U.S. patent application number 12/275164 was filed with the patent office on 2009-05-21 for substituted benzoazole pde4 inhibitors for treating pulmonary and cardiovascular disorders.
This patent application is currently assigned to DECODE GENETICS EHF. Invention is credited to Alex Burgin, Mark E. Gurney, Alexander Kiselyov, Munagala Rao, Jasbir Singh.
Application Number | 20090130076 12/275164 |
Document ID | / |
Family ID | 40229764 |
Filed Date | 2009-05-21 |
United States Patent
Application |
20090130076 |
Kind Code |
A1 |
Singh; Jasbir ; et
al. |
May 21, 2009 |
SUBSTITUTED BENZOAZOLE PDE4 INHIBITORS FOR TREATING PULMONARY AND
CARDIOVASCULAR DISORDERS
Abstract
The invention relates to substituted benzothiazoles,
benzoxazoles--and their counterparts having pyridine and pyrimidine
rings replacing the benzene ring--that are PDE4 inhibitors useful
for treating stroke, myocardial infarct, and cardiovascular
inflammatory conditions, to pharmaceutical compositions comprising
these compounds, and to methods for the treatment of stroke,
myocardial infarct, and cardiovascular inflammatory conditions in a
mammal. The compounds have general formula I: ##STR00001## in which
A and B are carbocycles or heterocycles. A particular embodiment is
##STR00002##
Inventors: |
Singh; Jasbir; (Naperville,
IL) ; Gurney; Mark E.; (Grand Rapids, MI) ;
Burgin; Alex; (Kingston, WA) ; Kiselyov;
Alexander; (San Diego, CA) ; Rao; Munagala;
(Westmont, IL) |
Correspondence
Address: |
HESLIN ROTHENBERG FARLEY & MESITI PC
5 COLUMBIA CIRCLE
ALBANY
NY
12203
US
|
Assignee: |
DECODE GENETICS EHF
Reykjavik
IS
|
Family ID: |
40229764 |
Appl. No.: |
12/275164 |
Filed: |
November 20, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60989557 |
Nov 21, 2007 |
|
|
|
Current U.S.
Class: |
424/94.1 ;
514/210.18; 514/254.03; 544/368; 546/270.1 |
Current CPC
Class: |
A61K 31/423 20130101;
C07D 513/04 20130101; C07D 263/58 20130101; A61P 9/06 20180101;
A61P 19/10 20180101; A61P 9/10 20180101; A61P 19/00 20180101; C07D
417/04 20130101; A61P 35/04 20180101; A61P 29/00 20180101; C07D
417/06 20130101; A61P 35/00 20180101; A61K 31/4439 20130101; C07D
263/56 20130101; A61P 11/00 20180101; A61P 19/08 20180101; C07D
417/14 20130101; A61K 31/519 20130101; A61P 11/06 20180101; A61P
13/10 20180101; C07D 277/64 20130101; A61K 31/454 20130101; C07D
277/82 20130101; A61P 1/00 20180101; A61K 31/496 20130101; C07D
498/04 20130101; A61P 13/02 20180101; A61P 43/00 20180101; A61P
9/00 20180101; A61K 31/428 20130101 |
Class at
Publication: |
424/94.1 ;
544/368; 514/254.03; 514/210.18; 546/270.1 |
International
Class: |
A61K 31/122 20060101
A61K031/122; C07D 417/08 20060101 C07D417/08; A61K 31/4965 20060101
A61K031/4965; A61P 35/00 20060101 A61P035/00; A61K 31/4439 20060101
A61K031/4439; C07D 417/14 20060101 C07D417/14 |
Claims
1. A compound of formula Ia, Ib or Ic: ##STR00085## or salt thereof
wherein U is selected from the group consisting of --S-- and --O--;
V is selected from the group consisting of H, CH.sub.3, NH.sub.2,
and CF.sub.3; X is selected from the group consisting of CH, C--F,
C--Cl, C--Br, C--I, C--NH.sub.2, C--OH, C--OCH.sub.3, N, and N--O;
Y is selected from the group consisting of N, CH, CF and C-lower
alkyl; R.sup.1 is H or lower alkyl; R.sup.2 is selected from the
group consisting of H, alkyl, OH, NH.sub.2, and OCH.sub.3; B is an
optionally substituted, mono- or bicyclic aryl or heteroaryl; A is
an optionally substituted heterocycle or an optionally substituted
carbocycle; and A.sup.1 is chosen from (a) a residue chosen from
##STR00086## wherein R.sup.40 is chosen from H, halogen, OH,
NH.sub.2 and CH.sub.3; (b) a substituted heterocycle of three or
fewer rings or substituted carbocycle of three or fewer rings; and
(c) a heterocycle that is itself substituted with a heterocycle
carrying a further substituent; wherein substituents on the
heterocycle or carbocycle are chosen from hydroxy, carboxy,
carboxyalkyl, carboxyalkoxy, carboxyalkylthio, alkoxycarbonyl,
carboxyalkylcarbonylamino, carboxyalkylaminocarbonylamino,
guanidino, the residue of an amino acid and the residue of an
N-methylated amino acid.
2. A compound or salt according to claim 1 wherein B is phenyl
which has a substituent at the 3-position, the 4-position or at
both the 3- and 4-positions.
3. A compound or salt according to claim 2 wherein B is selected
from 3-chlorophenyl, 3-nitrophenyl, 3-cyanophenyl, 3-bromophenyl,
3-acetylphenyl, 3-trifluoromethylphenyl, and
3-methylthiophenyl.
4. A compound or salt according to claim 1 wherein B is
benzo[c][1,2,5]oxadiazol-5-yl or benzo[d][1,3]dioxol-5-yl.
5. A compound or salt according to claim 1 wherein R.sup.1 is H and
R.sup.2 is chosen from H and OH.
6. A compound of formula Ia, Ib or Ic or salt thereof according to
claim 1 wherein A or A.sup.1 is optionally substituted phenyl.
7. A compound of formula Ia, Ib or Ic or salt thereof according to
claim 1 wherein A or A.sup.1 is selected from the group consisting
of optionally substituted 5- and 6-membered ring nitrogen
heterocycles.
8. A compound or salt according to claim 7 wherein A or A.sup.1 is
selected from the group consisting of optionally substituted
pyridinyl, morpholin-4-yl, piperazin-1-yl, piperidiny-1-yl,
imidazol-1-yl, pyrazol-1-yl, and pyrazol-5-yl.
9. A compound or salt according to claim 1 wherein X is selected
from the group consisting of CH, C--F, C--OH and N.
10. A compound or salt according to claim 1 wherein X is selected
from the group consisting of CH, C--F, C--OH, N and N--O; Y is N or
CH; U is O; V is selected from H, CH.sub.3 and NH.sub.2; B is
benzo[c][1,2,5]oxadiazol-5-yl or phenyl which has a substituent at
the 3-position, the 4-position or at both the 3- and 4-positions;
R.sup.1 is H; R.sup.2 is chosen from H and OH; and A or A.sup.1 is
selected from the group consisting of optionally substituted phenyl
and optionally substituted 5- and 6-membered ring nitrogen
heterocycles.
11. A compound or salt according to claim 1 wherein X is selected
from the group consisting of CH, C--F, C--OH, N and N--O; Y is N or
CH; U is S; V is selected from H, CH.sub.3 and NH.sub.2; B is
benzo[c][1,2,5]oxadiazol-5-yl or phenyl which has a substituent at
the 3-position, the 4-position or at both the 3- and 4-positions;
R.sup.1 is H; R.sup.2 is chosen from H and OH; and A or A.sup.1 is
selected from the group consisting of optionally substituted phenyl
and optionally substituted 5- and 6-membered ring nitrogen
heterocycles.
12. A compound or salt according to claim 6 or 7 wherein B is
selected from benzo[d][1,3]dioxol-5-yl, 3-chlorophenyl,
3-nitrophenyl, 3-cyanophenyl, 3-bromophenyl, 3-acetylphenyl,
3-trifluoromethylphenyl, and 3-methylthiophenyl.
13. A compound or salt according to claim 12 wherein A or A.sup.1
is selected from the group consisting of optionally substituted
pyridinyl, morpholin-4-yl, piperazin-1-yl, piperidiny-1-yl,
imidazol-1-yl, pyrazol-1-yl, and pyrazol-5-yl.
14. A compound or salt according to claim 1 of formula ##STR00087##
wherein A.sup.2 is chosen from phenyl, five-membered heteroaryl,
six-membered heteroaryl, 4-7 membered non-aryl heterocycle and
fused bicycle; R.sup.7 is H or F; R.sup.8 is chosen from halogen,
nitro, acetyl, hydroxyethyl, amino, methylthio, trifluoromethyl,
methoxymethyl, methoxycarbonyl, trifluoromethoxy, cyano and
1,3,4-thiadiazol-2-yl, or taken together R.sup.7 and R.sup.8 are
methylenedioxy, .dbd.N--O--N.dbd., --NH--CH.dbd.N-- or
difluoromethylenedioxy; R.sup.14 is chosen from H, halogen,
haloalkyl, alkyl, acyl, alkoxyalkyl, hydroxyalkyl, carbonyl,
phenyl, heteroaryl, benzenesulfonyl, hydroxy, alkoxy, haloalkoxy,
oxaalkyl, carboxy, alkoxycarbonyl, alkoxycarbonylalkyl,
alkoxycarbonylamino, carboxyalkyl, carboxyalkoxy, carboxyalkylthio,
alkoxycarbonylaminoalkyl, carboxyalkylcarbonylamino, carboxamido,
aminocarbonyloxy, alkylaminocarbonyl, dialkylaminocarbonyl,
aminocarbonylalkyl, cyano, acetoxy, nitro, amino, alkylamino,
dialkylamino, aminoalkyl, (alkyl)(aryl)aminoalkyl, alkylaminoalkyl,
dialkylaminoalkyl, dialkylaminoalkoxy, alkyl(hydroxyalkyl)amino,
heterocyclylalkoxy, mercapto, alkylthio, alkylsulfonyl,
alkylsulfonylamino, alkylsulfinyl, alkylsulfonyl, arylthio,
arylsulfonyl, arylsulfonylamino, arylsulfinyl, arylsulfonyl,
acylaminoalkyl, acylaminoalkoxy, acylamino, amidino, aryl, benzyl,
heterocyclyl, heterocyclylalkyl, phenoxy, benzyloxy, heteroaryloxy,
heterocyclylamino, hydroxyimino, alkoxyimino, oxaalkyl,
aminosulfonyl, trityl, amidino, guanidino, ureido,
--NHC(.dbd.O)NHalkyl, --NHC(.dbd.O)NH-heterocyclyl,
-alkyl-NHC(.dbd.O)N(alkyl).sub.2, heterocyclylalkylcarbonylamino,
benzyloxyphenyl, benzyloxy, the residues of amino acids, amino acid
amides, protected residues of aminoacids, protected residues of
amino acid amides, N-methylated amino acids and N-methylated amino
acid amides and monocyclic heterocycle substituted with any of the
foregoing; R.sup.14a is chosen from hydroxy, carboxy,
alkoxycarbonyl, carboxyalkylcarbonylamino, carboxyalkyl,
carboxyalkoxy, carboxyalkylthio, carboxyalkylaminocarbonylamino,
guanidino, the residue of an amino acid and the residue of an
N-methylated amino acid, 5-tetrazolyl and monocyclic heterocycle
substituted with any of the foregoing; R.sup.15 is chosen from H,
NO.sub.2, OH, NH.sub.2, and --NHSO.sub.2NH.sub.2; or R.sup.15
together with R.sup.14 forms methylene dioxy.
15. A compound or salt according to claim 1 of formula ##STR00088##
wherein A.sup.2 is phenyl, five-membered heteroaryl, six-membered
heteroaryl, 4-7 membered non-aryl heterocycle or fused bicycle;
R.sup.7 is H or F; R.sup.8 is chosen from halogen, nitro, acetyl,
hydroxyethyl, amino, methylthio, trifluoromethyl, methoxymethyl,
methoxycarbonyl, trifluoromethoxy, cyano and 1,3,4-thiadiazol-2-yl,
or taken together R.sup.7 and R.sup.8 are methylenedioxy,
.dbd.N--O--N.dbd., --NH--CH.dbd.N-- or difluoromethylenedioxy;
R.sup.14 is chosen from H, --CH.sub.3, --CH.sub.2CF.sub.3,
--CF.sub.3, --CHO, --COOH, --CN, halogen, --OH, --OEt,
--C(.dbd.O)NH.sub.2, --C(.dbd.O)NHEt,
--C(.dbd.O)NMe.sub.2-COOCH.sub.3, --COOEt,
--CH.sub.2NHC(.dbd.O)NH.sub.2, --CH(CH.sub.3)NHC(.dbd.O)NH.sub.2,
--CH.sub.2NHC(.dbd.O)H, --CH.sub.2NHC(.dbd.O)CH.sub.3,
--CH.sub.2C(.dbd.O)NH.sub.2, --CH.sub.2COOH, --CH.sub.2COOEt,
--CH.sub.2NHC(.dbd.O)OEt, --CH.sub.2NHC(.dbd.O)O--C.sub.6H.sub.5,
--CH.sub.2NHC(.dbd.O)C(.dbd.O)NH.sub.2, --CH.sub.2NHC(.dbd.O)NHEt,
--C(CH.sub.3).sub.2OH, --CH.sub.2NHC(.dbd.O)N(CH.sub.3).sub.2,
--CH.sub.2NHC(.dbd.O)NHCH.sub.3, --CH.sub.2NH.sub.2,
--CH(CH.sub.3)NH.sub.2, --C(CH.sub.3).sub.2NH.sub.2, --CH.sub.2OH,
--CH.sub.2CH.sub.2OH, --CH.sub.2NHSO.sub.2CH.sub.3,
--CH.sub.2C(.dbd.O)NHEt, --OCH.sub.3, --OC(.dbd.O)NH.sub.2,
--OCH.sub.2CH.sub.2N(CH.sub.3).sub.2, --OCH.sub.2CH.sub.2OCH.sub.3,
--OCH(CH.sub.3)COOH, --SCH.sub.2COOH, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHEt, --NHCH.sub.3, --NHEt, --NH(tBoc),
--NHCH.sub.2COOH, --N(CH.sub.3)CH.sub.2COOH,
--NHC(.dbd.O)NHCH.sub.2CH.sub.2Cl, --NHSO.sub.2NH.sub.2, --NHEt,
--N(CH.sub.3).sub.2, --NH.sub.2, --NH(CH.sub.3)C(.dbd.O)NH.sub.2,
--NHSO.sub.2CH.sub.3, --N(SO.sub.2CH.sub.3).sub.2,
--NHC(.dbd.O)OCH.sub.3, --NHC(.dbd.O)OtBu, --NHC(.dbd.O)CH.sub.3,
--SO.sub.2NH.sub.2, --NHC(.dbd.O)CH.sub.2CH.sub.2COOH,
--NHC(.dbd.O)NHCH.sub.2COOH, --CH.sub.2NHCHO,
--NHC(.dbd.O)NHCH.sub.2COOEt, --NHC(.dbd.O)NH(CH.sub.2).sub.3COOEt,
--NHC(.dbd.O)NH(CH.sub.2).sub.2COOEt,
--N(CH.sub.3)CH.sub.2CH.sub.2OH, --NHC(.dbd.O)OEt,
--N(Et)C(.dbd.O)OEt, --NHC(.dbd.O)NH(CH.sub.2).sub.2COOH,
--NHC(.dbd.O)CH.sub.2N(CH.sub.3).sub.2,
--NHC(.dbd.O)NH(CH.sub.2).sub.3COOH, --NHC(.dbd.O)CH.sub.2NH.sub.2,
--NHC(.dbd.O)CH.sub.2CH.sub.2NH.sub.2,
--NHC(.dbd.O)CH.sub.2NH(tBoc), ##STR00089## and monocyclic
heterocycle substituted with any of the foregoing; R.sup.14a is
chosen from --COOH, --OH, --COOCH.sub.3, --COOEt, --CH.sub.2COOH,
--CH.sub.2COOEt, --OCH(CH.sub.3)COOH, --SCH.sub.2COOH,
--CH.sub.2NHC(.dbd.O)OEt, --CH.sub.2NHC(.dbd.O)C(.dbd.O)NH.sub.2,
--NHCH.sub.2COOH, --N(CH.sub.3)CH.sub.2COOH, --NHSO.sub.2NH.sub.2,
--NHC(.dbd.O)CH.sub.2CH.sub.2COOH, --NHC(.dbd.O)NHCH.sub.2COOH,
--NHC(.dbd.O)NHCH.sub.2COOEt, --NHC(.dbd.O)NH(CH.sub.2).sub.3COOEt,
--NHC(.dbd.O)NH(CH.sub.2).sub.2COOEt,
--NHC(.dbd.O)NH(CH.sub.2).sub.2COOH,
--NHC(.dbd.O)NH(CH.sub.2).sub.3COOH, 5-tetrazolyl and monocyclic
heterocycle substituted with any of the foregoing; R.sup.15 is
chosen from H, NO.sub.2, OH, NH.sub.2, and --NHSO.sub.2NH.sub.2; or
R.sup.15 together with R.sup.14 forms methylene dioxy.
16. A compound or salt according to claim 1, wherein Y is N.
17. A compound or salt according to claim 1, wherein Y is CH.
18. A compound or salt according to claim 1, wherein U is S.
19. A compound or salt according to claim 1, wherein U is O.
20. A compound or salt according to claim 1, wherein V is selected
from H, CH.sub.3 and NH.sub.2.
21. A salt of a compound of claim 1 wherein the salt is a
pharmaceutically acceptable salt.
22. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a compound or pharmaceutically acceptable
salt according to claim 1.
23. A pharmaceutical composition comprising (a) a pharmaceutically
acceptable carrier; (b) a compound or pharmaceutically acceptable
salt according to claim 1; and (c) a second agent chosen from
cholinesterase inhibitors, NMDA antagonists, calpain inhibitors and
antioxidants.
24. A pharmaceutical composition according to claim 23 wherein said
second agent is chosen from tacrine, huperzine, donepezil,
lanicemine, remacemide, neramexane, memantine, vitamin E and
coenzyme Q10.
25. A method for the treatment or prophylaxis of a disease or
condition mediated by peripheral phosphodiesterase-4 comprising
administering to a mammal a therapeutically effective amount of a
compound according to claim 1.
26. A method according to claim 25 wherein said disease or
condition is chosen from stroke, myocardial infarct, and
cardiovascular inflammatory conditions.
27. A method according to claim 25 wherein said disease or
condition is cancer.
28. A method according to claim 25 wherein said disease or
condition is chosen from asthma and COPD.
29. A method for treating or preventing bone loss comprising
administering to a mammal a therapeutically effective amount of a
compound according to claim 1.
30. A method for treating bladder inflammation, bladder
overactivity and pain arising from bladder inflammation comprising
administering to a mammal a therapeutically effective amount of a
compound according to claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. provisional
application 60/989,557, filed Nov. 21, 2007, the entire disclosure
of which is incorporated herein by reference. The application is
related to, but does not claim priority from, four other US
non-provisional applications filed of even date herewith and having
Jasbir Singh as a common inventor. The applications are titled
"BIARYL PDE4 INHIBITORS FOR TREATING INFLAMMATORY, CARDIOVASCULAR
AND CNS DISORDERS", "BIARYL PDE4 INHIBITORS FOR TREATING PULMONARY
AND CARDIOVASCULAR DISORDERS", "SUBSTITUTED BENZOAZOLE PDE4
INHIBITORS FOR TREATING INFLAMMATORY, CARDIOVASCULAR AND CNS
DISORDERS" and "4-(OR 5-) SUBSTITUTED CATECHOL DERIVATIVES". Their
disclosures are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to substituted benzothiazoles,
benzoxazoles--and their counterparts having pyridine and pyrimidine
rings replacing the benzene ring--that are useful for treating
stroke, myocardial infarct, and cardiovascular inflammatory
conditions, to pharmaceutical compositions comprising these
compounds, and to methods for the treatment of stroke, myocardial
infarct, and cardiovascular inflammatory conditions in a
mammal.
BACKGROUND OF THE INVENTION
[0003] PDE4 is the major cAMP-metabolizing enzyme found in
inflammatory and immune cells. PDE4 inhibitors have proven
potential as anti-inflammatory drugs, especially in inflammatory
pulmonary diseases such as asthma, COPD and rhinitis. They suppress
the release of cytokines and other inflammatory signals and inhibit
the production of reactive oxygen species. A large number of PDE4
inhibitors have been developed for a variety of clinical
indications (Torphy and Page. 2000. TIPS 21, 157-159; Burnouf and
Pruniaux. 2002. Curr. Pharm. Design 8, 1255-1296; Lipworth. 2005.
Lancet 365, 167-175). To quote from a recent article in the British
Journal of Pharmacology, "PDE4 inhibitors have been in development
as a novel anti-inflammatory therapy since the 1980s with asthma
and chronic obstructive pulmonary disease (COPD) being primary
indications. Despite initial optimism, none have yet reached the
market. In most cases, the development of PDE4 inhibitors of
various structural classes, including cilomilast, filaminast,
lirimilast, piclamilast, tofimilast . . . has been discontinued due
to lack of efficacy. A primary problem is the low therapeutic ratio
of these compounds, which severely limits the dose that can be
given. Indeed, for many of these compounds it is likely that the
maximum tolerated dose is either sub-therapeutic or at the very
bottom of the efficacy dose-response curve. Therefore, the
challenge is to overcome this limitation." [Giembycz, Brit. J.
Pharmacol. 155, 288-290 (2008)]. Many of the PDE4 inhibitors of the
prior art have not reached the market because of the adverse side
effect of emesis (Giembycz 2005. Curr. Opin. Pharm. 5, 238-244).
Analysis of all known PDE4 inhibitors suggests that they are
competitive with cAMP and bind within the active site (Houslay et
al. 2005. DDT 10, 1503-1519); this may explain their narrow
therapeutic ratio. The compounds of the present invention are
non-competitive inhibitors of cAMP while being gene-specific
inhibitors (PDE4D), and, based on the target rationale and in vitro
potency, a person of skill in the art would expect the compounds to
be useful as anti-inflammatory agents for the treatment,
amelioration or prevention of inflammatory diseases and of
complications arising therefrom and useful as CNS agents for
amelioration of the cognitive decline in Alzheimer's disease,
Parkinson's disease, the treatment of schizophrenia and depression,
and neuroprotective in Huntington's disease.
SUMMARY OF THE INVENTION
[0004] The present invention relates to compounds exhibiting PDE4
enzyme inhibition, having the formulae Ia, Ib or Ic:
##STR00003##
In these compounds U is selected from the group consisting of --S--
and --O--; V is selected from the group consisting of H, CH.sub.3,
NH.sub.2, and CF.sub.3; X is selected from the group consisting of
CH, C--F, C--Cl, C--Br, C--I, C--NH.sub.2, C--OH, C--OCH.sub.3, N,
and N--O; Y is selected from the group consisting of N, CH, CF and
C-lower alkyl; R.sup.1 is H or lower alkyl; R.sup.2 is selected
from the group consisting of H, alkyl, OH, NH.sub.2, and OCH.sub.3;
B is an optionally substituted, mono- or bicyclic aryl or
heteroaryl; A is an optionally substituted heterocycle or an
optionally substituted carbocycle; and A.sup.1 is chosen from
[0005] (a) a residue chosen from
##STR00004##
wherein R.sup.40 is chosen from H, halogen, OH, NH.sub.2 and
CH.sub.3;
[0006] (b) a substituted heterocycle of three or fewer rings or
substituted carbocycle of three or fewer rings; and
[0007] (c) a heterocycle that is itself substituted with a
heterocycle carrying a further substituent;
wherein substituents on the heterocycle or carbocycle are chosen
from hydroxy, carboxy, carboxyalkyl, carboxyalkoxy,
carboxyalkylthio, alkoxycarbonyl, carboxyalkylcarbonylamino,
carboxyalkylaminocarbonylamino, guanidino, the residue of an amino
acid and the residue of an N-methylated amino acid.
[0008] There is also provided, in accordance with embodiments of
the invention, a pharmaceutical composition comprising a compound
as described herein, and a pharmaceutically acceptable carrier,
excipient or diluent therefore. When the compound is present as a
salt, the salt should be a pharmaceutically acceptable salt.
[0009] In a third aspect, the invention relates to methods for the
treatment or prophylaxis of a disease or condition mediated by
peripheral (i.e. outside the CNS) phosphodiesterase-4. The methods
comprise administering to a mammal a therapeutically effective
amount of a compound having the general formula I. The disease or
condition may be related to allergic, acute or chronic
inflammation. The disease may be, for example, atherosclerosis,
thrombosis, stroke, acute coronary syndrome, stable angina,
peripheral vascular disease, critical leg ischemia, intermittent
claudication, abdominal aortic aneurysm or myocardial
infarction.
[0010] Selective PDE4 inhibitors of the invention are also useful
for treating asthma and Chronic Obstructive Pulmonary Disease
(COPD). Compounds of the invention, which inhibit tumor growth and
metastases, also find utility in the treatment and prevention of
cancer, including esophageal cancer, brain cancer, pancreatic
cancer, and colon cancer.
[0011] These and other embodiments of the present invention will
become apparent in conjunction with the description and claims that
follow.
DETAILED DESCRIPTION OF THE INVENTION
[0012] Throughout this specification the substituents are defined
when introduced and retain their definitions.
[0013] Unless otherwise specified, alkyl is intended to include
linear, branched, or cyclic hydrocarbon structures and combinations
thereof. A combination would be, for example, cyclopropylmethyl.
Lower alkyl refers to alkyl groups of from 1 to 6 carbon atoms.
Examples of lower alkyl groups include methyl, ethyl, propyl,
isopropyl, butyl, s- and t-butyl and the like. Preferred alkyl
groups are those of C.sub.20 or below; C.sub.1 to C.sub.8 are more
preferred. Cycloalkyl is a subset of alkyl and includes cyclic
hydrocarbon groups of from 3 to 8 carbon atoms. Examples of
cycloalkyl groups include c-propyl, c-butyl, c-pentyl, norbornyl
and the like.
[0014] C.sub.1 to C.sub.20 hydrocarbon includes alkyl, cycloalkyl,
polycycloalkyl, alkenyl, alkynyl, aryl and combinations thereof.
Examples include benzyl, phenethyl, cyclohexylmethyl, camphoryl and
naphthylethyl. Hydrocarbon refers to any substituent comprised of
hydrogen and carbon as the only elemental constituents.
[0015] Unless otherwise specified, the term "carbocycle" is
intended to include ring systems in which the ring atoms are all
carbon but of any oxidation state. Thus (C.sub.3-C.sub.10)
carbocycle refers to both non-aromatic and aromatic systems,
including such systems as cyclopropane, benzene, cyclopentene and
cyclohexene; (C.sub.8-C.sub.12) carbopolycycle refers to such
systems as norbornane, decalin, indane and naphthalene. Carbocycle,
if not otherwise limited, refers to monocycles, bicycles and
polycycles.
[0016] Alkoxy or alkoxyl refers to groups of from 1 to 8 carbon
atoms of a straight, branched or cyclic configuration and
combinations thereof attached to the parent structure through an
oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy,
cyclopropyloxy, cyclohexyloxy and the like. Lower-alkoxy refers to
groups containing one to four carbons. For the purpose of this
application, alkoxy and lower alkoxy include methylenedioxy and
ethylenedioxy. Alkoxyalkyl refers to ether groups of from 3 to 8
atoms of a straight, branched, cyclic configuration and
combinations thereof attached to the parent structure through an
alkyl. Examples include methoxymethyl, methoxyethyl, ethoxypropyl,
and the like. Alkoxyaryl refers to alkoxy substituents attached to
an aryl, wherein the aryl is attached to the parent structure.
Arylalkoxy refers to aryl substituents attached to an oxygen,
wherein the oxygen is attached to the parent structure. Substituted
arylalkoxy refers to a substituted aryl substituent attached to an
oxygen, wherein the oxygen is attached to the parent structure.
[0017] Oxaalkyl refers to alkyl residues in which one or more
carbons (and their associated hydrogens) have been replaced by
oxygen. Examples include methoxypropoxy; 3,6,9-trioxadecyl;
2,6,7-trioxabicyclo[2.2.2]octane and the like. The term oxaalkyl is
intended as it is understood in the art [see Naming and Indexing of
Chemical Substances for Chemical Abstracts, published by the
American Chemical Society, 196, but without the restriction of
127(a)], i.e. it refers to compounds in which the oxygen is bonded
via a single bond to its adjacent atoms (forming ether bonds); it
does not refer to doubly bonded oxygen, as would be found in
carbonyl groups. Similarly, thiaalkyl and azaalkyl refer to alkyl
residues in which one or more carbons has been replaced by sulfur
or nitrogen, respectively. Examples include ethylaminoethyl and
methylthiopropyl.
[0018] Unless otherwise specified, acyl refers to formyl and to
groups of 1, 2, 3, 4, 5, 6, 7 and 8 carbon atoms of a straight,
branched, cyclic configuration, saturated, unsaturated and aromatic
and combinations thereof, attached to the parent structure through
a carbonyl functionality. One or more carbons in the acyl residue
may be replaced by nitrogen, oxygen or sulfur as long as the point
of attachment to the parent remains at the carbonyl. Examples
include acetyl, benzoyl, propionyl, isobutyryl, t-butoxycarbonyl,
benzyloxycarbonyl and the like. Lower-acyl refers to groups
containing one to four carbons. The double bonded oxygen, when
referred to as a substituent itself is called "oxo".
[0019] Aryl and heteroaryl mean (i) a phenyl group (or benzene) or
a monocyclic 5- or 6-membered heteroaromatic ring containing 1-4
heteroatoms selected from O, N, or S; (ii) a bicyclic 9- or
10-membered aromatic or heteroaromatic ring system containing 0-4
heteroatoms selected from O, N, or S; or (iii) a tricyclic 13- or
14-membered aromatic or heteroaromatic ring system containing 0-5
heteroatoms selected from O, N, or S. Aryl, as understood herein,
includes residues in which one or more rings are aromatic, but not
all need be. Thus aromatic 6- to 14-membered carbocyclic rings
include, e.g., benzene, naphthalene, indane, tetralin, and fluorene
and the 5- to 10-membered aromatic heterocyclic rings include,
e.g., imidazole, pyridine, indole, thiophene, benzopyranone,
thiazole, furan, benzimidazole, quinoline, isoquinoline,
quinoxaline, pyrimidine, pyrazine, tetrazole and pyrazole.
[0020] Arylalkyl refers to a substituent in which an aryl residue
is attached to the parent structure through alkyl. Examples are
benzyl, phenethyl and the like. Heteroarylalkyl refers to a
substituent in which a heteroaryl residue is attached to the parent
structure through alkyl. In one embodiment, the alkyl group of an
arylalkyl or a heteroarylalkyl is an alkyl group of from 1 to 6
carbons. Examples include, e.g., pyridinylmethyl, pyrimidinylethyl
and the like.
[0021] Heterocycle means a cycloalkyl or aryl carbocycle residue in
which from one to three carbons is replaced by a heteroatom
selected from the group consisting of N, O and S. The nitrogen and
sulfur heteroatoms may optionally be oxidized, and the nitrogen
heteroatom may optionally be quaternized. Unless otherwise
specified, a heterocycle may be non-aromatic or aromatic. It is to
be noted that heteroaryl is a subset of heterocycle in which the
heterocycle is aromatic. Examples of heterocyclic residues that
fall within the scope of the invention include pyrazole, pyrrole,
indole, quinoline, isoquinoline, tetrahydroisoquinoline,
benzofuran, benzodioxan, benzodioxole (commonly referred to as
methylenedioxyphenyl, when occurring as a substituent), morpholine,
thiazole, pyridine (including 2-oxopyridine), pyridine N-oxide,
pyrimidine, thiophene (i.e. thiene), furan, oxazole, oxazoline,
oxazolidine, isoxazolidine, isoxazole, dioxane, azetidine,
piperazine, piperidine, pyrrolidine, pyridazine, azepine,
pyrazolidine, imidazole, imidazoline, imidazolidine,
imidazolopyridine, pyrazine, thiazolidine, isothiazole,
1,2-thiazine-1,1-dioxide, quinuclidine, isothiazolidine,
benzimidazole, thiadiazole, benzopyran, benzothiazole,
benzotriazole, benzoxazole, tetrahydrofuran, tetrahydropyran,
benzothiene, thiamorpholine, thiamorpholine sulfoxide,
thiamorpholine sulfone, oxadiazole, triazole, tetrazole, isatin
(dioxoindole), phthalimide (dioxoisoindole), pyrrolopyridine,
triazolopyridine and the dihydro and tetrahydro congeners of the
fully unsaturated ring systems among the foregoing.
[0022] An oxygen heterocycle is a heterocycle containing at least
one oxygen in the ring; it may contain additional oxygens, as well
as other heteroatoms. Oxygen heterocycles found in the examples of
the invention include tetrahydrofuran, benzodioxole, morpholine,
isoxazole and 2,6,7-trioxabicyclo[2.2.2]octane. A sulphur
heterocycle is a heterocycle containing at least one sulphur in the
ring; it may contain additional sulphurs, as well as other
heteroatoms. A nitrogen heterocycle is a heterocycle containing at
least one nitrogen in the ring; it may contain additional
nitrogens, as well as other heteroatoms.
