U.S. patent application number 10/974893 was filed with the patent office on 2005-05-26 for 2,3-diaryl-pyrazolo[1,5-b]pyridazines derivatives, their preparation and their use as cyclooxygenase, 2 (cox-2) inhibitors.
Invention is credited to Beswick, Paul John, Bountra, Charanjit, Campbell, Ian Baxter, Mathews, Neil, Naylor, Alan.
Application Number | 20050113377 10/974893 |
Document ID | / |
Family ID | 26312181 |
Filed Date | 2005-05-26 |
United States Patent
Application |
20050113377 |
Kind Code |
A1 |
Beswick, Paul John ; et
al. |
May 26, 2005 |
2,3-diaryl-pyrazolo[1,5-b]pyridazines derivatives, their
preparation and their use as cyclooxygenase, 2 (COX-2)
inhibitors
Abstract
The invention provides a pharmaceutical composition comprising a
compound of formula (I) 1 or a pharmaceutically acceptable salt,
solvate, ester or salt or solvate of such ester, of a compound of
formula (I) in which: R.sup.0 is halogen, C.sub.1-6alkyl,
C.sub.1-6alkoxy, C.sub.1-6alkoxy substituted by one or more
fluorine atoms, or O(CH.sub.2).sub.nNR.sup.4R.- sup.5; R.sup.1 and
R.sup.2 are independently selected from H, C.sub.1-6alkyl,
C.sub.1-6alkyl substituted by one or more fluorine atoms,
C.sub.1-6alkoxy, C.sub.1-6hydroxyalkyl, SC.sub.1-6alkyl, C(O)H,
C(O)C.sub.1-6alkyl, C.sub.1-6alkylsulphonyl, C.sub.1-6alkoxy
substituted by one or more fluorine atoms,
O(CH.sub.2).sub.nCO.sub.2C.sub.1-6alkyl,
O(CH.sub.2).sub.nSC.sub.1-6alkyl, (CH.sub.2).sub.nNR.sup.4R.sup.5,
(CH.sub.2).sub.nSC.sub.1-6alkyl or C(O)NR.sup.4R.sup.5; with the
proviso that when R.sup.0 is at the 4-position and is halogen, at
least one of R.sup.1 and R.sup.2 is C.sub.1-6alkylsulphonyl,
C.sub.1-6alkoxy substituted by one or more fluorine atoms,
O(CH.sub.2).sub.nCO.sub.2C.sub- .1-6alkyl,
O(CH.sub.2).sub.nSC.sub.1-6alkyl, (CH.sub.2).sub.nNR.sup.4R.sup- .5
or (CH.sub.2).sub.nSC.sub.1-6alkyl, C(O)NR.sup.4R.sup.5; R.sup.3 is
C.sub.1-6alkyl or NH.sub.2; R.sup.4 and R.sup.5 are independently
selected from H and C.sub.1-6alkyl or, together with the nitrogen
atom to which they are attached, form a 4-8 membered saturated
ring; and n is 1-4; and an inhibitor of the release, or action, of
tumour necrosis factor .alpha..
Inventors: |
Beswick, Paul John;
(Stevenage, GB) ; Bountra, Charanjit; (Harlow,
GB) ; Campbell, Ian Baxter; (Stevenage, GB) ;
Mathews, Neil; (London, GB) ; Naylor, Alan;
(Stevenage, GB) |
Correspondence
Address: |
DAVID J LEVY, CORPORATE INTELLECTUAL PROPERTY
GLAXOSMITHKLINE
FIVE MOORE DR., PO BOX 13398
RESEARCH TRIANGLE PARK
NC
27709-3398
US
|
Family ID: |
26312181 |
Appl. No.: |
10/974893 |
Filed: |
October 27, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10974893 |
Oct 27, 2004 |
|
|
|
10212513 |
Aug 5, 2002 |
|
|
|
6861429 |
|
|
|
|
Current U.S.
Class: |
514/248 ;
544/235 |
Current CPC
Class: |
A61P 31/00 20180101;
A61P 17/02 20180101; A61P 25/16 20180101; A61P 1/00 20180101; A61P
9/00 20180101; A61P 29/02 20180101; A61P 19/06 20180101; Y02P 20/55
20151101; A61P 3/00 20180101; A61P 27/16 20180101; A61P 25/28
20180101; A61P 25/06 20180101; A61P 27/02 20180101; A61P 5/00
20180101; A61P 7/06 20180101; A61P 11/00 20180101; A61P 21/00
20180101; A61P 29/00 20180101; A61K 31/50 20130101; A61P 3/10
20180101; A61P 17/00 20180101; A61P 19/00 20180101; A61P 25/00
20180101; A61P 37/00 20180101; C07D 487/04 20130101; A61P 15/00
20180101; A61P 19/02 20180101; A61P 11/06 20180101; A61P 1/04
20180101; A61P 7/00 20180101; A61P 35/00 20180101 |
Class at
Publication: |
514/248 ;
544/235 |
International
Class: |
A61K 031/503; C07D
487/04 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 5, 1997 |
GB |
9718792.6 |
Dec 23, 1997 |
GB |
9727116.7 |
Claims
1-13. (canceled)
14. A pharmaceutical composition comprising a compound of formula
(I) 11or a pharmaceutically acceptable salt, solvate, ester or salt
or solvate of such ester, of a compound of formula (I) in which:
R.sup.0 is halogen, C.sub.1-6alkyl, C.sub.1-6alkoxy,
C.sub.1-6alkoxy substituted by one or more fluorine atoms, or
O(CH.sub.2).sub.nNR.sup.4R.sup.5; R.sup.1 and R.sup.2 are
independently selected from H, C.sub.1-6alkyl, C.sub.1-6alkyl
substituted by one or more fluorine atoms, C.sub.1-6alkoxy,
C.sub.1-6hydroxyalkyl, SC.sub.1-6alkyl, C(O)H, C(O)C.sub.1-6alkyl,
C.sub.1-6alkylsulphonyl, C.sub.1-6alkoxy substituted by one or more
fluorine atoms, O(CH.sub.2).sub.nCO.sub.2C.sub.1-6alkyl,
O(CH.sub.2).sub.nSC.sub.1-6alkyl, (CH.sub.2).sub.nNR.sup.4R.sup.5,
(CH.sub.2).sub.nSC.sub.1-6alkyl or C(O)NR.sup.4R.sup.5; with the
proviso that when R.sup.0 is at the 4-position and is halogen, at
least one of R.sup.1 and R.sup.2 is C.sub.1-6alkylsulphonyl,
C.sub.1-6alkoxy substituted by one or more fluorine atoms,
O(CH.sub.2).sub.nCO.sub.2C.sub- .1-6alkyl,
O(CH.sub.2).sub.nSC.sub.1-6alkyl, (CH.sub.2).sub.nNR.sup.4R.sup- .5
or (CH.sub.2).sub.nSC.sub.1-6 alkyl, C(O)NR.sup.4R.sup.5; R.sup.3
is C.sub.1-6alkyl or NH.sub.2; R.sup.4 and R.sup.5 are
independently selected from H and C.sub.1-6alkyl; and n is 1-4; and
an inhibitor of the release, or action, of tumour necrosis factor
.alpha..
15. The pharmaceutical composition according to claim 14 further
comprising one or more physiologically acceptable carriers or
excipients.
16. A method of treating an animal subject suffering from a
condition which is mediated by selective inhibition of COX-2, said
method comprising administering to said subject an effective amount
of the pharmaceutical composition according to claim 14.
17. The method according to claim 16, wherein said animal subject
is a human.
18. The method according to claim 16, wherein said condition which
is mediated by selective inhibition of COX-2 is selected from the
group consisting of pain, fever and inflammation.
19. The method according to claim 16, wherein said condition which
is mediated by selective inhibition of COX-2 is selected from the
group consisting of rheumatic fever, influenza, cold, lower back
pain, neck pain, headache, toothache, sprains, strains, myositis,
neuralgia, synovitis, arthritis, rheumatoid arthritis, degenerative
joint diseases, osteoarthristis, gout, ankylosing spondylitis,
tendinitis, bursitis, psoriasis, eczema, burns, dermatitis, sports
injuries, and injuries arising from surgical and dental
procedures.
20. The method according to claim 16, wherein said condition which
is mediated by selective inhibition of COX-2 is selected from the
group consisting of colonic cancer, stroke, epilepsy, epileptic
seizures, dysmenorrhoea, premature labour, asthma, allergic
rhinitis, respiratory distress syndrome, inflammatory bowel
disease, Crohn's disease, gastritis, irritable bowel syndrome,
ulcerative colitis, inflammation in vascular disease, inflammation
in migraine, inflammation in periarteritis, inflammation in nodosa,
inflammation in thyroiditis, inflammation in aplastic anemia,
inflammation in Hodgkin's disease, inflammation in sclerodoma,
inflammation in type 1 diabetes, inflammation in myasthenia gravis,
inflammation in multiple sclerosis, inflammation in sorcoidosis,
inflammation in nephrotic syndrome, inflammation in Bechet's
syndrome, inflammation in polymyositis, inflammation in gingivitis,
inflammation in conjunctivitis, inflammation in myocardial
ischemia, retinitis, retinopathies, uveitis and acute injury to the
eye.
21. The method according to claim 16, wherein said condition which
is mediated by selective inhibition of COX-2 is selected from the
group consisting of dementia, degenerative dementia, senile
dementia, Alzheimer's disease, Pick's disease, Huntington's chorea,
Parkinson's disease, Creutzfeldt-Jakob disease, vascular dementia,
dementia associated with intracranial space occupying lesions,
dementia associated with trauma, dementia associated with
infections, dementia associated with HIV infections, dementia
associated with metabolism, dementia associated with toxins,
dementia associated with anoxia, dementia associated with vitamin
deficiency, mild cognitive impairment associated with aging and Age
Associated Memory Impairment.
22. The method according to claim 16 wherein said condition which
is mediated by selective inhibition of COX-2 is an inflammatory
disorder.
