 mL) at 0.degree. C. The solution
was stirred for 15 min, and the ketone (2.1 g, 12.3 mmol) in
toluene (50 mL) was added. The solution was stirred at 0.degree. C.
for 1 h and then heated at 100.degree. C. overnight. Solvent was
removed, and the product was purified by flash chromatography
(Biotage, Horizon) 0 to 10% ethyl acetate/hexanes. The product was
dissolved in methanol (150 mL) and stirred over palladium on carbon
(5%, 1 g) under an atmosphere of hydrogen overnight. The solution
was filtered through celite and solvent was removed. The product
was dissolved in THF/MeOH/3N HCl (50 mL/20 mL/10 mL) for 36 h. The
mixture was neutralized with saturated sodium bicarbonate, and the
solvent was removed. The solution was washed with ethyl acetate,
and the resulting organic layer was washed with brine and dried
over Na.sub.2SO.sub.4. The product was purified by flash
chromatography (Biotage, Horizon) 0 to 20% ethyl
acetate/hexanes.
[0420] To a solution of the ketone (796 m g, 5.2 mmol) in anhydrous
THF (25 mL) cooled to -78.degree. C. under a N.sub.2 atmosphere,
was added LiHMDS (6.2 mL, 6.2 mmol, 1.0 M in THF). After 30 min,
methyl cyanoformate (0.538 mL, 6.7 mmol) was added, and the
reaction mixture was allowed to warm to 0.degree. C. over several
hours. The mixture was quenched with 1N HCl and extracted with
EtOAc (2.times.). The organic layer was washed with brine and dried
over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. This
material was used in the next step without any further
purification.
[0421] To a solution of the ketoester (1095 mg, 5.2 mmol) in
anhydrous THF (50 mL) was added NaH (309 mg, 7.7 mmol, 60%). After
15 min, 2-[N,N-Bis(trifluromethylsulfonyl)amino]-5-chloropyridine
(2.02 g, 5.2 mmol) was added. The reaction mixture was stirred at
room temperature for 18 h and then quenched with water. The
resulting mixture was extracted with EtOAc (2.times.). The organic
layer was washed with brine, dried over Na.sub.2SO.sub.4, filtered,
and concentrated in vacuo. The crude material was purified by flash
chromatography (Biotage, Horizon) (0% EtOAc/Hexane to 20%
EtOAc/Hexane) to give the desired product.
[0422] To a solution of the vinyl triflate (200 mg, 0.58 mmol) in
anhydrous dioxane (11 mL) was added the amide (15 mg, 0.07 mmol),
XANTPHOS (32 mg, 0.05 mmol), cesium carbonate (22 mg, 0.17 mmol)
and Pd.sub.2(dba).sub.3 (20 mg, 0.02 mmol). The resulting mixture
was de-gassed for 2 min by bubbling gaseous N.sub.2. The mixture
was heated at 60.degree. C. under a N.sub.2 atmosphere for 18 h.
The reaction mixture was cooled to room temperature, and filtered
through celite. The filtrate was concentrated in vacuo, and the
residue was purified by reverse phase HPLC (Gilson) to give the
desired product.
[0423] To a solution of the methyl ester in dioxane (3 mL) was
added MeOH (1 mL) and 1N LiOH (1 miL). The resulting mixture was
stirred at room temperature for 18 h, and then neutralized to pH=7
by the addition of 1N HCl, and purified by reverse phase HPLC
(Gilson) to provide Example 41. .sup.1H NMR (500 MHz, DMSO-d.sub.6)
.delta. 7.65 (d, 1H), 7.58 (d, 1H), 7.56 (s, 1H), 7.28 (d, 1H),
7.06-7.02 (m, 2H), 2.97-2.88 (m, 3H), 2.65-2.63 (m, 3H), 2.45-2.33
(m, 2H), 1.84-1.71 (m, 1H), 1.65-1.62 (m, 1H), 1.51-1.15 (m, 4H),
0.901 (d, 3H), 0.86 (t, 3H); LCMS m/z 396 (M+1).
Biological Assays
[0424] The activity of the compounds of the present invention
regarding niacin receptor affinity and function can be evaluated
using the following assays:
.sup.3H-Niacin Binding Assay:
1. Membrane: Membrane preps are stored in liquid nitrogen in:
[0425] 20 mM HEPES, pH 7.4
[0426] 0. 1 mM EDTA
[0427] Thaw receptor membranes quickly and place on ice. Resuspend
by pipetting up and down vigorously, pool all tubes, and mix well.
Use clean human at 15 .mu.g/well, clean mouse at 10 ug/well, dirty
preps at 30 ug/well. [0428] 1a. (human): Dilute in Binding Buffer.
[0429] 1b. (human+4% serum): Add 5.7% of 100% human serum stock
(stored at -20.degree. C.) for a final concentration of 4%. Dilute
in Binding Buffer. [0430] 1c. (mouse): Dilute in Binding Buffer. 2.