[0023] As used herein, the term "optionally substituted" may be
used interchangeably with "unsubstituted or substituted". The term
"substituted" refers to the replacement of one or more hydrogen
atoms in a specified group with a specified radical. For example,
substituted alkyl, aryl, cycloalkyl, heterocyclyl etc. refer to
alkyl, aryl, cycloalkyl, or heterocyclyl wherein up to three H
atoms in each residue are replaced with halogen, haloalkyl, alkyl,
acyl, alkoxyalkyl, hydroxyalkyl, carbonyl (i.e. oxo), phenyl,
heteroaryl, benzenesulfonyl, hydroxy, alkoxy, haloalkoxy, oxaalkyl,
carboxy, carboxyalkyl, carboxyalkoxy, carboxyalkylthio,
alkoxycarbonyl [--C(.dbd.O)O-alkyl], alkoxycarbonylamino
[--NHC(.dbd.O)O-alkyl], alkoxycarbonylaminoalkyl
[-alkyl-NHC(.dbd.O)O-alkyl], carboxyalkylcarbonylamino
[--NHC(.dbd.O)-alkyl-COOH], carboxamido [--C(.dbd.O)NH.sub.2],
aminocarbonyloxy [--OC(.dbd.O)NH.sub.2], alkylaminocarbonyl
[--C(.dbd.O)NH-alkyl], dialkylaminocarbonyl
[--C(.dbd.O)N(alkyl).sub.2], aminocarbonylalkyl
[-alkyl-C(.dbd.O)NH.sub.2], cyano, acetoxy, nitro, amino,
alkylamino, dialkylamino, aminoalkyl, (alkyl)(aryl)aminoalkyl,
alkylaminoalkyl (including cycloalkylaminoalkyl),
dialkylaminoalkyl, dialkylaminoalkoxy, alkyl(hydroxyalkyl)amino,
heterocyclylalkoxy, mercapto, alkylthio, alkylsulfonyl,
alkylsulfonylamino, alkylsulfinyl, alkylsulfonyl, arylthio,
arylsulfonyl, arylsulfonylamino, arylsulfinyl, arylsulfonyl,
acylaminoalkyl, acylaminoalkoxy, acylamino, amidino, aryl, benzyl,
heterocyclyl, heterocyclylalkyl, phenoxy, benzyloxy, heteroaryloxy,
heterocyclylamino, hydroxyimino, alkoxyimino, oxaalkyl,
aminosulfonyl, trityl, amidino, guanidino, ureido,
--NHC(.dbd.O)NHalkyl, --NHC(.dbd.O)NH-heterocyclyl,
-alkyl-NHC(.dbd.O)N(alkyl).sub.2, heterocyclylalkylcarbonylamino,
benzyloxyphenyl, and benzyloxy. Although oxo is included among the
substituents referred to in "optionally substituted", it will be
appreciated by persons of skill in the art that, because oxo is a
divalent radical, there are circumstances in which it will not be
appropriate as a substituent (e.g. on phenyl). Additional
substituents that are considered within the scope of the term,
particularly for R.sup.1, are the are the residues of amino acids,
amino acid amides, protected residues of aminoacids and their
amides, and N-methylated (mono- or di-, as appropriate) amino acids
and amino acid amides.
[0024] For the purpose of ring A or A.sup.1, the substituents
alkyl, acyl, alkoxyalkyl, hydroxyloweralkyl, phenyl, heteroaryl,
benzenesulfonyl, loweralkoxy, haloalkoxy, oxaalkyl, alkoxycarbonyl,
alkoxycarbonylamino, carboxamido, alkylaminocarbonyl, amino,
alkylamino, (alkyl)(aryl)aminoalkyl, alkylaminoalkyl,
heterocyclylalkoxy, alkylthio, sulfonylamino, alkylsulfinyl,
alkylsulfonyl, acylaminoalkyl, acylaminoalkoxy, acylamino, amidino,
aryl, benzyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkoxy,
phenoxy, benzyloxy, heteroaryloxy, heterocyclylamino, oxaalkyl,
aminosulfonyl, amidino, guanidino, ureido, benzyloxyphenyl, and
benzyloxy may be further substituted with one or two substituents
from the list of substituents above. Substituents that are
considered within the scope of the term, particularly for A, are
the are the residues of amino acids, amino acid amides and
protected residues of aminoacids and their amides, as well as the
following specific residues: --CH.sub.3, --CH.sub.2CF.sub.3,
--CF.sub.3, --CHO, --COOH, --CN, halogen, --OH, --OEt,
--C(.dbd.O)NH.sub.2, --C(.dbd.O)NHEt,
--C(.dbd.O)NMe.sub.2-COOCH.sub.3, --COOEt,
--CH.sub.2NHC(.dbd.O)NH.sub.2, --CH(CH.sub.3)NHC(.dbd.O)NH.sub.2,
--CH.sub.2NHC(.dbd.O)H, --CH.sub.2NHC(.dbd.O)CH.sub.3,
--CH.sub.2C(.dbd.O)NH.sub.2, --CH.sub.2COOH, --CH.sub.2COOEt,
--CH.sub.2NHC(.dbd.O)OEt, --CH.sub.2NHC(.dbd.O)O--C.sub.6H.sub.5,
--CH.sub.2NHC(.dbd.O)C(.dbd.O)NH.sub.2, --CH.sub.2NHC(.dbd.O)NHEt,
--C(CH.sub.3).sub.2OH, --CH.sub.2NHC(.dbd.O)N(CH.sub.3).sub.2,
--CH.sub.2NHC(.dbd.O)NHCH.sub.3, --CH.sub.2NH.sub.2,
--CH(CH.sub.3)NH.sub.2, --C(CH.sub.3).sub.2NH.sub.2, --CH.sub.2OH,
--CH.sub.2CH.sub.2OH, --CH.sub.2NHSO.sub.2CH.sub.3,
--CH.sub.2C(.dbd.O)NHEt, --OCH.sub.3, --OC(.dbd.O)NH.sub.2,
--OCH.sub.2CH.sub.2N(CH.sub.3).sub.2, --OCH.sub.2CH.sub.2OCH.sub.3,
--OCH(CH.sub.3)COOH, --SCH.sub.2COOH, --NHC(.dbd.O)NH.sub.2,
--NHC(.dbd.O)NHEt, --NHCH.sub.3, --NHEt, --NH(tBoc),
--NHCH.sub.2COOH ("residue of glycine"), --N(CH.sub.3)CH.sub.2COOH
("residue of N-methylglycine"), --NHC(.dbd.O)NHCH.sub.2CH.sub.2Cl,
--NHSO.sub.2NH.sub.2, --NHEt, --N(CH.sub.3).sub.2, --NH.sub.2,
--NH(CH.sub.3)C(.dbd.O)NH.sub.2, --NHSO.sub.2CH.sub.3,
--N(SO.sub.2CH.sub.3).sub.2, --NHC(.dbd.O)OCH.sub.3,
--NHC(.dbd.O)OtBu, --NHC(.dbd.O)CH.sub.3, --SO.sub.2NH.sub.2,
--NHC(.dbd.O)CH.sub.2CH.sub.2COOH, --NHC(.dbd.O)NHCH.sub.2COOH,
--CH.sub.2NHCHO, --NHC(.dbd.O)NHCH.sub.2COOEt,
--NHC(.dbd.O)NH(CH.sub.2).sub.3COOEt,
--NHC(.dbd.O)NH(CH.sub.2).sub.2COOEt,
--N(CH.sub.3)CH.sub.2CH.sub.2OH, --NHC(.dbd.O)OEt,
--N(Et)C(.dbd.O)OEt, --NHC(.dbd.O)NH(CH.sub.2).sub.2COOH,
--NHC(.dbd.O)CH.sub.2N(CH.sub.3).sub.2,
--NHC(.dbd.O)NH(CH.sub.2).sub.3COOH, --NHC(.dbd.O)CH.sub.2NH.sub.2,
--NHC(.dbd.O)CH.sub.2CH.sub.2NH.sub.2,
--NHC(.dbd.O)CH.sub.2NH(tBoc),
##STR00005##
The term "a residue of an amino acid, amino acid amide", etc.
refers to an amino acid etc. minus the functional groups that are
considered part of the bond to the parent structure. For example,
in the molecule BB-03 illustrated below:
##STR00006##
after one subtracts the hydrogen that connects D-proline to the
phenyl ring, the structure of A that remains is:
##STR00007##
This is not sensu stricto proline, since it lacks the hydrogen on
the ring nitrogen. This and similar structures that lack atoms at
the points of attachment (e.g. the H of NH.sub.2) are referred to
herein as "residues" of their respective parents.
[0025] The terms "haloalkyl" and "haloalkoxy" mean alkyl or alkoxy,
respectively, substituted with one or more halogen atoms. The terms
"alkylcarbonyl" and "alkoxycarbonyl" mean --C(.dbd.O)alkyl or
--C(O)alkoxy, respectively.
[0026] The term "halogen" means fluorine, chlorine, bromine or
iodine. In one embodiment, halogen may be fluorine or chlorine.
[0027] Substituents R.sup.n are generally defined when introduced
and retain that definition throughout the specification and in all
independent claims.
[0028] In the characterization of some of the substituents, it is
recited that certain substituents may combine to form rings. Unless
stated otherwise, it is intended that such rings may exhibit
various degrees of unsaturation (from fully saturated to fully
unsaturated), may include heteroatoms and may be substituted with
lower alkyl or alkoxy.
[0029] It will be recognized that the compounds of this invention
can exist in radiolabeled form, i.e., the compounds may contain one
or more atoms containing an atomic mass or mass number different
from the atomic mass or mass number usually found in nature.
Radioisotopes of hydrogen, carbon, phosphorous, fluorine, and
chlorine include .sup.2H, .sup.3H, .sup.13C, .sup.14C, .sup.15N,
.sup.35S, .sup.18F, and .sup.36Cl, respectively. Compounds that
contain those radioisotopes and/or other radioisotopes of other
atoms are within the scope of this invention. Tritiated, i.e.
.sup.3H, and carbon-14, i.e., .sup.14C, radioisotopes are
particularly preferred for their ease in preparation and
detectability. Compounds that contain isotopes .sup.11C, .sup.13N,
.sup.15O and .sup.18F are well suited for positron emission
tomography. Radiolabeled compounds of formula I of this invention
and prodrugs thereof can generally be prepared by methods well
known to those skilled in the art. Conveniently, such radiolabeled
compounds can be prepared by carrying out the procedures disclosed
in the Examples and Schemes by substituting a readily available
radiolabeled reagent for a non-radiolabeled reagent.
[0030] As used herein (particularly in the claims), and as would be
understood by the person of skill in the art, the recitation of "a
compound" is intended to include salts, solvates, co-crystals and
inclusion complexes of that compound as well as any stereoisomeric
form, or a mixture of any such forms of that compound in any ratio.
Thus, in accordance with some embodiments of the invention, a
compound as described herein, including in the contexts of
pharmaceutical compositions, methods of treatment, and compounds
per se, is provided as the salt form. Thus, for example, the
recitation "a compound of formula I" as depicted above, in which
R.sup.1 is imidazolyl, would include imidazolium salts. In a
particular embodiment, the term "compound of formula I" refers to
the compound or a pharmaceutically acceptable salt thereof.
[0031] The compounds described herein may contain asymmetric
centers and may thus give rise to enantiomers, diastereomers, and
other stereoisomeric forms. Each chiral center may be defined, in
terms of absolute stereochemistry, as (R)- or (S)-. The present
invention is meant to include all such possible isomers, in any
ratio from racemic to optically pure forms. Optically active (R)-
and (S)-isomers may be prepared using chiral synthons or chiral
reagents, or resolved using conventional techniques. The prefix
"rac" refers to a racemate. When the compounds described herein
contain olefinic double bonds or other centers of geometric
asymmetry, and unless specified otherwise, it is intended that the
compounds include both E and Z geometric isomers. The
representation of the configuration of any carbon-carbon double
bond appearing herein is selected for convenience only, and unless
explicitly stated, is not intended to designate a particular
configuration. Thus a carbon-carbon double bond depicted
arbitrarily as E may be Z, E, or a mixture of the two in any
proportion. Likewise, all tautomeric forms are also intended to be
included.
[0032] The term "solvate" refers to a compound of Formula I in the
solid state, wherein molecules of a suitable solvent are
incorporated in the crystal lattice. A suitable solvent for
therapeutic administration is physiologically tolerable at the
dosage administered. Examples of suitable solvents for therapeutic
administration are ethanol and water. When water is the solvent,
the solvate is referred to as a hydrate. In general, solvates are
formed by dissolving the compound in the appropriate solvent and
isolating the solvate by cooling or using an antisolvent. The
solvate is typically dried or azeotroped under ambient conditions.
Inclusion complexes are described in Remington: The Science and
Practice of Pharmacy 19.sup.th Ed. (1995) volume 1, page 176-177,
which is incorporated herein by reference. The most commonly
employed inclusion complexes are those with cyclodextrins, and all
cyclodextrin complexes, natural and synthetic, are specifically
encompassed within the claims.
[0033] The term "pharmaceutically acceptable salt" refers to salts
prepared from pharmaceutically acceptable non-toxic acids or bases
including inorganic acids and bases and organic acids and bases.
When the compounds of the present invention are basic, salts may be
prepared from pharmaceutically acceptable non-toxic acids including
inorganic and organic acids. Suitable pharmaceutically acceptable
anions for the compounds of the present invention include acetate,
benzenesulfonate (besylate), benzoate, bicarbonate, bisulfate,
carbonate, camphorsulfonate, citrate, ethanesulfonate, fumarate,
gluconate, glutamate, glycolate, bromide, chloride, isethionate,
lactate, maleate, malate, mandelate, methanesulfonate, mucate,
nitrate, pamoate, pantothenate, phosphate, succinate, sulfate,
tartrate, trifluoroacetate, p-toluenesulfonate, acetamidobenzoate,
adipate, alginate, aminosalicylate, anhydromethylenecitrate,
ascorbate, aspartate, calcium edetate, camphorate, camsylate,
caprate, caproate, caprylate, cinnamate, cyclamate,
dichloroacetate, edetate (EDTA), edisylate, embonate, estolate,
esylate, fluoride, formate, gentisate, gluceptate, glucuronate,
glycerophosphate, glycolate, glycollylarsanilate, hexylresorcinate,
hippurate, hydroxynaphthoate, iodide, lactobionate, malonate,
mesylate, napadisylate, napsylate, nicotinate, oleate, orotate,
oxalate, oxoglutarate, palmitate, pectinate, pectinate polymer,
phenylethylbarbiturate, picrate, pidolate, propionate, rhodanide,
salicylate, sebacate, stearate, tannate, theoclate, tosylate and
the like. The desired salt may be obtained by ion exchange of
whatever counter ion is obtained in the synthesis of the quat.
These methods are well known to persons of skill. Although
pharmaceutically acceptable counter ions will be preferred for
preparing pharmaceutical formulations, other anions are quite
acceptable as synthetic intermediates. When the compounds contain
an acidic side chain, suitable pharmaceutically acceptable base
addition salts for the compounds of the present invention include
metallic salts made from aluminum, calcium, lithium, magnesium,
potassium, sodium and zinc or organic salts made from lysine,
N,N'-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and
procaine.
[0034] The graphic representations of racemic, ambiscalemic and
scalemic or enantiomerically pure compounds used herein are taken
from Maehr J. Chem. Ed. 62, 114-120 (1985): solid and broken wedges
are used to denote the absolute configuration of a chiral element;
wavy lines and single thin lines indicate disavowal of any
stereochemical implication which the bond it represents could
generate; solid and broken bold lines are geometric descriptors
indicating the relative configuration shown but denoting racemic
character; and wedge outlines and dotted or broken lines denote
enantiomerically pure compounds of indeterminate absolute
configuration.
[0035] Terminology related to "protecting", "deprotecting" and
"protected" functionalities occurs throughout this application.
Such terminology is well understood by persons of skill in the art
and is used in the context of processes that involve sequential
treatment with a series of reagents. In that context, a protecting
group refers to a group, which is used to mask a functionality
during a process step in which it would otherwise react, but in
which reaction is undesirable. The protecting group prevents
reaction at that step, but may be subsequently removed to expose
the original functionality. The removal or "deprotection" occurs
after the completion of the reaction or reactions in which the
functionality would interfere. Thus, when a sequence of reagents is
specified, as it is in the processes of the invention, the person
of ordinary skill can readily envision those groups that would be
suitable as "protecting groups". Suitable groups for that purpose
are discussed in standard textbooks in the field of chemistry, such
as Protective Groups in Organic Synthesis by T. W. Greene [John
Wiley & Sons, New York, 1991], which is incorporated herein by
reference.
[0036] A comprehensive list of abbreviations utilized by organic
chemists appears in the first issue of each volume of the Journal
of Organic Chemistry. The list, which is typically presented in a
table entitled "Standard List of Abbreviations", is incorporated
herein by reference.
[0037] In general, the compounds of the present invention may be
prepared by the methods illustrated in the general reaction schemes
as, for example, described below, or by modifications thereof,
using readily available starting materials, reagents and
conventional synthesis procedures. In these reactions, it is also
possible to make use of variants that are in themselves known, but
are not mentioned here. The starting materials, are either
commercially available, synthesized as described in the examples or
may be obtained by the methods well known to persons of skill in
the art.
[0038] PDE4 inhibitors have been shown to be effective therapeutic
agents in clinical studies. For example, administration of
cilomilast and roflumilast (PDE4 inhibitors) to patients suffering
from asthma and COPD showed initially excellent results, although
the effect of cilomilast disappeared on long-term trial [Lipworth,
Lancet 365, 167-175 (2005)]. Genetic studies have clearly
demonstrated an association between PDE4D and ischemic stroke
(Gretarsdottir et al. 2003. Nature Genetics. 35, 1-8). Selective
PDE4 inhibitors (e.g. rolipram) are also useful for treating bone
loss [Yao et al., J. Musculoskelet. Neuronal Interact. 7, 119-130
(2007)].
[0039] Additionally, a PDE4 inhibitor, YM976, was shown to
ameliorate the effects of experimentally-induced interstitial
cystitis in rats, resulting in a decrease in the frequency of
urination and an increase in the volume of urine at each time of
urination [Kitta et al., BJU Int. 102, 1472-1476 (2008)]. Another
PDE4 inhibitor, IC485, was shown to be equally efficacious as
tolteradine tartrate, a marketed drug for treating overactive
bladder, in a rodent model of obstructive bladder [Kaiho et al. BJU
Int. 101, 615-20 (2008)]. These findings suggest that PDE4
inhibitors will be useful in treating symptoms of bladder
overactivity, inflammation and pain.
[0040] Furthermore, the compounds, compositions and methods of the
present invention may be useful in treating cancer.
Phosphodiesterase activity has been shown to be associated with
hematological malignancies [Lerner et al., Biochem. J. 393, 21-41
(2006); Ogawa et al., Blood 99, 3390-3397 (2002)].
[0041] Furthermore, the compounds, compositions and methods of the
present invention, particularly when radiolabeled as described
above or labeled by methods well-known in the art with florescent
and spin labels, may be employed as imaging agents and in other
ways for diagnosis and/or treatment. Moreover, immobilization of
compounds of the invention on solid support could be of utility for
affinity purification and modification of compounds of the
invention with chemically active groups may be used for protein
labeling.
[0042] For many of the utilities outlined above, it may be
advantageous to administer compounds of the general formula I
together with cholinesterase inhibitors (e.g. tacrine, huperzine,
donepezil); NMDA antagonists (e.g. lanicemine, remacemide,
neramexane, memantine); calpain inhibitors (e.g. CEP-3122);
antioxidants (e.g. vitamin E, coenzyme Q10) and agents that have
shown clinical efficacy but whose mechanism is unclear (e.g.
dimebon). Compounds of formula I may also be administered together
with one or more of the following agents to improve cognition:
amisulpride, atomoxetine, bromocryptine, buspirone, caffeine,
chlorpromazine, clonidine, clozapine, diazepam, flumazenil,
fluoxetine, galantamine, guanfacine, methylphenidate, idazoxan,
modafinil, olanzapine, paroxetine, pergolide, phenserine,
quetiapine, risperidone, rivastigmine, SGS742 and sulpiride.
[0043] The terms "methods of treating or preventing" mean
amelioration, prevention or relief from the symptoms and/or effects
associated with disorders. The term "preventing" as used herein
refers to administering a medicament beforehand to forestall or
obtund an acute episode. The person of ordinary skill in the
medical art (to which the present method claims are directed)
recognizes that the term "prevent" is not an absolute term. In the
medical art it is understood to refer to the prophylactic
administration of a drug to substantially diminish the likelihood
or seriousness of a condition, and this is the sense intended in
applicants' claims. As used herein, reference to "treatment" of a
patient is intended to include prophylaxis.
[0044] The term "mammal" is used in its dictionary sense. Humans
are included in the group of mammals, and humans would be the
preferred subjects of the methods.
[0045] While it may be possible for compounds of formula I to be
administered as the raw chemical, it will often be preferable to
present them as part of a pharmaceutical composition. In accordance
with an embodiment of the present invention there is provided a
pharmaceutical composition comprising a compound of formula I or a
pharmaceutically acceptable salt thereof, together with one or more
pharmaceutically carriers thereof and optionally one or more other
therapeutic ingredients. The carrier(s) must be "acceptable" in the
sense of being compatible with the other ingredients of the
formulation and not deleterious to the recipient thereof.
Furthermore, when reference is made in an independent claim to a
compound or a pharmaceutically acceptable salt thereof, it will be
understood that claims which depend from that independent claim
which refer to such a compound also include pharmaceutically
acceptable salts of the compound, even if explicit reference is not
made to the salts in the dependent claim.
[0046] The formulations include those suitable for oral, parenteral
(including subcutaneous, intradermal, intramuscular, intravenous
and intraarticular), rectal and topical (including dermal, buccal,
sublingual and intraocular) administration. The most suitable route
may depend upon the condition and disorder of the recipient. The
formulations may conveniently be presented in unit dosage form and
may be prepared by any of the methods well known in the art of
pharmacy. Such methods include the step of bringing into
association a compound of formula I or a pharmaceutically
acceptable salt or solvate thereof ("active ingredient") with the
carrier, which constitutes one or more accessory ingredients. In
general, the formulations are prepared by uniformly and intimately
bringing into association the active ingredient with liquid
carriers or finely divided solid carriers or both and then, if
necessary, shaping the product into the desired formulation.
[0047] Formulations suitable for oral administration may be
presented as discrete units such as capsules, cachets or tablets
each containing a predetermined amount of the active ingredient; as
a powder or granules; as a solution or a suspension in an aqueous
liquid or a non-aqueous liquid; or as an oil-in-water liquid
emulsion or a water-in-oil liquid emulsion. The active ingredient
may also be presented as a bolus, electuary or paste.
[0048] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared by compressing in a suitable machine the active ingredient
in a free-flowing form such as a powder or granules, optionally
mixed with a binder, lubricant, inert diluent, lubricating, surface
active or dispersing agent. Molded tablets may be made by molding
in a suitable machine a mixture of the powdered compound moistened
with an inert liquid diluent. The tablets may optionally be coated
or scored and may be formulated so as to provide sustained, delayed
or controlled release of the active ingredient therein. The
pharmaceutical compositions may include a "pharmaceutically
acceptable inert carrier", and this expression is intended to
include one or more inert excipients, which include starches,
polyols, granulating agents, microcrystalline cellulose, diluents,
lubricants, binders, disintegrating agents, and the like. If
desired, tablet dosages of the disclosed compositions may be coated
by standard aqueous or nonaqueous techniques, "Pharmaceutically
acceptable carrier" also encompasses controlled release means.
[0049] Pharmaceutical compositions may also optionally include
other therapeutic ingredients, anti-caking agents, preservatives,
sweetening agents, colorants, flavors, desiccants, plasticizers,
dyes, and the like. Any such optional ingredient must be compatible
with the compound of formula I to insure the stability of the
formulation. The composition may contain other additives as needed,
including for example lactose, glucose, fructose, galactose,
trehalose, sucrose, maltose, raffinose, maltitol, melezitose,
stachyose, lactitol, palatinite, starch, xylitol, mannitol,
myoinositol, and the like, and hydrates thereof, and amino acids,
for example alanine, glycine and betaine, and peptides and
proteins, for example albumen.
[0050] Examples of excipients for use as the pharmaceutically
acceptable carriers and the pharmaceutically acceptable inert
carriers and the aforementioned additional ingredients include, but
are not limited to binders, fillers, disintegrants, lubricants,
anti-microbial agents, and coating agents.
[0051] The dose range for adult humans is generally from 0.005 mg
to 10 g/day orally. Tablets or other forms of presentation provided
in discrete units may conveniently contain an amount of compound of
formula I which is effective at such dosage or as a multiple of the
same, for instance, units containing 5 mg to 500 mg, usually around
10 mg to 200 mg. The precise amount of compound administered to a
patient will be the responsibility of the attendant physician.
However, the dose employed will depend on a number of factors,
including the age and sex of the patient, the precise disorder
being treated, and its severity.
[0052] A dosage unit (e.g. an oral dosage unit) can include from,
for example, 1 to 30 mg, 1 to 40 mg, 1 to 100 mg, 1 to 300 mg, 1 to
500 mg, 2 to 500 mg, 3 to 100 mg, 5 to 20 mg, 5 to 100 mg (e.g. 1
mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg,
12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 25
mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg,
75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 150 mg, 200 mg, 250 mg,
300 mg, 350 mg, 400 mg, 450 mg, 500 mg) of a compound described
herein.
[0053] For additional information about pharmaceutical compositions
and their formulation, see, for example, Remington: The Science and
Practice of Pharmacy, 20.sup.th Edition, 2000. The agents can be
administered, e.g., by intravenous injection, intramuscular
injection, subcutaneous injection, intraperitoneal injection,
topical, sublingual, intraarticular (in the joints), intradermal,
buccal, ophthalmic (including intraocular), intranasally (including
using a cannula), or by other routes. The agents can be
administered orally, e.g., as a tablet or cachet containing a
predetermined amount of the active ingredient, gel, pellet, paste,
syrup, bolus, electuary, slurry, capsule, powder, granules, as a
solution or a suspension in an aqueous liquid or a non-aqueous
liquid, as an oil-in-water liquid emulsion or a water-in-oil liquid
emulsion, via a micellar formulation (see, e.g. WO 97/11682) via a
liposomal formulation (see, e.g., EP 736299, WO 99/59550 and WO
97/13500), via formulations described in WO 03/094886 or in some
other form. The agents can also be administered transdermally (i.e.
via reservoir-type or matrix-type patches, microneedles, thermal
poration, hypodermic needles, iontophoresis, electroporation,
ultrasound or other forms of sonophoresis, jet injection, or a
combination of any of the preceding methods (Prausnitz et al. 2004,
Nature Reviews Drug Discovery 3:115)). The agents can be
administered locally, for example, at the site of injury to an
injured blood vessel. The agents can be coated on a stent. The
agents can be administered using high-velocity transdermal particle
injection techniques using the hydrogel particle formulation
described in U.S. 20020061336. Additional particle formulations are
described in WO 00/45792, WO 00/53160, and WO 02/19989. An example
of a transdermal formulation containing plaster and the absorption
promoter dimethylisosorbide can be found in WO 89/04179. WO
96/11705 provides formulations suitable for transdermal
administration. The agents can be administered in the form a
suppository or by other vaginal or rectal means. The agents can be
administered in a transmembrane formulation as described in WO
90/07923. The agents can be administered non-invasively via the
dehydrated particles described in U.S. Pat. No. 6,485,706. The
agent can be administered in an enteric-coated drug formulation as
described in WO 02/49621. The agents can be administered
intranasally using the formulation described in U.S. Pat. No.
5,179,079. Formulations suitable for parenteral injection are
described in WO 00/62759. The agents can be administered using the
casein formulation described in U.S. 20030206939 and WO 00/06108.
The agents can be administered using the particulate formulations
described in U.S. 20020034536.
[0054] The agents, alone or in combination with other suitable
components, can be administered by pulmonary route utilizing
several techniques including but not limited to intratracheal
instillation (delivery of solution into the lungs by syringe),
intratracheal delivery of liposomes, insufflation (administration
of powder formulation by syringe or any other similar device into
the lungs) and aerosol inhalation. Aerosols (e.g., jet or
ultrasonic nebulizers, metered-dose inhalers (MDIs), and dry-Powder
inhalers (DPIs)) can also be used in intranasal applications.
Aerosol formulations are stable dispersions or suspensions of solid
material and liquid droplets in a gaseous medium and can be placed
into pressurized acceptable propellants, such as hydrofluoroalkanes
(HFAs, i.e. HFA-134a and HFA-227, or a mixture thereof),
dichlorodifluoromethane (or other chlorofluorocarbon propellants
such as a mixture of Propellants 11, 12, and/or 114), propane,
nitrogen, and the like. Pulmonary formulations may include
permeation enhancers such as fatty acids, and saccharides,
chelating agents, enzyme inhibitors (e.g., protease inhibitors),
adjuvants (e.g., glycocholate, surfactin, span 85, and nafamostat),
preservatives (e.g., benzalkonium chloride or chlorobutanol), and
ethanol (normally up to 5% but possibly up to 20%, by weight).
Ethanol is commonly included in aerosol compositions as it can
improve the function of the metering valve and in some cases also
improve the stability of the dispersion. Pulmonary formulations may
also include surfactants which include but are not limited to bile
salts and those described in U.S. Pat. No. 6,524,557 and references
therein. The surfactants described in U.S. Pat. No. 6,524,557,
e.g., a C.sub.8-C.sub.16 fatty acid salt, a bile salt, a
phospholipid, or alkyl saccharide are advantageous in that some of
them also reportedly enhance absorption of the compound in the
formulation. Also suitable in the invention are dry powder
formulations comprising a therapeutically effective amount of
active compound blended with an appropriate carrier and adapted for
use in connection with a dry-Powder inhaler. Absorption enhancers
which can be added to dry powder formulations of the present
invention include those described in U.S. Pat. No. 6,632,456. WO
02/080884 describes new methods for the surface modification of
powders. Aerosol formulations may include U.S. Pat. No. 5,230,884,
U.S. Pat. No. 5,292,499, WO 017/8694, WO 01/78696, U.S. 2003019437,
U.S. 20030165436, and WO 96/40089 (which includes vegetable oil).
Sustained release formulations suitable for inhalation are
described in U.S. 20010036481A1, 20030232019A1, and U.S.
20040018243A1 as well as in WO 01/13891, WO 02/067902, WO
03/072080, and WO 03/079885. Pulmonary formulations containing
microparticles are described in WO 03/015750, U.S. 20030008013, and
WO 00/00176. Pulmonary formulations containing stable glassy state
powder are described in U.S. 20020141945 and U.S. Pat. No.
6,309,671. Other aerosol formulations are described in EP 1338272A1
WO 90/09781, U.S. Pat. No. 5,348,730, U.S. Pat. No. 6,436,367, WO
91/04011, and U.S. Pat. No. 6,294,153 and U.S. Pat. No. 6,290,987
describes a liposomal based formulation that can be administered
via aerosol or other means. Powder formulations for inhalation are
described in U.S. 20030053960 and WO 01/60341. The agents can be
administered intranasally as described in U.S. 20010038824.
[0055] Solutions of medicament in buffered saline and similar
vehicles are commonly employed to generate an aerosol in a
nebulizer. Simple nebulizers operate on Bernoulli's principle and
employ a stream of air or oxygen to generate the spray particles.
More complex nebulizers employ ultrasound to create the spray
particles. Both types are well known in the art and are described
in standard textbooks of pharmacy such as Sprowls' American
Pharmacy and Remington's The Science and Practice of Pharmacy.
Other devices for generating aerosols employ compressed gases,
usually hydrofluorocarbons and chlorofluorocarbons, which are mixed
with the medicament and any necessary excipients in a pressurized
container, these devices are likewise described in standard
textbooks such as Sprowls and Remington.
[0056] The agent can be incorporated into a liposome to improve
half-life. The agent can also be conjugated to polyethylene glycol
(PEG) chains. Methods for pegylation and additional formulations
containing PEG-conjugates (i.e. PEG-based hydrogels, PEG modified
liposomes) can be found in Harris and Chess, Nature Reviews Drug
Discovery 2:214-221 and the references therein. The agent can be
administered via a nanocochleate or cochleate delivery vehicle
(BioDelivery Sciences International). The agents can be delivered
transmucosally (i.e. across a mucosal surface such as the vagina,
eye or nose) using formulations such as that described in U.S. Pat.
No. 5,204,108. The agents can be formulated in microcapsules as
described in WO 88/01165. The agent can be administered
intra-orally using the formulations described in U.S. 20020055496,
WO 00/47203, and U.S. Pat. No. 6,495,120. The agent can be
delivered using nanoemulsion formulations described in WO
01/91728A2.
[0057] In general, compounds of formula I may be prepared by the
methods illustrated in the general reaction schemes as, for
example, described below, or by modifications thereof, using
readily available starting materials, reagents and conventional
synthesis procedures. In these reactions, it is also possible to
make use of variants that are in themselves known, but are not
mentioned here.
[0058] The invention relates to compounds of formula Ia, Ib or
Ic:
##STR00008##
as described above.
[0059] In accordance with some embodiments of the invention, Y is
CH. In accordance with other embodiments, Y is N.
[0060] In accordance with some embodiments of the invention, U is
S. In accordance with other embodiments, U is O.
[0061] In accordance with some embodiments of the invention, V is
selected from H, CH.sub.3 and NH.sub.2. In accordance with some
embodiments, V is H. In accordance with some embodiments, V is
CH.sub.3. In accordance with some embodiments, V is NH.sub.2.
[0062] In accordance with some embodiments of the invention, B is
phenyl which has a substituent at the 3-position, the 4-position
and at both the 3- and 4-positions. In some embodiments, B is
selected from 3-chlorophenyl, 3-nitrophenyl, 3-cyanophenyl,
3-bromophenyl, 3-acetylphenyl, 3-trifluoromethylphenyl, and
3-methylthiophenyl. In some embodiments B is
benzo[c][1,2,5]oxadiazol-5-yl and benzo[d][1,3]dioxol-5-yl.