23. The method according to claim 16 wherein said condition which
is mediated by selective inhibition of COX-2 is arthritis.
24. The method according to claim 16 wherein said condition which
is mediated by selective inhibition of COX-2 is rheumatoid
arthritis or osteoarthritis.
25. The method according to claim 16 wherein said condition which
is mediated by selective inhibition of COX-2 is inflammation in
migraine.
26. The method according to claim 16 wherein said condition which
is mediated by selective inhibition of COX-2 is inflammation in
multiple sclerosis.
27. A pharmaceutical composition comprising a compound selected
from the group consisting of:
3-(4-methanesulfonyl-phenyl)-2-(4-methoxy-phenyl)-py-
razolo[1,5-b]pyridazine;
6-difluoromethoxy-2-(4-fluoro-phenyl)-3-(4-methan-
esulfonyl-phenyl)-pyrazolo[1,5-b]pyridazine;
2-(4-ethoxy-phenyl)-3-(4-meth-
anesulfonyl-phenyl)-pyrazolo[1,5-b]pyridazine;
2-(4-fluoro-phenyl)-6-metha-
nesulfonyl-3-(4-methanesulfonyl-phenyl)-pyrazolo[1,5-b]pyridazine;
2-(4-difluoromethoxy-phenyl)-3-(4-methanesulfonyl-phenyl)-pyrazolo[1,5-b]-
pyridazine;
4-[2-(4-ethoxy-phenyl)-pyrazolo[1,5-b]pyridazin-3-yl]-benzenes-
ulfonamide; and
6-difluoromethoxy-2-(3-fluoro-phenyl)-3-(4-methanesulfonyl-
-phenyl)-pyrazolo[1,5-b]pyridazine; and pharmaceutically acceptable
salts, solvates, esters and salts or solvates of such esters,
thereof and an inhibitor of the release, or action, of tumour
necrosis factor .alpha..
28. The pharmaceutical composition according to claim 27 further
comprising one or more physiologically acceptable carriers or
excipients.
29. The pharmaceutical composition according to claim 27, wherein
the compound is selected from the group consisting of
2-(4-ethoxy-phenyl)-3-(-
4-methanesulfonyl-phenyl)-pyrazolo[1,5-b]pyridazine;
6-difluoromethoxy-2-(3-fluoro-phenyl)-3-(4-methanesulfonyl-phenyl)-pyrazo-
lo[1,5-b]pyridazine; and pharmaceutically acceptable salts,
solvates, esters and salts or solvates of such ester, thereof.
30. A method of treating an animal subject suffering from a
condition which is mediated by selective inhibition of COX-2, said
method comprising administering to said subject an effective amount
of the pharmaceutical composition according to claim 29.
31. The method according to claim 30 wherein said condition which
is mediated by selective inhibition of COX-2 is an inflammatory
disorder.
32. The method according to claim 30 wherein said condition which
is mediated by selective inhibition of COX-2 is arthritis.
33. The method according to claim 30 wherein said condition which
is mediated by selective inhibition of COX-2 is rheumatoid
arthritis or osteoarthritis.
34. The method according to claim 30 wherein said condition which
is mediated by selective inhibition of COX-2 is inflammation in
migraine.
35. The method according to claim 30 wherein said condition which
is mediated by selective inhibition of COX-2 is inflammation in
multiple sclerosis.
Description
[0001] This invention relates to pyrazolo[1,5-b]pyridazine
derivatives, to processes for their preparation, to pharmaceutical
compositions containing them and to their use in medicine.
[0002] The enzyme cyclooxygenase (COX) has recently been discovered
to exist in two isoforms, COX-1 and COX-2. COX-1 corresponds to the
originally identified constitutive enzyme while COX-2 is rapidly
and readily inducible by a number of agents including mitogens,
endotoxin, hormones, cytokines and growth factors. Prostaglandins
generated by the action of COX have both physiological and
pathological roles. It is generally believed that COX-1 is
responsible for the important physiological functions such as
maintenance of gastrointestinal integrity and renal blood flow. In
contrast the inducible form, COX-2, is believed to be responsible
for the pathological effects of prostaglandins where rapid
induction of the enzyme occurs in response to such agents as
inflammatory agents, hormones, growth factors and cytokines. A
selective inhibitor of COX-2 would therefore have
anti-inflammatory, anti-pyretic and analgesic properties, without
the potential side effects associated with inhibition of COX-1. We
have now found a novel group of compounds which are both potent and
selective inhibitors of COX-2.
[0003] The invention thus provides the compounds of formula (I)
2
[0004] and pharmaceutically acceptable derivatives thereof in
which:
[0005] R.sup.0 is halogen, C.sub.1-6alkyl, C.sub.1-6alkoxy,
C.sub.1-6alkoxy substituted by one or more fluorine atoms, or
O(CH.sub.2).sub.nNR.sup.4R.sup.5;
[0006] R.sup.1 and R.sup.2 are independently selected from H,
C.sub.1-6alkyl, C.sub.1-6alkyl substituted by one or more fluorine
atoms, C.sub.1-6alkoxy, C.sub.1-6hydroxyalkyl, SC.sub.1-6alkyl,
C(O)H, C(O)C.sub.1-6alkyl, C.sub.1-6alkylsulphonyl, C.sub.1-6alkoxy
substituted by one or more fluorine atoms,
O(CH.sub.2).sub.nCO.sub.2C.sub.1-6alkyl,
O(CH.sub.2).sub.nSC.sub.1-6alkyl, (CH.sub.2).sub.nNR.sup.4R.sup.5,
(CH.sub.2).sub.nSC.sub.1-6alkyl or C(O)NR.sup.4R.sup.5; with the
proviso that when R.sup.0 is at the 4-position and is halogen, at
least one of R.sup.1 and R.sup.2 is C.sub.1-6alkylsulphonyl,
C.sub.1-6alkoxy substituted by one or more fluorine atoms,
O(CH.sub.2).sub.nCO.sub.2C.sub- .1-6alkyl,
O(CH.sub.2).sub.nSC.sub.1-6alkyl, (CH.sub.2).sub.nNR.sup.4R.sup- .5
or (CH.sub.2).sub.nSC.sub.1-6alkyl, C(O)NR.sup.4R.sup.5;
[0007] R.sup.3 is C.sub.1-6alkyl or NH.sub.2;
[0008] R.sup.4 and R.sup.5 are independently selected from H, or
C.sub.1-6alkyl or, together with the nitrogen atom to which they
are attached, form a 4-8 membered saturated ring; and
[0009] n is 1-4.
[0010] By pharmaceutically acceptable derivative is meant any
pharmaceutically acceptable salt, solvate or ester, or salt or
solvate of such ester, of the compounds of formula (I), or any
other compound which upon administration to the recipient is
capable of providing (directly or indirectly) a compound of formula
(I) or an active metabolite or residue thereof.
[0011] It will be appreciated that, for pharmaceutical use, the
salts referred to above will be the physiologically acceptable
salts, but other salts may find use, for example in the preparation
of compounds of formula (I) and the physiologically acceptable
salts thereof.
[0012] Suitable pharmaceutically acceptable salts of the compounds
of formula (I) include acid addition salts formed with inorganic or
organic acids, preferably inorganic acids, e.g. hydrochlorides,
hydrobromides and sulphates.
[0013] The term halogen is used to represent fluorine, chlorine,
bromine or iodine.
[0014] The term `alkyl` as a group or part of a group means a
straight or branched chain alkyl group, for example a methyl,
ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl group.
[0015] Preferably, R.sup.0 is at the 3- or 4-position of the phenyl
ring, as defined in formula (I).
[0016] Preferably, R.sup.0 is at the 6-position of the pyridazine
ring, as defined in formula (I).
[0017] Preferably, R.sup.0 is F, C.sub.1-3alkyl, C.sub.1-3alkoxy,
C.sub.1-3alkoxy substituted by one or more fluorine atoms, or
O(CH.sub.2).sub.1-3NR.sup.4R.sup.5. More preferably R.sup.0 is F,
C.sub.1-3alkoxy or C.sub.1-3alkoxy substituted by one or more
fluorine atoms.
[0018] Preferably, R.sup.1 is C.sub.1-4alkylsulphonyl,
C.sub.1-4alkoxy substituted by one or more fluorine atoms,
O(CH.sub.2).sub.1-3CO.sub.2C.s- ub.1-4alkyl,
O(CH.sub.2).sub.1-3SC.sub.1-4alkyl, (CH.sub.2).sub.1
NR.sup.4R.sup.5, (CH.sub.2).sub.1-3SC.sub.1-4alkyl or
C(O)NR.sup.4R.sup.5 or, when R.sup.0 is C.sub.1-6alkyl,
C.sub.1-4alkoxy, O(CH.sub.2).sub.nNR.sup.4R.sup.5, may also be H.
More preferably R.sup.1 is C.sub.1-4alkylsulphonyl, C.sub.1-4alkoxy
substituted by one or more fluorine atoms or, when R.sup.0 is
C.sub.1-6alkyl, C.sub.1-6alkoxy, C.sub.1-6alkoxy substituted by one
or more fluorine atoms, or O(CH.sub.2).sub.nNR.sup.4R.sup.5, may
also be H.
[0019] Preferably, R.sup.2 is H.
[0020] Preferably, R.sup.3 is methyl or NH.sub.2.
[0021] Preferably R.sup.4 and R.sup.5 are independently
C.sub.1-3alkyl or, together with the nitrogen atom to which they
are attached, form a 5-6 membered saturated ring.
[0022] Preferably, n is 1-3, more preferably 1 or 2.