Wash buffer and dilution buffer: Make 10 liters of ice-cold Binding
Buffer:
[0431] 20 mM HEPES, pH 7.4
[0432] 1 mM MgCl.sub.2
[0433] 0.01% CHAPS (w/v)
[0434] use molecular grade or ddH.sub.2O water
3. [5,6.sup.-H]-nicotinic acid: American Radiolabeled Chemicals,
Inc. (cat # ART-689). Stock is .about.50 Ci/mmol, 1 mCi/ml, 1 ml
total in ethanol.fwdarw.20 .mu.M
[0435] Make an intermediate .sup.3H-niacin working solution
containing 7.5% EtOH and 0.25 .mu.M tracer. 40 .mu.L of this will
be diluted into 200 .mu.L total in each well-1.5% EtOH, 50 nM
tracer final.
4. Unlabeled nicotinic acid:
[0436] Make 100 mM, 110 mM, and 80 .mu.M stocks; store at
-20.degree. C. Dilute in DMSO.
5. Preparing Plates:
[0437] 1) Aliquot manually into plates. All compounds are tested in
duplicate. 10 mM unlabeled nicotinic acid must be included as a
sample compound in each experiment. [0438] 2) Dilute the 10 mM
compounds across the plate in 1:5 dilutions (8 .mu.l:40 .mu.l).
[0439] 3) Add 195 .mu.L binding buffer to all wells of Intermediate
Plates to create working solutions (250 .mu.M.fwdarw.0). There will
be one Intermediate Plate for each Drug Plate. [0440] 4) Transfer
51 .mu.L from Drug Plate to the Intermediate Plate. Mix 4-5 times.
6. Procedure: [0441] 1) Add 140 .mu.L of appropriate diluted 19CD
membrane to every well. There will be three plates for each drug
plate: one human, one human+serum, one mouse. [0442] 2) Add 20
.mu.L of compound from the appropriate intermediate plate [0443] 3)
Add 40 .mu.L of 0.25 .mu.M .sup.3H-nicotinic acid to all wells.
[0444] 4) Seal plates, cover with aluminum foil, and shake at RT
for 3-4 hours, speed 2, titer plate shaker. [0445] 5) Filter and
wash with 8.times.200 .mu.L ice-cold binding buffer. Be sure to
rinse the apparatus with >1 liter of water after last plate.
[0446] 6) Air dry overnight in hood (prop plate up so that air can
flow through). [0447] 7) Seal the back of the plate [0448] 8) Add
40 .mu.l Microscint-20 to each well. [0449] 9) Seal tops with
sealer. [0450] 10) Count in Packard Topcount scintillation counter.
[0451] 11) Upload data to calculation program, and also plot raw
counts in Prism, determining that the graphs generated, and the
IC.sub.50 values agree.
[0452] The compounds of the invention generally have an IC.sub.50
in the .sup.3H-nicotinic acid competition binding assay within the
range of 1 nM to about 25 .mu.M.
.sup.35S-GTP.gamma.S binding assay:
[0453] Membranes prepared from Chinese Hamster Ovary (CHO)-K1 cells
stably expressing the niacin receptor or vector control (7
.mu.g/assay) were diluted in assay buffer (100 mM HEPES, 100 mM
NaCl and 10 mM MgCl.sub.2, pH 7.4) in Wallac Scintistrip plates and
pre-incubated with test compounds diluted in assay buffer
containing 40 .mu.M GDP (final [GDP] was 10 .mu.M) for .about.10
minutes before addition of .sup.35S-GTP.gamma.S to 0.3 nM. To avoid
potential compound precipitation, all compounds were first prepared
in 100% DMSO and then diluted with assay buffer resulting in a
final concentration of 3% DMSO in the assay. Binding was allowed to
proceed for one hour before centrifuging the plates at 4000 rpm for
15 minutes at room temperature and subsequent counting in a
TopCount scintillation counter. Non-linear regression analysis of
the binding curves was performed in GraphPad Prism.
Membrane Preparation
Materials:
[0454] CHO-K1 cell culture medium: F-12 Kaighn's Modified Cell
Culture Medium with 10% FBS, 2 mM L-Glutamine, 1 mM Sodium Pyruvate
and 400 .mu.g/ml G418 [0455] Membrane Scrape Buffer: 20 mM HEPES
[0456] 10 mM EDTA, pH 7.4 [0457] Membrane Wash Buffer: 20 mM HEPES
[0458] 0.1 mM EDTA, pH 7.4 [0459] Protease Inhibitor Cocktail:
P-8340, (Sigma, St. Louis, Mo.) Procedure:
[0460] (Keep everything on ice throughout prep; buffers and plates
of cells) [0461] Aspirate cell culture media off the 15 cm.sup.2
plates, rinse with 5 mL cold PBS and aspirate. [0462] Add 5 ml
Membrane Scrape Buffer and scrape cells. Transfer scrape into 50 mL
centrifuge tube. Add 50 uL Protease Inhibitor Cocktail. [0463] Spin
at 20,000 rpm for 17 minutes at 4.degree. C. [0464] Aspirate off
the supernatant and resuspend pellet in 30 mL Membrane Wash Buffer.