[0063] In accordance with some embodiments of the invention,
R.sup.1 and R.sup.2 are both H. In some embodiments, R.sup.1 is H
and R.sup.2 is OH.
[0064] In accordance with some embodiments of the invention, A is
optionally substituted phenyl; in other embodiments A is selected
from the group consisting of optionally substituted 5- and
6-membered ring nitrogen heterocycles. In accordance with some
embodiments of the invention, A is selected from the group
consisting of optionally substituted pyridinyl, phenyl,
morpholin-4-yl, piperazin-1-yl, piperidiny-1-yl, imidazol-1-yl,
pyrazol-1-yl, and pyrazol-5-yl.
[0065] In accordance with some embodiments of the invention, X is
selected from the group consisting of CH, C--F, C--OH and N.
[0066] In accordance with some embodiments of the invention, the
compounds are of formula
##STR00009##
wherein A.sup.2 is phenyl, five-membered heteroaryl, six-membered
heteroaryl, 4-7 membered non-aryl heterocycle or fused bicycle;
R.sup.7 is H or F;
[0067] R.sup.8 is chosen from halogen, nitro, acetyl, hydroxyethyl,
amino, methylthio, trifluoromethyl, methoxymethyl, methoxycarbonyl,
trifluoromethoxy, cyano and 1,3,4-thiadiazol-2-yl, or taken
together R.sup.7 and R.sup.8 are methylenedioxy, .dbd.N--O--N.dbd.,
--NH--CH.dbd.N-- or difluoromethylenedioxy; R.sup.14 is chosen from
H, halogen, haloalkyl, alkyl, acyl, alkoxyalkyl, hydroxyalkyl,
carbonyl, phenyl, heteroaryl, benzenesulfonyl, hydroxy, alkoxy,
haloalkoxy, oxaalkyl, carboxy, alkoxycarbonyl, alkoxycarbonylalkyl,
alkoxycarbonylamino, carboxyalkyl, carboxyalkoxy, carboxyalkylthio,
alkoxycarbonylaminoalkyl, carboxyalkylcarbonylamino, carboxamido,
aminocarbonyloxy, alkylaminocarbonyl, dialkylaminocarbonyl,
aminocarbonylalkyl, cyano, acetoxy, nitro, amino, alkylamino,
dialkylamino, aminoalkyl, (alkyl)(aryl)aminoalkyl, alkylaminoalkyl,
dialkylaminoalkyl, dialkylaminoalkoxy, alkyl(hydroxyalkyl)amino,
heterocyclylalkoxy, mercapto, alkylthio, alkylsulfonyl,
alkylsulfonylamino, alkylsulfinyl, alkylsulfonyl, arylthio,
arylsulfonyl, arylsulfonylamino, arylsulfinyl, arylsulfonyl,
acylaminoalkyl, acylaminoalkoxy, acylamino, amidino, aryl, benzyl,
heterocyclyl, heterocyclylalkyl, phenoxy, benzyloxy, heteroaryloxy,
heterocyclylamino, hydroxyimino, alkoxyimino, oxaalkyl,
aminosulfonyl, trityl, amidino, guanidino, ureido,
--NHC(.dbd.O)NHalkyl, --NHC(.dbd.O)NH-heterocyclyl,
-alkyl-NHC(.dbd.O)N(alkyl).sub.2, heterocyclylalkylcarbonylamino,
benzyloxyphenyl, benzyloxy, the residues of amino acids, amino acid
amides, protected residues of aminoacids, protected residues of
amino acid amides, N-methylated amino acids and N-methylated amino
acid amides; alternatively, R.sup.14 may be chosen from H,
--CH.sub.3, --CH.sub.2CF.sub.3, --CF.sub.3, --CHO, --COOH, --CN,
halogen, --OH, --OEt, --C(.dbd.O)NH.sub.2, --C(.dbd.O)NHEt,
--C(.dbd.O)NMe.sub.2-COOCH.sub.3, --COOEt,
--CH.sub.2NHC(.dbd.O)NH.sub.2, --CH(CH.sub.3)NHC(.dbd.O)NH.sub.2,
--CH.sub.2NHC(.dbd.O)H, --CH.sub.2NHC(.dbd.O)CH.sub.3,
--CH.sub.2C(.dbd.O)NH.sub.2, --CH.sub.2COOH, --CH.sub.2COOEt,
--CH.sub.2NHC(.dbd.O)OEt, --CH.sub.2NHC(.dbd.O)O--C.sub.6H.sub.5,
--CH.sub.2NHC(.dbd.O)C(.dbd.O)NH.sub.2, --CH.sub.2NHC(.dbd.O)NHEt,
--C(CH.sub.3).sub.2OH, --CH.sub.2NHC(.dbd.O)N(CH.sub.3).sub.2,
--CH.sub.2NHC(.dbd.O)NHCH.sub.3, --CH.sub.2NH.sub.2,
--CH(CH.sub.3)NH.sub.2, --C(CH.sub.3).sub.2NH.sub.2, --CH.sub.2OH,
--CH.sub.2CH.sub.2OH, --CH.sub.2NHSO.sub.2CH.sub.3,
--CH.sub.2C(.dbd.O)NHEt, --OCH.sub.3, --OC(.dbd.O)NH.sub.2,
--OCH.sub.2CH.sub.2N(CH.sub.3).sub.2, --OCH.sub.2CH.sub.2OCH.sub.3,
--NHC(.dbd.O)NH.sub.2, --NHC(.dbd.O)NHEt, --NHCH.sub.3, --NHEt,
--NH(tBoc), --NHCH.sub.2COOH, --N(CH.sub.3)CH.sub.2COOH,
--NHC(.dbd.O)NHCH.sub.2CH.sub.2Cl, --NHSO.sub.2NH.sub.2, --NHEt,
--N(CH.sub.3).sub.2, --NH.sub.2, --NH(CH.sub.3)C(.dbd.O)NH.sub.2,
--NHSO.sub.2CH.sub.3, --N(SO.sub.2CH.sub.3).sub.2,
--NHC(.dbd.O)OCH.sub.3, --NHC(.dbd.O)OtBu, --NHC(.dbd.O)CH.sub.3,
--SO.sub.2NH.sub.2, --NHC(.dbd.O)CH.sub.2CH.sub.2COOH,
--NHC(.dbd.O)NHCH.sub.2COOH, --CH.sub.2NHCHO, --OCH(CH.sub.3)COOH,
--SCH.sub.2COOH, --NHC(.dbd.O)NHCH.sub.2COOEt,
--NHC(.dbd.O)NH(CH.sub.2).sub.3COOEt,
--NHC(.dbd.O)NH(CH.sub.2).sub.2COOEt,
--N(CH.sub.3)CH.sub.2CH.sub.2OH, --NHC(.dbd.O)OEt,
--N(Et)C(.dbd.O)OEt, --NHC(.dbd.O)NH(CH.sub.2).sub.2COOH,
--NHC(.dbd.O)CH.sub.2N(CH.sub.3).sub.2,
--NHC(.dbd.O)NH(CH.sub.2).sub.3COOH, --NHC(.dbd.O)CH.sub.2NH.sub.2,
--NHC(.dbd.O)CH.sub.2CH.sub.2NH.sub.2,
--NHC(.dbd.O)CH.sub.2NH(tBoc),
##STR00010##
and monocyclic heterocycle substituted with any of the foregoing;
R.sup.14a is chosen from hydroxy, carboxy, alkoxycarbonyl,
carboxyalkylcarbonylamino, carboxyalkylaminocarbonylamino,
guanidino, the residue of an amino acid and the residue of an
N-methylated amino acid, 5-tetrazolyl and monocyclic heterocycle
substituted with any of the foregoing; alternatively, R.sup.14a may
be chosen from --COOH, --OH, --COOCH.sub.3, --COOEt,
--CH.sub.2COOH, --CH.sub.2COOEt, --CH.sub.2NHC(.dbd.O)OEt,
--CH.sub.2NHC(.dbd.O)C(.dbd.O)NH.sub.2, --NHCH.sub.2COOH,
--OCH(CH.sub.3)COOH, --SCH.sub.2COOH, --N(CH.sub.3)CH.sub.2COOH,
--NHSO.sub.2NH.sub.2, --NHC(.dbd.O)CH.sub.2CH.sub.2COOH,
--NHC(.dbd.O)NHCH.sub.2COOH, --NHC(.dbd.O)NHCH.sub.2COOEt,
--NHC(.dbd.O)NH(CH.sub.2).sub.3COOEt,
--NHC(.dbd.O)NH(CH.sub.2).sub.2COOEt,
--NHC(.dbd.O)NH(CH.sub.2).sub.2COOH,
--NHC(.dbd.O)NH(CH.sub.2).sub.3COOH, 5-tetrazolyl and monocyclic
heterocycle substituted with any of the foregoing; R.sup.15 is
chosen from H, NO.sub.2, OH, NH.sub.2, and --NHSO.sub.2NH.sub.2; or
R.sup.15 together with R.sup.14 forms methylene dioxy.
[0068] An example of the embodiment in which A.sup.2 is a
monocyclic heterocycle attached to a monocyclic heterocycle
substituted with a carboxylic acid is found in example BB-01
below:
##STR00011##
Exemplary carboxyalkoxy and carboxyalkylthio are lactic acid and
thioglycolic acid respectively. Exemplary amino acids are glycine,
alanine and proline.
[0069] In some embodiments of the invention, the compound is
selected from the following:
TABLE-US-00001 TABLE 1 ##STR00012## CmpNo X R1 BB-01 CF N-azetidinc
(R/S)-2-carboxylic acid BB-03 CF N-(R) Proline BB-04 CF N-azetidine
(R)-2-carboxylic acid BB-05 CF N-azetidine (S)-2-carboxylic acid
BB-06 CF N--CO--CH2--N(O)Me2 BB-07 CF N(CH3)--CH2--COOH BB-08 CH
N-azetidine (R)-2-carboxylic acid BB-09 CH N-azetidine
(S)-2-carboxylic acid
[0070] Other exemplary compounds that do not fall within the
claimed genus but that illustrate the generality of the syntheses
are found in Tables 2 and 3.
TABLE-US-00002 TABLE 2 ##STR00013## CmpdNo V U X Y W A M P Q
R.sup.14 R3 R4 BA-01 NH2 S CH CH CH2 C CH CH CH F NO2 H BA-02 NH2 S
CH CH CH2 N CH N -- H NO2 H BA-03 NH2 S CH CH CH2 C CH CH CH F
N--O--N BA-04 NH2 S CH CH CH2 C CH CH CH F O--CH2--O BA-05 NH2 S CH
CH CH(OH) C CH CH CH F O--CH2--O BA-06 NH2 S CH CH CH(OH) C NMe N
-- H O--CH2--O BA-07 NH2 S CH CH CH2 C CH CH CH F CF3 H BA-08 NH2 S
CH CH CH2 C NMe N -- H O--CH2--O BA-09 NH2 S CH CH CH2 N N CH -- H
O--CH2--O BA-11 NH2 S N N CH2 C CH CH CH F O--CH2--O BA-10 NH2 S CH
CH CH2 N N CH -- H NO2 H BA-12 NH2 S CH CH CH2 N N CH -- H COCH3 H
BA-13 NH2 S CH CH CH2 C CH CH CH F Br H BA-14 NH2 S CH CH CH2 N N
CH -- H SCH3 H BA-15 NH2 S C--OH CH CH2 C CH CH CH NH2 NO2 H BA-16
NH2 S CF CH CH2 C CH CH CH NH2 Cl H BA-18 NH2 S CH CH CH2 C CH CH
CH NHSO2CH3 Cl H BA-19 NH2 S CF CH CH2 C CH CH CH NHSO2CH3 Cl H
BA-20 NH2 O CH CH CH2 C CH CH CH NHSO2CH3 Cl H BA-21 NH2 S CF CH
CH2 C CH CH CH NHCONH2 Cl H BA-22 NH2 O CH CH CH2 C CH CH CH NH2 Cl
H BA-23 H S CF CH CH2 C CH CH CH NH2 Cl H BA-25 H S CH CH CH2 C CH
CH CH NH2 Cl H BA-26 NH2 S CH CH CH2 C CH CH CH NH2 Cl H BA-27 NH2
S CH CH CH2 C CH CH CH NHCONH2 Cl H BA-28 H S CH CH CH2 C CH CH CH
NHCONH2 Cl H BA-24 CH3 O CH CH CH2 C CH CH CH NHCONH2 Cl H BA-29
NH2 S CF CH CH2 C CH CH CH NHCONH-Et Cl H BA-30 H S CF CH CH2 C CH
CH CH NHCONH-Et Cl H BA-32 NH2 O CH CH CH2 C CH CH CH NH2 Cl H
BA-33 H S CF CH CH2 C CH N CH NHCONH-Et Cl H BA-38 H S CF CH CH2 C
CH N CH OCH3 Cl H BA-39 H S CF CH CH2 C CH C(OH) CH H Cl H BA-40 H
S CF CH CH2 C CH N CH NHCOO-Et Cl H BA-51 H S CF CH CH2 C CH CH CH
NHCONH2 Cl H BA-53 H S CF CH CH2 C CH N CH NHCONHEt CN H BA-55 H S
CF CH CH2 C CH CH CH NMe2 Cl H BA-57 H S CF CH CH2 C CH CH CH
N-Pyrrolidine Cl H BA-58 H S CH CH CH2 C CH CH CH NMe2 Cl H BA-59 H
S CH CH CH2 C CH CH CH N-Pyrrolidine Cl H BA-60 H S CF CH CH2 C CH
CH CH CN Cl H BA-61 H S CF CH CH2 C CH CH CH N-azetidine Cl H BA-63
H S CF CH CH2 C CH CH CH NH2 Cl H BA-64 H S CF CH CH2 C CH CH CH
NHCO--CH2--NMe2 Cl H BA-66 CH3 S CH CH CH2 C CH CH CH NH2 Cl H
BA-67 CH3 S CH CH CH2 C CH CH CH NHCO--CH2--NMe2 Cl H BA-74 H S CF
CH CH2 C CH CH CH N-azetidine-(R)- Cl H 2-carboxamide BA-75 H S CF
CH CH2 C CH CH CH N-azetidine-(S)- Cl H 2-carboxamide
TABLE-US-00003 TABLE 3 ##STR00014## Cmpd no M Q L G R.sup.14
R.sup.8 R.sup.7 BA-31 CH2 CH2 CH2 N CONH2 Cl H BA-43 CH2 CH2 CH2 O
-- Cl H BA-44 CH2 CH2 CH2 N COCH3 Cl H BA-45 CH2 CH2 CH2 CH
NH--CO2-Et Cl H BA-46 CH2 CH2 CH2 CH NCONH2 Cl H BA-47 CH2 CH2 CH2
CH NH2 Cl H BA-48 CH2 CH2 CH2 N H Cl H BA-49 CH2 CH2 CH2 N CO2Et Cl
H BA-50 CH2 CH2 CH2 N CH3 Cl H BA-52 CH2 CH2 CH2 N CO--NHEt Cl H
BA-54 CH2 -- CH2 CH H Cl H BA-71 CH2 CO CH2 N H Cl H BA-72 CO CH2
CH2 N CONH-Et Cl H BA-73 CO CH2 CH2 N H Cl H
[0071] All of the compounds falling within the foregoing genera.
Ia, Ib and Ic are useful as PDE4 inhibitors. It may be found upon
examination that species and genera not presently excluded are not
patentable to the inventors in this application because of prior
art. In this case, the exclusion of species and genera in
applicants' claims are to be considered artifacts of patent
prosecution and not reflective of the inventors' concept or
description of their invention. The invention, in a composition
aspect, is all active compounds of formula Ia, Ib and Ic, except
those that are in the public's possession.
[0072] In general, compounds of formula Ia, Ib and Ic may be
prepared by the methods illustrated in the general reaction schemes
as, for example, described below, or by modifications thereof,
using readily available starting materials, reagents and
conventional synthesis procedures. In these reactions, it is also
possible to make use of variants that are in themselves known, but
are not mentioned here.
[0073] Table 1 above lists compounds representative of embodiments
of the invention. Processes for obtaining compounds of formula I
are presented below. Other compounds of formula I may be prepared
in analogous fashion to those whose synthesis is exemplified
herein. The procedures below illustrate such methods. Furthermore,
although the syntheses depicted herein may result in the
preparation of enantiomers having a particular stereochemistry,
included within the scope of the present invention are compounds of
formula I in any stereoisomeric form, and preparation of compounds
of formula I in stereoisomeric forms other than those depicted
herein would be obvious to one of ordinary skill in the chemical
arts based on the procedures presented herein.
Synthetic Methods
[0074] Generally compounds of the Formula I, can be prepared by
sequential introduction of substitution at the 4 and 6 positions of
the benzoazoles. The introduction of the substituents at the C4
position to form a C--C bond can be accomplished by organometallic
coupling protocols (e.g. Suzuki, Stille reaction) or by
displacement of a halogen using metal assisted displacement with a
cyclic or heterocyclic NH compound forming a C--N bond at the C4
position of the benzoazole. The atom numberings referenced in this
section are shown in G1 (scheme 1). These reactions can be
performed with benzoazole derivatives bearing a variety of
functionalities at the C-2 which may include V=H, CH3, a protected
or derivatized amine or ether. The 2-amino group of benzoazole
(V=NH2) can be converted thru intermediacy of a diazo
(--N.sup.+.ident.N) group to from V=H (for example). The
substituent at the C-6 position can be introduced by a wide variety
of approaches. These chemistries employed depend of the 1-carbon
functionality (CH3, CHO, COOR', CN etc.) present at the C-6. The
benzoazoles bearing diverse functional groups which are amenable to
standard function group interconversion, for example alkyl, ester,
nitrile which could provide alcohol, alkyl halide could be
manipulated by standard and these may include aldehyde, nitrile and
esters, which allow generation of alcohol which could be converted
to a carbonate or alkyl halide. These functionalities allow
introduction of aryl, heteroaryl substituents through C--C forming
chemistries. Alternatively nucleophilic displacement of the alkyl
halide, OTs, OTf etc. allow incorporation of substituents via C--N
bond forming approach to introduce cyclic, acylic, amine derived
functional groups (G7). This strategy allows incorporation of
acyclic, heterocyclic or heteroaryl derived substituents at the C6
position of the benzoazole nucleus.
##STR00015##
Moreover, introduction of substituents at C6 or C4 could be carried
in either sequence, i.e. formation of C4 substituent followed by C6
(route A, G1->G2) or vice versa (route B, G1-.G5). Either of
these substituents may carry additional functional groups which
could be further derivatized through standard functional group
transformation chemistries that are well know in the art. Some of
these include formation of amide, sulfonamide, ureas, imidazolone,
oxazolones, and carbamates from appropriate amine, carboxylic acid,
alcohols or phenol groups. Additionally, when the R1 group contains
an ortho-halo N-heterocycles (e.g 2-halo pyridine or
2-halo-pyrimidine) G8, a nucleophilic displacement of the halo (or
-OTf, ONf derived from pyridin-2-one) groups. Examples of these
nucleophile include an amine (primary, sec. tert.; acyclic or
cyclic including) or NH-containing heteroaryl (for example,
substituted imidazole or pyrazole); or alcohol/thiol allowing
incorporation of additional --O, --S or --N linked substituents to
provide G9. Alternatively, an appropriately functionalized pyridine
can be converted to corresponding 2-OTf or 2-ONf which could then
participate in similar chemistries.
##STR00016##
The R1 group could also be assembled form an acyclic intermediate
(Scheme A3) to form a heterocylic or heteroaromatic ring. Examples
of these chemistries include formation of 5-membered heteroaryls
(G12) such as oxadiazole, thiadiazole, triazole form acyl hydrazide
(G11); thiazole from 2-halo-ketone or dipolar cycloaddition
reactions when the C4 or C6 substituent is an olefin or acetylic
group (G10->G13)). Alternatively the 6-membered heteroaryl or
heterocyclic rings could be formed using Diels-Alder or
hetero-Diels-Alder chemistries using appropriately substituted
alkyl aryl ether bearing either a dienophile or a diene at C4 or C6
position.
##STR00017##
##STR00018##
With an aldehyde, ketone, nitrile or an ester at C6, addition of an
organometallic (Grignard or organozinc reagent) allows formation of
heteroatom containing substituents where Ra or Rb bear a
heteroatom. Alternatively, when C6 is C--H, Friedle-Craft acylation
provide a ketone (G17), which is subsequently transformed to sec.
alcohol (via reduction), tert. alcohol (thru addition of an
alkyl/aryl using RMgX or R2Zn) or sec. or tertiary amine thru
mediation of an imine/oxime; or to --CH2-, providing variations at
the linker position (G16).
##STR00019##
[0075] The above approaches describe means to decorate a benzoazole
nucleus. On the other hand, one may also be able to start with 1,3
functionalized phenyl or heteroaryl (G20 or G22) which is then
elaborated through construction of the fused five membered ring to
assemble the benzoazole nucleus later stages in the synthesis of
analogs corresponding to the genus 1. This approach is depicted in
scheme A5. Example of this strategy is also provided in some
non-limiting specific example in the later section.
##STR00020##
[0076] The diverse selection of substituents present in R1 could be
formed by standard functional group transformations that are well
know in the art. Some of these include formation of amide,
sulfonamide, ureas, imidazolone, oxazolones, carbamates from the
alkoxy-biaryl fragments bearing and appropriate amine, carboxylic
acid, alcohols or phenol groups. When the R1 group contains an
ortho-halo pyridine or pyrimidine for example, the nucleophilic
displacement of the halo (or -OTf, ONf derived from pyridone)
groups. Examples of the nucleophile include an amine (primary, sec.
tert.; acyclic or cyclic including), alcohol or HN-containing
heterocylic groups (for example, substituted imidazole or
pyrazole). These displacement reactions could be carried out using
alkali or tert. amine base; or could be mediated thru use of an
organometallic reagent such as Pd, or Al reagent.
[0077] Scheme 1 provides an outline of the synthesis of example
1.
##STR00021## ##STR00022##
[0078] BA-01
(2,6-dibromo-4-cyano-phenyl)-thiourea (2)
[0079] To a suspension of 4-amino-3,5-dibromobenzonitrile (11 g, 40
mmol) in toluene (80 ml) was added thiophosgene (5.06 g, 44 mmol,
1.1 eq.). The reaction mixture was stirred under reflux for 16 h.
After cooling to RT, the volatile material was removed in-vacuo,
residue was suspended in dioxane (80 mL) and treated with ammonium
(27 w/w %, 9.85 g) with stirring at room temperature After for 30
min., dioxane was removed in-vacuo. The solid obtained was washed
with ether (50 mL), water (50 mL) and ether (50 mL), dried in vacuo
to provide 9.6 g (70% yield) of the title compound (2).
.sup.1H-NMR-(400 MHz, CDCl.sub.3)
2-amino-4-bromo-benzothiazole-6-carbonitrile (3)
[0080] A reaction mixture of (2) compound (1.3 g, 3.66 mmol), CuI
(70 mg, 0.366 mmol, 0.1 eq.), 1, 10-phenanthroline (70 mg, 0.366
mmol), and Cs.sub.2CO.sub.3 (1.8 g, 5.5 mmol, 1.5 eq.) in dioxane
(20 mL) was stirred under reflux for 2 h under Ar. After cooling to
room temperature, water (100 mL) was added; solid was filtered,
washed with water (100 mL.times.3) followed by ether (50
mL.times.3). The solid was dried in vacuo at room temperature
overnight, to provide 1 g (quantitative yield) of the desired
product (3). .sup.1H-NMR-(400 MHz, CDCl.sub.3)
2-amino-4-(3-nitro-phenyl)-benzothiazole-6-carbonitrile. (4)
[0081] A mixture of compound (3) (750 mg, 3 mmol),
3-nitrophenyl-boronic acid (752 mg, 4.5 mmol), triphenylphosphine
(470 mg, 1.8 mmol, 0.6 eq.), Pd(OAc).sub.2 (130 mg, 0.6 mmol, 0.2
eq.) in dioxane (30 mL), ethanol (9 mL) and (aqueous) 1N
Na.sub.2CO.sub.3 (9 mL, 3 eq.) was stirred at under reflux
overnight under Argon. The reaction mixture was diluted with water
(100 mL) and extracted with ethyl acetate (3.times.100 mL). The
combined organic phase was passed through a filtration funnel.
Evaporation of solvent gave a residue, which was treated with
dichloromethane (3.times.20 mL) to afford the desired product (4)
530 mg (45% yield). 1H-NMR-(400 MHz, DMSO-d6). MS (ESI+): 301.7
(M+1). LC-MS: 90%.
[6-cyano-4-(3-nitro-phenyl)-benzothiazol-2-yl]-carbamic Acid
tert-butyl Ester (5)
[0082] A reaction mixture of compound (4) (230 mg, 0.77 mmol),
(Boc).sub.2O (220 mg, 1 mmol, 1.3 eq.) and DMAP (15 mg, 0.08 mmol,
0.1 eq.) in MeCN (10 mL) was stirred at room temperature for 48 h.
The volatile material was removed under reduced pressure to give
300 mg (98% yield) of desired product (5) as yellow solid.
[6-formyl-4-(3-nitro-phenyl)-benzothiazol-2-yl]-carbamic Acid
tert-butyl Ester. (6)
[0083] To a solution of compound (5) (150 mg, 0.38 mmol) in
dichloromethane (8 mL) was added DIBAL-H (1 mL, 1N in hexane) by a
syringe at -60.degree. C. After the addition was complete, the
reaction mixture was allowed to warm to room temperature over 2 h.
Then 1 mL of aqueous 3 N HCl was added and the mixture was stirred
at room temperature for 20 min before additional 1 mL of aqueous 3
N HCl was added. After the resulting mixture was stirred for 30
min. dichloromethane (20 mL) was added followed by water (20 mL).
The organic layer was separated and the aqueous layer was extracted
with dichloromethane (3.times.15 mL). The combined organics was
dried over Na.sub.2SO.sub.4. Evaporation of solvent under reduced
pressure gave 120 mg (79% yield) of crude product (6). The product
thus obtained was forward to the next step without any further
purification.
[6-[(4-fluorophenyl)-hydroxymethyl]-4-(3-nitrophenyl)-benzothiazol-2-yl]-c-
arbamic Acid tert-butyl Ester (7)
[0084] To a solution of aldehyde (6) (120 mg, 0.3 mmol) in THF (4
mL) was added 4-fluorophenyl magnesium bromide (1 ml, 1N in THF)
dropwise by a syringe at -78.degree. C. The mixture was allowed to
warm to room temperature and the reaction was monitored by TLC
analysis. After the starting aldehyde was consumed, the reaction
was quenched by addition of saturated NH.sub.4Cl (aq) followed by
aqueous 3 N HCl to bring the mixture to pH=3. The mixture was
extracted with EtOAc (3.times.15 mL). The combined organics were
dried over Na.sub.2SO.sub.4 and removal of solvent gave a residue,
which was purified by chromatography on silica gel using
hexanes/dichloromethane (1:2) followed by dichloromethane as eluent
to give 65 mg (44% yield) of product (7).
6-(4-fluorobenzyl)-4-(3-nitrophenyl)-benzothiazol-2-yl Amine.
BA-01
[0085] A solution of compound (7) (32.5 mg, 0.066 mmol) was treated
with Et.sub.3SiH (500 uL, 3 mmol, 47 eq.). The mixture was stirred
at room temperature for 130 min. until starting material XM-17 was
consumed. The volatile material was removed under reduced pressure.
The residue was washed with sodium bicarbonate solution, extracted
with EtOAc (3.times.15 mL), dried. Evaporation gave a residue,
which was purified chromatography on silica gel using
dichloromethane/hexanes (1:1, 2:1, 3:1, 4:1, 20 mL each) followed
by dichloromethane as eluent to give 21 mg of product, which
contained impurities. The product thus obtained was triturated with
hexanes to give 7.6 mg of desired product. BA-01. 1H NMR. LCMS:
99%.
[0086] In similar fashion the following schemes provide outlines of
the syntheses as applied to examples.
[0087] BA-02,
##STR00023##
[6-Hydroxymethyl-4-(3-nitro-phenyl)-benzothiazol-2-yl]-carbamic
Acid tert-butyl Ester. (8)
[0088] To a solution of aldehyde (6) (2 g, 4.3 mmol, 1 eq) in a
mixture of DCM (20 ml) and MeOH (10 ml) was added NaBH.sub.4 (760
mg, 20 mmol, 5 eq.) portion-wise at 0 C. The mixture was stirred at
0 C.about.rt until starting material was consumed. The mixture was
added sat. NH.sub.4Cl aq. (200 ml) and extracted with EtOAc (20
ml.times.3). The combined organic layers were dried over MgSO4.
Removal of solvent under reduced pressure gave a residue, which was
purified by chromatography on silica gel using DCM followed by
DCM/MeOH with a ratio of 100:1 as eluent to give 500 mg of alcohol
(8) in 29% yield.
[6-Bromomethyl-4-(3-nitro-phenyl)-benzothiazol-2-yl]-carbamic Acid
tert-butyl Ester. (9)
[0089] To a solution of (8) (500 mg, 1.25 mmol, 1 eq.) in DCM (12
ml) was added Ph.sub.3P (340 mg, 1.25 mmol, 1 eq.). After the
mixture was stirred at rt for 20 min., NBS (230 mg, 1.25 mmol, 1
eq.) was added in one portion. The mixture was stirred at rt until
(8) was consumed. The volatile material was removed under reduced
pressure to afford a residue, which was purified by chromatography
on silica gel using hexane/DCM (1:1 then 1:1.5) as eluent to give
126 mg of desired bromide (9) in 22% yield.
[6-imidazol-1-ylmethyl-4-(3-nitro-phenyl)-benzothiazol-2-yl]-carbamic
Acid tert-butyl Ester. (10)
[0090] To a 20 mL vial which contained (9) (46 mg, 0.13 mmol) in
DMF (5 mL) was added imidazole (200 mg, excess) at 0.degree. C. The
reaction mixture was allowed to warm to rt and stir at rt for 16 h.
The mixture was poured onto 30 mL ice-water which was extracted
with ethyl acetate (3.times.20 mL), washed with water (2.times.20
mL), brine (20 mL) and dried over Na2SO4. After removal of solvent,
the residue was purified by silica gel column chromatography with
ethyl acetate/Hexane as eluent to give 28 mg of product (10) in 60%
yield. .sup.1H-NMR (400 MHz, CDCl.sub.3)
6-imidazol-1-ylmethyl-4-(3-nitro-phenyl)-benzothiazol-2-ylamine HCl
Salt. BA-02
[0091] To a 20 mL vial which contained (10) (28 mg, 0.07 mmol) was
added HCl (4 N in dioxane, 1 mL) at it. The mixture was allowed to
stir at it for 16 h. Diethyl ether (20 mL) was added and the solid
which formed was filtered out, washed with diethyl ether (20 mL),
dried to yield 23 mg (70%) of the title product BA-02. .sup.1H-NMR
(400 MHz, DMSO)
[0092] BA-03
##STR00024##
(4-bromo-6-cyano-benzothiazol-2-yl)-carbamic Acid tert-butyl Ester
(11)
[0093] A reaction mixture of compound (3) (25 g, 100 mmol),
(Boc).sub.2O (26.8 g, 123 mmol, 1.23 eq.) and DMAP (352 mg, 2.9
mmol, 0.03 eq.) in THF (260 mL) was stirred at room temperature
over night. 5 g of additional of (Boc).sub.2O and 350 mg of DMAP
was added and the resulting mixture was stirred for 48 h. The
volatile material was removed under reduced pressure and the
residue was purified by chromatography on silica gel (300 g,
regular) using Hex/DCM (2:1 then 1:1) as eluent to give the product
(24 g), which was further triturated with hexane to give the
desired product (11) (20 g, 56% yield). as light yellow solid.
.sup.1H-NMR (400 MHz, CDCl.sub.3)
(4-bromo-6-formyl-benzothiazol-2-yl)-carbamic Acid tert-butyl
Ester. (12)
[0094] To a solution of compound (11) (10 g, 28 mmol) in
dichloromethane (200 mL) was added DIBAL-H (84 mL, 1N in hexane, 3
eq.) dropwise using a addition funnel keeping the temperature
around 0.degree. C. After the addition was complete, the reaction
mixture was stirred at room temperature over night. The reaction
mixture was poured into 1.2 N HCl (200 ml) with stirring. The
yellow precipitate was collected by filtration and washed with
water (3.65 g of de-Boc product was obtained). The mother liquid
was separated and dried over Na.sub.2SO.sub.4. Removal of solvent
gave the crude desired product (12) (4.5 g, 45% yield). The
compound thus obtained was used next step without any further
purification. 1H-NMR (400 MHz, DMSO-d6) MS (ESI+): 301.7 (M+1)
{4-bromo-6-[(4-fluoro-phenyl)-hydroxy-methyl]-benzothiazol-2-yl}-carbamic
Acid tert-butyl Ester. (13)
[0095] To a solution of the aldehyde (12) (4.5 g, 10 mmol) in
anhydrous THF (200 mL) was added dropwise a solution of
4-fluorophenyl magnesium bromide in THF (30 ml, 1N) at -50.degree.
C. After the addition was complete, the resulting mixture was
allowed to warm to room temperature over night. Then saturated
NH.sub.4Cl aq. was added. The mixture was extracted with EtOAc
(3.times.20 ml). The combined organic layers were dried over
Na.sub.2SO.sub.4. Removal of solvent gave a residue, which was
purified by chromatography on silica gel (100 g, regular) using
DCM/hexane (1:2 to 3:1) followed by DCM as eluents to give the
recovered starting aldehyde (12) (1.7 g) and the desired product
(13) (0.82 g, 25% yield).