[0023] Within the invention there is provided one group of
compounds of formula (I) (group A) wherein: R.sup.0 is F,
C.sub.1-3alkyl, C.sub.1-3alkoxy, C.sub.1-3alkoxy substituted by one
or more fluorine atoms, or O(CH.sub.2).sub.nNR.sup.4R.sup.5;
R.sup.1 is C.sub.1-4alkylsulphonyl, C.sub.1-4alkoxy substituted by
one or more fluorine atoms,
O(CH.sub.2).sub.nCO.sub.2C.sub.1-4alkyl,
O(CH.sub.2).sub.nSC.sub.1-4alkyl, (CH.sub.2).sub.nNR.sup.4R.sup.5,
(CH.sub.2).sub.nSC.sub.1-4alkyl or C(O)NR.sup.4R.sup.5 or, when
R.sup.0 is C.sub.1-3alkyl, C.sub.1-3alkoxy, C.sub.1-3alkoxy
substituted by one or more fluorine atoms, or
O(CH.sub.2).sub.nNR.sup.4R.sup.5, may also be H; R.sup.2 is H;
R.sup.3 is methyl or NH.sub.2; R.sup.4 and R.sup.5 are
independently C.sub.1-3alkyl or, together with the nitrogen atom to
which they are attached, form a 5-6 membered saturated ring; and n
is 1-3.
[0024] Within group A, there is provided another group of compounds
(group A1) wherein R.sup.0 is F, methyl, C.sub.1-2alkoxy,
OCHF.sub.2, or O(CH.sub.2).sub.nNR.sup.4R.sup.5; R.sup.1 is
methylsulphonyl, OCHF.sub.2,
O(CH.sub.2).sub.nCO.sub.2C.sub.1-4alkyl,
O(CH.sub.2).sub.nSCH.sub.3, (CH.sub.2).sub.nNR.sup.4R.sup.5,
(CH.sub.2).sub.nSCH.sub.3 or C(O)NR.sup.4R.sup.5 or, when R.sup.0
is methyl, C.sub.1-2alkoxy, OCHF.sub.2, or
O(CH.sub.2).sub.nN(CH.sub.3).sub.2, may also be H; R.sup.2 is H;
R.sup.3 is methyl or NH.sub.2; R.sup.4 and R.sup.5 are both methyl
or, together with the nitrogen atom to which they are attached,
form a 5-6 membered saturated ring; and n is 1-2.
[0025] Within group A, there is provided a further group of
compounds (group A2) wherein R.sup.0 is F, C.sub.1-3alkoxy or
C.sub.1-3alkoxy substituted by one or more fluorine atoms; R.sup.1
is C.sub.1-4alkylsulphonyl, C.sub.1-4alkoxy substituted by one or
more fluorine atoms or, when R.sup.0 C.sub.1-3alkoxy or
C.sub.1-3alkoxy substituted by one or more fluorine atoms, may also
be H; R.sup.2 is H; and R.sup.3 is methyl or NH.sub.2.
[0026] Within groups A, A1 and A2, R.sup.0 is preferably at the 3-
or 4-position of the phenyl ring and R.sup.2 is preferably at the
6-position of the pyridazine ring.
[0027] It is to be understood that the present invention
encompasses all isomers of the compounds of formula (I) and their
pharmaceutically acceptable derivatives, including all geometric,
tautomeric and optical forms, and mixtures thereof (e.g. racemic
mixtures).
[0028] Particularly preferred compounds of the invention are:
[0029]
3-(4-methanesulfonyl-phenyl)-2-(4-methoxy-phenyl)-pyrazolo[1,5-b]py-
ridazine;
[0030]
6-difluoromethoxy-2-(4-fluoro-phenyl)-3-(4-methanesulfonyl-phenyl)--
pyrazolo[1,5-b]pyridazine;
[0031]
2-(4-ethoxy-phenyl)-3-(4-methanesulfonyl-phenyl)pyrazolo[1,5-b]pyri-
dazine;
[0032]
2-(4-fluoro-phenyl)-6-methanesulfonyl-3-(4-methanesulfonyl-phenyl)--
pyrazolo[1,5-b]pyridazine;
[0033]
2-(4-difluoromethoxy-phenyl)-3-(4-methanesulfonyl-phenyl)-pyrazolo[-
1,5-b]pyridazine;
[0034]
4-[2-(4-ethoxy-phenyl)-pyrazolo[1,5-b]pyridazin-3-yl]-benzenesulfon-
amide;
[0035]
6-difluoromethoxy-2-(3-fluoro-phenyl)-3-(4-methanesulfonyl-phenyl)--
pyrazolo[1,5-b]pyridazine;
[0036] and pharmaceutically acceptable derivatives thereof.
[0037] Compounds of the invention are potent and selective
inhibitors of COX-2. This activity is illustrated by their ability
to selectively inhibit COX-2 over COX-1.
[0038] In view of their selective COX-2 inhibitory activity, the
compounds of the present invention are of interest for use in human
and veterinary medicine, particularly in the treatment of the pain
(both chronic and acute), fever and inflammation of a variety of
conditions and diseases. Such conditions and diseases are well
known in the art and include rheumatic fever; symptoms associated
with influenza or other viral infections, such as the common cold;
lower back and neck pain; headache; toothache; sprains and strains;
myositis; neuralgia; synovitis; arthritis, including rheumatoid
arthritis; degenerative joint diseases, including osteoarthritis;
gout and ankylosing spondylitis; tendinitis; bursitis; skin related
conditions, such as psoriasis, eczema, burns and dermatitis;
injuries, such as sports injuries and those arising from surgical
and dental procedures.
[0039] The compounds of the invention may also be useful for the
treatment of other conditions mediated by selective inhibition of
COX-2.
[0040] For example, the compounds of the invention may inhibit
cellular and neoplastic transformation and metastatic tumour growth
and hence be useful in the treatment of certain cancerous diseases,
such as colonic cancer.
[0041] Compounds of the invention may also prevent neuronal injury
by inhibiting the generation of neuronal free radicals (and hence
oxidative stress) and therefore may be of use in the treatment of
stroke; epilepsy; and epileptic seizures (including grand mal,
petit mal, myoclonic epilepsy and partial seizures).
[0042] Compounds of the invention also inhibit prostanoid-induced
smooth muscle contraction and hence may be of use in the treatment
of dysmenorrhoea and premature labour.
[0043] Compounds of the invention inhibit inflammatory processes
and therefore may be of use in the treatment of asthma, allergic
rhinitis and respiratory distress syndrome; gastrointestinal
conditions such as inflammatory bowel disease, Chron's disease,
gastritis, irritable bowel syndrome and ulcerative colitis; and the
inflammation in such diseases as vascular disease, migraine,
periarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin's
disease, sclerodoma, type I diabetes, myasthenia gravis, multiple
sclerosis, sorcoidosis, nephrotic syndrome, Bechet's syndrome,
polymyositis, gingivitis, conjunctivitis and myocardial
ischemia.
[0044] Compounds of the invention may also be useful in the
treatment of ophthalmic diseases such as retinitis, retinopathies,
uveitis and of acute injury to the eye tissue.
[0045] Compounds of the invention may also be useful for the
treatment of cognitive disorders such as dementia, particularly
degenerative dementia (including senile dementia, Alzheimer's
disease, Pick's disease, Huntington's chorea, Parkinson's disease
and Creutzfeldt-Jakob disease), and vascular dementia (including
multi-infarct dementia), as well as dementia associated with
intracranial space occupying lesions, trauma, infections and
related conditions (including HIV infection), metabolism, toxins,
anoxia and vitamin deficiency; and mild cognitive impairment
associated with ageing, particularly Age Associated Memory
Impairment.
[0046] According to a further aspect of the invention, we provide a
compound of formula (I) or a pharmaceutically acceptable derivative
thereof for use in human or veterinary medicine.
[0047] According to another aspect of the invention, we provide a
compound of formula (I) or a pharmaceutically acceptable derivative
thereof for use in the treatment of a condition which is mediated
by selective inhibition of COX-2.
[0048] According to a further aspect of the invention, we provide a
method of treating a human or animal subject suffering from a
condition which is mediated by selective inhibition of COX-2 which
comprises administering to said subject an effective amount of a
compound of formula (I) or a pharmaceutically acceptable
derivative.
[0049] According to another aspect of the invention, we provide the
use of a compound of formula (I) or a pharmaceutically acceptable
derivative thereof for the manufacture of a therapeutic agent for
the treatment of a condition which is mediated by selective
inhibition of COX-2, such as an inflammatory disorder.
[0050] According to a further aspect of the invention, we provide a
method of treating a human or animal subject suffering from an
inflammatory disorder, which method comprises administering to said
subject an effective amount of a compound of formula (I) or a
pharmaceutically acceptable derivative thereof.
[0051] It is to be understood that reference to treatment includes
both treatment of established symptoms and prophylactic treatment,
unless explicitly stated otherwise.
[0052] It will be appreciated that the compounds of the invention
may advantageously be used in conjunction with one or more other
therapeutic agents. Examples of suitable agents for adjunctive
therapy include pain relievers such as a glycine antagonist, a
sodium channel inhibitor (e.g. lamotrigine), a substance P
antagonist (e.g. an NK.sub.1 antagonist), acetaminophen or
phenacetin; a matrix metalloproteinase inhibitor; a nitric oxide
synthase (NOS) inhibitor (e.g. an iNOS or an nNOS inhibitor); an
inhibitor of the release, or action, of tumour necrosis factor
.alpha.; an antibody therapy (e.g. a monoclonal antibody therapy);
a stimulant, including caffeine; an H.sub.2-antagonist, such as
ranitidine; a proton pump inhibitor, such as omeprazole; an
antacid, such as aluminium or magnesium hydroxide; an
antiflatulent, such as simethicone; a decongestant, such as
phenylephrine, phenylpropanolamine, pseudoephedrine, oxymetazoline,
epinephrine, naphazoline, xylometazoline, propylhexedrine, or
levo-desoxyephedrine; an antitussive, such as codeine, hydrocodone,
carmiphen, carbetapentane, or dextramethorphan; a diuretic; or a
sedating or non-sedating antihistamine. It is to be understood that
the present invention covers the use of a compound of formula (I)
or a pharmaceutically acceptable derivative thereof in combination
with one or more other therapeutic agents.