Add 50 .mu.L Protease Inhibitor Cocktail. [0465] Spin at 20,000 rpm
for 17 minutes at 4.degree. C. [0466] Aspirate the supernatant off
the membrane pellet. The pellet may be frozen at -80.degree. C. for
later use or it can be used immediately. Assay Materials: [0467]
Guanosine 5'-diphosphate sodium salt (GDP, Sigma-Aldrich Catalog
#87127) [0468] Guanosine 5'-[.gamma..sup.35S]thiotriphosphate,
triethylammonium salt ([.sup.35S]GTP.gamma.S, Amersham Biosciences
Catalog #SJ1320, .about.1000 Ci/mmol) [0469] 96 well Scintiplates
(Perkin-Elmer #1450-501) [0470] Binding Buffer: 20 mM HEPES, pH 7.4
[0471] 100 mM NaCl [0472] 10 mM MgCl.sub.2 [0473] GDP Buffer:
binding buffer plus GDP, ranging from 0.4 to 40 .mu.M, make fresh
before assay Procedure: [0474] (total assay volume=100 .mu.well)
[0475] 25 .mu.L GDP buffer with or without compounds (final GDP 10
.mu.M--so use 40 .mu.M stock) [0476] 50 .mu.L membrane in binding
buffer (0.4 mg protein/mL) [0477] 25 .mu.L [.sup.35S]GTP-.gamma.S
in binding buffer. This is made by adding 5 .mu.l
[.sup.35S]GTP.gamma.S stock into 10 mL binding buffer (This buffer
has no GDP) [0478] Thaw compound plates to be screened (daughter
plates with 5 .mu.L compound@ 2 mM in 100% DMSO) [0479] Dilute the
2 mM compounds 1:50 with 245 .mu.L GDP buffer to 40 .mu.M in 2%
DMSO. (Note: the concentration of GDP in the GDP buffer depends on
the receptor and should be optimized to obtain maximal signal to
noise; 40 .mu.M). [0480] Thaw frozen membrane pellet on ice. (Note:
they are really membranes at this point, the cells were broken in
the hypotonic buffer without any salt during the membrane prep
step, and most cellular proteins were washed away) [0481]
Homogenize membranes briefly (few seconds--don't allow the
membranes to warm up, so keep on ice between bursts of
homogenization) until in suspension using a POLYTRON PT3100 (probe
PT-DA 3007/2 at setting of 7000 rpm). Determine the membrane
protein concentration by Bradford assay. Dilute membrane to a
protein concentrations of 0.40 mg/ml in Binding Buffer. (Note: the
final assay concentration is 20 .mu.g/well). [0482] Add 25 .mu.L
compounds in GDP buffer per well to Scintiplate. [0483] Add 50
.mu.L of membranes per well to Scintiplate. [0484] Pre-incubate for
5-10 minutes at room temperature. (cover plates with foil since
compounds may be light sensitive) [0485] Add 25 .mu.L of diluted
[.sup.35S]GTP.gamma.S. Incubate on shaker (Lab-Line model #1314,
shake at setting of 4) for 60 minutes at room temperature. Cover
the plates with foil since some compounds might be light sensitive.
[0486] Assay is stopped by spinning plates sealed with plate covers
at 2500 rpm for 20 minutes at 22.degree. C. [0487] Read on TopCount
NXT scintillation counter -.sup.35S protocol.
[0488] The compounds of the invention generally have an EC.sub.50
in the functional in vitro GTP.gamma.S binding assay within the
range of about less than 1 .mu.M to as high as about 100 .mu.M.
Flushing via Laser Doppler
[0489] Male C57B16 mice (.about.25 g) are anesthetized using 10
mg/ml/kg Nembutal sodium. When antagonists are to be administered
they are co-injected with the Nembutal anesthesia. After ten
minutes the animal is placed under the laser and the ear is folded
back to expose the ventral side. The laser is positioned in the
center of the ear and focused to an intensity of 8.4-9.0 V (with is
generally .about.4.5 cm above the ear). Data acquisition is
initiated with a 15 by 15 image format, auto interval, 60 images
and a 20 sec time delay with a medium resolution. Test compounds
are administered following the 10th image via injection into the
peritoneal space. Images 1-10 are considered the animal's baseline
and data is normalized to an average of the baseline mean
intensities.
Materials and Methods--Laser Doppler Pirimed Pimil; Niacin (Sigma);
Nembutal (Abbott labs).
[0490] All patents, patent applications and publications that are
cited herein are hereby incorporated by reference in their
entirety. While certain preferred embodiments have been described
herein in detail, numerous alternative embodiments are seen as
falling within the scope of the invention.
* * * * *