4-bromo-6-(4-fluorobenzyl)-benzothiazol-2-yl Amine. (14)
[0096] To a solution of compound (13) (205 mg, 0.45 mmol) in TFA (3
ml) was added Et.sub.3SiH (1 mL, 6.25 mmol, 14 eq.) in one portion.
The mixture was stirred at room temperature over night. The
volatile material was removed under reduced pressure. The residue
was triturated with DCM/hexane (1:5). After drying, the desired
product (14) was obtained as light yellow solid (145 mg, 96%
yield).
4-Benzol[1,2,5]oxadiazol-5-yl-6-(4-fluoro-benzyl)-benzothiazol-2-yl
Amine. BA-03
[0097] A mixture of compound (14) (118 mg, 0.35 mmol), the boronic
acid (86 mg, 0.52 mmol, 1.5 eq.), triphenylphosphine (70 mg, 0.26
mmol, 0.6 eq.), Pd(OAc).sub.2 (20 mg, 0.09 mmol, 0.2 eq.) in
dioxane (4 mL), and (aqueous) 1N Na.sub.2CO.sub.3 (1 mL, 3 eq.) was
stirred under reflux overnight under Ar. After cooling to room
temperature, The volatile material was removed under reduced
pressure to give a residue, which was purified by chromatography on
silica gel using DCM/hexane (1:1) followed by DCM as eluent to
afford the product (20 mg), which was further triturated with
DCM/hexane (1:1) to give the desired product BA-03 (13.5 mg, 10%
yield). 1H-NMR (400 MHz, DMSO-d6) MS (ESI+): 301.7 (M+1) LC-MS:
96.8%.
[0098] BA-04
##STR00025##
4-Benzo[1,3]dioxol-5-yl-6-(4-fluoro-benzyl)-benzothiazol-2-ylamine.
BA-04
[0099] To a mixture of compound (15) (100 mg, 0.2 mmol) in TFA (2
ml) was added Et.sub.3SiH (0.5 ml, 15 eq.) in one portion by a
syringe. The mixture was stirred at room temperature over night.
The volatile material was removed under reduced pressure. The
residue was washed with hexane and then purified by chromatography
on silica gel using dichloromethane followed by
dichloromethane/methanol (200:1) as eluent to give the desired
product, which was triturated with hexane/dichloromethane (10:1) to
give the desired product BA-04 (31 mg, 41% yield). 1H-NMR (400 MHz,
DMSO-d6) MS (ESI+): (M+1) LC-MS: 99%.
[0100] BA-05
##STR00026##
[4-Benzo[1,3]dioxol-5-yl-6-[(4-fluoro-phenyl)-hydroxy-ethyl]-benzothiazol-
-2-yl]-carbamic Acid tert-butylester. (15)
[0101] A mixture of compound (13) (316 mg, 0.7 mmol), the boronic
acid (175 mg, 1.05 mmol, 1.5 eq.), triphenylphosphine (120 mg, 0.42
mmol, 0.6 eq.), Pd(OAc).sub.2 (40 mg, 0.14 mmol, 0.2 eq.) in
dioxane (9 mL), and (aqueous) 1N Na.sub.2CO.sub.3 (3 mL, 3 eq.) was
stirred under reflux overnight under Ar. After cooling to room
temperature, The volatile material was removed under reduced
pressure to give a residue, which was purified by chromatography on
silica gel using hexane/dichloromethane (from 2:1 to 1:5) followed
by DCM as eluent to afford the product (15) (236 mg, 68%
yield).
(2-Amino-4-benzo[1,3]dioxol-5-yl-benzothiazol-6-yl)-(4-fluoro-phenyl)-meth-
anol. BA-05
[0102] Compound (15) (70 mg, 0.14 mmol) in dichloromethane (1 ml)
was added TFA (0.6 ml). The mixture was stirred at room temperature
over night. The volatile material was removed under reduced
pressure. The residue was purified by chromatography on silica gel
using dichloromethane followed by dichloromethane/methanol (100:1)
as eluent to give the desired product BA-05 (18.5 mg, 34% yield) as
gray solid. 1H-NMR (400 MHz, DMSO-d6) MS (ESI+): (M+1) LC-MS:
95%.
[0103] BA-06
##STR00027##
{4-Bromo-6-[hydroxy-(2-methyl-2H-pyrazol-3-yl)-methyl]-benzothiazol-2-yl}-
-carbamic Acid tert-butyl Ester. (16)
[0104] To a solution of 1-methylpyrazole (1.13 g, 13.8 mmol) in THF
(20 ml) at -78.degree. C. was added n-BuLi (2.5 N in hexane, 4 ml)
dropwise by a syringe. The resulting mixture was stirred at
-78.degree. C..about.rt over 1.5 h. The solution was stored at a
refrigerator for further use. To a solution of aldehyde (12) (357
mg, 1 mmol) in THF (10 ml) was added dropwise above prepared
lithium reagent (8 ml, 3.2 mmol, 1.6 eq.) by a syringe at
-58.degree. C. with stirring. After the addition was complete, the
mixture was allowed to warm to room temperature over 2 h. The
mixture was added to aqueous NH.sub.4Cl solution and extracted with
ethyl acetate. The organic layers were separated and dried over
MgSO4. Removal of solvent gave a residue, which was purified by
chromatography on silica gel using DCM/MeOH (100:1) as eluent to
give the desired product (16) (416 mg, 95% yield).
{4-Benzo[1,3]dioxol-5-yl-6-[hydroxy-(2-methyl-2H-pyrazol-3-yl)-methyl]-ben-
zothiazol-2-yl}-carbamic Acid tert-butyl Ester. (17)
[0105] A mixture of (16) (206 mg, 0.47 mmol), boronic acid (120 mg,
0.705 mmol, 1.5 eq.), Pd(OAc).sub.2 (21 mg, 0.098 mmol, 0.2 eq.),
PPh.sub.3 (75 mg, 0.282 mmol, 0.6 eq.) in 1N Na.sub.2CO.sub.3 (2
ml) and dioxane (6 ml) was stirred at 100.degree. C. (oil bath)
under Ar over night. After cooling to room temperature, the
volatile material was removed and the residue was partitioned
between ethyl acetate and water. The EA layer was separated and
passed through a plug of MgSO.sub.4. Solvent was removed to give
crude product (17) (260 mg, 115% yield). No further purification
was done.
(2-Amino-4-benzo[1,3]dioxol-5-yl-benzothiazol-6-yl)-(2-methyl-2H-pyrazol-3-
-yl)-methanol. BA-06
[0106] To a solution of compound (17) (260 mg, 0.47 mmol) in TFA (1
ml) was added Et.sub.3SiH (1 ml, 6 mmol, 30 eq.) in one portion by
a syringe. The mixture was stirred at room temperature over night.
The volatile material was removed under reduced pressure. The
residue was washed with hexane and then purified by chromatography
on silica gel using dichloromethane/methanol (100:1) as eluent to
give the desired product BA-06 (43 mg, 25% yield). 1H-NMR (400 MHz,
DMSO-d6); MS (ESI+): (M+1), LC-MS: 97%.
[0107] BA-07
##STR00028##
[6-[(4-Fluoro-phenyl)-hydroxy-methyl]-4-(3-trifluoromethyl-phenyl)-benzot-
hiazol-2-yl]-carbamic Acid tert-butyl Ester. (18)
[0108] A mixture of compound (13) (170 mg, 0.375 mmol), the boronic
acid (110 mg, 0.563 mmol, 1.5 eq.), triphenylphosphine (60 mg,
0.225 mmol, 0.6 eq.), Pd(OAc).sub.2 (20 mg, 0.075 mmol, 0.2 eq.) in
dioxane (6 mL), and (aqueous) 1N Na.sub.2CO.sub.3 (2 mL, 3 eq.) was
stirred under reflux overnight under Ar. After cooling to room
temperature, The volatile material was removed under reduced
pressure to give a residue, which was purified by chromatography on
silica gel using dichloromethane/methanol with a ratio of 100:1 as
eluent to afford the product (18) (136 mg, 70% yield).
6-(4-Fluoro-benzyl)-4-(3-trifluoromethyl-phenyl)-benzothiazol-2-ylamine.
BA-07
[0109] To a solution of compound (18) (91 mg, 0.17 mmol) in TFA
(0.5 ml) was added Et.sub.3SiH (0.5 ml). The mixture was stirred at
room temperature over night. The volatile material was removed
under reduced pressure. The residue was triturated with hexane. The
solid was filtered and washed with hexane. After dried, 60 mg of
desired product was obtained. the product thus obtained was passed
through a plug of silica gel using dichloromethane/methanol (100:1)
as eluent to afford the desired product BA-07 (50 mg, 73% yield).
1H-NMR (400 MHz, DMSO-d6); MS (ESI+): (M+1), LC-MS: 100%.
[0110] BA-08
##STR00029##
4-Benzo[1,3]dioxol-5-yl-6-(2-methyl-2H-pyrazol-3-ylmethyl)-benzothiazol-2-
-ylamine. BA-08
[0111] Compound BA-06 (20 mg, 0.05 mmol) was treated with a mixture
of TFA (ml) and Et.sub.3SiH (1 ml) at 70.degree. C. with stirring
over night. After cooling to room temperature, the volatile
material was removed under reduced pressure to give a residue,
which was purified by chromatography on silica gel using
dichloromethane/methanol with a ratio of 100:1 as eluent to give
the desired product BA-08 (15 mg, 76% yield). 1H-NMR (400 MHz,
DMSO-d6) MS (ESI+): (M+1) LC-MS: 93%.
##STR00030##
[0112] BA-09
[0113] BA-10
[0114] BA-12
[0115] BA-14
Synthesis 2-Amino-4-bromo-benzothiazole-6-carboxylic Acid. (19)
[0116] To a suspension of compound (3) (2.54 g, 10 mmol) in acetic
acid (5 ml) was added sulfuric acid (5 ml) and water (5 ml). The
resulting mixture was stirred at 100.degree. C. overnight. After
cooling to room temperature, the mixture was added water up to 500
ml. The precipitate was collected by filtration and washed with
water until the washing is neutral (pH=7). After dried in the air,
1.93 g (71% yield) of desired product (19) was obtained as
off-white solid.
2-Amino-4-bromo-benzothiazole-6-carboxylic Acid Methyl Ester.
(24)
[0117] Compound (19) (1.48 g, 5.4 mmol) was suspended in a mixture
of tetrahydrofuran (THF, 160 ml) and methanol (MeOH, 40 ml). To the
mixture was added TMSCHN.sub.2 (2M in ether) dropwise by a syringe.
After the addition was complete, the mixture was stirred at room
temperature for 1 hour. The volatile material was removed under
reduced pressure to give the crude product (24) (1.6 g, 100%
yield). The product thus obtained was forwarded to the next step
without further purification.
(2-Amino-4-bromo-benzothiazol-6-yl)-methanol. (21)
[0118] To a suspension of (20) (1.6 g, 5.4 mmol) in THF (120 ml)
was added lithium aluminum hydride (0.93 g, 24.5 mmole, 4.5 eq.).
The mixture was stirred at room temperature for 30 minutes before
H.sub.2O (0.93 ml), NaOH (0.93 ml, 12.5%) and H.sub.2O (0.93
ml.times.3) was added successively. After the mixture was stirred
for 10 minutes, the insoluble material was filtered and washed
tetrahydrofuran (20 ml.times.3). The organic layer was evaporated
to give the crude product (21) (2.24 g, 100% yield), which was
forwarded to the next step without further purification.
(4-Bromo-6-hydroxymethyl-benzothiazol-2-yl)-carbamic Acid
tert-butyl Ester. (22)
[0119] To a solution of (21) (2.2 g, 8.5 mmole) in THF (50 ml) was
added (Boc).sub.2O (5.7 g, 25.5 mmole, 3 eq.) The mixture was
stirred at rt overnight. Then 1N NaOH solution (50 ml) and methanol
(20 ml) were added. The mixture was stirred at room temperature
over night. The volatile material was removed and water layer was
neutralized by addition of 1N HCl to pH=7. The precipitate was
filtered and washed with water. Dried in the air, 0.394 g of the
desired product (22) was obtained (13% yield). This compound was
also prepared by reduction of its corresponding aldehyde with
NaBH.sub.4 in DCM/MeOH. (1.2 g of (22) was obtained on a 1.2 g
scale reaction.
(4-Bromo-6-bromomethyl-benzothiazol-2-yl)-carbamic Acid tert-butyl
Ester. (23)
[0120] To a suspension of (22) (394 mg, 1.1 mmole, 1 eq.) in
dichloromethane (DCM, 10 ml) was added triphenylphosphine (390 mg,
1.1 mmole, 1 eq.) followed by NBS (200 mg, 1.1 mmole, 1.0 eq.) in
one portion. The mixture was stirred 0.degree. C. for 2 hours. The
volatile material was removed and the residue was purified by
chromatography on silica gel using DCM/Methanol (200:1) as eluent
to give 200 mg of desired product (23) (43% yield) along with 100
mg of recovered starting (22) (25%). In a 1.2 scale reaction under
the same condition described above, 936 mg of desired product (23)
was isolated in 67% yield.
(4-Bromo-6-pyrazol-1-ylmethyl-benzothiazol-2-yl)-carbamic Acid
tert-Butyl Ester. (24)
[0121] To a solution of (23) (1.5 g, 3.6 mmole, 1 eq.) in DMF (8
ml) was added pyrazole (1.5 g, 21 mmole, 6 eq.). The resulting
mixture was stirred at room temperature until SM was consumed. The
mixture was poured into water and the precipitate was filtered and
washed with water. The product was dried under N.sub.2 flow over
night, the solid was purified by chromatography on silica gel using
DCM as eluent to give 925 mg of product (24) in 63% yield.
[0122] General Procedure A: Suzuki Coupling of (24) with Boronic
Acids: Examples BA-09, BA-10, BA-12 and BA-14.
[0123] A mixture of compound (24) (1 eq.), boronic acid (1.5 eq.),
triphenylphosphine (0.6 eq.), Pd(OAc).sub.2 (0.2 eq.) in dioxane
(6.times.mL), and (aqueous) 1N Na.sub.2CO.sub.3 (X mL, 3 eq.) was
stirred under Ar at 80.degree. C. overnight. The reaction mixture
was diluted with water and extracted with ethyl acetate. The
combined organic phase was passed through a filtration funnel.
Evaporation of solvent gave a residue, which was purified by
chromatography on silica gel using DCM/MeOH (100:1) as eluent to
give desired coupling product. Removal of Boc group was effected by
treatment of Suzuki coupling product with TFA/DCM (1:1). Volatile
material was removed and the residue was treated with 2N HCl in
ether to give target compounds, Examples 003, 011, 012 and 014 as
HCl salt.
[0124] BA-09.
4-Benzo[1,3]dioxol-5-yl-6-pyrazol-1-ylmethyl-benzothiazol-2-ylamine
Started with 70 mg of (24), 34.7 mg of BA-09 was obtained. LC-MS:
97.6%.
[0125] BA-10.
4-(3-Nitro-phenyl)-6-pyrazol-1-ylmethyl-benzothiazol-2-ylamine.
Started with 90 mg of (24), 35 mg of BA-10 was obtained. LC-MS:
93%.
[0126] BA-12.
1-[3-(2-Amino-6-pyrazol-1-ylmethyl-benzothiazol-4-yl)-phenyl]-ethanone
Started with 120 mg of (24), 93.7 mg of BA-12 was obtained. LC-MS:
94%.
[0127] BA-14.
4-(3-Methylsulfanyl-phenyl)-6-pyrazol-1-ylmethyl-benzothiazol-2-ylamine
Started with 120 mg of (24), 60 mg of BA-14 was obtained. LC-MS:
99%.
[0128] BA-11
##STR00031## ##STR00032##
2-Chloro-4,6-dimethoxy-5-nitro-pyrimidine. (25)
[0129] To a suspended tetramethyl-ammonium nitrate (11.2 g, 39.5
mmol) in methylene chloride (100 mL) was added triflic anhydride
(5.4 g, 39.5 mmol), and stirred at room temperature for 2 hour.
After the resultant was cooled to -78.degree. C., a solution of
2-chloro-4,6-dimethoxypyrimidine (5.0 g, 36.0 mmol) in methylene
chloride (50 mL) was added at -78.degree. C., and continued to
stirred at room temperature for 24 hours. The reaction was diluted
with brine, extracted with ethyl acetate, washed with brine, dried
over Na.sub.2SO.sub.4, concentrated to yield
2-chloro-4,6-dimethoxy-5-nitro-pyrimidine (25) (6.0 g, 76%). 1HNMR
(DMSO-d6)
2-(4-Fluoro-benzyl)-4,6-dimethoxy-5-nitro-pyrimidine. (26)
[0130] A solution of compound (25) (1.2 g, 5.5 mmol),
4-fluorobenzylzinc chloride (13.1 mL, 0.5 M in THF, 6.6 mmol),
tetrakis(triphenylphosphine)palladium (0.13 g, 0.1 mmol) in THF (50
mL) was heated to refluxed for 3 hours under nitrogen. After it
cooled to room temperature, the THF was removed in vacuo. The
residue was diluted with ethyl acetate, washed with water and
brine, dried over Na.sub.2SO.sub.4. After concentrated, the residue
was purified by a column chromatography on silica gel eluting with
5% ethyl acetate in hexane to yield
2-(4-fluoro-benzyl)-4,6-dimethoxy-5-nitro-pyrimidine (26) (1.1 g,
63%). 1HNMR (CDCl.sub.3)
2-(4-Fluoro-benzyl)-5-nitro-pyrimidine-4,6-diol. (27)
[0131] A mixture of compound (26) (1.0 g, 3.4 mmol) and pyridine
hydrochloride (3.9 g, 34 mmol) was heated to 140 C for 1 hour.
After it cooled to room temperature, the resultant was diluted with
water. The product was collected on a filter and dried in vacuo.
2-(4-Fluoro-benzyl)-5-nitro-pyrimidine-4,6-diol (27) was obtained
in a yield of 62% (1.1 g). 1HNMR (DMSO-d.sub.6)
4,6-Dichloro-2-(4-fluoro-benzyl)-5-nitro-pyrimidine. (28)
[0132] To a mixture of compound (27) (0.7 g, 2.6 mmol) in
phosphorus oxylchloride (3 mL) was added N,N-dimethylaniline (0.42
g, 3.4 mmol), and heated refluxed for 2 hours. After it cooled to
room temperature, the reaction resultant was poured into ice-water
and stirred for 5 minutes. The mixture was extracted with ethyl
acetate, washed brine, dried over Na.sub.2SO.sub.4, concentrated to
yield (28) (0.5 g, 62%). 1HNMR (CDCl.sub.3)
4-Benzo[1,3]dioxol-5-yl-6-chloro-2-(4-fluorobenzyl)-5-nitro-pyrimidine.
(29)
[0133] A solution of compound (28) (0.1 g, 3.3 mmol),
3,4-methylenedioxyphenyl-boronic acid (49 mg, 0.29 mmol), sodium
carbonate (0.11 g, 0.1 mmol) and
tetrakis(triphenyl-phosphine)palladium (0.13 g, 0.1 mmol) in
toluene/water (1:1, 4 mL) was heated to refluxed for 3 hours under
nitrogen. After it cooled to room temperature, the THF was removed
in vacuo. The residue was diluted with ethyl acetate, washed with
water and brine, dried over Na.sub.2SO.sub.4. After concentrated,
the residue was purified by a column chromatography on silica gel
eluting with 5% ethyl acetate in hexane to yield (29) (0.12 g,
90%). 1 HNMR (CDCl.sub.3)
4-Benzo[1,3]dioxol-5-yl-6-chloro-2-(4-fluoro-benzyl)-pyrimidin-5-ylamine.
(30)
[0134] A mixture of compound (29) (0.12 g, 0.31 mmol) in ethanol (5
mL) was added Raney nickel (0.05 g), and hydrogenated under 50 Psi
for 2 hours. After the catalyst was filtered off, the solution was
concentrated to yield (30) (0.1 g, 90%). 1HNMR (CDCl.sub.3)
7-Benzo[1,3]dioxol-5-yl-5-(4-fluorobenzyl)-thiazolo[5,4-d]pyrimidin-2-ylam-
ine. BA-11
[0135] To a solution of compound (30) (0.1 g, 0.28 mmol) in acetic
acid (1 mL) was added potassium thiocynate (27 mg, 0.29 mmol). The
mixture was heated at 60.degree. C. for 16 hours. After it cooled
to room temperature, the reaction mixture was diluted with water,
extracted with ethyl acetate, washed with water and brine, dried
over Na.sub.2SO.sub.4. After concentrated, the residue was purified
by a column chromatography on silica gel eluting with 20% ethyl
acetate in hexane to yield BA-11 (33 mg, 30%). 1HNMR
(DMSO-d.sub.6). LC/MS: 99%
[0136] BA-13
##STR00033##
(4-bromo-6-cyano-benzothiazol-2-yl)-carbamic Acid Tert-butyl Ester.
(11)
[0137] See BA-03.
[4-(3-Bromo-phenyl)-6-cyano-benzothiazol-2-yl]-carbamic Acid
tert-butyl Ester. (31)
[0138] A reaction mixture of compound (11) (1.23 g, 3 mmole, 3
eq.), the boronic acid (200 mg, 1 mmol, 1 eq.), triphenylphosphine
(80 mg, 0.3 mmol, 0.3 eq.), Pd(OAc).sub.2 (22 mg, 0.1 mmol, 0.1
eq.) in dioxane (20 mL), and (aqueous) 1N Na.sub.2CO.sub.3 (6 mL, 6
eq.) was stirred under reflux overnight under Ar. Only very small
amount of less polar product was formed. 0.2 eq of additional of
Pd(OAc)2 (44 mg) and 0.6 eq. of Ph.sub.3P (160 mg) were added. The
resulting mixture was stirred at 85.degree. C. for 6 hours. After
cooling to room temperature, The volatile material was removed
under reduced pressure to give a residue, which was purified by
chromatography on silica gel using DCM/hexane (3:1) as eluent to
afford the product (31) (262 mg, 61% yield) along with (11) (560
mg). .sup.1H-NMR (400 MHz, CDCl.sub.3).
[4-(3-Bromo-phenyl)-6-formyl-benzothiazol-2-yl]-carbamic Acid
tert-butyl Ester (32)
[0139] To a solution of compound (31) (242 mg, 0.56 mmol, 1 eq.) in
dichloromethane (10 mL) was added DIBAL-H (2 mL, 1N in hexane, 3
eq.) dropwise by a syringe at -78.degree. C. After the addition was
complete, the reaction mixture was stirred at room temperature over
night. The reaction mixture was poured into 1 N HCl (5 ml) with
stirring at 0.degree. C. The mixture was extracted with DCM. DCM
layer was separated and dried over MgSO.sub.4. Removal of solvent
gave a residue, which was purified by chromatography on silica gel
using DCM/hex as eluent to give desired product (32) (54.8 mg, 23%
yield). 1H-NMR (400 MHz, DMSO-d6). MS (ESI+): 301.7 (M+1)
[4-(3-Bromo-phenyl)-6-[(4-fluoro-phenyl)-hydroxy-methyl]-benzothiazol-2-yl-
]-carbamic Acid tert-butyl Ester. (33)
[0140] To a solution of the aldehyde (32) (55 mg, 0.13 mmol, 1 eq.)
in anhydrous THF (4 mL) was added dropwise a solution of
4-fluorophenyl magnesium bromide in THF (0.8 ml, 1N, 3 eq.) at
-78.degree. C. After the addition was complete, the resulting
mixture was allowed to warm to room temperature over night. Then
saturated NH.sub.4Cl aq. was added. The mixture was extracted with
EtOAc (3.times.5 ml). The combined organic layers were dried over
Na.sub.2SO.sub.4. Removal of solvent gave a residue, which was
purified by chromatography on silica gel using DCM/MeOH (200:1) to
give the desired product (33) (24 mg, 35% yield).
4-(3-bromophenyl)-6-(4-fluorobenzyl)-1,3-benzothiazol-2-amine.
BA-13
[0141] Compound (33) (24 mg, 0.045 mmol) was treated with
Et.sub.3SiH (0.8 mL, 3 mmol, 100 eq.) in TFA (0.8 ml). The mixture
was stirred at room temperature for 130 min. until starting
material XM-45 was consumed. The volatile material was removed
under reduced pressure. The residue was washed with sodium
bicarbonate solution, extracted with EtOAc (3.times.15 mL), dried.
Evaporation gave a residue, which was purified by chromatography on
prep. TLC plate using dichloromethane/hexanes (1:1) as developing
system to give 20 mg of XM-46, which was passed through a plug of
silica gel using DCM/hexane (1:1) followed by dichloromethane as
eluent to give BA-13 (12 mg, 65% yield). 1H NMR. LCMS: 99%.
[0142] BA-15
##STR00034## ##STR00035##
2-Amino-5-methoxy-benzothiazole-6-carboxylic Acid Methyl Ester.
(35)
[0143] To a suspension of methyl-4-amino-2-methoxybenzoate (3 g,
16.55 mmole), potassium thiocyanate (6.45 g, 66.23 mmole) in 30 ml
acetic acid was added bromine at room temperature (2.65 g, 16.55
mmole). During the bromine addition suspension became solution, and
then a precipitate formed. The reaction mixture was stirred at room
temperature, overnight. A sample (1 ml) was taken and concentrated,
submitted for H NMR. This showed the intermediate (34). The
suspension was then heated at 60.degree. C., overnight. The
suspension was concentrated, the solid was taken into a mixture of
50% saturated NaHCO3 sol./water (50 ml) and stirred for 30 minutes.
The suspension was filtered, the solid was washed with water and
dried to afford (35) 3.2 g (82% yield). .sup.1H-NMR-(400 MHz,
DMSO)
2-Amino-4-bromo-5-methoxy-benzothiazole-6-carboxylic Acid Methyl
Ester. (36)
[0144] A suspension of (35) (1.1 g, 4.62 mmole) in 15 ml acetic
acid was heated at 70.degree. C., when a solution of bromine (1.3
g, 8.08 mmole) in 5 ml acetic acid was added. The suspension became
solution during bromine addition, then a solid precipitated out.
The reaction mixture was heated at 70.degree. C. for 4 hours. The
mixture was concentrated to dryness, the residue was taken into 50
ml mixture of 1:1=saturated NaHCO3/water, stirred for one hour, the
suspension was filtered, solid was washed with water, dried to give
1.4 g product (36). .sup.1H-NMR-(400 MHz, DMSO)
4-Bromo-2-tert-butoxycarbonylamino-5-methoxy-benzothiazole-6-carboxylic
Acid Methyl Ester. (37)
[0145] To a suspension of (36) (1.3 g, 4.1 mmole), DMAP (500 mg,
4.1 mmloe) in 40 ml methylene chloride, was added at room
temperature (Boc).sub.2O (1.075 g, 4.92 mmole). The reaction
mixture was stirred at rt overnight. Mixture was concentrated down,
diluted with 100 ml ethyl acetate and washed with water, 10% HCl
solution, water and brine, dried over sodium sulfate, filtered,
concentrated to give 1.5 g brown solid. Trituration with a mixture
of 5% methanol/ether afforded. 1.3 g (37). .sup.1H-NMR-(400 MHz,
DMSO)
2-tert-Butoxycarbonylamino-5-methoxy-4-(3-nitro-phenyl)-benzothiazole-6-ca-
rboxylic Acid Methyl Ester. (38)
[0146] A suspension of (37) (1.3 g, 3.11 mmole), sodium carbonate
(990 mg, 9.33 mmole), 3-nitrophenyl boronic acid (778 mg, 4.665
mmole) in 50 ml dry dioxane was degassed for 10 min using argon,
then palladium acetate (140 mg, 0.622 mmole) and triphenyl
phosphine (490 mg, 1.86 mmole) were added and the mixture was
heated at 95.degree. C. for 24 hours. The reaction mixture was
cooled to room temperature, diluted with 50 ml water and extracted
with ethylacetate (3.times.60 ml). Combined organic layers were
washed with water, brine, dried over sodium sulfate, filtered and
concentrated. The residue was purified by column chromatography
using 10% t0 15% ethyl acetate in hexane to obtain 330 mg product
(38) and 452 mg unreacted (37). .sup.1H-NMR-(400 MHz,
CDCl.sub.3)
[6-Hydroxymethyl-5-methoxy-4-(3-nitro-phenyl)-benzothiazol-2-yl]-carbamic
Acid tert-butyl Ester. (39)
[0147] To a solution of (38) (326 mg, 0.71 mmole) in 8 ml dry THF
was added at 0.degree. C. 1.1 ml lithium borohydride (2M solution
in THF). The reaction mixture was stirred at room temperature
overnight. The reaction mixture was cooled to 0.degree. C. and
quenched with 0.5% HCl solution to a pH=6, then extracted with
ethyl acetate (3.times.20 ml). Combined organic layers were washed
with water, brine, dried over sodium sulfate, filtrated and
concentrated to give the crude, which was purified by column
chromatography using 20% to 30% ethylacetate/hexane. 180 mg (39)
was obtained. .sup.1H-NMR-(400 MHz, CDCl.sub.3)
[6-(Diethoxy-phosphoryloxymethyl)-5-methoxy-4-(3-nitro-phenyl)-benzothiazo-
l-2-yl]-carbamic Acid tert-butyl Ester. (40)
[0148] To a solution of (39) (140 mg, 0.325 mmole) in 2 ml dry THF
were added triethyl amine (50 mg, 0.48 mmole), DMAP (4 mg, 0.01
eq.) and diethylchlorophosphate (58 mg, 0.325 mmole) The resulting
suspension was stirred at room temperature, overnight. Mixture was
concentrated down, diluted with 5 ml water, then 5% HCl solution
was added to pH=6 and extracted with ethyl acetate 3.times.5 ml.
Combined organic layers were washed with brine, dried over sodium
sulfate, filtered and concentrated. The crude was purified using
preparative TLC and 10% acetone/methylene chloride to afford 110 mg
product, (40). .sup.1H-NMR-(400 MHz, CDCl.sub.3)
[6-(4-tert-Butoxycarbonylamino-benzyl)-5-methoxy-4-(3-nitro-phenyl)-benzot-
hiazol-2-yl]-carbamic Acid tert-butyl Ester. (41)
[0149] A suspension of (40) (110 mg, 0.194 mmole), potassium
phosphate (46 mg, 0.194 mmole), boronic acid (51 mg, 0.213 mmole)
in 1.5 ml dry toluene was degassed for 10 min using argon, then
palladium acetate (4.5 mg, 0.1 eq.) and triphenyl phosphine (20 mg,
0.2 eq.) were added and the mixture was heated at 90.degree. C. for
7 hours. The reaction mixture was cooled to room temperature,
diluted with 2 ml water and extracted with ethylacetate (3.times.5
ml). Combined organic layers were washed with water, brine, dried
over sodium sulfate, filtered and concentrated. The residue was
purified by prep TLC using 4% MeOH/methylene chloride to obtain 55
mg product (41). .sup.1H-NMR-(400 MHz, CDCl.sub.3)
2-Amino-6-(4-amino-benzyl)-4-(3-nitro-phenyl)-benzothiazol-5-ol.
BA-015
[0150] To a solution of (41) (50 mg, 0.082 mmole) in 1 ml dry
methylene chloride was added at 0.degree. C. BBr3, 1M solution in
methylene chloride (0.2 ml, 2 eq.). The reaction mixture was let it
stirred at RT for 24 hrs. Mixture was concentrated, diluted with 5
ml MeOH, concentrated, then 2 ml of water was added to the residue,
neutralized with 30% ammonium hydroxide, extracted with
ethylacetate (3.times.5 ml). Combined organic layers were washed
with water, dried over sodium sulfate, concentrated to give 40 mg
crude. Purification by preparative TLC gave 13 mg of the desired
product, BA-15. .sup.1H-NMR-(400 MHz, CDCl.sub.3). LCMS (APCI+):
393 (M+1), 85%.
[0151] BA-16
##STR00036## ##STR00037##
5-fluoro-6-methyl-benzothiazol-2-ylamine. (42)
[0152] To a stirred solution of 3-fluoro-4-methyl-phenylamine (10.0
g, 79.9 mmol) and potassium thiocyanide (31.06 g, 319.62 mmol) in
acetic acid (130 mL) was added a solution of bromine (12.77 g, 79.9
mmol) in acetic acid (20 mL) over 20 min. The reaction mixture was
stirred at room temperature for 20 h, poured on to crushed
ice-water (800 mL). Ammonium hydroxide solution (28%) was added to
pH 8, stirred for 2 h. Filtered, washed with water, dried to afford
(42) 14.27 g (98%) as light yellow solid.
4-bromo-5-fluoro-6-methyl-benzothiazol-2-ylamine. (43)
[0153] To a heated (80.degree. C.) and stirred solution of (42)
(10.0 g, 54.88 mmol) in acetic acid (210 mL) was added a solution
of bromine (17.54 g, 109.76 mmol) in acetic acid (40 mL) over 30
min. The reaction mixture was stirred at 80.degree. C. for 20 h,
cooled to room temperature than poured on to crushed ice-water (400
mL). Ammonium hydroxide solution (28%) was added to pH 8, stirred
for 2 h. Filtered, washed with water, dried to afford (43) 12.08 g
(84%) as light orange-brown solid.
(4-bromo-5-fluoro-6-methyl-benzothiazol-2-yl)-carbamic Acid
tert-butyl Ester. (44)
[0154] To a stirred solution of (43) (3.0 g, .+-.1.49 mmol) and
di-tert-butyl dicarbonate (2.5 g, 11.49 mmol) in dichloromethane
(350 mL) was added DMAP (0.2 g, 1.64 mmol). The reaction was
stirred at room temperature for 20 h, than concentrated. The
residue was purified by silica gel column chromatography using 1:1
dichloromethane in hexanes to afford (44) 3.45 g (73%) as off-white
solid.