[0053] The compounds of formula (I) and their pharmaceutically
acceptable derivatives are conveniently administered in the form of
pharmaceutical compositions. Thus, in another aspect of the
invention, we provide a pharmaceutical composition comprising a
compound of formula (I) or a pharmaceutically acceptable derivative
thereof adapted for use in human or veterinary medicine. Such
compositions may conveniently be presented for use in conventional
manner in admixture with one or more physiologically acceptable
carriers or excipients.
[0054] The compounds of formula (I) and their pharmaceutically
acceptable derivatives may be formulated for administration in any
suitable manner. They may, for example, be formulated for topical
administration or administration by inhalation or, more preferably,
for oral, transdermal or parenteral administration. The
pharmaceutical composition may be in a form such that it can effect
controlled release of the compounds of formula (I) and their
pharmaceutically acceptable derivatives.
[0055] For oral administration, the pharmaceutical composition may
take the form of, for example, tablets (including sub-lingual
tablets), capsules, powders, solutions, syrups or suspensions
prepared by conventional means with acceptable excipients.
[0056] For transdermal administration, the pharmaceutical
composition may be given in the form of a transdermal patch, such
as a transdermal iontophoretic patch.
[0057] For parenteral administration, the pharmaceutical
composition may be given as an injection or a continuous infusion
(e.g. intravenously, intravascularly or subcutaneously). The
compositions may take such forms as suspensions, solutions or
emulsions in oily or aqueous vehicles and may contain formulatory
agents such as suspending, stabilising and/or dispersing agents.
For administration by injection these may take the form of a unit
dose presentation or as a multidose presentation preferably with an
added preservative.
[0058] Alternatively for parenteral administration the active
ingredient may be in powder form for reconstitution with a suitable
vehicle.
[0059] The compounds of the invention may also be formulated as a
depot preparation. Such long acting formulations may be
administered by implantation (for example subcutaneously or
intramuscularly) or by intramuscular injection. Thus, for example,
the compounds of the invention may be formulated with suitable
polymeric or hydrophobic materials (for example as an emulsion in
an acceptable oil) or ion exchange resins, or as sparingly soluble
derivatives, for example, as a sparingly soluble salt.
[0060] As stated above, the compounds of the invention may also be
used in combination with other therapeutic agents. The invention
thus provides, in a further aspect, a combination comprising a
compound of formula (I) or a pharmaceutically acceptable derivative
thereof together with a further therapeutic agent.
[0061] The combinations referred to above may conveniently be
presented for use in the form of a pharmaceutical formulation and
thus pharmaceutical formulations comprising a combination as
defined above together with a pharmaceutically acceptable carrier
or excipient comprise a further aspect of the invention. The
individual components of such combinations may be administered
either sequentially or simultaneously in separate or combined
pharmaceutical formulations.
[0062] When a compound of formula (I) or a pharmaceutically
acceptable derivative thereof is used in combination with a second
therapeutic agent active against the same disease state the dose of
each compound may differ from that when the compound is used alone.
Appropriate doses will be readily appreciated by those skilled in
the art.
[0063] A proposed daily dosage of a compound of formula (I) for the
treatment of man is 0.01 mg/kg to 500 mg/kg, such as 0.05 mg/kg to
100 mg/kg, e.g. 0.1 mg/kg to 50 mg/kg, which may be conveniently
administered in 1 to 4 doses. The precise dose employed will depend
on the age and condition of the patient and on the route of
administration. Thus, for example, a daily dose of 0.25 mg/kg to 10
mg/kg may be suitable for systemic administration.
[0064] Compounds of formula (I) and pharmaceutically acceptable
derivatives thereof may be prepared by any method known in the art
for the preparation of compounds of analogous structure.
[0065] Suitable methods for the preparation of compounds of formula
(I) and pharmaceutically acceptable derivatives thereof are
described below. In the formulae that follow R.sup.0 to R.sup.5 and
n are as defined in formula (I) above unless otherwise stated; Hal
is a halogen, such as Br or I; X.sup.- is a counterion, such as
I.sup.-; and alkyl is as previously defined.
[0066] Thus according to a first process (A), compounds of formula
(I) may be prepared by reacting a compound of formula (II) 3
[0067] or a protected derivative thereof with a boronic acid of
formula (III) 4
[0068] or a suitable derivative thereof in the presence of a
suitable transition metal catalyst. Suitable derivatives of formula
(III) include boronic acid esters, such as those described in R.
Miyaura et al, J. Org. Chem., 1995, 60, 7508-7510. Conveniently,
the reaction is carried out in a solvent, such as an ether (e.g.
1,2 dimethoxyethane); in the presence of a base, such as an
inorganic base (e.g. sodium carbonate); and employing a palladium
catalyst, such as tetrakis(triphenylphosphine)palla- dium(0).
[0069] According to a another process (B), compounds of formula (I)
wherein R.sup.3 is C.sub.1-6alkyl may be prepared by oxidising a
compound of formula (IV) 5
[0070] or a protected derivative thereof under conventional
conditions. Conveniently the oxidation is effected using a
monopersulfate compound, such as potassium peroxymonosulfate (known
as Oxone.TM.) and the reaction is carried out in a solvent, such as
an aqueous alcohol, (e.g. aqueous methanol), and at between
-78.degree. C. and ambient temperature.
[0071] According to a another process (C), compounds of formula (I)
wherein R.sup.1 is C.sub.1-6alkylsulphonyl may be prepared by
oxidising a compound of formula (V) 6
[0072] or a protected derivative thereof under conventional
conditions. Conveniently the oxidation is effected in the manner
described just above for process (B).
[0073] According to a another process (D), compounds of formula (I)
wherein R.sup.1 is C.sub.1-6alkoxy substituted by one or more
fluorine atoms may be prepared by reacting an alcohol of formula
(VI) 7
[0074] or a protected derivative thereof with a halofluoroalkane
under conventional conditions. Conveniently the reaction is
effected in a solvent, such as a polar solvent (e.g.
N,N-dimethylformamide), in the presence of a strong base, such as
an inorganic hydride (e.g. sodium hydride), at about ambient
temperature and using the appropriate bromofluoroalkane to give the
desired compound of formula (I).
[0075] According to another process (E) compounds of formula (I)
may be prepared by interconversion, utilising other compounds of
formula (I) as precursors. The following procedures are
illustrative of suitable interconversions.
[0076] Compounds of formula (I) wherein R.sup.1 or R.sup.2
represent C.sub.1-4alkyl substituted by one or more fluorine atoms
may be prepared from the appropriate compound of formula (I)
wherein R.sup.1 or R.sup.2 is C.sub.1-6hydroxyalkyl, C(O)H or
C(O)C.sub.1-6alkyl, by treatment with a suitable source of
fluorine. Suitable sources of fluorine include, for example,
diethylaminosulphur trifluoride. Conveniently the reaction is
effected in the presence of a solvent, such as a halogenated
hydrocarbon (e.g. dichloromethane), and at reduced temperature,
such as -78.degree. C.
[0077] Compounds of formula (I) wherein R.sup.1 or R.sup.2
represent C(O)H may be prepared from the corresponding compound of
formula (I) wherein R.sup.1 or R.sup.2 represent CH.sub.2OH by
oxidation. Suitable oxidising agents include, for example,
manganese (IV) oxide. Conveniently the oxidation is effected in the
presence of a solvent, such as a halogenated hydrocarbon (e.g.
chloroform), and at elevated temperature (e.g. reflux).
[0078] Compounds of formula (I) wherein R.sup.1 or R.sup.2
represent C.sub.1-6hydroxyalkyl, and wherein the hydroxy group is
attached to the carbon linked to the pyridazine ring, may be
prepared by reduction of the compound of formula (I) wherein
R.sup.1 or R.sup.2 represent the corresponding aldehyde or ketone.
Suitable reducing agents include hydride reducing agents, such as
diisobutylaluminium hydride.
[0079] Conveniently the reduction is effected in the presence of a
solvent, such as a halogenated hydrocarbon (e.g. dichloromethane),
and at reduced temperature, such as -78.degree. C.
[0080] As will be appreciated by those skilled in the art it may be
necessary or desirable at any stage in the synthesis of compounds
of formula (I) to protect one or more sensitive groups in the
molecule so as to prevent undesirable side reactions.
[0081] Another process (F) for preparing compounds of formula (I)
thus comprises deprotecting protected derivatives of compounds of
formula (I).
[0082] The protecting groups used in the preparation of compounds
of formula (I) may be used in conventional manner. See, for
example, those described in `Protective Groups in Organic
Synthesis` by Theodora W. Greene and Peter G. M. Wuts, second
edition, (John Wiley and Sons, 1991), which also describes methods
for the removal of such groups.
[0083] Compounds of formula (II) may be prepared by halogenating
compounds of formula (VII) 8
[0084] by conventional means.
[0085] Thus esters of formula (VI) are first hydrolysed to their
corresponding acids, for example by treatment with a strong base
(e.g. sodium hydroxide), in the present of a solvent (e.g. ethanol)
and at elevated temperature. The corresponding acid is then treated
with a halogenating agent, conveniently at ambient temperature and
in a solvent (e.g. chlorinated hydrocarbon), under which conditions
the acid undergoes both halogenation and decarboxylation.
Conveniently, the halogenating agent is a brominating agent, such
as bromine in the presence of a strong acid (e.g. hydrobromic acid
in acetic acid) or N-bromosuccinimide, to yield the corresponding
compound of formula (II) wherein Hal is bromine.
[0086] Esters of formula (VII) may be prepared by reacting a
compound of formula (VIII) 9
[0087] with an aminopyridazinium complex of formula (IX) 10
[0088] under conventional conditions. Conveniently the reaction is
effected in the presence of a base, such as potassium carbonate, a
solvent, such as N,N-dimethylformamide and at ambient
temperature.
[0089] Boronic acids of formula (III) are either known compounds or
may be prepared by literature methods such as those described in,
for example, EPA publication No. 533268.