[4-(3-chloro-phenyl)-5-fluoro-6-methyl-benzothiazol-2-yl]-carbamic
Acid tert-butyl Ester. (45)
[0155] To a mixture of (44) (3.3 g, 9.14 mmol),
3-chlorophenylboronic acid (2) (2.14 g, 13.7 mmol), PPh.sub.3 (1.17
g, 4.48 mmol), K.sub.2CO.sub.3 (0.49 g, 3.56 mmol) and
Pd(OAc).sub.2 (0.25 g, 1.1 mmol) was added dioxane (90 mL), and
EtOH--H.sub.2O (1:1, 45 mL). Ar gas was bubbled through the stirred
reaction for 15 min. The reaction was stirred at 80.degree. C.
under Ar for 20 h. The reaction was cooled to room temperature,
concentrated and H.sub.2O (60 mL) and dichloromethane (80 mL) were
added. The layers were separated and the aqueous was extracted with
dichloromethane (2.times.40 mL). The combined organic extracts were
dried with Na.sub.2SO.sub.4, filtered, and concentrated. The
residue was purified by silica gel column chromatography using 1:1
dichloromethane-hexanes to afford (45) 2.8 g (78%) as off-white
solid.
[6-bromomethyl-4-(3-chloro-phenyl)-5-fluoro-benzothiazol-2-yl]-carbamic
Acid tert-butyl Ester. (46)
[0156] To a mixture of (45) (1.0 g, 2.54 mmol) and NBS (0.5 g, 2.8
mmol) in CCl.sub.4 (50 .mu.mL) was added benzoylperoxide (0.1 g,
0.41 mmol). The reaction was stirred at 80.degree. C. under N.sub.2
for 18 h. The reaction was cooled to room temperature and
concentrated. The residue was dissolved in mixture of
dichloromethane and hexanes (1:1, 8 mL) and purified by silica gel
column chromatography using 1:1 dichloromethane hexanes to afford
(46) 0.63 g (53%) as a light brown solid.
[4-(3-chloro-phenyl)-5-fluoro-6-(4-nitro-benzyl)-benzothiazol-2-yl]-carbam-
ic Acid tert-butyl Ester. (47)
[0157] To a mixture of (46) (0.3 g, 0.64 mmol) and
tributyl-(4-nitro-phenyl)-stannane (3) (0.39 g, 0.95 mmol) in
dioxane (10 mL) was added bis-triphenylphosphine palladium
dichloride (0.02 g, 0.03 mmol). Ar gas was bubbled through the
stirred reaction for 2 min. The reaction was stirred at 80.degree.
C. under Ar for 10 h. The reaction was cooled to room temperature,
concentrated. The residue was purified by silica gel column
chromatography using 1:1 dichloromethane-hexanes to afford (47)
0.21 g (64%) as light yellow solid.
6-(4-amino-benzyl)-4-(3-chloro-phenyl)-5-fluoro-benzothiazol-2-ylamine
Hydrochloride
[0158] BA-16.
[0159] To a mixture of (47) (0.19 g, 0.36 mmol), Fe (0.08 g, 1.44
mmol) and ammonium chloride (0.12 g, 2.16 mmol) in ethanol (10 mL)
was added water (3.3 mL). The reaction was stirred at 80.degree. C.
for 16 h. The reaction mixture was cooled to room temperature,
filtered over Celite, concentrated. The residue was purified by
prep TLC using 40% ethyl acetate in hexanes afforded 0.48 g (28%)
of (48) as off-white solid (less polar compound Rf 0.45) and 0.085
g (62%) of BA-16 as off-white solid (polar compound Rf 0.21). 1H
NMR-(400 MHz, CDCl3); Yes MS (APCI+): 384.0 (M+1), LC-MS: >99%.
To BA-16 (0.02 g, 0.05 mmol) in ether (1.0 mL) was added 2M HCl in
ether (0.5 mL). The reaction mixture was stirred for 1 h. The ether
layer was decanted, triturated with ether (2.times.2 mL), dried to
afford 0.017 g (78%) BA-16, HCl salt, as light yellow solid.
.sup.1H NMR-(400 MHz, CDCl.sub.3); Yes, MS (APCI+): 384.0 (M+1),
LC-MS: 79%.
[0160] BA-18
##STR00038## ##STR00039##
[4-(3-Chloro-phenyl)-6-formyl-benzothiazol-2-yl]-carbamic Acid
tert-butyl Ester. (49)
[0161] To a mixture of boronic acid (800 mg, 5 mmol, 1.1 eq.),
palladium catalyst (500 mg, 0.43 mmol, 0.1 eq.), K.sub.2CO.sub.3
(4.7 g, 35 mmol, 8 eq.) and (12) (1.54 g, 4.3 mmol, 1 eq.) was
added toluene (30 ml) and EtOH (30 ml). The mixture was stirred at
95 C under Ar overnight. After cooling to room temperature, water
was added. The mixture was extracted with EtOAc (25 ml.times.3).
The organic layers were dried over MgSO.sub.4. Removal of solvent
under reduced pressure gave a residue, which was purified by
chromatography on silica gel using hexane/DCM (from 1.5:1 to 1:2)
as eluent to afford (49) 960 mg in 57% yield. .sup.1H-NMR (400 MHz,
CDCl.sub.3)
[4-(3-Chloro-phenyl)-6-hydroxymethyl-benzothiazol-2-yl]-carbamic
Acid tert-butyl Ester. (50)
[0162] To a solution of compound (49) (540 mg, 1.38 mmol) in
dichloromethane (8 mL) and MeOH (6 ml) was added sodium borohydride
(80 mg, 2.1 mmol, 1.5 eq.) in one portion at 0 C. The mixture was
stirred at 0 C.about.rt for 1 hr. Then water was added followed by
1N HCl aq. to bring the mixture to pH=3. Solid was filtered and
washed with water. After dried in the air, 475 mg of product (50)
was obtained in 88% yield. 1H-NMR (400 MHz).
[6-Bromomethyl-4-(3-chloro-phenyl)-benzothiazol-2-yl]-carbamic Acid
tert-butyl Ester. (51)
[0163] To a suspension of alcohol (50) (360 mg, 0.92 mmol, 1 eq.)
in DCM (12 mL) was added Ph.sub.3P (340 mg, 1.3 mmol, 1.4 eq.). The
mixture was stirred at rt for 5 min. and then cooled to 0 C. NBS
(230 mg, 1.3 mmol, 1.4 eq.) was added in one portion. The resulting
mixture was stirred at rt for 1 hr. The volatile material was
removed under reduced pressure and the residue was purified by
chromatography on silica gel using DCM/hexane (1:2) as eluent to
give product (51) 305 mg in 73% yield.
[4-(3-Chloro-phenyl)-6-(4-nitro-benzyl)-benzothiazol-2-yl]-carbamic
Acid tert-butyl Ester. (52)
[0164] To a mixture of compound (51) (200 mg, 0.44 mmol),
tributyl-(4-nitro-phenyl)-stannane (363 mg, 0.88 mmol, 2 eq.), and
palladium catalyst (Pd(Ph3P)2Cl2, 70 mg, 0.1 mmol, 0.2 eq.) was
added dioxane (8 ml). The resulting mixture was refluxed under Ar
for 4 hrs. After cooling to rt, the volatile material was removed
under reduced pressure to afford a residue, which was purified by
chromatography on silica gel using DCM/hexane (1:1.5) as eluent to
give 82 mg of compound (52) in 38% yield.
[6-(4-Amino-benzyl)-4-(3-chloro-phenyl)-benzothiazol-2-yl]-carbamic
Acid tert-butyl Ester. (53)
[0165] A mixture of compound (52) (82 mg, 0.165 mmol), Pd/C (105
mg) in EtOAc (5 ml) was stirred under one atmosphere of H.sub.2 for
1 hr. The reaction mixture was passed through a plug of celite,
washed with EtOAc. The organics was evaporated under reduced
pressure to give 76 mg of compound (53) in quantitative yield.
[4-(3-Chloro-phenyl)-6-(4-methanesulfonylamino-benzyl)-benzothiazol-2-yl]--
carbamic Acid tert-butyl Ester. (54)
[0166] To a solution of compound (53) (76 mg, 0.163 mmol, 1 eq.) in
pyridine (1 ml) was added MeSO.sub.2Cl (0.014 ml, 0.17 mmol, 1.05
eq.) by a syringe at 0 C. The resulting mixture was stirred at 0
C.about.rt for 1 hr. Then the mixture was poured into water and
extracted with EtOAc. The EtOAc layer was washed with 1N HCl aq.
and dried over MgSO.sub.4. Removal of solvent gave a residue, which
was purified by chromatography on silica gel using DCM/hexane (1:1)
as eluent to afford 83 mg of (54) in 94% yield.
N-{4-[2-Amino-4-(3-chloro-phenyl)-benzothiazol-6-ylmethyl]-phenyl}-methane-
sulfonamide. BA-18
[0167] To a solution of (54) (84 mg, 0.153 mmol) in DCM (1 ml) was
added TFA (1 ml). After the mixture was stirred at rt for 1 hr, the
volatile material was removed under reduced pressure to give a
residue, which was purified by chromatography on silica gel using
EtOAc/hexane with a ratio of 1:1.5 follow by 1:1 as eluent to give
62 mg of desired product, which was further triturated with a
mixture of DCM (1 ml) and hexane (7 ml) to give 59 mg of product
BA-18 in 87% yield. LCMS: 100%. 1H NMR.
[0168] BA-19
##STR00040##
N-{4-[2-amino-4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-pheny-
l}-methanesulfonamide Hydrochloride. BA-19
[0169] To (48) (0.044 g, 0.09 mmol) in pyridine (1.5 mL) was added
methanesulfonyl chloride (0.31 g, 0.27 mmol). The reaction mixture
was stirred for 1 h, concentrated. Water (20 mL) and ether (20 mL)
were added. The organic layer was separated and the aqueous was
extracted with ether (2.times.20 mL). The combined organic extracts
were dried with Na2SO4, filtered, and concentrated to afford 0.05 g
(98%) of (55) as off-white solid To a solution of (55) (0.05 g,
0.09 mmol) in dichloromethane (1 mL) was added trifluoroacetic acid
(1 mL). The reaction mixture was stirred for 1.5 h, concentrated.
Water (2 mL) and sat. Na.sub.2HCO.sub.3 (3 mL) were added,
extracted with ether (2.times.20 mL). The combined organic extracts
were dried with Na.sub.2SO.sub.4, filtered, and concentrated. The
residue was suspended in ether (1 mL) and dioxane (1 mL), 2M HCl in
ether (0.5 mL) was added. The reaction mixture was stirred for 1 h.
The ether layer was decanted, triturated with ether (2.times.2 mL),
dried to afford 0.03 g (70%) of BA-19 as off-white solid. .sup.1H
NMR-(400 MHz, CDCl.sub.3); MS (APCI+): (M+1) LC-MS: >98%.
[0170] BA-20
##STR00041## ##STR00042##
4-Amino-3-methoxy-benzoic Acid Methyl Ester. (56)
[0171] A solution of 4-amino-3-methoxybenzoic acid (1.5 g, 8.97
mmole) in 15 ml methanol and 4.8 ml sulfuric acid 95-98% was heated
at 70.degree. C. for 24 hrs. Upon completion, the mixture was
concentrated down and basified with 2N NaOH solution to pH=8. A
solid precipitated out. The suspension was filtered off, solid was
washed with water, dried to afford 1.62 g tan solid (56), 100%
yield. .sup.1H-NMR (400 MHz, DMSO)
4-Amino-3-bromo-5-methoxy-benzoic Acid Methyl Ester. (57)
[0172] To a solution of (56) (1.6 g, 8.83 mmole) in 15 ml methanol
was added slowly at room temperature a solution of bromine (0.45
ml, 8.83 mmole) in 4 mL acetic acid. The reaction mixture was
stirred at room temperature for 7 hrs. The reaction mixture was
concentrated down to a solid residue. The residue was taken in 50
mL saturated solution of NaHCO3 and extracted 3.times.100 mL ethyl
acetate. Combined organic layers were washed with water, brine,
dried over Na2SO4, filtered and concentrated to give 2.1 g brown
solid (57), 95.1% yield. .sup.1H-NMR (400 MHz, DMSO)
6-Amino-3'-chloro-5-methoxy-biphenyl-3-carboxylic Acid Methyl
Ester. (58)
[0173] A suspension of (57) (2.14 g, 8.26 mmole),
3-chlorophenylboronic acid (1.95 g, 12.39 mmole), sodium carbonate
(2.63 g, 24.78 mmole) in a mixture of solvents: 70 mL toluene, 12
mL ethanol and 30 mL water was degassed for 10 min before the
catalyst addition. After the catalyst Pd(PPh.sub.3).sub.4 (960 mg,
0.826 mmole) was added, the reaction mixture was refluxed for 7
hrs. The reaction mixture was concentrated down, diluted with water
and extracted with ethylacetate. Combined organic layers were
washed with water, brine, dried over Na2SO4, filtered and
concentrated to give 3.8 g crude. Purification by column
chromatography using a mixture of methylene chloride/hexane=1:1
gave 2.12 g product (58), 88.3% yield. .sup.1H-NMR (400 MHz,
DMSO)
6-Amino-3'-chloro-5-hydroxy-biphenyl-3-carboxylic Acid Methyl
Ester. (59)
[0174] To a solution of (58) (2.12 g, 7.27 mmole) in 75 mL
anhydrous methylene chloride was added at -70.degree. C. neat BBr3
(2.22 mL, 21.81 mmole). The reaction mixture was slowly warmed up
to room temperature and stirred for 5 hrs. Mixture was collected to
0.degree. C. and diluted by slowly addition of 100 mL of methanol.
The mixture was stirred at room temperature, overnight, then
concentrated down, diluted with 50 ml water and neutralized to pH=6
by addition of 30% ammonium hydroxide. The mixture was extracted
with ethylacetate. Combined organic layers were washed with water,
brine, dried over Na.sub.2SO.sub.4, filtered and concentrated to
give 2.1 g crude product (59), quantitative yield, pure enough to
use it next. .sup.1H-NMR (400 MHz, DMSO)
2-Amino-4-(3-chloro-phenyl)-benzooxazole-6-carboxylic Acid Methyl
Ester. (60)
[0175] To a solution of cyanogen bromide (920 mg, 8.7 mmole) in a
mixture of methanol/water=7:3 (12 mL) was added a solution of (59)
(2.1 g, 7.56 mmole) in 12 mL methanol. The reaction mixture was
stirred at room temperature for 24 hrs. More cyanogen bromide (100
mg) was added and mixture stirred for one more day. The mixture was
concentrated down, diluted with 15 mL 2N NaOH solution, stirred for
20 minutes, and filtered off. The solid was washed with water,
trituration with 5% MeOH/ether gave 1.45 g light pink solid (60),
57% yield. .sup.1H-NMR (400 MHz, DMSO)
[2-Amino-4-(3-chloro-phenyl)-benzooxazol-6-yl]-methanol. (61)
[0176] To a solution of (60) (260 mg, 0.86 mmole) in 10 mL
anhydrous THF at 0.degree. C. was added LiBH.sub.4 (2M in THF, 1.75
ml, 4 eq.). The reaction mixture was stirred at room temperature
for 3 days. The reaction was quenched with 5 ml saturated solution
of NH.sub.4Cl, diluted with 5 mL water and extracted with
2.times.20 mL ethylacetate. Combined organic layers were washed
with water, brine, dried over Na.sub.2SO.sub.4, filtered and
concentrated to give 200 mg crude. Trituration with
ether/hexane=1:1 gave 140 mg off white solid (61), 59% yield.
.sup.1H-NMR (400 MHz, DMSO)
Carbonic Acid 2-amino-4-(3-chloro-phenyl)-benzooxazol-6-ylmethyl
Ester Methyl Ester. (62)
[0177] To a solution of (61) (140 mg, 0.51 mmole), pyridine (0.103
ml, 2.5 eq.) in 2 mL anhydrous THF at 0.degree. C. was added
methylchloroformate (102 mg, 1.071 mmole). The reaction mixture was
stirred at room temperature for 24 hrs. The reaction was diluted
with 1.5 mL water and extracted 3.times. with methylene chloride.
Combined organic layers were washed with water, brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated to give 196 mg crude
(62). This was pure enough for next step. .sup.1H-NMR (400 MHz,
DMSO)
N-{4-[2-Amino-4-(3-chloro-phenyl)-benzooxazol-6-ylmethyl]-phenyl}-methanes-
ulfonamide. BA-20
[0178] To a solution of (62) (190 mg, 0.48 mmole), 3-chlorophenyl
boronic acid (157 mg, 0.73 mmole), K.sub.2CO.sub.3 (153 mg, 1.1
mmole), [Pd(n.sup.3-C.sub.3H.sub.5)Cl].sub.2 (9 mg, 0.024 mmole),
1,5-bis(diphenylphosphino)pentane (22 mg, 0.048 mmole) in 1.5 mL
anhydrous DMF was heated at 80.degree. C. for 24 hrs. The reaction
was diluted with 5 mL water and extracted 3.times. with ethyl
acetate. Combined organic layers were washed with water, brine,
dried over Na.sub.2SO.sub.4, filtered and concentrated to give 200
mg crude. Purification by silica gel prep plate gave 10 mg white
solid BA-20. .sup.1H-NMR (400 MHz, DMSO)
[0179] BA-21
##STR00043##
[6-(4-amino-benzyl)-4-(3-chloro-phenyl)-5-fluoro-benzothiazol-2-yl]-carba-
mic Acid tert-butyl Ester. (47)
[0180] To (47) (0.11 g, 0.21 mmol) in ethyl acetate (3.0 mL) was
added Pd--C (0.16 g, 10%, 50% wet). The reaction mixture was
stirred under H.sub.2 atmosphere for 5 h, filtered over Celite,
concentrated to afford 0.09 g (95%) of (48) as light brown
solid.
{4-[2-amino-4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-phenyl}--
urea Hydrochloride. BA-21
[0181] To (48) (0.1 g, 0.19 mmol) in acetic acid-water (1:2, 1 mL)
was added sodium cyanate (0.05 g, 0.77 mmol). The reaction mixture
was stirred for 72 h at room temperature. Water (4 mL) was added,
basified with 28% NH.sub.4OH solution to pH8, extracted with ethyl
acetate (3.times.6 mL). The combined organic extracts were dried
with Na.sub.2SO.sub.4, filtered, and concentrated. The residue was
triturated with 1:1 dichloromethane-hexanes (2.times.2 mL), than
treated with 4M hydrochloric acid in dioxane at 80.degree. C. for
40 min. The reaction was concentrated, triturated with ether
(2.times.2 mL), dried to afford BA-21 0.03 g (33%) as light
grayish-brown solid. .sup.1H NMR-(400 MHz, CDCl.sub.3). MS (APCI+):
427.0 (M+1), LC-MS: 84%.
[0182] BA-22, and
[0183] BA-24.
##STR00044## ##STR00045##
Methyl 4-(3-chlorophenyl)-2-methyl-1,3-benzoxazole-6-carboxylate.
(63)
[0184] A solution of (59) (150 mg, 0.54 mmole) in 1 mL
triethylorthoformate was heated at 100.degree. C. for 4 hours. The
reaction mixture was cooled to room temperature, diluted with water
and extracted with ethylacetate. Combined organic layers were
washed with brine, dried over Na.sub.2SO.sub.4, concentrated to
give 160 mg crude. After trituration with 50% ether in hexane 130
mg (63) was obtained. .sup.1H-NMR (400 MHz, DMSO)
[4-(3-chlorophenyl)-2-methyl-1,3-benzoxazol-6-yl]methanol. (64)
[0185] To a solution of (63) (125 mg, 0.414 mmole) in 5 mL
anhydrous THF at 0.degree. C. was added LiBH.sub.4 (2M in THF, 0.62
ml, 3 eq.). The reaction mixture was stirred at room temperature
for 3 days. The reaction was quenched with 5N HCl, diluted with
water and extracted with 3.times.10 mL ethylacetate. Combined
organic layers were washed with water, brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated to give 130 mg crude.
Purification by silica gel prep TLC afforded 98 mg (64).
.sup.1H-NMR (400 MHz, DMSO)
6-(bromomethyl)-4-(3-chlorophenyl)-2-methyl-1,3-benzoxazole.
(65)
[0186] To a solution of (64) (90 mg, 0.33 mmole),
triphenylphosphine (87 mg, 0.33 mmole) was added at 0.degree. C.
NBS (59 mg, 0.33 mmole). The reaction mixture was stirred at room
temperature, overnight. The mixture was concentrated and purified
by column chromatography using 50% methylene chloride in hexane to
give 55 mg (65). .sup.1H-NMR (400 MHz, DMSO)
4-(3-chlorophenyl)-2-methyl-6-(4-nitrobenzyl)-1,3-benzoxazole.
(66)
[0187] To a solution of (65) (50 mg, 0.148 mmole), 3-nitrophenyl
tributyl stannane (91 mg, 0.22 mmole), bis(triphenyl
phosphine)dichloride (11 mg, 10%) in 2 mL anhydrous dioxane was
heated at 90.degree. C. for 24 hrs. The reaction was cooled to RT,
diluted with 2 mL methylene chloride and purified by silica gel
prep plate and gave 40 mg (66) yellow solid. .sup.1H-NMR (400 MHz,
DMSO)
4-{[4-(3-chlorophenyl)-2-methyl-1,3-benzoxazol-6-yl]methyl}aniline.
BA-22
[0188] A suspension of (66) (40 mg, 0.102 mmole), 10% Pd/C (12 mg,
10%) in 3 mL ethylacetate was stirred under H.sub.2 (1 atm) for 5
hours. Mixture was diluted with ethylacetate and filtered off.
After solvent evaporation 40 mg crude was obtained. Purification by
silica gel prep TLC afforded 30 mg product BA-22.
[0189] .sup.1H-NMR (400 MHz, DMSO). HPLC 97%
N-(4-{[4-(3-chlorophenyl)-2-methyl-1,3-benzoxazol-6-yl]methyl}phenyl)urea.
BA-24
[0190] A suspension of BA-22 (20 mg, 0.057 mmole), sodium cyanate
(15 mg, 0.23 mmole) in a mixture of acetic acid/water=1:2 (2 mL)
was stirred at room temperature for 3 days. Mixture was
concentrated down, diluted with water and extracted several times
with ethylacetate. Combined organic layers were washed with brine,
dried over Na.sub.2SO.sub.4, filtered and concentrated. Crude was
purified by silica gel prep TLC to afford 11 mg BA-24. .sup.1H-NMR
(400 MHz, DMSO). 2.63 (s, 3H), 4.028 (s, 2H), 5.78 (s, 2H), 7.155
(d, J=8.8 Hz, 2H), 7.29 (d, J=8.8 Hz, 2H), 7.44-7.47 (m, 1H),
7.51-7.55 (m, 3H), 7.93 (d, J=8 Hz, 1H), 8.08 (dd, J=4 Hz, 2 Hz,
8.41 (s, 1H).
[0191] BA-23
##STR00046##
4-(3-chloro-phenyl)-5-fluoro-6-(4-nitro-benzyl)-benzothiazol-2-ylamine.
(67)
[0192] To a solution of (47) (0.05 g, 0.09 mmol) in dichloromethane
(1 mL) was added trifluoroacetic acid (1 mL). The reaction mixture
was stirred for 1.5 h, concentrated. Water (2 mL) was added,
basified with 28% NH.sub.4OH solution to pH8, extracted with
dichloromethane (2.times.3 mL). The combined organic extracts were
dried with Na.sub.2SO.sub.4, filtered, and concentrated to afford
0.035 g (97%) of (67) as yellow solid.
4-(3-chloro-phenyl)-5-fluoro-6-(4-nitro-benzyl)-benzothiazole.
(68)
[0193] To a cooled (-8.degree. C.) solution of (67) (0.035 g, 0.08
mmol) in phosphoric acid (0.8 mL, 85% wt solution) was added a
solution of sodium nitrite (0.035 g, 0.51 mmol) in water (0.1 mL)
over min. The reaction mixture was stirred for 5 min at -4.degree.
C., than hypophosphorous acid (0.6 mL) was added, warmed to room
temperature. Water (5 mL) was added, basified with Na.sub.2CO.sub.3
solution to pH8, extracted with dichloromethane (2.times.20 mL).
The combined organic extracts were dried with Na.sub.2SO.sub.4,
filtered, and concentrated. The residue was purified by prep TLC
using dichloromethane to afford 0.018 g (53%) of (68) as light
yellowish-brown solid.
4-[4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-phenylamine
Hydrochloride. BA-23
[0194] To (68) (0.018 g, 0.045 mmol) in ethyl acetate (2.5 mL) was
added Pd--C (0.08 g, 10%, 50% wet). The reaction mixture was
stirred under H.sub.2 atmosphere for 5 h, filtered over Celite,
concentrated. The residue was dissolved in ether (0.5 mL), than 2M
HCl in ether (0.5 mL) was added. The reaction mixture was stirred
for 1 h. The ether layer was decanted, triturated with ether
(2.times.1 mL), dried to afford 0.007 g (38%) of BA-23 as off-white
solid. .sup.1H NMR-(400 MHz, CDCl.sub.3); MS (APCI+): 369.0 (M+1),
LC-MS: 92%.
[0195] BA-26
[0196] BA-27
##STR00047##
4-(4-Nitro-benzyl)-phenylamine. (69)
[0197] A reaction mixture of 4-nitrobenzyl chloride (2.44 g, 20
mmole, 1 eq.),
4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenylamine (5 g,
22.8 mmol, 1.14 eq.), Pd(PPh.sub.3).sub.4 (2.31 g, 2.2 mmol, 0.1
eq.) K.sub.3PO.sub.4 (8.5 g, 45 mmol, 2 eq.) in DME (100 mL), EtOH
(30 ml) and water (30 ml) was stirred at 65 C for 3 hours. After
cooling to room temperature, the volatile material was removed
under reduced pressure to give a residue, which was partitioned
between EtOAc and water. Organic layer was separated and dried.
Solvent was removed and the crude product was purified by
chromatography on silica gel using DCM/hexane (1:1) as eluent to
afford the product (69) (1.4 g, 30% yield).
6-(4-Nitro-benzyl)-benzothiazol-2-ylamine. (70)
[0198] To a reaction mixture of compound (69) (0.7 g, 3.1 mmole),
KSCN (600 mg, 6.2 mmol, 2 eq.) in acetic acid (10 ml) was added
bromine dropwise by a syringe at 0.degree. C. with stirring. After
the addition was complete, the resulting mixture was stirred at
room temperature until starting material was consumed (monitored by
TLC). The mixture was diluted with water. The solid was collected
by filtration and washed with water and dried under N2 flow to give
the desired product (70) (526 mg, 60% yield). .sup.1H-NMR (400 MHz,
CDCl.sub.3)
4-Bromo-6-(4-nitro-benzyl)-benzothiazol-2-ylamine. (71)
[0199] Compound (70) (906 mg, 3 mmol) was suspended in acetic acid
(10 mL) and bromine was added dropwise by a syringe at room
temperature. After the addition was complete, the reaction mixture
was stirred at room temperature over night. The reaction mixture
was poured into water and the solid was collected by filtration and
dried. 870 mg of (71) was obtained in 79% yield.
4-(3-Chloro-phenyl)-6-(4-nitro-benzyl)-benzothiazol-2-ylamine.
(72)
[0200] A reaction mixture of compound (71) (435 mg, 1.2 mmole), the
boronic acid (230 mg, 1.44 mmol, 1.2 eq.), Pd(PPh.sub.3).sub.4 (140
mg, 0.71 mmol, 0.1 eq.) in dioxane (4 mL), and 2N Na.sub.2CO.sub.3
aqueous solution (2.5 mL, 6 eq.) was stirred under reflux overnight
under Ar. After cooling to room temperature, the volatile material
was removed under reduced pressure to give a residue, which was
partitioned between water (15 mL) and ethyl acetate (15 mL). The
organic layer was separated. The aqueous layer was extracted with
ethyl acetate (10 ml.times.3). The combined organic layers were
dried and evaporation of solvent gave a residue, which was purified
by chromatography on silica gel using DCM/hexane (1:1) as eluent to
afford the product (72) (488 mg, 73% yield).
[4-(3-Chloro-phenyl)-6-(4-nitro-benzyl)-benzothiazol-2-yl]-carbamic
Acid tert-butyl Ester. (73)
[0201] Compound (72) (379 mg, 0.95 mmol) was dissolved in THF (5
mL). (Boc).sub.2O (230 mg, 1.1 mmol, 1.1 eq.) was added followed by
DMAP (12 mg, 0.1 mmol, 0.1 eq:). The mixture was stirred at room
temperature overnight. Then 0.5 additional eq. of (Boc).sub.2O was
added. After the resulting mixture was stirred for 4 hours, the
volatile material was removed under reduced pressure. The residue
was purified by chromatography on silica gel using
dichloromethane/hexanes (1:1) as eluent to give 281 mg of (73),
which contains bis-Boc product.
[6-(4-Amino-benzyl)-4-(3-chloro-phenyl)-benzothiazol-2-yl]-carbamic
Acid tert-butyl Ester. (74)
[0202] Compound (73) containing Bis-Boc protected counterpart (211
mg, 0.95 mmol) was dissolved in ethyl acetate (10 mL). Pd/C (300
mg, 10%, wet) was added and the mixture was stirred under H.sub.2
(1 atm) for 3 h. The catalyst was filtered and washed with ethyl
acetate (10 mL.times.3). Removal of solvent under reduced pressure
gave 200 mg of desired (74) in quantitative yield.
6-(4-Amino-benzyl)-4-(3-chloro-phenyl)-benzothiazol-2-ylamine.
BA-26
[0203] Compound (74) (30 mg, 0.06 mmol) was dissolved in
dichloromethane (1 mL). Trifluoroacetic acid (1 mL) was added. The
mixture was stirred at rt for 1 hr. The volatile material was
removed and the residue was partitioned between ethyl acetate (EA)
and sodium bicarbonate. The EA layer was separated and dried over
MgSO4. Removal of solvent gave a residue, which was treated with 2N
HCl in ether. The precipitate was collected by filtration. 20 mg of
desired product BA-26 was obtained as HCl salt, which is a white
solid (83% yield). LCMS: 97.8%. 1H-NMR (400 MHz, DMSO-d.sub.6)
[4-(3-Chloro-phenyl)-6-(4-ureido-benzyl)-benzothiazol-2-yl]-carbamic
Acid tert-butyl Ester (75)
[0204] A mixture of compound (74) (33 mg, 0.06 mmol), NaOCN (36 mg,
0.13 mmol, 2 eq.) in HOAc (0.2 mL) and water (0.2 mL) was stirred
at rt overnight. Then the mixture was diluted with addition of
water. Solid was collected by filtration and purified by Prep TLC
plate using DCM/MeOH (30:1) as developing system to give 18 mg of
product (75) in 59% yield.
{4-[2-Amino-4-(3-chloro-phenyl)-benzothiazol-6-ylmethyl]-phenyl}-urea.
BA-27
[0205] Compound (75) (18 mg, 0.036 mmol) was dissolved in
dichloromethane (1 mL). Trifluoroacetic acid (1 mL) was added. The
mixture was stirred at rt for 1 hr. The volatile material was
removed and the residue was partitioned between ethyl acetate (EA)
and sodium bicarbonate. The EA layer was separated and dried over
MgSO.sub.4. Removal of solvent gave a residue, which was treated
with 2N HCl in ether. The precipitate was collected by filtration.
20 mg of desired product BA-27 was obtained as HCl salt, which is a
white solid (100% yield). LCMS: 97.6%. 1H-NMR (400 MHz,
DMSO-d6)
[0206] BA-25
[0207] BA-28
##STR00048##
4-(3-Chloro-phenyl)-6-(4-nitro-benzyl)-benzothiazole. (76)
[0208] Compound (72) (109 mg, 0.27 mmol) was dissolved in dioxane
(1 mL). To the solution was added t-butyl nitrite (60 mg, 0.55
mmol, 2 eq.) The mixture was stirred at 60 C for 30 min. After
cooling to room temperature, the volatile material was removed
under reduced pressure. The residue was purified by chromatography
on silica gel using dichloromethane/hexane (1:1 then 1.5:1) as
eluent to give 40 mg of (76) in 39% yield.
4-[4-(3-Chloro-phenyl)-benzothiazol-6-ylmethyl]-phenylamine.
BA-25
[0209] Compound (76) (40 mg, 0.1 mmol) was dissolved in ethyl
acetate (5 mL). Pd/C (100 mg, 10%, wet) was added and the mixture
was stirred under H.sub.2 (1 atm) for 2 h (the reaction was not
complete). The catalyst was filtered and washed with ethyl acetate
(5 mL.times.3). Removal of solvent under reduced pressure gave a
residue, which was purified by chromatography on silica gel using
DCM/Hexane (1:1) as eluent to give 10 mg of desired BA-25 (31%
yield). The product was converted to its HCl salt by treatment with
2N HCl in ether. LCMS: 98%. .sup.1H NMR
{4-[4-(3-Chloro-phenyl)-benzothiazol-6-ylmethyl]-phenyl}-urea.
BA-28
[0210] A mixture of compound BA-25 (8 mg, 0.02 mmol, HCl salt),
NaOCN (10 mg, 0.15 mmol, 7 eq.) in HOAc (0.1 mL) and water (0.1 mL)
was stirred at rt overnight. Then the mixture was diluted with
addition of water. Solid was collected by filtration and purified
by Prep TLC plate using DCM/MeOH (30:1) as developing system to
give 3.6 mg of product BA-28 in 40% yield. 1H-NMR (400 MHz,
DMSO-d.sub.6).
[0211] BA-29
[0212] BA-30
##STR00049##
4-(3-chloro-phenyl)-6-[4-(3-ethyl-ureido)-benzyl]-5-fluoro-benzothiazol-2-
-yl}-carbamic Acid tert-butyl Ester. (77)
[0213] To (48) (0.06 g, 0.13 mmol) in pyridine (1.5 mL) was added
ethylisocyanate (0.1 mL). The reaction mixture was stirred at room
temperature for 72 h, and then concentrated. Water (20 mL) was
added, stirred for 0.5 h, filtered, washed with water, dried. The
crude white solid was purified by prep TLC using 1:1 ethyl acetate
in hexanes to afford 0.057 g (80%) of (77) as off-white solid.
1-{4-[2-Amino-4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-phenyl-
}-3-ethyl-urea. BA-29
[0214] To (77) (0.055 g, 0.1 mmol) in dichloromethane (0.5 mL) was
added trifluoroacetic acid (0.5 mL). The reaction mixture was
stirred for 1.5 h, concentrated. Water (2 mL) was added, basified
with 28% NH.sub.4OH solution to pH8, stirred for 1 h, filtered,
washed with water, dried to afford 0.038 g (85%) of BA-29 as
off-white solid. .sup.1H NMR-(400 MHz, CDCl.sub.3); YesMS (APCI+):
455.1 (M+1), LC-MS: >99%.
1-{4-[4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-phenyl}-3-ethy-
l-urea. BA-30
[0215] To BA-29 (0.016 g, 0.035 mmol) in dioxane (1.5 mL) was added
tert-butyl nitrite (0.007 g. 0.07 mmol). The reaction mixture was
stirred and heated at 60.degree. C. for 1.0 h, concentrated. The
residue was purified by prep TLC using dichloromethane to afford
0.0082 g (53%) of BA-30 as light yellow solid. .sup.1H NMR-(400
MHz, CDCl.sub.3); MS (ESI+): 441.1 (M+1) LC-MS: >99%.
[0216] BA-32
##STR00050##
Methyl
2-[(tert-butoxycarbonyl)amino]-4-(3-chlorophenyl)-1,3-benzoxazole--
6-carboxylate. (78)
[0217] To a suspension of (60) (1.43 g, 4.72 mmole) in 35 mL
methylene chloride were added DMAP (60 mg, 10%) and BOC anhydride
(1.08 g, 4.956 mmole). The reaction mixture was stirred at RT, for
24 hours. TLC shows 40% conversion. More DMAP (500 mg) and BOC
anhydride (250 mg) were added and reaction mixture was stirred for
one more day. Suspension was filtered off, solid was washed with
methylene chloride. 695 mg of starting material was recovered. The
filtrate was concentrated and purified by silica gel column
chromatography using 20% to 50% ethylacetate/hexane to afford 755
mg product (78). .sup.1H-NMR (400 MHz, DMSO)
tert-Butyl
4-(3-chlorophenyl)-6-(hydroxymethyl)-1,3-benzoxazol-2-ylcarbama-
te. (79)
[0218] To a solution of (78) (560 mg, 1.39 mmole) in 15 mL
anhydrous THF at 0.degree. C. was added LiBH.sub.4 (2M in THF, 2
mL, 3 eq.). The reaction mixture was stirred at room temperature
for 3 days. TLC shows 40% conversion. More LiBH.sub.4 (2 mL) was
added and reaction mixture stirred at RT for one more day. The
reaction was quenched with saturated solution of NH.sub.4Cl,
diluted with 5 mL water and extracted with ethylacetate. Combined
organic layers were washed with water, brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated to give 790 mg crude.
Purification by silica gel column chromatography gave 400 mg
product (79) and 110 mg starting material (78). .sup.1H-NMR (400
MHz, DMSO)
[4-(3-Chloro-phenyl)-6-(diethoxy-phosphoryloxymethyl)-benzooxazol-2-yl]-ca-
rbamic Acid tert-butyl Ester. (80)
[0219] To a solution of (79) (100 mg, 0.266 mmole), TEA (0.055 ml,
1.5 eq.) and DMAP (4 mg, 10%) in 1 mL anhydrous THF at 0.degree. C.
was added diethylchlorophosphate (46 mg, 0.266 mmole). The reaction
mixture was stirred at room temperature for 3 days. The reaction
was diluted with 5% HCl and extracted 3.times. with ethyl acetate.
Combined organic layers were washed with water, brine, dried over
Na2SO4, filtered and concentrated to give 200 mg crude (80). This
was used for next step. .sup.1H-NMR (400 MHz, DMSO)
[6-(4-tert-Butoxycarbonylamino-benzyl)-4-(3-chloro-phenyl)-benzooxazol-2-y-
l]-carbamic Acid tert-butyl Ester. (81)
[0220] A suspension of (80) (200 mg, 0.39 mmole), potassium
phosphate (91 mg, 0.429 mmole), boronic acid (102 mg, 0.429 mmole)
in 4 mL dry toluene was degassed for 10 min using argon, then
palladium acetate (5 mg, 5% eq.) and triphenyl phosphine (21 mg,
20% eq.) were added and the mixture was heated at 90.degree. C. for
5 hours. The reaction mixture was cooled to room temperature,
diluted with water and extracted with ethylacetate. Combined
organic layers were washed with water, brine, dried over sodium
sulfate, filtered and concentrated. The residue was purified by
prep TLC using 5% MeOH/methylene chloride to obtain 120 mg product
(81). .sup.1H-NMR (400 MHz, CDCl.sub.3)
6-(4-aminobenzyl)-4-(3-chlorophenyl)-1,3-benzoxazol-2-amine.
BA-32
[0221] To a solution of (81) (100 mg, 0.22 mmole) in 1.5 mL
methylene chloride was added 0.55 mL HCl 4N in dioxane. Solution
became suspension and this was stirred at RT overnight. The mixture
was diluted with diethylether and filtered off. The solid was
triturated with ether and after filtration gave 35 mg BA-32 as HCl
salt. .sup.1H-NMR (400 MHz, DMSO). LCMS (APCI+): 350 (M+1),
87%.
[0222] BA-33
##STR00051##
4-(3-chloro-phenyl)-5-fluoro-6-methyl-benzothiazol-2-ylamine.
(82)
[0223] To (43) (0.4 g, 1.53 mmol), 3-chlorophenylboronic acid (1)
(0.29 g, 1.84 mmol), PPh.sub.3 (0.2 g, 0.79 mmol), K.sub.2CO.sub.3
(0.08 g, 0.6 mmol) and Pd(OAc).sub.2 (0.04 g, 0.181 mmol) was added
dioxane (8 mL), and EtOH--H.sub.2O (1:1, 4 mL). Ar gas was bubbled
through the stirred reaction for 15 min. The reaction was stirred
at 180.degree. C. for 15 minutes using microwave oven (Biotage
Intiator II). Three more runs (0.5 g scale MR-49, total 1.9 g) were
done under similar conditions. The reaction mixtures from all four
runs were concentrated. Water (80 mL) and dichloromethane (80 mL)
were added. The organic layer was separated and the aqueous layer
was extracted with dichloromethane (2.times.40 mL). The combined
organic extracts were dried with Na.sub.2SO.sub.4, filtered, and
concentrated. The residue was purified by silica gel column
chromatography using 30% ethyl acetate-hexanes to afford 1.18 g
(55%) of (82) as a viscous liquid.
4-(3-chloro-phenyl)-5-fluoro-6-methyl Benzothiazole (83)
[0224] To (82) (0.986 g, 3.35 mmol) in dioxane (40 mL) was added
tert-butyl nitrite (0.52 g. 5.02 mmol). The reaction mixture was
stirred and heated at 60.degree. C. for 1.0 h, concentrated. The
residue was purified by silica gel column chromatography using 1:1
dichloromethane-hexanes to afford 0.57 g (61%) of (83) as light
orange solid.
6-bromomethyl-4-(3-chloro-phenyl)-5-fluoro-benzothiazole. (84)
[0225] To (83) (0.57 g, 2.05 mmol) and NBS (0.37 g, 2.05 mmol) in
CCl.sub.4 (30 mL) was added benzoylperoxide (0.01 g, 0.04 mmol).
The reaction was stirred at 80.degree. C. under N.sub.2 for 18 h.
The reaction was cooled to room temperature and concentrated. The
residue was dissolved in mixture of dichloromethane and hexanes
(1:1, 8 mL) and purified by silica gel column chromatography using
10% ethyl acetate-hexanes to afford 0.44 g (49%) of (84) as a light
yellow solid.
1-[5-[4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-pyridin-2-yl]--
3-ethyl-urea Hydrochloride. BA-33
Step-1. 5-tributylstannanyl-pyridin-2-yl)-urea. (85)
[0226] To 5-iodo-pyridin-2-ylamine (0.5 g, 2.27 mmol) in pyridine
(2.5 mL) was added ethylisocyanate (0.24 g, 3.4 mmol)). The
reaction mixture was stirred at room temperature for 72 h,
concentrated. Water (40 mL) was added, stirred for 0.5 h, filtered,
washed with water, dried to afford 0.61 g (92%) of
1-ethyl-3-(5-iodo-pyridin-2-yl)-urea, MR-66, as white solid. To
MR-66 (0.5 g, 1.72 mmol) and bis-tributyltin (5.6 g, 9.64 mmol) in
dioxane (25 mL) was added bis-triphenylphosphine palladium
dichloride (0.28 g, 0.4 mmol). Ar gas was bubbled through the
stirred reaction for 5 min. The reaction was stirred at 90.degree.
C. under Ar for 20 h. The reaction was cooled to room temperature,
concentrated. The residue was purified by silica gel column
chromatography using 30% ethyl acetate in hexanes to afford 0.41 g
(53%) of (85) as viscous liquid.
Step-2. BA-33
[0227] To (84) (0.065 g, 0.18 mmol) and (85) (0.08 g, 0.22 mmol) in
dimethoxyethane (2 mL) was added bis-triphenylphosphine palladium
dichloride (0.006 g, 0.009 mmol). Ar gas was bubbled through the
stirred reaction for 2 min. The reaction was stirred at 120.degree.
C. for 15 minutes using microwave oven (Biotage Intiator II). The
reaction was cooled to room temperature, concentrated. The residue
was purified by silica gel column chromatography using 30% ethyl
acetate in hexanes dichloromethane-hexanes afforded 0.042 g of
light orange solid. The solid was suspended in ether (1.5 mL), than
2M HCl in ether (0.7 mL) was added. The reaction mixture was
stirred for 1 h. The ether layer was decanted, triturated with
ether (2.times.1 mL), dried to afford 0.022 g (25%) of BA-33 as
yellow solid. .sup.1H NMR-(400 MHz, CDCl.sub.3); MS (APCI+): 441.00
(M+1), LC-MS: 97%.
[0228] BA-38
##STR00052##
4-(3-chloro-phenyl)-5-fluoro-6-(6-methoxy-pyridin-3-ylmethyl)-benzothiazo-
le Hydrochloride. (BA-38)
[0229] To (84) (0.06 g, 0.17 mmol), 2-methoxy-5-pyridineboronic
acid (1) (0.04 g, 0.25 mmol), (PPh.sub.3).sub.4Pd (0.02 g, 0.017
mmol) and K.sub.2PO.sub.4 (0.07 g, 0.034 mmol) was added DME (1.5
mL), and EtOH--H.sub.2O (1:1, 0.5 mL). Ar gas was bubbled through
the stirred reaction for 5 min. The reaction was stirred at
150.degree. C. for 15 minutes using microwave oven (Biotage
Initiator II). The reaction was cooled to room temperature,
concentrated. The residue was purified by silica gel column
chromatography using dichloromethane to afford 0.036 g (56%) of the
target compound (BA-38, free-base) as viscous liquid. To the
free-base (0.034 g, 0.088 mmol) in ether (2.0 mL) was added 2M HCl
in ether (0.5 mL). The reaction mixture was stirred for 1 h. The
ether layer was decanted, triturated with ether (2.times.2 mL),
dried to afford 0.035 g (94%) of BA-38, HCl salt, as off-white
solid. .sup.1H NMR-(400 MHz, CDCl.sub.3); MS (APCI+): 385.0 (M+1),
LC-MS: 96%.
[0230] BA-39
##STR00053##
3-[4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-phenol.
BA-39
[0231] To (84) (0.08 g, 0.22 mmol), 3-hydroxyphenylboronic acid (1)
(0.046 g, 0.34 mmol), (PPh.sub.3).sub.4Pd (0.026 g, 0.02 mmol) and
K.sub.2PO.sub.4 (0.095 g, 0.045 mmol) was added DME (3 mL), and
EtOH--H.sub.2O (1:1, 1.0 mL). Ar gas was bubbled through the
stirred reaction for 5 min. The reaction was stirred at 160.degree.
C. for 20 minutes using microwave oven (Biotage Intiator II). The
reaction was cooled to room temperature, concentrated. The residue
was purified by silica gel column chromatography using
dichloromethane to afford 0.056 g (68%) of BA-39 as viscous liquid.
.sup.1H NMR-(400 MHz, CDCl.sub.3); MS (APCI+): 371.1 (M+1), LC-MS:
>99%.
[0232] BA-40
##STR00054##
{5-[4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-pyridin-2-yl}-c-
arbamic Acid Ethyl Ester, BA-40
[0233] Step-1 (86). To
5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyridin-2-ylamine
(0.2 g, 0.91 mmol) in pyridine (2.5 mL) was added ethyl
chloroformate (0.15 g, 1.36 mmol)). The reaction mixture was
stirred at room temperature for 18 b, concentrated. Water (1.0 mL)
and saturated NaHCO.sub.3 solution (1.0 mL) was added, stirred for
1.0 h, filtered, washed with water, dried to afford 0.16 g (60%) of
(86), as white solid.
[0234] BA-40: To (84) (0.08 g, 0.22 mmol), MR-75 (0.1 g, 0.34
mmol), (PPh.sub.3).sub.4Pd (0.026 g, 0.02 mmol) and K.sub.2PO.sub.4
(0.095 g, 0.45 mmol) was added DME (3.0 mL), and EtOH--H.sub.2O
(1:1, 1.0 mL). Ar gas was bubbled through the stirred reaction for
5 min. The reaction was stirred at 160.degree. C. for 20 minutes
using microwave oven (Biotage Initiator II). The reaction was
cooled to room temperature, concentrated. The residue was purified
by silica gel column chromatography using dichloromethane followed
by triturating with methanol to afford 0.03 g (30%) of BA-40 as
off-white solid. .sup.1H NMR-(400 MHz, CDCl.sub.3); MS (APCI+):
442.0 (M+1), LC-MS: 86%.
[0235] Followed the same procedure as for BA-40 using (84).
[0236] Scheme. piperazine analogs
[0237] BA-31,
[0238] BA-43,
[0239] BA-44,
[0240] BA-45,
[0241] BA-46,
[0242] BA-47,
[0243] BA-48,
[0244] BA-49,
[0245] BA-50.
##STR00055##
[0246] BA-48
4-(3-Chloro-phenyl)-5-fluoro-6-piperazin-1-ylmethyl-benzothiazole.
BA-48
[0247] To a solution of piperazine (821 mg, 9.53 mmol, 10 eq.) in
15 mL anh. THF, at room temperature was added a solution of (84)
(340 mg, 0.953 mmol) in 5 mL anh. THF. The reaction mixture was
stirred at room temperature for 2 hours. The mixture was
concentrated down, diluted with water, and filtrated to give solid
crude. This was triturated with 5 mL ether, and after filtration
and drying afforded 280 mg (81.4%) of BA-48 as a white solid.
.sup.1H-NMR-(400 MHz, DMSO). LCMS (APCI+): 362 (M+1), 99%.
[0248] BA-49
4-[4-(3-Chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-piperazine-1-carb-
oxylic Acid Ethyl Ester. BA-49
[0249] To a solution of BA-48 980 mg, 0.22 mmole) in 1.5 mL anh.
THF was added at room temperature pyridine (35 mg, 36 .mu.L, 2
eq.). This mixture was cooled to 0.degree. C. and
ethylchloroformate (48 mg, 0.44 mmole) was added. Reaction mixture
was stirred at room temperature for 24 hrs. Mixture was
concentrated, diluted with water and extracted 3 times with
methylene chloride. Combined organic layers were washed with brine,
dried over Na.sub.2SO.sub.4, filtered and concentrated to give
about 100 mg crude. Purification by silica gel prep plate provided
80 mg (84.21%) of BA-49 as white foam. .sup.1H-NMR-(400 MHz, DMSO).
LCMS (APCI+): 434 (M+1), 100%.
[0250] BA-31
4-[4-(3-Chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-piperazine-1-carb-
oxylic Acid Amide. BA-31
[0251] To a solution of BA-48 (70 mg, 0.2 mmole) in 1.5 mL
methylene chloride was added at 0.degree. C. trimethyl silyl
isocyanate (46 mg, 0.4 mmole). The reaction mixture was stirred at
room temperature for 24 hrs. Mixture was quenched by slowly
addition of saturated sol of NaHCO.sub.3 and extracted 3 times with
methylene chloride. Combined organic layers were washed with brine,
dried over Na.sub.2SO.sub.4, filtered and concentrated to give
about 100 mg crude. Purification by silica gel preparative plate
provided 20 mg (25.6%) of BA-31 as a white solid, 25.
.sup.1H-NMR-(400 MHz, DMSO). LCMS (APCI+): 405 (M+1), 92%.
[0252] BA-43
4-(3-Chloro-phenyl)-5-fluoro-6-morpholin-4-ylmethyl-benzothiazole.
BA-43
[0253] To a solution of BA-48 (45 mg, 0.126 mmol) in 1.5 mL
methylene chloride was added diisopropyl ethylamine (25 mg, 0.19
mmol) and morpholine (12 mg, 0.138 mmol). The reaction mixture was
stirred at room temperature for 24 hrs. Mixture was concentrated
down, redissolved in ethyl acetate and washed with water. Organic
layer was washed with water, brine, dried over Na.sub.2SO.sub.4,
filtered and concentrated to give about 50 mg solid BA-43.
.sup.1H-NMR-(400 MHz, CDCl.sub.3). LCMS (APCI+): 363 (M+1),
97%.
[0254] BA-44
1-{4-[4-(3-Chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-piperazin-1-yl-
}-ethanone. BA-44
[0255] To a solution of BA-48 (80 mg, 0.22 mmole) in 1.5 mL
methylene chloride was added at 0.degree. C. pyridine (26 mg, 0.33
mmole) followed by addition of acetyl chloride (26 mg, 0.33 mmole).
The reaction mixture was stirred at room temperature for 3 hrs.
Mixture was diluted with water and extracted 3 times with methylene
chloride. Combined organic layers were washed with brine, dried
over Na.sub.2SO.sub.4, filtered and concentrated to give about 80
mg crude. Purification by silica gel prep plate using 5%
methanol/methylene chloride provided 40 mg (45%) of BA-44.
.sup.1H-NMR-(400 MHz, DMSO). LCMS (APCI+): 405 (M+1), 100%.
[0256] BA-50
4-(3-Chloro-phenyl)-5-fluoro-6-(4-methyl-piperazin-1-ylmethyl)-benzothiazo-
le. BA-50
[0257] A small vial containing BA-48 (60 mg, 0.165 mmole), 0.1 mL
formic acid and 0.1 mL formaldehyde was sealed and heated 40
minutes @ 160.degree. C. using microwave Biotage Initiator. The
reaction mixture was basified to pH=8 using saturated solution of
NaHCO.sub.3 and extracted 3 times with methylene chloride. Combined
organic layers were washed with brine, dried over Na.sub.2SO.sub.4,
filtered and concentrated to give about 60 mg crude. Purification
by silica gel prep plate using 5% methanol/methylene chloride
provided 50 mg (79%) of BA-50. .sup.1H-NMR-(400 MHz, CDCl.sub.3).
LCMS (APCI+): 376 (M+1), 92.5%.
[0258] BA-47
Step-1:
[1-[4-(3-Chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-piperidi-
n-4-yl]-carbamic Acid tert-butyl Ester. (87)
[0259] To a solution of (84) (114 mg, 0.32 mmole) in 3 ml methylene
chloride was added diisopropyl ethylamine (62 mg, 0.48 mmole) and
4(N--BOC amino) piperidine (68 mg, 0.32 mmole). The reaction
mixture was stirred at room temperature for 24 hrs. Mixture was
concentrated down to afford 200 mg foamy crude, (87).
.sup.1H-NMR-(400 MHz, CDCl3)
Step-2:
[1-[4-(3-Chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-piperidi-
n-4-yl]-carbamic Acid tert-butyl Ester. BA-47
[0260] To a solution of (87) (195 mg, 0.41 mmole) in 2 mL methylene
chloride was added at 0.degree. C. 1.1 mL of HCl 4N in dioxane. The
reaction mixture was stirred at room temperature for 8 hrs. Mixture
was concentrated down and solid residue was triturated with 5 mL
ether to give 190 mg the desired product, BA-47, as HCl salt. A
small amount was converted to free base using saturated
NaHCO.sub.3. .sup.1H-NMR-(400 MHz, CDCl3). LCMS (APCI+): 376 (M+1),
100%.
[0261] BA-45
{1-[4-(3-Chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-piperidin-4-yl}--
carbamic Acid Ethyl Ester. BA-45
[0262] To a solution of BA-47 (100 mg, 0.25 mmol) in 1.5 mL
anhydrous THF was added pyridine (60 mg, 0.75 mmol) and at
0.degree. C. ethylchloroformate (60 mg, 0.5 mmol). The reaction
mixture was stirred at room temperature for 24 hrs. Mixture was
concentrated down, diluted with water and extracted 3 times with
ethyl acetate. Combined organic layers were washed with brine,
dried over Na.sub.2SO.sub.4, filtered and concentrated. Trituration
of the crude with ether afforded 30 mg solid BA-45.
.sup.1H-NMR-(400 MHz, CDCl3). LCMS (APCI+): 448 (M+1), 100%.
[0263] BA-46
[1-[4-(3-Chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-piperidin-4-yl]--
urea. BA-46
[0264] To a solution of BA-47 (60 mg, 0.145 mmole) in 1.5 mL
methylene chloride was added triethylamine (30 mg, 0.29 mmole) and
at 0.degree. C. trimethyl silyl isocyanate (50 mg, 0.435 mmole).
The reaction mixture was stirred at room temperature for 24 hrs.
Mixture was quenched by slowly addition of saturated sol. of
NaHCO.sub.3 and extracted 3 times with methylene chloride. Combined
organic layers were washed with brine, dried over Na2SO, filtered
and concentrated. Purification by silica gel prep plate provided 15
mg product BA-46 as white solid. .sup.1H-NMR-(400 MHz, CDCl3). LCMS
(APCI+): 419 (M+1), 100%.
[0265] BA-52
##STR00056##
4-(3-chloro-phenyl)-5-fluoro-6-piperazin-1-ylmethyl-benzothiazole:
BA-48
[0266] To (84) (0.1 g, 0.28 mmol) in tetrahydrofuran (7.0 mL) was
added a solution of piperazine (1) (0.24 g, 2.8 mmol) in
tetrahydrofuran (1.0 mL) over 10 min. The reaction was stirred at
room temperature for 18 h. The reaction mixture was concentrated,
washed with saturated NaHCO.sub.3 solution (2 mL), water (2.times.2
mL) than ether ((2.times.2 mL). The residue was dissolved in
dichloromethane, dried (Na.sub.2SO.sub.4), filtered and
concentrated to afford 0.086 g (85%) of BA-48 as light yellow
solid. .sup.1H NMR (400 MHz, CDCl.sub.3).
4-[4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-piperazine-1-carb-
oxylic Acid Ethylamide Hydrochloride. BA-52
[0267] To BA-48 (0.08 g, 0.22 mmol) in pyridine (1.5 mL) was added
ethylisocyanate (0.05 mL). The reaction mixture was stirred at room
temperature for 18 h, concentrated. Water (10 mL) was added,
stirred for 0.5 h, filtered, washed with water (5 mL). The residue
was dissolved in dichloromethane, dried (Na.sub.2SO.sub.4),
filtered and concentrated. The crude yellow solid was purified by
prep TLC using 5% methanol in dichloromethane to afford 0.06 g of
light yellow solid. The light yellow solid was dissolved in ether
(2.0 mL) was added 2M HCl in ether (1.0 mL). The reaction mixture
was stirred for 2 h at room temperature, concentrate under N.sub.2
flow, than dried under vacuum to afford 0.06 g (63%) of BA-52 as
off-white solid. .sup.1H NMR (400 MHz, CDCl.sub.3); MS (APCI+):
433.1 (M+1), LC-MS: 88%.
[0268] BA-51
##STR00057##
(MR 77) Synthesis of
{5-[4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-pyridin-2-yl}-c-
arbamic Acid Ethyl Ester
[0269] To benzyl bromide (84) (0.08 g, 0.22 mmol),
[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-urea (1)
(0.07 g, 0.27 mmol), (PPh.sub.3).sub.4Pd (0.026 g, 0.02 mmol) and
K.sub.2PO.sub.4 (0.095 g, 0.45 mmol) was added DME (3.0 mL), and
EtOH--H.sub.2O (1:1, 1.5 mL). Ar gas was bubbled through the
stirred reaction for 5 min. The reaction was stirred at 120.degree.
C. for 20 minutes using microwave oven (Biotage Intiator II). The
reaction was cooled to room temperature, concentrated. The residue
was purified by silica gel column chromatography using 5% methanol
in dichloromethane followed by prep TLC using 5% methanol in
dichloromethane to afford 0.015 g (16%) of BA-51 as light brown
solid. 1H NMR (DMSO-d6, 400 MHz): 9.39 (s, 1H), 8.45 (s, 1H), 8.1
(d, J=7.2 Hz, 1H), 7.66 (s, 1H), 7.5-7.59 (m, 4H), 7.32 (d, J=8.5
Hz, 2H), 7.14 (d, J=8.5 Hz, 2H), 5.77 (s, 2H), 4.06 ppm (s, 2H); MS
(APCI+): 412.0 (M+1), LC-MS: 97%.
[0270] BA-53)
##STR00058## ##STR00059##
(MR 91) Synthesis of 5-fluoro-6-methyl-benzothiazol-2-ylamine
[0271] To MR 49 (1.0 g, 3.83 mmol) in dioxane (30 mL) was added
tert-butyl nitrite (0.59 g. 5.12 mmol). The reaction mixture was
stirred and heated at 60.degree. C. for 1.5 h, concentrated. The
residue was purified by silica gel column chromatography using 1:1
dichloromethane-hexanes to afford 0.45 g (47%) of MR 91 as reddish
orange solid.
(MR 92) Synthesis of
3-(5-fluoro-6-methyl-benzothiazol-4-yl)-benzonitrile
[0272] To MR 91 (0.44 g, 1.79 mmol), 3-cyanophenylboronic acid (1)
(0.32 g, 2.15 mmol), PPh.sub.3 (0.23 g, 0.89 mmol), K.sub.2CO.sub.3
(0.1 g, 0.72 mmol) and Pd(OAc).sub.2 (0.05 g, 0.21 mmol) was added
dioxane (8 mL), and EtOH--H.sub.2O (1:1, 4 mL). Ar gas was bubbled
through the stirred reaction for 15 min. The reaction was stirred
at 180.degree. C. for 15 minutes using microwave oven (Biotage
Intiator II). The reaction mixture was concentrated. Water (50 mL)
and dichloromethane (50 mL) were added. The organic layer was
separated and the aqueous layer was extracted with dichloromethane
(2.times.40 mL). The combined organic extracts were dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The residue was
purified by silica gel column chromatography using 1:1
dichloromethane-hexanes than 80% dichloromethane in to afford 0.39
g (81%) of MR 92 as off-white solid.
(MR 93) Synthesis of
3-(6-bromomethyl-5-fluoro-benzothiazol-4-yl)-benzonitrile
[0273] To MR 92 (0.39 g, 1.45 mmol) and NBS (0.27 g, 1.53 mmol) in
CCl.sub.4 (20 mL) was added benzoylperoxide (0.04 g, 0.14 mmol).
The reaction was stirred at 80.degree. C. under N.sub.2 for 5 h.
The reaction was cooled to room temperature and concentrated. The
residue was dissolved in mixture of dichloromethane and hexanes
(1:1, 8 mL) and purified by silica gel column chromatography using
20% ethyl acetate-hexanes to afford 0.21 g (41%) of MR 93 as white
solid.
(MR 94) Synthesis of
3-[6-(6-amino-pyridin-3-ylmethyl)-5-fluorobenzothiazol-4-yl]-benzonitrile
[0274] To MR 93 (0.2 g, 0.58 mmol),
5-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyridin-2-ylamine
(2) (0.15 g, 0.7 mmol), (PPh.sub.3).sub.4Pd (0.07 g, 0.06 mmol) and
K.sub.3PO.sub.4 (0.24 g, 1.15 mmol) was added DME (8.0 mL), and
EtOH--H.sub.2O (1:1, 4.0 mL). Ar gas was bubbled through the
stirred reaction for 5 min. The reaction was stirred at 120.degree.
C. for 20 minutes using microwave oven (Biotage Intiator II). The
reaction was cooled to room temperature, concentrated. The residue
was purified by silica gel column chromatography using 5% methanol
in dichloromethane followed by prep TLC using 6% methanol in
dichloromethane to afford 0.08 g (39%) of MR 94 as off-white
solid.
(MR 95) Synthesis of
1-{5-[4-(3-cyano-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-pyridin-2-yl}--
3-ethyl-urea
[0275] To MR 94 (0.075 g, 0.21 mmol) in pyridine (1.5 mL) was added
ethylisocyanate (0.044 mL). The reaction mixture was stirred at
room temperature for 18 h, concentrated. Water (10 mL) was added,
stirred for 0.5 h, filtered, washed with water (5 mL), ethyl
acetate (2.times.5 mL), than ether (10 mL), dried to afford 0.036 g
(40%) of MR 95 as white solid.
(MR 96). Synthesis of
1-{5-[4-(3-cyano-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-pyridin-2-yl}--
3-ethyl-urea Hydrochloride (BA-53)
[0276] To MR 95 (0.032 g, 0.07 mmol) in ether (1.0 mL) was added 2M
HCl in ether (0.2 mL, 0.4 mmol). The reaction mixture was stirred
for 2 h at room temperature, concentrate under N.sub.2 flow, than
dried under vacuum to afford 0.036 g (98%) of MR 96 as off-white
solid. 1H NMR (DMSO-d6, 400 MHz): 9.44 (s, 1H), 8.16-8.2 (m, 2H),
8.09 (s, 1H), 7.92-7.98 (m, 3H), 7.78-7.86 (m, 2H), 7.7-7.76 (m,
2H), 7.32 (d, J=9.2 Hz, 1H), 4.15 (s, 2H), 3.14-3.22 (m, 2H), 1.08
ppm (t, J=7.2 Hz, 3H); MS (APCI+): 432.0 (M+1), LC-MS: 92%.
[0277] BB-01
##STR00060##
(MR 97) Synthesis of
4-(3-chloro-phenyl)-5-fluoro-6-(6-fluoro-pyridin-3-ylmethyl)-benzothiazol-
e
[0278] To MR 70 (0.6 g, 1.68 mmol), 2-fluoro-5-pyridineboronic acid
(2) (0.24 g, 1.68 mmol) and (PPh.sub.3).sub.4Pd (0.1 g, 0.08 mmol)
was added toluene (20.0 mL), and EtOH (5.0 mL). The reaction
mixture stirred for 5 min, than Na2CO3 (2M soln, 1.7 mL, 3.36 mmol)
was added. Ar gas was bubbled through the stirred reaction for 15
min. Then the reaction was stirred at 80.degree. C. for 18 h. The
reaction was cooled to room temperature, concentrated. The residue
was diluted with water (40 mL), extracted with ethyl acetate
(2.times.30 mL), washed with brine (30 mL), dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The residue was
purified by silica gel column chromatography using dichloromethane
to afford 0.48 g (76%) of MR 97 as off-white solid.
(MR 98) Synthesis of 1-{5-[4-(3-chloro-phenyl)-5-fluoro
benzothiazol-6-ylmethyl]-pyridin-2-yl}-azetidine-2-carboxylic Acid
(BB-0)
[0279] To MR 97 (0.07 g, 0.19 mmol) and D,L-azetidine-2-carboxylic
acid (2) (0.06 g, 0.56 mmol) was added
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.14 g, 0.94 mmol). The
reaction mixture was stirred and heated at 160.degree. C. for 20
min. Cooled to room temperature, diluted with dichloromethane (6
mL), washed with 0.5 N HCl (2.times.2 mL), dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The residue was
purified by silica gel column chromatography using 5% methanol in
dichloromethane to afford 0.022 g (23%) of BB-01 as light yellow
solid. 1H NMR (DMSO-d6, 400 MHz): 9.35 (s, 1H), 8.12 (d, J=7.2 Hz,
1H), 8.05 (s, 1H), 7.66 (s, 1H), 7.45-7.59 (m, 4H), 6.41 (d, J=8.4
Hz, 1H), 4.58 (dd, J=8, 6.8 Hz, 1H), 4.03 (s, 2H), 3.72-3.8 (m,
2H), 2.3-2.41 (m, 2H); MS (APCI+): 454.1 (M+1), LC-MS: 99%.