[0090] Compounds of formulae (IV), (V) and (VI) may be prepared by
methods analogous to those described for the preparation of the
compound of formula (I) from compounds of formula (II).
[0091] Compounds of formula (VIII) are either known compounds or
may be prepared by literature methods such as those described in,
for example, D H Wadsworth et al, J Org Chem, (1987), 52(16),
3662-8 and J. Morris and D. G. Wishka, Synthesis (1994), (1),
43-6.
[0092] Compounds of formula (IX) are either known compounds or may
be prepared by literature methods such as those described in, for
example, Y Kobayashi et al., Chem Pharm Bull, (1971), 19(10),
2106-15; T. Tsuchiya, J. Kurita and K. Takayama, Chem. Pharm. Bull.
28(9) 2676-2681 (1980) and K Novitskii et al., Khim Geterotskil
Soedin, 1970 2, 57-62.
[0093] Certain intermediates described above are novel compounds,
and it is to be understood that all novel intermediates herein form
further aspects of the present invention. Compounds of formula (II)
are key intermediates and represent a particular aspect of the
present invention.
[0094] Conveniently, compounds of the invention are isolated
following work-up in the form of the free base. Pharmaceutically
acceptable acid addition salts of the compounds of the invention
may be prepared using conventional means.
[0095] Solvates (e.g. hydrates) of a compound of the invention may
be formed during the work-up procedure of one of the aforementioned
process steps.
[0096] The following Examples illustrate the invention but do not
limit the invention in any way. All temperatures are in .degree. C.
Flash column chromatography was carried out using Merck 9385
silica. Thin layer chromatography (Tlc) was carried out on silica
plates. NMR was carried out on a Brucker 250 MHz spectrometer.
Chemical shifts are given, with respect to tetramethylsilane as
internal chemical shift reference, in .delta. ppm. The following
abbreviations are used: Me=methyl, s=singlet, d=doublet, t=triplet
and m=multiplet.
EXAMPLE 1
6-Difluoromethoxy-2-(4-fluoro-phenyl)-3-(4-methanesulfonyl-phenyl)-pyrazol-
o[1,5-b]pyridazine
(i)
6-Methoxy-2-(4-fluoro-phenyl-pyrazolo[1,5-b]pyridazine-3-carboxylic
Acid Methyl Ester
[0097] 1,8-Diazabicyclo[5.4.0]undec-7-ene (3.39 ml) was added to a
mixture of 3-(4-fluorophenyl)-prop-2-ynoic acid methyl ester (3.36
g) and 1-amino-3-methoxy-pyridazin-1-ium mesitylene
sulphonate.sup.1 (6.1419 g) in acetonitrile (125 ml) and the
mixture was stirred at ambient temperature for 48 hours. During the
first 2 hours a stream of air was passed through the reaction. The
mixture was concentrated in vacuo, dissolved in ethyl acetate (150
ml), washed with water (3.times.25 ml), dried (MgSO.sub.4),
filtered and evaporated in vacuo to give the title compound as a
brown solid (4.77 g).
[0098] .sup.1H NMR (CDCl.sub.3): 8.4 (d, 1H, J=10 Hz) 7.85-7.90 (m,
2H) 7.1-7.2 (m, 2H) 6.9-7.0 (d, 1H, J=10 Hz) 4.1 (s, 3H) 3.9 (s,
3H)
[0099] MH.sup.+ 302
[0100] Ref:.sup.1 T. Tsuchiya, J. Kurita and K. Takayama, Chem.
Pharm. Bull. 28(9) 2676-2681 (1980).
(ii)
6-Methoxy-2-(4-fluoro-phenyl-pyrazolo[1,5-b]pyridazine-3-carboxylic
Acid
[0101] A mixture of
6-methoxy-2-(4-fluoro-phenyl-pyrazolo[1,5-b]pyridazine-
-3-carboxylic acid methyl ester (4.469 g), 2N sodium hydroxide (50
ml) and methanol (90 ml) was heated at reflux for 2 hours. The
cooled solution was added to 2N hydrochloric acid (200 ml) and the
title compound was isolated by filtration as a beige solid (3.639
g).
[0102] .sup.1H NMR (DMSO-d.sub.6): 12.8 (br. s, 1H) 8.4 (d, 1H,
J=10 Hz) 7.8-7.9 (m, 2H) 7.21-7.32 (m, 2H) 7.15-7.2 (d, 1H, J=10
Hz) 4.0 (s, 3H)
[0103] MH.sup.+ 288
(iii)
2-(4-Fluoro-phenyl)-3-(4-methanesulfonyl-phenyl)-6-methoxy-pyrazolo[-
1,5-b]pyridazine
[0104] A mixture of
6-methoxy-2-(4-fluoro-phenyl-pyrazolo[1,5-b]pyridazine-
-3-carboxylic acid (869 mg) and sodium bicarbonate (756 mg) in
dimethylformamide (10 ml) was treated with N-bromosuccinimide (587
mg) and stirred at ambient temperature for 1 hour, then added to
water (50 ml) and extracted with ethyl acetate (3.times.50 ml),
dried (MgSO.sub.4), and evaporated in vacuo. The resulting brown
solid (1.612 g) was dissolved in 1,2 dimethoxyethane (20 ml). 2N
Aqueous sodium carbonate solution (10 ml) was added together with
4-(methanesulphonyl)phenyl boronic acid (660 mg) and
tetrakis(triphenylphosphine)palladium (0) (100 mg) and the mixture
was heated at reflux for 20 hours. The reaction was poured into
water (50 ml), extracted with dichloromethane (3.times.100 ml). The
combined organic extracts were dried (MgSO.sub.4) and evaporated in
vacuo to give a brown solid (1.116 g) which was purified by flash
column chromatography on silica, eluting with cyclohexane/ethyl
acetate (4:1 then 2:1), to give the title compound as a yellow
solid (390 mg).
[0105] Tic, SiO.sub.2, R.sub.f 0.3 (1:1 cyclohexane/ethyl acetate),
detection UV
[0106] MH.sup.+ 398
(iv)
2-(4-Fluoro-phenyl)-3-(4-methanesulfonyl-phenyl)-pyrazolo[1,5-b]pyrid-
azin-6-ol
[0107] A mixture of
2-(4-fluoro-phenyl)-3-(4-methanesulfonyl-phenyl)-6-met-
hoxy-pyrazolo[1,5-b]pyridazine (321 mg) and pyridine hydrochloride
(1.4 g) was heated to and at 200.degree. C. in a sealed vessel
(Reactivial.TM.) for 3 hours. The cooled reaction was poured into
water (20 ml), and extracted with ethyl acetate (3.times.30 ml).
The combined organic extracts dried (MgSO.sub.4), filtered and
evaporated in vacuo to give a solid which was triturated with
diethyl ether to give the title compound as a beige solid (119
mg).
[0108] Tlc, SiO.sub.2, R.sub.f 0.07 (1:2 cyclohexane/ethyl
acetate), detection UV.
[0109] MH.sup.+ 384
(v)
6-Difluoromethoxy-2-(4-fluoro-phenyl)-3-(4-methanesulfonyl-phenyl)-pyr-
azolo[1,5-b]pyridazine
[0110] A solution of
2-(4-fluoro-phenyl)-3-(4-methanesulfonyl-phenyl)-pyra-
zolo[1,5-b]pyridazin-6-ol (0.2 g) in anhydrous dimethyl formamide
(5 ml) was treated with sodium hydride (0.046 g, 60% dispersion in
mineral oil), after effervescence ceased a stream of
bromodifluoromethane gas was passed through the mixture at ambient
temperature for 30 minutes. The reaction mixture was then poured
into water (50 ml) and extracted with ethyl acetate (50 ml), the
organic extract was washed with water (3.times.50 ml), dried and
concentrated in vacuo. The residue was purified by chromatography
to give the title compound as a white solid (0.17 g).
[0111] MH.sup.+ =434
[0112] .sup.1HNMR(CDCl.sub.3):.delta.8.05-8.0(d,J=10 HZ,2H)
8.0-7.95(d,J=10 HZ,1H) 7.6-7.5(m,4H) 7.8-7.2(t,J=70 HZ,1H)
7.1-7.05(t,J=11 HZ,2H) 6.9-6.85(d,J=10 HZ,1H) 3.15(s,3H)
[0113] Tlc, SiO.sub.2, R.sub.f 0.35(ethyl
acetate/cyclohexane(1/1))
EXAMPLE 2
3-(4-Methanesulfonyl-phenyl)-2-(4-methoxy-phenyl)-pyrazolo[1,5-b]pyridazin-
e
(i) 2-(4-Methoxy-phenyl)-pyrazolo[1,5-b]pyridazine-3-carboxylic
Acid Methyl Ester
[0114] Diazabicyclo[5.4.0]undec-7-ene (22.76 ml, 2 eq) was added
dropwise to a solution of methyl 3-(4-methoxy-phenyl)-prop-2-ynoic
acid.sup.1 (14.46 g, 76 mM) and 1-amino pyridazinium iodide.sup.2
(2 eq) in acetonitrile under nitrogen and stirred for 6 h.
Purification by chromatography on silica gel eluting with toluene,
then toluene:ethyl acetate (9:1) gave the title compound (2.76 g)
as a brown solid.