[0280] BB-03
##STR00061##
(MR 100). Synthesis
of(S)-1-{5-[4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-pyridin-
-2-yl}-pyrrolidine-2-carboxylic Acid (BB-03)
[0281] To MR 97 (0.15 g, 0.4 mmol) and (S)-Pyrrolidine-2-carboxylic
acid (2) (0.1 g, 0.8 mmol) was added
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.31 g, 2.01 mmol). The
reaction mixture was stirred and heated at 160.degree. C. for 30
min. Cooled to room temperature, diluted with dichloromethane (10
mL), washed with 0.5 N HCl (2.times.4 mL), dried with
Na.sub.2SO.sub.4, filtered, and concentrated to afford 0.18 g (94%)
of BB-03 as light yellowish-brown solid. 1H NMR (DMSO-d6, 400 MHz):
9.42 (s, 1H), 8.16 (d, J=6.8 Hz, 1H), 8.0 (s, 1H), 7.72-7.82 (br s,
1H), 7.67 (s, 1H), 7.5-7.59 (m, 4H), 6.8-6.88 (br s, 1H), 4.64 (br
s, 1H), 4.11 (s, 2H), 3.4-3.65 (m, 2H), 1.88-2.3 (m, 4H); MS
(APCI+): 468.1 (M+1), LC-MS: 99%
[0282] BA-54
##STR00062##
(MR 101) Synthesis of
1-[4-(3-Chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-pyrrolidin-2-one
(BA-54)
[0283] To a cooled (0.degree. C.) and stirred suspension of NaH
(0.034 g, 0.84 mmol) in DMF (2.0 mL) was added a solution of
pyrrolidine-2-one (0.07 g, 0.84 mmol) in DMF (0.5 ml). The reaction
mixture was slowly warmed to room temperature, stirred for 0.5 h.
Again cooled (0.degree. C.), then a solution of MR 70 (0.15 g, 0.42
mmol) in DMF (0.5 mL) was added over 5 min. The reaction mixture
was slowly warmed to room temperature, stirred for 2 h. Poured on
to crushed ice-water, extracted with ethyl acetate (2.times.40 mL).
The combined organic extracts were dried with Na.sub.2SO4,
filtered, and concentrated. The residue was purified by preparative
thin layer chromatography using 1% methanol in dichloromethane to
afford 0.087 g (58%) of BA-54 as a viscous liquid. 1H NMR (DMSO-d6,
400 MHz): 9.44 (s, 1H), 8.11 (d, J=6.8 Hz, 1H), 7.69 (s, 1H),
7.5-7.62 (m, 4H), 4.6 (s, 2H), 3.37 (t, J=7.2 Hz, 2H), 2.33 (t,
J=7.2 Hz, 2H), 1.95-2.06 (m, 2H); MS (APCI+): 361.0 (M+1), LC-MS:
100%.
[0284] BA-55
##STR00063##
(MR 102). Synthesis of
{5-[4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-pyridin-2-yl}-d-
imethyl-amine (BA-55)
[0285] To MR 97 (0.1 g, 0.27 mmol) was added 2M solution of
dimethylamine in methanol (2 mL, 4 mmol). Vial was sealed and
heated at 140.degree. C. for 40 min. using Biotage intiator 11
microwave oven. The reaction mixture was concentrated, the residue
was purified by preparative thin layer chromatography using 5%
methanol in dichloromethane to afford 0.06 g (56%) of BA-55 as
off-white solid. 1H NMR (DMSO-d6, 400 MHz): 9.39 (s, 1H), 8.11 (d,
J=6.8 Hz, 1H), 8.05 (d, J=2.0 Hz, 1H), 7.6 (s, 1H), 7.5-7.59 (m,
3H), 7.41 (dd, J=8.4, 2.4 Hz, 1H), 6.59 (d, J=8.4 Hz, 1H), 4.0 (s,
2H), 2.97 (s, 6H); MS (APCI+): 398.1 (M+1), LC-MS: 100%.
[0286] BA-57
##STR00064##
(MR 103) Synthesis of
4-(3-chloro-phenyl)-5-fluoro-6-(6-pyrrolidin-1-yl-pyridin-3-ylmethyl)-ben-
zothiazole Hydrochloride (BA-57)
[0287] To MR 97 (0.13 g, 0.35 mmol) and Pyrrolidine (0.07 g, 1.05
mmol) was added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.27 g,
1.74 mmol). The reaction mixture was stirred and heated at
100.degree. C. for 1 h. Cooled to room temperature, diluted with
dichloromethane (6 mL), washed with 0.5 N HCl (2.times.4 mL), dried
with Na.sub.2SO.sub.4, filtered, and concentrated. The residue was
purified by silica gel column chromatography using 3% methanol in
dichloromethane to afford 0.06 g (41%) of foamy solid. The solid
was suspended in ether (2 mL), than 2M HCl-ether (0.5 mL, 1.0 mmol)
was added, stirred for 1 h, than concentrated, again triturated
with ether (2 mL), dried to afford 0.6 g (98%) of BA-57 as
off-white solid. 1H NMR (DMSO-d6, 400 MHz): 13.27 (br s, 1H), 9.44
(s, 1H), 8.16 (d, J=6.8 Hz, 1H), 7.85-7.95 (m, 2H), 7.66 (s, 1H),
7.51-7.58 (m, 3H), 7.07 (d, J=9.2 Hz, 1H), 4.14 (s, 2H), 3.48-3.56
(m, 4H), 1.92-2.7 (m, 4H); MS (APCI+): 424.1 (M+1), LC-MS:
87.4%.
[0288] BA-58
##STR00065##
(MR 104 Synthesis of 4-bromo-6-methyl-benzothiazol-2-ylamine
[0289] To a heated (80.degree. C.) and stirred solution of
6-methyl-benzothiazol-2-ylamine (10.0 g, 60.89 mmol) in acetic acid
(210 mL) was added a solution of bromine (19.46 g, 121.78 mmol) in
acetic acid (40 mL) over 30 min. The reaction mixture was stirred
at 80.degree. C. for 20 h, cooled to room temperature than poured
on to crushed ice-water (400 mL). Ammonium hydroxide solution (28%)
was added to pH 8, stirred for 2 h. Filtered, washed with water,
dried to afford 13.5 g (91%) of MR 104 as brown solid.
(MR 105) Synthesis of 4-bromo-6-methyl-benzothiazole
[0290] To MR 104 (13.4 g, 55.11 mmol) in dioxane (150 mL) was added
tert-butyl nitrite (6.25 g. 60.63 mmol). The reaction mixture was
stirred and heated at 60.degree. C. for 1.0 h, concentrated. The
residue was purified by silica gel column chromatography using 1:1
dichloromethane-hexanes to afford 4.45 g (34%) of MR 105 as orange
solid.
(MR 106) Synthesis of
4-(3-chloro-phenyl)-6-methyl-benzothiazole
[0291] To MR 105 (2.03 g, 8.77 mmol), 3-chlorophenylboronic acid
(2) (1.51 g, 9.64 mmol) and Pd(Ph.sub.3P).sub.4 (0.51 g, 0.44 mmol)
was added toluene (80 mL), EtOH (20 mL) and 2M NaCO.sub.3 solution
(8.8 mL, 17.54 mmol). Ar gas was bubbled through the stirred
reaction for 15 min. The reaction was stirred at 80.degree. C.
under Ar for 20 h. The reaction was cooled to room temperature,
H.sub.2O (60 mL) and ethyl acetate (80 mL) were added. The layers
were separated and the aqueous was extracted with ethyl acetate
(2.times.40 mL). The combined organic extracts were dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The residue was
purified by silica gel column chromatography using 20% ethyl
acetate in hexanes to afford 2.23 g (98%) of MR 106 as brown
viscous liquid.
(MR 107) Synthesis of
6-bromomethyl-4-(3-chloro-phenyl)-benzothiazole
[0292] To MR 106 (2.2 g, 8.47 mmol) and NBS (1.54 g, 8.55 mmol) in
CCl.sub.4 (50 mL) was added benzoylperoxide (0.1 g, 0.41 mmol). The
reaction was stirred at 80.degree. C. under N.sub.2 for 18 h. The
reaction was cooled to room temperature and concentrated. The
residue was triturated with 20% ethyl acetate in hexanes to afford
2.9 g (99%) of MR 107 as a light brown solid.
(MR 108) Synthesis of
4-(4-(3-chloro-phenyl)-6-(6-fluoro-pyridin-3-ylmethyl)-benzothiazole
[0293] To MR 107 (2.8 g, 8.27 mmol), 2-fluoro-5-pyridylboronic acid
(1.28 g, 9.09 mmol) and Pd(Ph.sub.3P).sub.4 (0.48 g, 0.41 mmol) was
added toluene (60 mL), EtOH (10 mL) and 2M NaCO.sub.3 solution (8.3
mL, 16.54 mmol). Ar gas was bubbled through the stirred reaction
for 15 min. The reaction was stirred at 80.degree. C. under Ar for
3 h. The reaction was cooled to room temperature, H.sub.2O (60 mL)
and ethyl acetate (80 mL) were added. The layers were separated and
the aqueous was extracted with ethyl acetate (2.times.40 mL). The
combined organic extracts were dried with Na.sub.2SO.sub.4,
filtered, and concentrated. The residue was purified by silica gel
column chromatography using dichloromethane to afford 1.53 g (53%)
of MR 108 as light yellow solid.
(MR 109) Synthesis of
{5-[4-(3-chloro-phenyl)-benzothiazol-6-ylmethyl]-pyridin-2-yl}-dimethyl-a-
mine Hydrochloride (BA-58)
[0294] To MR 108 (0.15 g, 0.42 mmol) was added 2M solution of
dimethylamine in methanol (2.2 mL, 4.4 mmol). Vial was sealed and
heated at 150.degree. C. for 1 h. The reaction mixture was
concentrated, the residue was purified by silica gel column
chromatography using dichloromethane to afford 0.116 g (75%) of
gummy solid. The solid was suspended in ether (2 mL), than 2M
HCl-ether (0.8 mL, 1.6 mmol) was added, stirred for 1 h, than
concentrated, again triturated with ether (2 mL), dried to afford
0.118 g (98%) of BA-58 as white solid. 1H NMR (DMSO-d6, 400 MHz):
13.4 (br s, 1H), 9.42 (s, 1H), 8.11 (d, J=1.6 Hz, 1H), 8.02 (s,
1H), 7.92-8.0 (m, 2H), 7.69 (d, J=1.6 Hz, 1H), 7.48-7.57 (m, 21H),
7.19 (d, J=9.24 Hz, 1H), 4.14 (s, 2H), 3.19 (s, 6H); MS (APCI+):
380.0 (M+1), LC-MS: 95.7%; HPLC 93.61% pure.
[0295] BA-59
##STR00066##
(MR 110) Synthesis of
4-(3-chloro-phenyl)-6-(6-pyrrolidin-1-yl-pyridin-3-ylmethyl)-benzothiazol-
e Hydrochloride (BA-59)
[0296] To MR 108 (0.15 g, 0.42 mmol) and Pyrrolidine (0.09 g, 1.27
mmol) was added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.32 g,
2.11 mmol). The reaction mixture was stirred and heated at
100.degree. C. for 1 h. Cooled to room temperature, diluted with
dichloromethane (6 mL), washed with 0.5 N HCl (2.times.4 mL), dried
with Na.sub.2SO.sub.4, filtered, and concentrated. The residue was
purified by silica gel column chromatography using 2% methanol in
dichloromethane to afford 0.103 g (60%) of viscous liquid. The
viscous liquid (0.09 g, 0.23 mmol) was suspended in ether (2 mL),
than 2M HCl-ether (0.5 mL, 1.0 mmol) was added, stirred for 1 h,
than concentrated, again triturated with ether (2 mL), dried to
afford 0.9 g (94%) of BA-59 as white solid. 1H NMR (DMSO-d6, 400
MHz): 13.25 (br s, 1H), 9.42 (s, 1H), 8.1 (d, J=1.6 Hz, 1H), 8.0
(d, J=1.6 Hz, 1H), 7.9-7.95 (m, 2H), 7.81 (dt, J=7.2, 1.6 Hz 1H),
7.67 (d, J=1.2 Hz), 7.481-7.56 (m, 2H), 7.0 (d, J=8.4 Hz, 1H), 4.11
(s, 2H), 3.4-3.56 (m, 4H), 1.95-2.17 (m, 4H); MS (APCI+): 406.1
(M+1), LC-MS: 98.1%.
[0297] BA-55
##STR00067##
(MR 102) Synthesis of
{5-[4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-pyridin-2-yl}-d-
imethyl-amine Hydrochloride (BA-55)
[0298] To MR 102 (0.1 g, 0.26 mmol) in ether (2 mL), than 2M
HCl-ether (1.0 mL, 2.0 mmol) was added, stirred for 1 h, than
concentrated, again triturated with ether (2 mL), dried to afford
0.11 g (98%) of BA-55 as white solid. 1H NMR (DMSO-d6, 400 MHz):
9.39 (s, 1H), 8.11 (d, J=6.8 Hz, 1H), 8.05 (d, J=2.0 Hz, 1H), 7.6
(s, 1H), 7.45-7.59 (m, 3H), 7.45 (d, J=6.8 Hz, 1H), 6.63 (d, J=8.8
Hz, 1H), 4.01 (s, 2H), 2.99 (s, 6H); MS (APCI+): 398.1 (M+1),
LC-MS: 98.9%; HPLC 98.5% pure.
[0299] BA-60
##STR00068##
(MR 112) Synthesis of
5-[4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-pyridine-2-carbo-
nitrile (BA-60)
[0300] To MR 70 (0.8 g, 2.24 mmol),
5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyridine-2-carbonitrile
(2) (0.57 g, 2.47 mmol) and (PPh.sub.3).sub.4Pd (0.13 g, 0.11 mmol)
was added toluene (30.0 mL), and EtOH (10.0 mL). The reaction
mixture stirred for 5 min, than Na2CO3 (2M soln, 2.5 mL, 5.0 mmol)
was added. Ar gas was bubbled through the stirred reaction for 15
min. Then the reaction was stirred at 80.degree. C. for 1 h. The
reaction was cooled to room temperature, concentrated. The residue
was diluted with water (40 mL), extracted with ethyl acetate
(2.times.30 mL), washed with brine (30 mL), dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The residue was
purified by silica gel column chromatography using dichloromethane
to afford 0.62 g (73%) of BA-60 as light brown solid. 1H NMR
(DMSO-d6, 400 MHz): 9.43 (s, 1H), 8.76 (d, J=1.6 Hz, 1H), 8.21 (d,
J=7.2 Hz, 1H), 7.92-7.81 (m, 2H), 7.66 (s, 1H), 7.5-7.59 (m, 3H),
4.32 (s, 2H); MS (APCI-): 378.0 (M-1), LC-MS: 100%.
[0301] BA-61
##STR00069##
(MR 113) Synthesis of
6-(6-azetidin-1-yl-pyridin-3-ylmethyl)-4-(3-chloro-phenyl)-5-fluoro-benzo-
thiazole Hydrochloride (BA-61)
[0302] To MR 97 (0.15 g, 0.45 mmol) and azetidine (0.07 g, 1.21
mmol) was added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.31 g,
2.01 mmol). The reaction mixture was stirred and heated at
85.degree. C. for 15 min. Cooled to room temperature, diluted with
dichloromethane (6 mL), washed with 0.5 N HCl (2.times.4 mL), dried
with Na.sub.2SO.sub.4, filtered, and concentrated. The residue was
purified by silica gel column chromatography using 5% methanol in
dichloromethane followed by preparative thin layer chromatography
using 1:1 ethyl acetate in hexanes to afford 0.022 g (13%) of off
white solid. The solid was suspended in ether (2 mL), than 2M
HCl-ether (0.5 mL, 1.0 mmol) was added, stirred for 1 h, than
concentrated, again triturated with ether (2 mL), dried to afford
0.023 g (98%) of BA-61 as off-white solid. 1H NMR (DMSO-d6, 400
MHz): 9.44 (s, 1H), 8.15 (d, J=7.2 Hz, 1H), 7.95 (s, 1H), 7.8-7.9
(m, 1H), 7.66 (s, 1H), 7.44-7.59 (m, 3H), 6.8 (d, J=8.8 Hz, 1H),
4.18-4.28 (m, 4H), 4.11 (s, 2H), 2.38-2.46 (m, 2H); MS (APCI+):
410.0 (M+1), LC-MS: 87%.
[0303] BA-63
##STR00070##
(MR 114) Synthesis of
5-[4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-pyridin-2-ylamin-
e (BA-63)
[0304] To MR 70 (0.81 g, 2.27 mmol), 2-amino-5-pyridineboronic acid
pinacol ester (2) (0.56 g, 2.5 mmol) and (PPh.sub.3).sub.4Pd (0.13
g, 0.11 mmol) and K.sub.3PO.sub.4 (0.96 g, 4.54 mmol) was added DME
(20.0 mL), and EtOH--H.sub.2O (1:1, 10.0 mL). Ar gas was bubbled
through the stirred reaction for 5 min. The reaction was stirred
and heated at 80.degree. C. for 4 h. The reaction was cooled to
room temperature, concentrated. Diluted with dichloromethane (60
mL), washed with water (2.times.50 mL), brine (30 mL), dried
(Na.sub.2SO.sub.4), filtered, concentrated. The residue was
purified by silica gel column chromatography using 5% methanol in
dichloromethane to afford 0.31 g (37%) of BA-63 as light brown
solid. 1H NMR (DMSO-d6, 400 MHz): 9.39 (s, 1H), 8.1 (d, J=6.8 Hz,
1H), 7.87 (d, J=2.0 Hz, 1H), 7.66 (s, 1H), 7.5-7.59 (m, 3H), 7.28
(dd, J=8.0, 2.4 Hz, 1H), 6.39 (d, J=8.4 Hz, 1H), 5.78 (s, 2H), 3.95
(s, 2H); MS (APCI+): 370.2 (M+1), LC-MS: 98%.
[0305] BA-64
##STR00071##
(MR 114) Synthesis of
N-{5-[4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-pyridin-2-yl}-
-2-dimethylamino-acetamide Hydrochloride (BA-64)
[0306] To dimethylamino-acetic acid (0.04 g, 0.41 mmol) in
dichloromethane (2 mL) was added thionyl chloride (0.1 g, 0.81
mmol). The reaction mixture was stirred for 3 h, concentrated. THF
(1 mL) was added under nitrogen atmosphere, than a solution of
MR114 (0.1 g, 0.27 mmol) in THF (1 mL) was added followed by
diisopropylethylamine (0.14 g, 1.08 mmol). Stirred for 3 h, than
water (4 mL) and ethyl acetate (10 mL) was added. The organic layer
was separated and the aqueous layer was again washed with ethyl
acetate (5 mL). The combined organic layers were washed with brine
(4 mL), dried (Na.sub.2SO.sub.4), filtered, concentrated. The
residue was purified by preparative thin layer chromatography using
5% methanol in dichloromethane to afford 0.027 g (22%) of off white
solid. The solid (0.026 g, 0.06 mmol) was suspended in ether (2
mL), than 2M HCl-ether (0.5 mL, 1.0 mmol) was added, stirred for 1
h, than concentrated, again triturated with ether (2 mL), dried to
afford 0.029 g (98%) of BA-64 as off-white solid. 1H NMR (DMSO-d6,
400 MHz): 11.13 (s, 1H), 9.86 (br s, 1H), 9.42 (s, 1H), 8.35 (s,
1H), 8.2 (d, J=7.2 Hz, 1H), 7.95-8.05 (m, 1H), 7.77 (dd, J=8, 2.4
Hz, 1H), 7.56 (s, 1H), 7.5-7.59 (m, 3H), 4.17 (s, 2H), 3.68 (s,
2H), 2.86 9 s, 6H); MS (APCI-): 411.2 (M-1), LC-MS: 96%.
[0307] BA-66
##STR00072##
(MR 116) Synthesis of N-(2,6-dibromo-4-methyl-phenyl)-acetamide
[0308] To 2,6-dibromo-4-methyl-phenylamine (10.0 g, 37.74 mmol) in
acetic acid (20 mL) was added acetic anhydride (5.0 g, 48.98 mmol).
The reaction mixture was stirred and heated at 90.degree. C. for 30
min, cooled to room temperature than poured on to crushed ice-water
(300 mL). The white solid was filtered, washed with water, dried to
afford 12.0 g (99%) of MR 116 as white solid.
(MR 117) Synthesis of
N-(2,6-dibromo-4-methyl-phenyl)-thioacetamide
[0309] To MR 116 (1.0 g, 3.26 mmol) in toluene (20 mL) was added
Lawesson's Reagent (0.66 g. 1.63 mmol). The reaction mixture was
stirred and heated at reflux for 2.5 h, concentrated. The residue
was purified by silica gel column chromatography using 1:1
dichloromethane-hexanes to afford 0.98 g (93%) of MR 117 as white
solid.
(MR 118) Synthesis of 4-bromo-2,6-dimethyl-benzothiazole
[0310] To MR 117 (0.63 g, 1.93 mmol) in DME (8 mL) was added
Copper(I)iodide (0.02 g. 0.1 mmol), 1,10-phenanthroline (0.04 g,
0.2 mmol) and cesium carbonate (0.95 g, 2.9 mmol). The reaction
mixture was stirred and heated at 85.degree. C. for 20 h, filtered
over Celite, concentrated. The residue was purified by silica gel
column chromatography using dichloromethane to afford 0.41 g (87%)
of MR 118 as light yellow viscous liquid.
(MR 119) Synthesis of
4-(3-chloro-phenyl)-2,6-dimethyl-benzothiazole
[0311] To MR 118 (0.65 g, 2.68 mmol), 3-chlorophenylboronic acid
(2) (0.46 g, 2.954 mmol) and Pd(Ph.sub.3P).sub.4 (0.16 g, 0.13
mmol) was added toluene (40 mL), EtOH (10 mL) and 2M NaCO.sub.3
solution (2.7 mL, 5.4 mmol). Ar gas was bubbled through the stirred
reaction for 15 min. The reaction was stirred at 85.degree. C.
under Ar for 3 h. The reaction was cooled to room temperature,
H.sub.2O (60 mL) and ethyl acetate (80 mL) were added. The layers
were separated and the aqueous was extracted with ethyl acetate
(2.times.40 mL). The combined organic extracts were dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The residue was
purified by silica gel column chromatography using 1:1
dichloromethane in hexanes to afford 0.7 g (98%) of MR 119 as
viscous liquid.
(MR 120) Synthesis of
6-bromomethyl-4-(3-chloro-phenyl)-2-methyl-benzothiazole
[0312] To MR 119 (0.3 g, 1.1 mmol) and NBS (0.2 g, 1.1 mmol) in
CCl.sub.4 (30 mL) was added benzoylperoxide (0.02 g, 0.08 mmol).
The reaction was stirred at 80.degree. C. under N.sub.2 for 1 h.
The reaction was cooled to room temperature and concentrated. The
residue was triturated with 1:1 dichloromethane in hexanes,
concentrated to afford 0.4 g (98%) of MR 120 as a light brown
solid.
(MR 121) Synthesis of
5-[4-(3-chloro-phenyl)-2-methyl-benzothiazol-6-ylmethyl]-pyridin-2-ylamin-
e (BA-66)
[0313] To MR 120 (0.4 g, 1.1 mmol), 2-amino-5-pyridineboronic acid
pinacol ester (2) (0.19 g, 1.2 mmol) and (PPh.sub.3).sub.4Pd (0.06
g, 0.05 mmol) and Na.sub.2CO.sub.3 solution (2M, 1.1 mL, 2.2 mmol)
was added toluene (40.0 mL), and EtOH (10.0 mL). Ar gas was bubbled
through the stirred reaction for 15 min. The reaction was stirred
and heated at 80.degree. C. for 3 h. The reaction was cooled to
room temperature, H.sub.2O (50 mL) and ethyl acetate (50 mL) were
added. The layers were separated and the aqueous was extracted with
ethyl acetate (2.times.40 mL). The combined organic extracts were
dried with Na.sub.2SO.sub.4, filtered, and concentrated. The
residue was purified by silica gel column chromatography using 5%
methanol in dichloromethane than by preparative thin layer
chromatography using 5% methanol in dichloromethane to afford 0.26
g (63%), of BA-66 as light yellow gummy liquid. 1H NMR (CDCl.sub.3,
400 MHz): 8.0 (d, J=2.0 Hz, 1H), 7.78-7.8 (m, 1H), 7.66-7.7 (m,
1H), 7.58 (d, J=2.0 Hz, 1H), 7.25-7.41 (m, 4H), 6.46 (d, J=8.4 Hz,
1H), 4.4 (s, 2H), 3.98 (s, 2H), 2.82 (s, 3H); MS (APCI+): 366.1
(M+1), LC-MS: 96.5%.
[0314] BA-67
##STR00073##
(MR 122) Synthesis of
1-{5-[4-(3-chloro-phenyl)-2-methyl-benzothiazol-6-ylmethyl]-pyridin-2-yl}-
-3-ethyl-urea (BA-67)
[0315] To MR 121 (0.25 g, 0.68 mmol) in pyridine (2.5 mL) was added
ethylisocyanate (0.15 g, 2.05 mmol). The reaction mixture was
stirred at room temperature for 72 h, concentrated. Water (10 mL)
was added, stirred for 0.5 h, filtered, washed with water (5 mL),
than ether (10 mL), dried. Again recrystallized from hot ethyl
acetate to afford 0.083 g (28%) of BA-67 as light yellow
crystalline solid. 1H NMR (DMSO-d6, 400 MHz): 9.08 (s, 1H), 8.16
(d, J=2.0 Hz, 1H), 8.06-8.12 (br s, 1H), 7.91 (d, J=1.6 Hz, 1H),
7.87 (t, J=2.4 Hz, 1H), 7.74-7.78 (m, 1H), 7.62 (dd, J=8.8, 2.4 Hz,
1H), 7.44-7.54 (m, 3H), 7.25 (d, J=8.8 Hz, 1H), 4.04 (s, 2H),
3.12-3.4 (m, 2H), 2.78 (s, 3H), 1.07 (t, J=7.2 Hz, 1H); MS (APCI+):
437.1 (M+1), LC-MS: 100%; HPLC 98.7% pure.
[0316] BB-04
##STR00074##
(MR 124) Synthesis of
(S)-1-{5-[4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-pyridin-2-
-yl}-azetidine-2-carboxylic Acid (BB-04)
[0317] To MR 97 (0.3 g, 0.8 mmol) and L-azetidine-2-carboxylic acid
(2) (0.16 g, 1.61 mmol) was added
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.61 g, 4.02 mmol). The
reaction mixture was stirred and heated at 150.degree. C. for 30
min. Cooled to room temperature, diluted with dichloromethane (8
mL), washed with 0.5 N HCl (2.times.4 mL), dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The residue was
purified by silica gel column chromatography using 5% methanol in
dichloromethane to afford 0.057 g (16%) of BB-04 as light yellow
solid. 1H NMR (DMSO-d6, 400 MHz): 9.4 (s, 1H), 8.13 (d, J=6.8 Hz,
1H), 8.05 (d, J=2.0 Hz, 1H), 7.66 (s, 1H), 7.5-7.59 (m, 4H), 6.47
(d, J=8.0 Hz, 1H), 4.6-4.7 (m, 1H), 4.04 (s, 2H), 3.8-3.96 (m, 2H),
2.3-2.6 (m, 2H); MS (APCI+): 454.0 (M+1), LC-MS: 100%.
[0318] BB-05
##STR00075##
(MR 125) Synthesis of
(R)-1-{5-[4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-pyridin-2-
-yl}-azetidine-2-carboxylic Acid (BB-05)
[0319] To MR 97 (0.3 g, 0.8 mmol) and D-azetidine-2-carboxylic acid
(2) (0.16 g, 1.61 mmol) was added
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.61 g, 4.02 mmol). The
reaction mixture was stirred and heated at 100.degree. C. for 30
min. Cooled to room temperature, diluted with dichloromethane (8
mL), washed with 0.5 N HCl (2.times.4 mL), dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The residue was
purified by silica gel column chromatography using 5% methanol in
dichloromethane to afford 0.074 g (20%) of BB-05 as off-white
solid. 1H NMR (DMSO-d6, 400 MHz): 12.98 (s, 1H), 9.4 (s, 1H), 8.12
(d, J=7.2 Hz, 1H), 8.05 (d, J=2.4 Hz, 1H), 7.66 (s, 1H), 7.46-7.58
(m, 4H), 6.41 (d, J=8.8 Hz, 1H), 4.52-4.63 (m, 1H), H), 4.03 (s,
2H), 3.75-3.85 (m, 2H), 2.3-2.58 (m, 2H); MS (APCI+): 454.0 (M+1),
LC-MS: 100%; HPLC 97.5% pure.
[0320] BA-74
##STR00076##
(MR 126) Synthesis of
(S)-1-{5-[4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-pyridin-2-
-yl}-azetidine-2-carboxylic Acid Amide Hydrochloride (BA-74)
[0321] To a cooled 0.degree. C. and stirred solution of MR 124
(0.28 g, 0.62 mmol) in THF (4 mL) was added diisopropylethylamine
(0.16 g, 1.23 mmol). The reaction mixture was stirred for 5 min,
than isobutylchloroformate (0.1 g, 0.74 mmol) was added, stirred at
0.degree. C. for 30 min. Ammonium hydroxide (28%, 2.0 mL) was
added, warmed to room temperature, stirred for 18 h. The organic
layer was separated, the aqueous layer was washed with ether (6
mL). The combined organic layers were dried with Na.sub.2SO.sub.4,
filtered, and concentrated. The residue was purified by silica gel
column chromatography using 5% methanol in dichloromethane than
preparative thin layer chromatography using 5% methanol in
dichloromethane to afford 0.059 g of white solid. The solid was
suspended in ether (2.0 mL) was added 2M HCl in ether (0.5 mL, 1.0
mmol). The reaction mixture was stirred for 2 h at room
temperature, concentrate under N.sub.2 flow, than dried under
vacuum to afford 0.06 g (21%) of BA-74 as white solid. 1H NMR
(DMSO-d6, 400 MHz): 9.43 (s, 1H), 8.16 (d, J=7.2 Hz, 1H), 8.01 (d,
J=2.0 Hz, 1H), 7.85 (br s, 1H), 7.66 (s, 1H), 7.5-7.58 (m, 4H),
7.36 (br s, 1H), 6.7 (br s, 1H), 4.82 (br s, 1H), 4.11 (s, 2H),
3.89-4.05 (m, 2H), 2.3-2.58 (m, 2H); MS (APCI+): 453.0 (M+1),
LC-MS: 99.50%; HPLC 98.7% pure.
[0322] BA-75
##STR00077##
(MR 128) Synthesis of
(R)-1-{5-[4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-pyridin-2-
-yl}-azetidine-2-carboxylic Acid Amide Hydrochloride (BA-75)
[0323] To a cooled 0.degree. C. and stirred solution of MR 125 (0.5
g, 1.1 mmol) in THF (10 mL) was added diisopropylethylamine (0.28
g, 2.2 mmol). The reaction mixture was stirred for 5 min, than
isobutylchloroformate (0.18 g, 1.3 mmol) was added, stirred at
0.degree. C. for 45 min. Ammonium hydroxide (28%, 4.0 mL) was
added, warmed to room temperature, stirred for 1.5 h. The reaction
mixture was diluted with water (5 mL). The organic layer was
separated, the aqueous layer was washed with ethyl acetate
(2.times.20 mL). The combined organic layers were dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The residue was
purified by silica gel column chromatography using 1:1 ethyl
acetate in hexanes to pure ethyl acetate than preparative thin
layer chromatography using 5% methanol in dichloromethane to afford
0.17 g of white solid. The solid was suspended in ether (3.0 mL)
was added 2M HCl in ether (1.0 mL, 2.0 mmol). The reaction mixture
was stirred for 1 h at room temperature, concentrate under N.sub.2
flow, than dried under vacuum to afford 0.18 g (36%) of BA-75 as
white solid. 1H NMR (DMSO-d6, 400 MHz): 9.43 (s, 1H), 8.16 (d,
J=7.2 Hz, 1H), 8.01 (d, J=2.0 Hz, 1H), 7.85 (br s, 1H), 7.7 (s,
1H), 7.66 (s, 1H), 7.5-7.58 (m, 3H), 7.38 (br s, 1H), 6.7 (br s,
1H), 4.86 (br s, 1H), 4.121 (s, 2H), 4.0-4.25 (m, 2H), 2.6-2.75 (m,
1H), 2.25-2.38 (m, 12H); MS (APCI+): 453.0 (M+1), LC-MS: 97.8%;
HPLC 97.4% pure.
[0324] BB-08
##STR00078##
(MR 129) Synthesis of
4-(3-chloro-phenyl)-6-(6-fluoro-pyridin-3-ylmethyl)-2-methyl-benzothiazol-
e
[0325] Prepared according to the procedure described in Scheme
17.
(MR 130) Synthesis of
(S)-1-{5-[4-(3-chloro-phenyl)-2-methyl-benzothiazol-6-ylmethyl]-pyridin-2-
-yl}-azetidine-2-carboxylic Acid Hydrochloride (BB-08)
[0326] To MR 129 (0.28 g, 0.76 mmol) and L-azetidine-2-carboxylic
acid (2) (0.15 g, 1.52 mmol) was added
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.58 g, 3.8 mmol). The
reaction mixture was stirred and heated at 150.degree. C. for 15
min. Cooled to room temperature, diluted with dichloromethane (8
mL), washed with 0.5 N HCl (2.times.4 mL), dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The residue was
purified by silica gel column chromatography using 5% methanol in
dichloromethane to afford 0.22 g of white solid. The solid was
suspended in ether (4.0 mL) was added 2M HCl in ether (1.8 mL, 3.6
mmol). The reaction mixture was stirred for 1 h at room
temperature, concentrate under N.sub.2 flow, than dried under
vacuum to afford 0.22 g (64%) of BB-08 as white solid. 1H NMR
(DMSO-d6, 400 MHz): 8.08 (d, J=1.6 Hz, 1H), 7.94 (d, J=1.6 Hz, 1H),
7.87 (t, J=2.0 Hz, 1H), 7.82 (br s, 1H), 7.75-7.79 (m, 1H),
7.46-7.58 (m 4H), 6.76 (br s, 1H), 4.86-5.07 (m, 1H), 4.05 (s, 2H),
3.98-4.2 (m, 2H), 2.79 (s, 3H), 2.65-2.75 (m, 1H), 2.36-2.45 (m,
1H); MS (APCI+): 450.0 (M+1), LC-MS: 98.3%.
[0327] BB-09
##STR00079##
Synthesis of
(R)-1-{5-[4-(3-chloro-phenyl)-2-methyl-benzothiazol-6-ylmethyl]-pyridin-2-
-yl}-azetidine-2-carboxylic Acid Hydrochloride (BB-09)
[0328] To MR 129 (0.37 g, 1.0 mmol) and D-azetidine-2-carboxylic
acid (2) (0.2 g, 2.01 mmol) was added
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.76 g, 5.02 mmol). The
reaction mixture was stirred and heated at 150.degree. C. for 15
min. Cooled to room temperature, diluted with dichloromethane (8
mL), washed with 0.5 N HCl (2.times.4 mL), dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The residue was
purified by silica gel column chromatography using 3% methanol in
dichloromethane to afford 0.214 g of white solid. The solid was
suspended in ether (4.0 mL) was added 2M HCl in ether (1.8 mL, 3.6
mmol). The reaction mixture was stirred for 1 h at room
temperature, concentrate under N.sub.2 flow, than dried under
vacuum to afford 0.21 g (47%) of BB-09 as white solid 1H NMR
(DMSO-d6, 400 MHz): 8.08 (d, J=1.6 Hz, 1H), 7.94 (d, J=1.6 Hz, 1H),
7.87 (t, J=2.0 Hz, 1H), 7.83 (br s, 1H), 7.75-7.79 (m, 1H),
7.46-7.58 (m 4H), 6.77 (br s, 1H), 4.86-5.07 (m, 1H), 4.05 (s, 2H),
3.9-4.25 (m, 2H), 2.79 (s, 3H), 2.65-2.75 (m, 1H), 2.36-2.45 (m,
1H); MS (APCI+): 450.0 (M+1), LC-MS: 98.3%.
[0329] BB-06
##STR00080##
[0330] (JB-159)--previously synthesized by Munagala Rao (see
experimental). Note--1 have a higher yielding procedure which
involves synthesizing the entire right hand portion as the boronic
ester and then performing the Pd coupling as the final step.
(JB-160) Synthesis of the N-oxide of
N-{5-[4-(3-chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-pyridin-2-yl}-
-2-dimethylamino-acetamide (BB-06)
[0331] In an 8 mL vial equipped with a stir bar was placed JB-159
(50 mg, 0.110 mmol) and dichloromethane (1.1 mL). The solution was
cooled to 0.degree. C. and then 3-chloroperbenzoic acid (77% max)
(12.3 mg, 0.0550 mmol) was added and the solution was warmed to
room temperature for 2 hours. The reaction was quenched with 5%
aqueous potassium carbonate (3 mL) and the layers were separated.
The aqueous portion was extracted with dichloromethane (4 mL) and
the organic portions were combined, washed with brine (4 mL), dried
(MgSO.sub.4) and concentrated. The crude material was purified by
preparative TLC (20.times.20 cm, 1500 microns) using 10%
methanol/DCM as the eluent to produce 10 mg of BB-06 as a yellow
solid in 19% yield. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
3.17 (s, 6H), 4.02 (s, 2H), 4.15 (s, 2H), 7.52-7.58 (m, 4H), 7.67
(s, 1H), 7.70 (dd, J=9, 4 Hz, 1H), 8.01 (d, J=9 Hz, 1H), 8.17 (d,
J=7 Hz, 1H), 8.29 (d, J=2 Hz, 1H), 9.41 (s, 1H). MS (APCI+): 471.0
(M+1) LC/MS: 95%
[0332] BA-73
##STR00081##
Synthesis of BOC-Protected Intermediate
[0333] To a suspension of sodium hydride (1.8 mmol, 1.2 eq) in 2 mL
of tetrahydrofuran at 0-5.degree. C. was added a suspension of
MO-64 (1.57 mmol, 1.05 eq), and MO-71 (1.50 mmol, 1.0 eq). The
resultant solution was stirred at 0-5.degree. C. for 20 minutes,
and them ambient temperature for 4 hours. The reaction was diluted
with 10 mL of 15% ammonium chloride, and the aqueous portion
extracted with 2 portions of ethyl acetate. The combined organics
were washed with successive portions of water and brine, dried over
magnesium sulfate, filtered and concentrated to afford the BOC
protected intermediate in 82% yield. .sup.1HNMR, CDCl.sub.3; 400
MHz): 1.46 (s, 9H), 3.46 (dd, J=5.6, 5.2 Hz, 2H), 3.66 (dd, J=5.6,
5.2 Hz, 2H), 4.17 (s, 2H), 4.84 (s, 2H), 7.41-7.48 (M, 2H),
7.54-7.57 (M, 1H), 7.66 (d, J=1.2 Hz, 1H), 8.01 (d, J=6.4 Hz,
1H)
Synthesis of
1-[4-(3-Chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-piperazin-2-one
(BA-73)
[0334] BOC-protected intermediate (1.24 mmol) was stirred with
excess trifluoroacetic acid in dichloromethane at ambient
temperature for 2.5 hours. The reaction was concentrated, the
residue taken into ethyl acetate, and washed with saturated sodium
bicarbonate until the pH of the aqueous was 8-9. The combined
organics were washed with successive portions of water and brine,
dried over magnesium sulfate, filtered and concentrated to afford
the title compound as an oil in 63% yield. .sup.1HNMR,
DMSO-d.sub.6; 400 MHz): 3.11 (dd, J=5.6, 5.2 Hz, 2H), 3.43 (dd,
J=5.6, 5.2 Hz, 2H), 3.63 (s, 2H), 4.83 (s, 2H), 7.41-7.48 (M, 2H),
7.56 (m, 1H), 7.66 (d, J=1.6 Hz, 1H), 7.17 (d, J=6.4 Hz, 1H), 9.03
(s, 1H) LC/MS (84.4%) APCI.sup.+- found: 376.0 calc'd 375.9 m/z
[0335] BA-72
##STR00082##
Synthesis of
4-[4-(3-Chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-3-oxo-piperazine-
-1-carboxylic Acid Ethylamide (BA-72)
[0336] To a solution of BA-73 (0.19 mmol, 1 eq) in 0.7 mL of
pyridine at ambient temperature was added ethyl isocyanate (0.58
mmol, 3 eq), and the resulting mixture stirred for 18 hours at
ambient temperature. The reaction was poured into water (10 mL),
the solids filtered and washed with 2 portions of water and dried
in vacuo over ethyl acetate vapors to afford the title compound
BA-72 as a solid in 50% yield. .sup.1HNMR, DMSO-d.sub.6; 400 MHz):
1.01 (t, J=7.2 Hz, 3H), 3.06 (dd, J=6.4 Hz, 2H), 3.38 (dd, J=5.6
Hz, 2H), 3.6 (t, J=5.6, 5.2 Hz, 2H), 4.03 (s, 2H), 4.75 (s, 2H),
6.62 (dd, J=5.6, 5.2 Hz, 1H), 7.52-7.60 (M, 3H), 7.69 (s, 1H), 8.12
(d, J=7.2 Hz, 1H), 9.44 (s, 1H). LC/MS (87.8%): APCI.sup.+ found:
447.1 calc'd: 446.9 m/z
[0337] BA-71
##STR00083##
Synthesis of
4-[4-(3-Chloro-phenyl)-5-fluoro-benzothiazol-6-ylmethyl]-piperazin-2-one
(BA-71)
[0338] A mixture of MO-64 (0.32 mmol, 1.0 eq), potassium carbonate
(1.8 mmol, 5.6 eq) and 2-oxo-piperazine (0.96 mmol, 3 eq) in 2 mL
of tetrahydrofuran was stirred at ambient temperature for 4 hours.
The reaction mixture was diluted with 50 mL of water, and stirred
for 20 minutes. The solids were collected by filtration, washed
with 2 portions of water, 2 portions of hexanes, and dried in vacuo
at 35-40.degree. C. for 18 hours to afford the title compound BA-71
as a solid in 62% yield. .sup.1HNMR, DMSO-d.sub.6; 400 MHz): 2.80
(dd, J=5.6, 5.2 Hz, 2H), 3.40-3.44 (M, 2H), 3.85 (d, 0.8 Hz, 2H),
5.95 (s, 1H), 7.41-7.47 (M, 2H), 7.56 (dd, J=7.2, 1.6 Hz, 1H), 7.67
(d, J=1.2 Hz, 1H), 8.00 (d, J=6.4 Hz, 1H), 9.04 (s, 1H). LC/MS
(92.8%): APCI.sup.+ found: 376.0 calc'd: 375.9 m/z
[0339] BB-07
##STR00084##
Synthesis of
({5-[4-(3-Chloro-phenyl)-2-methyl-benzothiazol-6-ylmethyl]-pyridin-2-yl}--
methyl-amino)-acetic Acid (BB-07)
[0340] To MR 129 (0.23 g, 0.62 mmol) and methylamino-acetic acid
(2) (0.11 g, 1.24 mmol) was added
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.44 ml 5 eq.). The
reaction mixture was stirred and heated at 150.degree. C. for 20
min. Cooled to room temperature, diluted with dichloromethane (8
mL), washed with 0.1 N HCl (2.times.4 mL), dried with
Na.sub.2SO.sub.4, filtered, and concentrated. The residue was
purified by silica gel preparative plate using 7.5% methanol in
dichloromethane to afford BB-07 (6.16 g) as a sticky solid. This
was triturated with 25% ether/heptane (4.0 mL) to afford 100 mg
product. 1H NMR (CDCl3, 400 MHz): 7.99 (s, 1H), 7.78 (t, J=4 Hz,
1H), 7.68 (dd, J=8, 1.6 Hz, 1H), 7.58 (s, 1H), 7.46 (dd, J=8.8, 2
Hz, 1H), 7.42-7.34 (m, 2H), 7.28 (s, 1H), 6.65 (d, J=8.8 Hz, zH1H),
4.15 (bs, 2H), 4.02 (s, 2H), 3.125 (s, 3H), 2.82 (s, 3H); MS
(APCI+): 438 (M+1), LC-MS: 98%.
TABLE-US-00004 CmpdNo NMR BA-01 1H NMR (CHLOROFORM-d, 400 MHz): d =
8.67 (s, 2 H), 7.57-7.62 (m, 2 H), 7.41 (d, J = 1.6 Hz, 2 H), 7.26
(s, 6 H), 7.24 (d, J = 1.7 Hz, 2 H), 7.17 (s, 4 H), 7.00 (t, J =
8.7 Hz, 4 H), 5.18 (s, 3 H), 4.06 (s, 4 H), 1.56 ppm (s, 22 H)
BA-02 1H NMR (DMSO-d6, 400 MHz): d = 9.30 (s, 1H), 8.64 (t, J = 2.0
Hz, 1 H), 8.27 (d, J = 8 Hz, 1 H), 8.22 (dd, J = 8.4, 1.6 Hz, 1 H),
7.86 (brs, 3 H), 7.76 (t, J = 8.0 Hz, 1 H), 7.69 (brs, 1 H), 7.64
(s, 1 H), 5.48 ppm (s, 2 H) BA-03 1H NMR (CHLOROFORM-d, 400 MHz): d
= 8.13 (s, 3 H), 7.83-7.94 (m, 6 H), 7.45 (d, J = 1.2 Hz, 3 H),
7.28 (d, J = 1.3 Hz, 3 H), 7.26 (s, 3 H), 7.19 (dd, J = 8.4, 5.4
Hz, 6 H), 6.95-7.06 (m, 6 H), 5.22 (s, 6 H), 4.06 (s, 6 H), 1.58
(s, 6 H), 1.26 ppm (s, 2 H) BA-04 1H NMR (CHLOROFORM-d, 400 MHz): d
= 7.25-7.28 (m, 11 H), 7.13-7.21 (m, 9 H), 6.97-7.04 (m, 6 H),
6.89-6.97 (m, 10 H), 6.01 (s, 7 H), 4.03 ppm (s, 7 H) BA-05 1H NMR
(CHLOROFORM-d, 400 MHz): d = 7.56 (d, J = 1.5 Hz, 2 H), 7.30-7.38
(m, 6 H), 7.25-7.30 (m, 9 H), 7.05 (s, 4 H), 6.90 (s, 6 H), 6.01
(s, 4 H), 5.91 (s, 2 H), 5.29-5.31 ppm (m, 3 H) BA-07 1H NMR
(CHLOROFORM-d, 400 MHz): d = 7.80 (s, 7 H), 7.59 (s, 3 H), 7.35 (s,
2 H), 7.26 (s, 4 H), 7.22 (d, J = 1.6 Hz, 3 H), 7.17 (dd, J = 8.4,
5.4 Hz, 5 H), 7.00 (t, J = 8.7 Hz, 5 H), 4.05 (s, 5 H), 2.73 (br.
s., 11 H), 1.25 ppm (s, 4 H) BA-08 1H NMR (CHLOROFORM-d, 400 MHz):
d = 7.42 (s, 1 H), 7.26 (d, J = 0.7 Hz, 7 H), 7.28 (s, 2 H), 7.15
(dt, J = 2.3, 1.2 Hz, 3 H), 6.89 (d, J = 8.1 Hz, 2 H), 6.08 (d, J =
1.7 Hz, 1 H), 6.00 (d, J = 0.7 Hz, 3 H), 5.16 (s, 3 H), 4.07 (s, 3
H), 3.75 (d, J = 0.7 Hz, 5 H), 1.57 (s, 15 H), 0.00 ppm (s, 5 H)
BA-09 1H NMR (DMSO-d6, 400 MHz): d = 7.86 (d, J = 2.1 Hz, 4 H),
7.60 (s, 4 H), 7.46 (d, J = 1.6 Hz, 4 H), 7.27 (s, 8 H), 7.09 (d, J
= 1.5 Hz, 5 H), 7.00-7.06 (m, 4 H), 6.27 (t, J = 1.9 Hz, 4 H), 6.07
(s, 8 H), 5.37 ppm (s, 8 H) BA-10 1H NMR (CHLOROFORM-d, 400 MHz): d
= 8.65-8.68 (m, 2 H), 8.18-8.22 (m, 2 H), 8.07 (dd, J = 7.7, 0.8
Hz, 3 H), 7.52-7.63 (m, 5 H), 7.47 (d, J = 2.3 Hz, 5 H), 7.21-7.32
(m, 6 H), 6.28-6.33 (m, 2 H), 5.42 (s, 5 H), 5.30 (s, 4 H), 1.60
(s, 13 H), 0.00 ppm (s, 3 H) BA-11 1H NMR (400 MHz, DMSO-d6) 8.32
(dd, J = 8.3, 1.5 Hz, 1 H), 8.21 (d, J = 1.5 Hz, 2 H), 7.39 (dd, J
= 8.4, 5.7 Hz, 2 H), 6.95-7.22 (m, 3 H), 6.11 (s, 2 H), 4.20 (s, 2
H) BA-12 1H NMR (DMSO-d6, 400 MHz): d = 8.23 (s, 4 H), 7.87 (s, 4
H), 7.62 (s, 7 H), 7.46 (s, 4 H), 7.34 (s, 3 H), 6.26 (s, 3 H),
5.39 (s, 7 H), 2.64 ppm (s, 11 H) BA-13 1 H NMR (CHLOROFORM-d, 400
MHz): d = 7.88 (t, J = 1.7 Hz, 3 H), 7.62-7.67 (m, 3 H), 7.44-7.50
(m, 3 H), 7.25-7.37 (m, 8 H), 7.14-7.22 (m, 8 H), 6.95-7.02 (m, 5
H), 5.23-5.34 (m, 6 H), 4.02 (s, 6 H), 1.57 (s, 6 H), 1.19-1.30 (m,
2 H), 0.00 ppm (s, 1 H) BA-14 1H NMR (DMSO-d6, 400 MHz): d = 7.86
(s, 4 H), 7.64 (s, 5 H), 7.46 (s, 9 H), 7.40 (s, 7 H), 7.30 (d, J =
1.6 Hz, 7 H), 6.27 (t, J = 2.0 Hz, 4 H), 5.76 (s, 6 H), 5.39 ppm
(s, 7 H) BA-15 (DMSO-d6, 400 MHz), 3.81 (s, 2H), 4.84 (bs, 2H),
6.49 (d, J = 8.4 Hz, 2H), 6.89(d, J = 8.4 Hz, 2H), 7.21 (s, 1H),
7.37 (bs, 2H), 7.68 (t, J = 8.4 Hz, 1H), 7.89 (d, J = 8 Hz, 1H),
8.15 (m, 1H), 8.23 (dd, 1H), 8.26 (s, 1H) BA-16 1H NMR (DMSO-d6,
400 MHz): 10.13 (br s, 2 H), 7.82 (br s, 1 H), 7.64 (d, J = 7.2 Hz,
1 H), 7.53 (s, 1 H), 7.4-7.5 (m, 3 H), 7.36 (d, J = 8.4 Hz, 2 H),
7.3 (d, J = 8.4 Hz, 2 H), 4.48 (br s, 2 H), 4.03 (s, 2H); BA-18 1H
NMR (DMSO-d6, 400 MHz): d = 9.58 (s, 2 H), 7.82 (s, 1 H), 7.53 (s,
4 H), 7.45 (s, 2 H), 7.23-7.27 (m, 5 H), 7.12 (d, J = 8.5 Hz, 3 H),
3.95 (s, 3 H), 3.57 (br. s., 24 H), 2.93 (s, 5 H), 0.00 ppm (s, 2
H) BA-19 1H NMR (DMSO-d6, 400 MHz): 9.62 (s, 1 H), 7.76 (br s, 2
H), 7.59 (d, J = 7.2 Hz, 1 H), 7.53 (s, 1 H), 7.4-7.5 (m, 3 H),
7.22 (d, J = 8.4 Hz, 2 H), 7.14 (d, J = 8.4 Hz, 2 H), 3.95 (s, 2H),
2.94 (s, 3 H); BA-20 (DMSO-d6, 400 MHz), 3.05 (s, 3H), 4.82 (s,
2H), 6.68 (d, J = 8.4 Hz, 2H), 7.14 (d, J = 8.4 Hz, 2H), 7.2 (s,
1H), 7.25 (s, 1H), 7.39 (d, J = 8 Hz, 1H), 7.47 (t, J = 8.4 Hz,
1H), 7.64 (bs, 2H), 7.8 (d, J = 8 Hz, 1H), 8.06 (s, 1H), 9.56 (s,
1H) BA-21 1H NMR (DMSO-d6, 400 MHz): 8.46 (s, 1 H), 7.69 (br s, 2
H), 7.55 (d, J = 7.2 Hz, 1 H), 7.53 (s, 1 H), 7.4-7.5 (m, 4 H),
7.31 (d, J = 8.4 Hz, 2 H), 7.09 (d, J = 8.4 Hz, 2 H), 3.9 (s, 2H);
BA-22 1H NMR (DMSO-d6, 400 MHz): d = 8.13 (s, 1 H), 7.99 (d, J =
7.6 Hz, 1 H), 7.50-7.63 (m, 4 H), 6.97-7.09 (m, J = 8.2 Hz, 2 H),
6.52-6.61 (m, J = 8.2 Hz, 2 H), 4.94 (s, 2 H), 3.99 (s, 2 H),
2.68-2.69 (m, 3 H), 2.69 ppm (s, 3 H) BA-23 1H NMR (DMSO-d6, 400
MHz): 9.42 (s, 1 H), 8.18 (d, J = 6.8 Hz, 1 H), 7.65 (s, 1 H),
7.5-7.59 (m, 3 H), 7.33 (d, J = 8.4 Hz, 2 H), 7.16 (d, J = 8.4 Hz,
2 H), 4.16 (s, 2H); BA-24 (DMSO-d6, 400 MHz), 2.63 (s, 3H), 4.02
(s, 2H), 5.78 (s, 2H), 7.16 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.4
Hz, 2H), 7.44-7.47 (m, 1H), 7.51-7.55 (s, 3H), 7.93 (d, J = 8 Hz,
1H), 8.07 (d, J = 2 Hz, 1H), 8.41 (s, 1H) BA-25 1H NMR
(METHANOL-d4, 400 MHz): d = 9.19 (s, 3 H), 7.95 (s, 3 H), 7.79 (s,
3 H), 7.66 (s, 3 H), 7.40-7.51 (m, 14 H), 7.34 (d, J = 8.3 Hz, 7
H), 4.26 ppm (s, 6 H) BA-26 1H NMR (DMSO-d6, 400 MHz): d = 7.79 (s,
3 H), 7.59 (s, 3 H), 7.40 (s, 13 H), 7.29 (d, J = 8.2 Hz, 10 H),
4.03 (s, 6 H), 3.25 (s, 4 H), 1.03 ppm (s, 4 H) BA-27 1H NMR
(METHANOL-d4, 400 MHz): d = 7.52 (s, 5 H), 7.32 (s, 4 H), 7.17 (s,
3 H), 4.05 (s, 3 H), 2.66 ppm (br. s., 19 H) BA-28 1H NMR (acetone,
400 MHz): d = 9.23 (s, 1 H), 7.98 (d, J = 1.6 Hz, 3 H), 7.97 (br.
s., 1 H), 7.82 (s, 1 H), 7.81 (t, J = 1.3 Hz, 1 H), 7.62 (d, J =
1.5 Hz, 1 H), 7.40-7.52 (m, 6 H), 7.20 (d, J = 8.5 Hz, 3 H), 5.36
(br. s., 3 H), 4.15 (s, 3 H), 2.81 (s, 8 H), 2.09 (s, 1 H), 2.05
(dt, J = 4.4, 2.2 Hz, 17 H), 1.29 ppm (s, 1 H) BA-29 1H NMR
(DMSO-d6, 400 MHz): 8.31 (s, 1 H), 7.65 (s, 2 H), 7.54 (d, J = 7.2
Hz, 1 H), 7.52 (s, 1 H), 7.4-7.48 (m, 3 H), 7.29 (d, J = 8.4 Hz, 2
H), 7.09 (d, J = 8.4 Hz, 2 H), 6.0-6.05 (m, 1 H), 3.89 (s, 2H),
3.2-3.6 (m, 2 H), 1.03 (t, J = 7.24 Hz, 3 H); BA-30 1H NMR
(DMSO-d6, 400 MHz): 10.59 (s, 1 H), 9.41 (s, 1 H), 7.17 (d, J = 6.8
Hz, 1 H), 7.67 (s, 1 H), 7.64 (d, J = 8.8 Hz, 1 H), 7.5-7.6 (m, 3
H), 7.3 (d, J = 8.4 Hz, 2 H), 4.15 (s, 2H), 3.82 (q, J = 6.8 Hz, 2
H), 0.98 (t, J = 6.8 Hz, 3 H); BA-31 (CDC13, 400 MHz), 2.57 (t,
6H), 3.45 (t, 6H), 3.78 (s, 2H), 4.44 (bs, 2H), 7.41-7.47(m, 2H),
7.55-7.57 (m, 1H), 7.66 (d, 2H), 8 (d, J = 6.4 Hz, 1H), 9.02 (s,
1H) BA-32 (DMSO-d6, 400 MHz), 4.05 (bs, 2H), 6.85 (d, J = 8.4 Hz,
1H), 7.18 (d, J = 8.4 Hz, 1H), 7.26 (d, J = 7.2 Hz, 1H), 7.29 (s,
1H), 7.37-7.45 (m, 3H), 7.47 (t, J = 8.4 Hz, 1 H), 7.65 (bs, 2H),
7.9 (d, J = 7.2 Hz, 1H), 8.13 (s, 1H) BA-33 1H NMR (DMSO-d6, 400
MHz): 10.4 (br s, 1 H), 9.43 (s, 1 H), 8.18 (d, J = 2.0 Hz, 1 H),
8.16 (d, J = 7.2 Hz, 1 H), 7.92 (d, 7 = 8.4 Hz, 1 H), 7.75 (br s, 1
H), 7.67 (s, 1 H), 7.5-7.6 (m, 3 H), 7.34 (d, J = 8.8 Hz, 1 H),
4.17 (s, 2 H), 3.15-3.24 (m, 2 H), 1.09 (t, J = 7.2 Hz, 3 H); BA-38
1H NMR (DMSO-d6, 400 MHz): 9.41 (s, 1 H), 8.14 (d, J = 4.4 Hz, 1
H), 8.14 (s, 1 H), 7.67 (s, 1 H), 7.63 (dd, J = 8.4, 2.4 Hz, 1 H),
7.5-7.59 (m, 3 H), 6.78 (d, J = 8.48 Hz, 1 H), 4.11 (s, 2 H), 3.82
(s, 3 H); BA-39 .sup.1H NMR (400 MHz, CDCl.sub.3): 8.98 (s, 1 H),
7.68 (d, J = 6.8 Hz, 1 H), 7.6 (s, 1 H), 7.67 (s, 1 H), 7.56 (dd, J
= 7.2, 1.2 Hz, 1 H), 7.38-7.46 (m, 2 H), 7.2 (t, J = 8.0 Hz, 1 H),
6.86 (d, J = 7.6 Hz, 1 H), 6.7-6.76 (m, 2 H), 5.3 (s, 1H), 4.13 (s,
2 H); BA-40 1H NMR (DMSO-d6, 400 MHz): 10.04 (s, 1 H), 9.41 (s, 1
H), 8.23 (d, J = 2.4 Hz, 1 H), 8.15 (d, J = 7.2 Hz, 1 H), 7.76 (d,
J = 8.8 Hz, 1 H), 7.64- 7.69 (m, 2 H), 7.5-7.58 (m, 3 H), 4.13 (s,
2 H), 4.09-4.16 (q, J = 7.2 Hz, 2 H), 1.22 (t, J = 7.2 Hz, 3 H);
BA-43 (CDCl3, 400 MHz), 2.57 (t, 6H), 3.68-3.75 (overlap, 8H),
7.41-7.47(m, 2H), 7.55-7.57 (m, 1H), 7.66 (d, 2H), 8.01 (d, J = 6.4
Hz, 1H), 9.02 (s, 1H) BA-44 (CDCl3, 400 MHz), 2.53-2.57 (m, 4H),
3.5 (t, 2H), 3.66 (t, 2H), 3.78 (s, 3H), 7.41-7.47(m, 2H),
7.55-7.58 (m, 1H), 7.66 (d, 1H), 8.0 (d, J = 6.4 Hz, 1H), 9.02 (s,
1H) BA-45 (DMSO-d6, 400 MHz), 1.13 (t, 3H), 1.69-1.97 (m, 4H), 3.15
(t, 2H), 3.48 (t, 2H), 3.98 (m, 3H), 4.48 (d, 2H), 7.32(d, 1H),
7.55-7.62 (m, 2H), 7.73 (s, 1H), 8.47 (d, J = 6.4 Hz, 1H), 9.57 (s,
1H), 10.02 (bs, 1H) BA-46 (CDCl3, 400 MHz), 1.45-1.55 (m, 2H), 1.98
(d, 2H), 2.25-2.31 (t, 2H), 2.9 (d, 2H), 3.58-3.6 (m, 1H), 3.74 (s,
2H), 4.27 (s, 2H), 4.36 (d, 1H), 7.39-7.47(m, 2H), 7.55-7.57 (m,
1H), 7.66 (d, 1H), 7.98 (d, J = 6.4 Hz, 1H), 9.01 (s, 1H) BA-47
(CDCl3, 400 MHz), 1.46-1.56 (m, 2H), 1.87 (d, 2H), 2.16-2.21
(overlap, 4H), 2.77 (m, 1H), 2.93 (d, 2H), 3.74 (s, 2H),
7.39-7.46(m, 2H), 7.54- 7.57 (m, 1H), 7.66 (d, J = 1.6 Hz, 1H),
8.01 (d, J = 6.4 Hz, 1H), 9 (s, 1H) BA-48 (CDCl3, 400 MHz), 2.55
(t, 4H), 2.94 (t, 4H), 3.75 (s, 2H), 7.39-7.47(m, 2H), 7.55-7.57
(m, 2H), 7.66 (d, 1H), 8.01 (d, J = 6.4 Hz, 1H), 9.01 (s, 1H) BA-49
(CDCl3, 400 MHz), 1.25 (t, 3H), 2.53 (t, 4H), 3.52 (t, 4H), 3.77
(s, 2H), 4.14 (q, 2H), 7.41-7.47(m, 2H), 7.55-7.57 (m, 1H), 7.66
(d, 1H), 8 (d, J = 6.4 Hz, 1H), 9.02 (s, 1H) BA-50 (CDCl3, 400
MHz), 2.31 (s, 3H), 2.51 (t, 4H), 2.61 (t, 4H), 3.77 (s, 2H),
7.39-7.46(m, 2H), 7.54-7.57 (m, 1H), 7.66 (d, 1H), 8 (d, J = 6.4
Hz, 1H), 9.01 (s, 1H) BA-51 1H NMR (DMSO-d6, 400 MHz): 9.39 (s, 1
H), 8.45 (s, 1 H), 8.1 (d, J = 7.2 Hz, 1 H), 7.66 (s, 1 H),
7.5-7.59 (m, 4 H), 7.32 (d, J = 8.5 Hz, 2 H), 7.14 (d, J = 8.5 Hz,
2 H), 5.77 (s, 2 H), 4.06 ppm (s, 2 H); BA-52 1H NMR (DMSO-d6, 400
MHz): 10.78 (s, 1H), 9.58 (s, 1 H), 8.52 (d, J = 6.8 Hz, 1 H, 7.75
(s, 1 H), 7.54-7.68 (m, 3 H), 6.73 (s, 1 H), 4.55 (s, 2 H), 3.47
(m, 2H), 3.0-3.2 (m, 6 H), 1.01 ppm (t, J = 1.2 Hz, 3 H);
[0341] Methods of the invention parallel the compositions and
formulations. The methods comprise administering to a patient in
need of treatment a therapeutically effective amount of a compound
according to the invention. The present invention also provides a
method for inhibiting phosphodiesterase 4.
[0342] In-vitro assay for PDE4 enzymes. The in-vitro activity of
PDE4 enzymes and the in-vitro potency of therapeutic agents
described in the present invention was measured using a real-time,
enzyme-coupled spectrophotometric assay. By using three different
coupling enzymes, the product of the PDE4 reaction is coupled to
the oxidation of the reduced form .beta.-nicotinamide adenine
dinucleotide (NADH), which dissipation can be monitored
spectrophotometrically at 340 nM.
[0343] Assay description. Buffer A containing 50 mM Tris, pH 8.0,
16 mM MgCl.sub.2 and 80 mM KCl is prepared and stored at room
temperature. Buffer B containing 50 mM Tris, pH 8.0 is prepared and
stored at room temperature. Stock solutions of the following
reagents are prepared in Buffer B and stored at -20.degree. C.:
Adenosine-5'-triphosphate (ATP), cyclic adenosine-5'-monophosphate
(cAMP), phosphoenolpyruvate (PEP) and NADH. An assay mix is
prepared by mixing Buffer A, trichloroethylphosphine (TCEP), ATP,
PEP, NADH, myokinase (MK), pyruvate kinase (PK), lactate
dehydroganese (LDH) and PDE4 to a final volume of 20 mL, which is
enough for a single 96-well assay plate. Assay mix (180 .mu.L) and
test article (10 .mu.L) in 1:1 DMSO/H.sub.2O mixture is
pre-incubated at room temperature for 10 min. The enzymatic
reaction is initiated by addition of cAMP (10 .mu.L). Final
concentration of all components in the assay (200 .mu.L/well) are
as follows: 10 mM MgCl.sub.2, 50 mM KCl, 5 mM TCEP, 2.5% DMSO, 0.4
mM NADH, 1 mM PEP, 0.04 mM ATP, 5 units MK, 1 unit PK, 1 unit LDH
and appropriate amount of PDE4. Reaction progress curves are
monitored in a plate reader capable of measuring light absorbance
at 340 nM. A decrease in light absorbance at 340 nm is due to
oxidation of NADH. Positive controls containing no test article and
negative controls containing no test article and no cAMP are
included on every assay plate. Reaction rates are determined from
the slopes of the linear portions of the progress curves. All data
is percent normalized with respect to controls and presented as
percent inhibition. Compounds BB-01, BB-02, BB-03, BB-04, BB-05,
BB-08, BB-09 gave IC50<5 uM for human PDE4D and PDE4B
isozymes.
[0344] The activity of PDE4 inhibitors described in the present
invention can be measured using in an ex-vivo assay measuring
leukotriene E4 (LTE4) in human whole blood after Sephadex
stimulation. The anti-inflammatory activity of therapeutic agents
of the present invention is demonstrated by the inhibition of
eosinophil activation as measured by sephadex bead stimulated LTE4
production in whole human blood. For each sample, 356 .mu.l of
heparinized human whole blood (Vacutainer tube #6480) is added to
wells of a 96 well plate. Then, 4 .mu.l of a series of compound
dilutions (in DMSO) are added in triplicates, suspension mixed and
allowed to incubate at 37.degree. C. for 15 min with gentle
shaking. After that, blood samples are stimulated by adding 40
.mu.L of Sephadex G-15 beads (Sigma-Aldrich, Sweden). The beads are
predissolved in PBS (0.16 g/mL PBS). After mixing, the suspension
is incubated at 37.degree. C. for 90 min. Then, 8 .mu.L of 15%
EDTA/PBS is added to each sample, mixed and plate centrifuged for 5
min at 115.times.g at 21.degree. C. and supernatants taken. In each
plate, 10 positive controls and 10 negative controls are used,
containing DMSO instead of compound solution. The positive controls
are stimulated with Sephadex as described for the samples, and in
the negative controls (unstimulated), Sephadex solution is replaced
by PBS. LTE.sub.4 levels in the resulting plasma samples are
determined using a commercial enzyme-linked immunoassay (Cayman
Chemical Company, Ann Arbor, Mich.) according to the manufacturer's
instructions.
* * * * *