[0115] MH.sup.+ 284
[0116] 1H NMR (CDCl.sub.3) .delta. 3.87 (3H, s) 3.9 (3H, s) 7.0
(2H, d, J=9 Hz) 7.25 (1H, dd, J=9 & 4 Hz) 7.90 (2H, d, J=9 Hz)
8.45 (1H, dd, J=4 & 2 Hz) 8.55 (1H, dd, J=9 & 2 Hz)
[0117] Ref: .sup.1 J. Morris and D. G. Wishka, Synthesis (1994),
(1), 43-6
[0118] Ref: .sup.2 Kobayashi et al Chem.Pharm.Bull. (1971), 19
(10), 2106-15
(ii)
3-(4-Methanesulfonyl-phenyl)-2-(4-methoxy-phenyl)-pyrazolo[1,5-b]pyri-
dazine
[0119] A mixture of
2-(4-methoxy-phenyl)-pyrazolo[1,5-b]pyridazine-3-carbo- xylic acid
methyl ester (2.76 g) and aq. sodium hydroxide (2N, 30 ml) in
ethanol (30 ml) was refluxed under nitrogen for 2 h. The cooled
mixture was acidified with hydrochloric acid (2N) and the resulting
white solid (2.53 g) isolated by filtration. This solid was
dissolved in DMF and sodium bicarbonate (2.67 g, 3.3 eq) added,
followed by N-bromosuccinimide (1.88 g, 1.1 eq) portionwise. After
stirring for 1 h under nitrogen, water was added and extracted into
ethyl acetate (2.times.25 ml). The dried organic phase was
concentrated and the residue taken up in DME (60 ml). Aqueous
sodium carbonate (2N, 15 ml) was added, followed by
4-methanesulfonyl-phenylboronic acid (3.12 g) and
tetrakis(triphenylphosp- hine)palladium(0) (250 mg). The mixture
was heated at reflux under nitrogen for 18 h, cooled, poured into
water and extracted into ethyl acetate (2.times.25 ml). The
combined organic phases were dried and concentrated onto silica
gel. Chromatography on silica gel eluting with toluene:ethyl
acetate (8:1) gave, on concentration, the title compound (3.58 g)
as a cream solid.
[0120] MH.sup.+ 380
[0121] 1H NMR (DMSO) .delta. 3.25 (3H, s) 3.75 (3H, s) 6.95 (2H, d,
J=8.5 Hz) 7.25 (1H, dd, J=9 & 5 Hz) 7.45 (2H, d, J=8.5 Hz) 7.60
(2H, d, J=8 Hz) 7.9 (2H, d, J=8.5 Hz) 8.15 (1H, dd, J=9&2 Hz)
8.49 (1H, dd, J=5&2 Hz)
EXAMPLE 3
2-(4-Ethoxy-phenyl)-3-(4-methanesulfonyl-phenyl)-pyrazolo[1,5-b]pyridazine
(i)
4-[3-(4-Methanesulfonyl-phenyl)-pyrazolo[1,5-b]pyridazin-2-yl]-phenol
[0122] Boron tribromide (1 M solution in CH.sub.2Cl.sub.2, 2.1 eq)
was added to
3-(4-methanesulfonyl-phenyl)-2-(4-methoxy-phenyl)-pyrazolo[1,5-b-
]pyridazine (3.58 g) in CH.sub.2Cl.sub.2 at -70.degree.. The
mixture was stirred for 10 min then warmed to 0.degree. and stirred
at 0.degree. overnight. The reaction mixture was made alkaline with
potassium carbonate then acidified with hydrochloric acid (2M),
poured into water and extracted into CH.sub.2Cl.sub.2. The organic
phase was dried, filtered and concentrated to give the title
compound (1.87 g) as a yellow solid.
[0123] MH.sup.+ 366
[0124] 1H NMR (DMSO) .delta. 3.30 (3H, s) 6.80 (2H, d, J=8.5 Hz)
7.30 (1H, dd, J=9 & 5 Hz) 7.35 (2H, d, J=8.5 Hz) 7.60 (2H, d,
J=8 Hz) 8.0 (2H, d, J=8.5 Hz) 8.20 (1H, dd, J=9& 2 Hz) 8.55
(1H, dd, J=5& 2 Hz) 9.75 (1H, s)
(ii)
2-(4-Ethoxy-phenyl)-3-(4-methanesulfonyl-phenyl)-pyrazolo[1,5-b]pyrid-
azine
[0125]
4-[3-(4-Methanesulfonyl-phenyl)-pyrazolo[1,5-b]pyridazin-2-yl]-phen-
ol (663 mg, 1.82), iodoethane (1 eq) and potassium carbonate (2 eq)
in acetonitrile (30 ml) were heated at reflux under nitrogen for 18
h. The cooled reaction mixture was partitioned between water (30
ml) and ethyl acetate (30 ml). The organic phase was collected,
dried and purified by chromatography to give the title compound
(547 mg) as a cream foam.
[0126] MH.sup.+ 394
[0127] 1H NMR (DMSO) .delta. 1.45 (3H, t, J=7 Hz) 3.10 (3H, s) 4.1
(2H, q, J=7 Hz) 6.87 (2H, d, J=9 Hz) 7.08 (1H, dd, J=9 & 5 Hz)
7.55 (4H, t, J=9 Hz) 7.92 (1H, dd, J=9 &2 Hz) 7.95 (2H, d, J=9
Hz) 8.20 (1H, dd, J=9& 2 Hz) 8.32 (1H, dd, J=5& 2 Hz)
EXAMPLE 4
2-(4-Fluoro-phenyl)-6-methanesulfonyl-3-(4-methanesulfonyl-phenyl)-pyrazol-
o[1,5-b]pyridazine
(i)
2-(4-Fluoro-phenyl)-6-methylsulfanyl-pyrazolo[1,5-b]pyridazine-3-carbo-
xylic Acid Methyl Ester
[0128] Solid
t-butoxycarbonyl-O-mesitylenesulfonylhydroxylamine.sup.1 (7.8 g)
was added portionwise with stirring to TFA (25 ml) over 10 min then
stirred for a further 20 minutes. The solution was poured onto ice
(.about.200 ml) and left until the ice melted. The resulting white
solid was filtered off, washed with water, and dissolved in DME
(100 ml). The solution was dried over 4 A mol. sieves for 1.5
hours, filtered then added to a solution of
3-methylthio-pyridazine.sup.2 (2.6 g) in dichloromethane (35 ml)
and the reaction stirred at room temperature for 20 h. The
intermediate salt was isolated by filtration as light brown
crystals (3.87 g), suspended in acetonitrile (100 ml) and methyl
3-(4-fluoro-phenyl)-prop-2-ynoic acid (2.02 g) added.
1,8-Diazabicyclo[5.4.0]undec-7-ene (2.1 ml) was added dropwise and
the reaction was stirred at room temperature for 20 hours. The
resulting crystalline precipitate was filtered off, washed and
dried (770 mg). Concentration of the filtrate gave a second crop
(430 mg). The residues were partioned between water and ethyl
acetate (100 ml each) and the aqueous layer was extracted with
ethyl acetate (20 ml). The combined organics were washed with
water, brine and dried. Removal of solvent gave a brown oil which
was purified by flash chromatography on silica (300 g) eluting with
cyclohexane/ethyl acetate (3:1) to give a further quantity of
product (247 mg). The three crops were combined to give the title
compound (1.45 g) as a light brown solid.
[0129] MH.sup.+ 318
[0130] 1H NMR (CDCl.sub.3) .delta. 2.70 (3H, s), 3.88 (3H, s)
7.08-7.18 (3H, m) 7.84 (2H, m) 8.31 (1H, d, J=10 Hz)
[0131] Ref: .sup.1 K Novitskii et al, Khim Geterotskil Soedin, 1970
2, 57-62
[0132] Ref: .sup.2 Barlin G. B., Brown, W. V., J Chem Soc (1968),
(12), 1435-45
(ii)
2-(4-Fluoro-phenyl)-3-(4-methanesulfonyl-phenyl)-6-methylsulfanyl-pyr-
azolo[1,5-b]pyridazine
[0133] A mixture of the
2-(4-fluoro-phenyl)-6-(methylthio)-pyrazolo[1,5-b]-
pyridazine-3-carboxylic acid methyl ester (1.45 g) potassium
carbonate (690 mg) in methanol (40 ml) and water (14 ml) was
stirred and heated under reflux for 20 hours under nitrogen. The
solvents were removed and the resulting solid partioned between
ethyl acetate (50 ml) and water (250 ml). The aqueous layer was
acidified to pH1 (2 MHCl) and a solid was filtered off (1.0 g,
MH.sup.+ 304). A mixture of the solid (1.0 g), sodium bicarbonate
(557 mg) and NBS (594 mg) were stirred at room temperature for 4
hours. The reaction was poured into water (150 ml) and extracted
with ethyl acetate (3.times.50 ml). The combined extracts were
washed with water (50 ml), brine (20 ml), dried and concentrated.
The resulting solid (1.015 g, MH.sup.+ 338,340),
4-(methanesulphonyl)phenyl boronic acid (902 mg), sodium carbonate
(740 mg) and tetrakis(triphenylphosphine)palladium(0) (175 mg) were
stirred and heated under nitrogen at reflux in DME (30 mls) and
water (15 ml) for 48 hours. The reaction was poured into water and
extracted with ethyl acetate (3.times.50 ml). The combined extracts
were dried and the solvent removed to give a brown solid. This was
purified on silica (300 g) eluting with cyclohexane, ethyl acetate
(1:1) to give the title compound (0.713 g) as a yellow solid.
[0134] MH.sup.+ 414
[0135] 1H NMR .delta. (DMSO) 2.65 (3H, s) 3.28 (3H, s) 7.20-7.30
(3H, m) 7.55 (2H, m) 7.62 (4H, d, J=8.5 Hz) 7.95-8.05 (3H, m)
(iii)
2-(4-Fluoro-phenyl)-6-methanesulfonyl-3-(4-methanesulfonyl-phenyl)-p-
yrazolo[1,5-b]pyridazine
[0136] A suspension of
2-(4-fluoro-phenyl)-3-(4-methanesulfonyl-phenyl)-6--
(methylthio)-pyrazolo[1,5-b]pyridazine (60 mg 0.145) in MeOH (5 ml)
and water (2 ml) was stirred with oxone (196 mg 0.32) for 20 hours.
The resulting solution was poured into water (50 ml) and extracted
with chloroform (3.times.20 ml). The combined extracts were dried
and the solvent removed. Crystallisation of the residue from
methanol gave the title compound (60 mg) as a white solid.
[0137] MH.sup.+ 446
[0138] 1H NMR (DMSO-d.sub.6) .delta. 3.34 (3H, s) 3.53 (3H, s) 7.33
(2H, t, J=9 Hz) 7.62 (2H, m) 7.68 (1H, d, J=8.5 Hz) 8.04 (1H, d,
J=10 Hz) 8.52 (1H, d, J=9 Hz)
[0139] TLC SiO.sub.2 Hexane:Ethyl acetate (1:1) R.sub.f 0.24 UV
EXAMPLE 5
2-(4-Difluoromethoxy-phenyl)-3-(4-methanesulfonyl-phenyl)-pyrazolo[1,5-b]p-
yridazine
[0140] Sodium hydride (48 mg, 60% disp. in oil, 1.2 mmol) was added
to a solution of
4-[3-(4-methanesulfonyl-phenyl)-pyrazolo[1,5-b]pyridazin-2-yl-
]-phenol (200 mg, 0.55 mmol) in anhydrous dimethylformamide (5 ml).
Bromodifluoromethane gas was gently bubbled through the solution
for 20 min, then diluted with CH.sub.2Cl.sub.2 (30 ml). Aqueous
workup followed by chromatography on silica gel with
CH.sub.2Cl.sub.2:ethyl acetate (3:1) as eluant then chromatography
with CH.sub.2Cl.sub.2:ethyl acetate (10:1) as eluant gave the title
compound (63 mg, 28%) as a white solid.
[0141] MH.sup.+ 416
[0142] NMR (CDCl.sub.3) .delta.8.38 (1H,dd, J=4 Hz), 8.01 (2H, d,
J=8.5 Hz), 7.94 (1H, dd, J=9 & 2 Hz), 7.65 (2H, d, J=8.5 Hz)
7.57 (2H, d, J=8 Hz), 7.10 (3H, m), 6.87-6.27 (1H, t, J=7.4 Hz)
3.15 (3H, s)
EXAMPLE 6
4-[2-(4-Ethoxy-phenyl)-pyrazolo[1,5-b]pyridazin-3-yl]-benzenesulfonamide
(i) 2-(4-Ethoxy-phenyl)-pyrazolo[1,5-b]pyridazine-3-carboxylic Acid
Methyl Ester
[0143] Diazabicyclo[5.4.0]undec-7-ene (1.47 ml, 2 eq) was added
dropwise to a solution of methyl 3-(4-ethoxy-phenyl)-prop-2-ynoic
acid (1.0 g) and 1-amino pyridazinium iodide.sup.2 (2.19 g) in
acetonitrile (10 ml) under nitrogen and stirred for 5 h.
Concentration and aqueous workup gave the title compound (1.2 g) as
a sticky brown solid.
[0144] MH.sup.+ 298
(ii) 2-(4-Ethoxy-phenyl)-pyrazolo[1,5-b]pyridazine-3-carboxylic
Acid
[0145] A mixture of
2-(4-ethoxy-phenyl)-pyrazolo[1,5-b]pyridazine-3-carbox- ylic acid
methyl ester (1.2 g), ethanol (10 ml) and 2N sodium hydroxide (10
ml) was heated to 800 for 1.5 h. The mixture was allowed to cool
and acidified to pH 1 with 2N hydrochloric acid. The title compound
was isolated by filtration as a brown solid (716 mg, 63%).
[0146] MH.sup.+ 284
(iii) 2-(4-Ethoxy-phenyl)-3-iodo-pyrazolo[1,5-b]pyridazine
[0147] A mixture of
2-(4-ethoxy-phenyl)-pyrazolo[1,5-b]pyridazine-3-carbox- ylic acid
(710 mg), N-iodosuccinimide (678 mg) and sodium bicarbonate (717
mg) in DMF (8 ml) was stirred for 4 h. A further quantity of
N-iodosuccinimide(100 mg) was added and stirring continued for 2 h.
Aqueous workup gave a dark brown solid which was purified by SPE
with dichloromethane as eluant. This gave the title compound as an
orange-brown solid (429 mg, 47%).
[0148] MH.sup.+ 366
(iv)
4-[2-(4-Ethoxy-phenyl)-pyrazolo[1,5-b]pyridazin-3-yl]-benzenesulfonam-
ide
[0149] A mixture of 4-iodobenzenesulphonamide (0.311 g),
dipinacoldiborane.sup.1 (0.279 g), potassium acetate (486 mg) and
[1,1'-bis(diphenylphosphino)-ferrocene]palladium(II) chloride
complex with dichloromethane (1:1) (0.45 g) in dimethylformamide (8
ml) was heated under nitrogen at 800 for 2 h. The cooled reaction
mixture was concentrated in vacuo and the residue suspended in 1,2
dimethoxyethane (10 ml),
2-(4-ethoxy-phenyl)-3-iodo-pyrazolo[1,5-b]pyridazine (0.4 g) was
added together with 2N sodium carbonate (4 ml) and
tetrakis(triphenylphosphine)palladium (0) (20 mg) and the mixture
heated at reflux under nitrogen for 18 hours. The cooled reaction
mixture was poured into water (60 ml) and the suspension extracted
with ethyl acetate (3.times.60 ml). The organic extracts were
combined, dried (Na.sub.2SO.sub.4) and concentrated. The residue
was purified by chromatography eluting with dichloromethane/ethyl
acetate (3:1) to give the title compound as a yellow solid (0.116
g, 27%).
[0150] MH.sup.+ 395
[0151] NMR (CDCL.sub.3) .delta. 8.32 (1H, dd, J=4 & 2 Hz), 7.97
(2H, d, J=8 Hz), 7.89 (1H, dd, J=9 & 2 Hz), 7.54 (4H, m), 7.04
(1H, dd, J=9 & 4 Hz), 6.88 (2H, d, J=9 Hz), 1.43 (3H, t, J=7
Hz)
[0152] Ref: 1 R. Miyaura et al J.Org.Chem., 1995,60,7508-7510.
EXAMPLE 7
6-Difluoromethoxy-2-(3-fluoro-phenyl)-3-(4-methanesulfonyl-phenyl)-pyrazol-
o[1,5-b]pyridazine
(i) 1-(2,2-Dibromo-vinyl)-3-fluoro-benzene
[0153] To a stirred cooled (ice/salt, 0.degree.) solution of carbon
tetrabromide (48.82 g) in anhydrous CH.sub.2Cl.sub.2 (200 ml) was
added portionwise over 3 minutes, triphenylphosphine (77.1 g),
maintaining the temperature below 100. The resulting orange
suspension was stirred at 0.degree. for 1 hour before adding to it,
3-fluorobenzaldehyde (7.8 ml). After the addition was complete, the
suspension was stirred at 0.degree. for 1 hour then quenched by the
addition of water (75 ml). The organic phase was separated and
washed with brine (75 ml), dried (Na.sub.2SO.sub.4) and evaporated
to dryness. The residual gum was poured into cyclohexane (1 L) and
stirred for 30 minutes. The organic phase was decanted and the
residue taken up into CH.sub.2Cl.sub.2 and poured into cyclohexane
(1 L). This procedure was repeated twice more and the combined
organic phases concentrated to .about.100 ml and passed through
silica gel. The filtrate was concentrated to give the title
compound as a mobile yellow oil (24 g, 100%).
[0154] MH.sup.+ 280, MH.sup.- 279
[0155] NMR (CDCl.sub.3) .delta. 7.05 (1H, tm, J=9 Hz) 7.3 (3H, m)
7.45 (1H, s)
(ii) (3-Fluoro-phenyl)-propynoic Acid Methyl Ester
[0156] To a stirred solution of
1-(2,2-dibromo-vinyl)-3-fluoro-benzene (23.8 g) in anhydrous THF
(350 ml) cooled to -780 was added dropwise over 30 minutes,
n-butyllithium (2.2 eq, 1.6M in hexanes). The mixture was stirred
for a further 30 minutes at -780 before methyl chloroformate (11.6
g, 9.5 ml) was added and the resultant mixture allowed to warm to
0.degree. for 1 hour before being diluted with 1:1 saturated
aqueous sodium bicarbonate:ammonium chloride (100 ml) and extracted
into ether (2.times.100 ml). The combined organic extract was
washed with brine (25 ml), dried (Na.sub.2SO.sub.4) and evaporated
to dryness to give the title compound as a brown oil (16.7 g,
100%).
[0157] MH.sup.- 173
[0158] NMR (CDCl.sub.3) .delta. 7.4-7.1 (4H, m) 3.85 (3H, s,
CO.sub.2Me)
(iii)
2-(3-Fluoro-phenyl)-6-methoxy-pyrazolo[1,5-b]pyridazine-3-carboxylic
Acid Methyl Ester
[0159] 1,8-Diazabicyclo[5.4.0]undec-7-ene (5 ml) was added to a
stirred, chilled, mixture of (3-fluoro-phenyl)-propynoic acid
methyl ester (2.67 g) and 1-amino-3-methoxy-pyridazin-1-ium
mesitylene sulphonate (4.89 g) in acetonitrile (80 ml) and the
mixture was stirred at 0.degree. for 1 hour then at ambient
temperature for 18 hours. The mixture was concentrated in vacuo,
and partitioned between ethyl acetate (150 ml) and water (150 ml).
The aqueous phase was separated and further extracted with ethyl
acetate (2.times.100 ml). The combined organic extracts were washed
with water (2.times.50 ml), brine (25 ml), dried (MgSO.sub.4),
filtered and evaporated in vacuo to give a solid which was
triturated with anhydrous ether: petroleum ether (1:0.5) to give
the title compound as a brown solid (2.4 g, 53%).
[0160] MH.sup.+ 302
[0161] 1H NMR (CDCl.sub.3) .delta. 12.8 (1H, br s); 8.4 (1H, d,
J=10 Hz) 7.7-7.6 (2H, m) 7.42 (1H, q, J=8 Hz) 7.15 (1H, td, J 8
& 3 Hz) 6.95 (1H, d, J=10 Hz) 4.1 (3H, s) 3.88 (3H, s)
(iv)
2-(3-Fluoro-phenyl)-6-methoxy-pyrazolo[1,5-b]pyridazine-3-carboxylic
Acid
[0162] 2N sodium hydroxide (50 ml) was added to a solution of
2-(3-fluoro-phenyl)-6-methoxy-pyrazolo[1,5-b]pyridazine-3-carboxylic
acid methyl ester (2.3 g) in absolute ethanol (50 ml) and the
resulting mixture heated to reflux for three hours. The cooled
reaction mixture was poured slowly into a stirred solution of 2N
hydrochloric acid (300 ml). The resulting suspension was stirred at
ambient temperature for 1 hour then filtered and the filter cake
washed with water and dried in vacuo at 60.degree. to give the
title compound as an off-white solid (2.0 g, 91%).
[0163] MH.sup.+ 288
[0164] 1H NMR (DMSO) .delta. 8.45 (1H, d, J=10 Hz); 7.67 (2H, m);
7.5 (1H, q, J=7 Hz); 7.3 (1H, td, J 7& 2 Hz); 7.21 (1H, d, J=10
Hz); 4.0 (3H, s)
(v)
3-Bromo-2-(3-fluoro-phenyl)-6-methoxy-pyrazolo[1,5-b]pyridazine
[0165] To a stirred solution of
2-(3-fluoro-phenyl)-6-methoxy-pyrazolo[1,5-
-b]pyridazine-3-carboxylic acid (2.0 g) in anhydrous DMF (20 ml)
was added n-bromosuccinimide (1.78 g) and the resulting solution
stirred at ambient temperature for 3 hours. The reaction mixture
was diluted with ethyl acetate (800 ml) and washed sequentially
with water (10.times.100 ml) and sat. brine (25 ml), dried
(Na.sub.2SO.sub.4), and concentrated to give the title compound as
a buff solid (2.1 g, 93%).
[0166] MH.sup.+ 323, MH.sup.- 321
[0167] 1H NMR (CDCl.sub.3) 7.9 (2H, m) 7.8 (1H, d, J=10 Hz); 7.45
(1H, m); 7.1 91H, td, J 8 & 2 Hz); 6.78 (1H, d, J=10 Hz); 4.1
(3H, s)
(vi)
6-Difluoromethoxy-2-(3-fluoro-phenyl)-pyrazolo[1,5-b]pyridazine
[0168] Portions of
3-bromo-2-(3-fluoro-phenyl)-6-methoxy-pyrazolo[1,5-b]py- ridazine
(400 mg, 2.1 g total) were placed in individual Reactivials
equipped with a magnetic stirrer bar. Pyridine hydrochloride (10
eq) was added to each vial, the vials sealed, and heated to 2000
for 3 hours. The vials were allowed to cool to .about.140.degree.
before opening and the contents poured into ice/water. The
resulting mixture was extracted into ethyl acetate (3.times.100 ml)
and the combined organic extracts washed with water (7.times.75
ml), dried (Na.sub.2SO.sub.4) and evaporated to give the des-bromo
phenol as a brown solid (1.0 g, MH.sup.+ 230). This solid was
dissolved in anhydrous DMF (10 ml) and sodium hydride (60%
dispersion in mineral oil, 200 mg) added portionwise. After
stirring for 20 minutes at ambient temperature the solution was
transferred to a small cooled autoclave and bromodifluoromethane (5
ml, xs, condensed at -30.degree.) added. The autoclave was then
sealed, allowed to warm to ambient temperature and stirred for 36
hours. The resulting solution was diluted with ethyl acetate (200
ml), washed with water (10.times.20 ml), dried (Na.sub.2SO.sub.4),
concentrated and the residual gum purified by flash column
chromatography with cyclohexane:ethyl acetate (4:1) as eluant. This
gave the title compound as a solid (652 mg, 60%).
[0169] MH.sup.+ 280 MH.sup.- 278
[0170] NMR (DMSO) .delta. 8.42(1H, d, J=10 Hz) 7.85 (1H, d, J=8 Hz)
7.78 (1H, t, J=70 Hz) 7.55 (1H, q, J=8 Hz) 7.38 (1H, s) 7.25 (1H,
m) 7.17 (1H, d, J=10 Hz)
(vii)
3-Bromo-6-difluoromethoxy-2-(3-fluoro-phenyl)-pyrazolo[1,5-b]pyridaz-
ine
[0171] N-bromo succinimide (195 mg) was added to a solution of
6-difluoromethoxy-2-(3-fluoro-phenyl)-pyrazolo[1,5-b]pyridazine
(251 mg) and sodium bicarbonate (185 mg) in anhydrous DMF (10 ml)
and stirred for 18 h. The reaction mixture was diluted with ethyl
acetate (300 ml) and washed with water (10.times.20 ml), brine (20
ml), dried (Na.sub.2SO.sub.4) and concentrated to give the title
compound as a solid (293 mg, 91%).
[0172] MH.sup.+ 359, MH.sup.- 356/357
[0173] NMR (DMSO) .delta. 8.36 (1H, d, J=10 Hz) 7.88 (1H, d, J=8
Hz) 7.78 (1H, t, J=70 Hz, OCHF.sub.2) 7.77 (1H, dm, J=10 Hz) 7.62
(1H, dt, J 8 & 6 Hz) 7.38 (1H, dt, J 9 & 2 Hz) 7.3 (1H, d,
J=10 Hz)
(viii)
6-Difluoromethoxy-2-(3-fluoro-phenyl)-3-(4-methanesulfonyl-phenyl)--
pyrazolo[1,5-b]pyridazine
[0174] To a stirred solution of
3-bromo-6-difluoromethoxy-2-(3-fluoro-phen-
yl)-pyrazolo[1,5-b]pyridazine (286 mg) in DMF(20 ml) was added 2N
aq sodium carbonate (10 ml). To this mixture was added
4-methanesulfonyl-phenylboronic acid (180 mg) and tetrakis
triphenylphosphine palladium (0) (34 mg). The resulting mixture was
stirred and heated to reflux for 18 hours. The cooled reaction
mixture was diluted with ethyl acetate (300 ml) and the organic
solution washed with water (10.times.30 ml) and brine (30 ml),
dried (Na.sub.2SO.sub.4) and evaporated to give a gum which was
purified by flash column chromatography with chloroform:ethyl
acetate (50:1 to 5:1) as eluant. Combination of appropriate
fractions and concentration gave the title compound as an off-white
solid (132 mg, 37%).
[0175] MH.sup.+ 434
[0176] 1H NMR(CDCl.sub.3) .delta. 8.02 (1H, d, J=9 Hz); 7.95 (2H,
d, J=10 Hz); 7.58 (1H, d, 9 Hz); 7.52 (1H, t, J=70 Hz); 7.32 (3H,
m); 7.08 (1H, m); 6.9 (1H, d, J=9 Hz); 3.15 (3H, s).
[0177] Biological Data
[0178] Inhibitory activity against human COX-1 and COX-2 was
assessed in COS cells which had been stably transfected with cDNA
for human COX-1 and human COX-2. 24 Hours prior to experiment, COS
cells were transferred from the 175 cm.sup.2 flasks in which they
were grown, onto 24-well cell culture plates using the following
procedure. The incubation medium (Dulbecco's modified eagles medium
(DMEM) supplemented with heat-inactivated foetal calf serum (10%
v/v), penicillin (100 IU/ml), streptomycin (100 .mu.g/ml) and
geneticin (600 .mu.g/ml)) was removed from a flask of confluent
cells (1 flask at confluency contains approximately
1.times.10.sup.7 cells). 10 ml of phosphate buffered saline (PBS)
was added to the flask to wash the cells. Having discarded the PBS,
cells were then rinsed in 10 ml trypsin for 20 seconds, after which
the trypsin was removed and the flask placed in an incubator
(37.degree.) for 1-2 minutes until cells became detached from the
flask. The flask was then removed from the incubator and cells
resuspended in 10 ml of fresh incubation medium. The contents of
the flask was transferred to a 250 ml sterile container and the
volume of incubation medium subsequently made up to 100 ml. 1 ml
cell suspension was pipetted into each well of 4.times.24-well cell
culture plates. The plates were then placed in an incubator
(37.degree. C., 95% air/5% CO.sub.2) overnight. If more than 1
flask of cells were required, the cells from the individual flasks
were combined before being dispensed into the 24-well plates.
[0179] Following the overnight incubation, the incubation medium
was completely removed from the 24-well cell culture plates and
replaced with 250 .mu.l fresh DMEM (37.degree. C.). The test
compounds were made up to 250.times. the required test
concentration in DMSO and were added to the wells in a volume of 1
.mu.l. Plates were then mixed gently by swirling and then placed in
an incubator for 1 hour (37.degree. C., 95% air/5% CO.sub.2).
Following the incubation period, 10 .mu.l of arachidonic acid (750
.mu.M) was added to each well to give a final arachidonic acid
concentration of 30 .mu.M. Plates were then incubated for a further
15 minutes, after which the incubation medium was removed from each
well of the plates and stored at -20.degree. C., prior to
determination of prostaglandin E.sub.2 (PGE2) levels using enzyme
immunoassay. The inhibitory potency of the test compound was
expressed as an IC.sub.50 value, which is defined as the
concentration of the compound required to inhibit the PGE2 release
from the cells by 50%. The selectivity ratio of inhibition of COX-1
versus COX-2 was calculated by comparing respective IC.sub.50
values.
[0180] The following IC.sub.50 values for inhibition of COX-2 and
COX-1 were obtained for compounds of the invention:
1 Example No. COX-2: IC.sub.50(nM) COX-1: IC.sub.50(nM) 1(v) 35
>100,000 2(ii) <10 3,880 3(ii) 3 >100,000 4(iii) 370
>100,000 5 21 >100,000 6(iv) 0.44 3828 7(viii) 16
>55,200
* * * * *