Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
2-(3 ,5-DIFLUOROPHENYL)-3-(4-(METHYLSULFONYL)PHENYL)-
2-CYCLOPENTEN-l-ONE USEFUL AS AN IN~BITOR OF COX-2
5 BACKGROUND OF THE INVENTION
This invention relates to methods of treating cyclooxygenase
mediated diseases and certain pharmaceutical compositions therefor.
Non-steroidal, antiinfl~mm~tory drugs exert most of their
antiinfl~mm~tory, analgesic and antipyretic activity and inhibit hormone-
10 induced uterine contractions and certain types of cancer growth throughinhibition of prostaglandin G/H synthase, also known as cyclooxygenase.
Initially, only one form of cyclooxygenase was known, this
corresponding to cyclooxygenase-l (COX-l) or the constitutive enzyme,
as originally identified in bovine seminal vesicles. More recently the
15 gene for a second inducible form of cyclooxygenase, cyclooxygenase-2
(COX-2) has been cloned, sequenced and characterized initially from
chicken, murine and human sources. This enzyme is distinct from the
COX-l which has been cloned, sequenced and characterized from various
sources including the sheep, the mouse and man. The second form of
20 cyclooxygenase, COX-2, is rapidly and readily inducible by a number of
agents including mitogens, endotoxin, hormones, cytokines and growth
factors. As prostaglandins have both physiological and pathological
roles, we have concluded that the constitutive enzyme, COX-l, is
responsible, in large part, for endogenous basal release of prostaglandins
25 and hence is important in their physiological functions such as the
maintenance of gastrointestinal integrity and renal blood flow. In
contrast, we have concluded that the inducible form, COX-2, is mainly
responsible for the pathological effects of prostaglandins where rapid
induction of the enzyme would occur in response to such agents as
30 infl~mm~tory agents, hormones, growth factors, and cytokines. Thus, a
selective inhibitor of COX-2 will have similar ~ntiinfl~mm~tory,
antipyretic and analgesic properties to a conventional non-steroidal
antiinfl~mm~tory drug, and in addition would inhibit hormone-induced
uterine contractions and have potential anti-cancer effects, but will have a
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~imini.shed ability to induce some of the mechanism-based side effects.
In particular, such a compound should have a reduced potential for
gastrointestinal toxicity, a reduced potential for renal side effects, a
reduced effect on bleeding times and possibly a lessened ability to induce
asthma attacks in aspirin-sensitive asthmatic subjects.
Furthermore, such a compound will also inhibit prostanoid-
induced smooth muscle contraction by preventing the synthesis of
contractile prostanoids and hence may be of use in the treatment of
dysmenorrhea, premature labour, asthma and eosinophil related disorders.
10 It will also be of use in the treatment of Alzheimer's disease, for
decreasing bone loss particularly in postmenopausal women (i.e.
treatment of osteoporosis) and for the treatment of glaucoma.
A brief description of the potential utility of
cyclooxygenase-2 inhibitors is given in an article by John Vane, Nature,
15 Vol. 367, pp. 215-216, 1994, and in an article in Drug News and
Perspectives, Vol. 7, pp. 501-512, 1994.
SUMMARY OF THE INVENTION
The invention encompasses the novel compound 2-(3,5-
20 dinuorophenyl)-3-(4-(methylsulfonyl)phenyl)-2-cyclopenten- 1-one,
pharmaceutical compositions and use of the compound in the preparation
of medicaments for the treatment of cyclooxygenase-2 mediated diseases.
DETAILED DESCRIPTION OF THE INVENTION
In one aspect the invention encompasses the compound 2-
(3,5-difluorophenyl)-3-(4-(methylsulfonyl)phenyl)-2-cyclopenten- 1 -one:
,~,SO2CH3
~F
Compound A
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In a second aspect the invention encompasses
pharmaceutical compositions useful for the treatment of COX-2 mediated
diseases comprising 2-(3,5-difluorophenyl)-3-(4-(methylsulfonyl)-
phenyl)-2-cyclopenten-1-one and a pharmaceutically acceptable carrier.
In a third aspect the invention encompasses pharmaceutical
compositions comprising 2-(3,5-difluorophenyl)-3-(4-(methylsulfonyl)-
phenyl)-2-cyclopenten-1-one and a pharmaceutically acceptable carrier.
In a fourth aspect the invention encompasses the use 2-(3,5-
difluorophenyl)-3-(4-(methylsulfonyl)phenyl)-2-cyclopenten-1-one in the
10 preparation of a medicament for the treatment of COX-2 mediated
dlseases.
In a further aspect compositions, methods of treatment, and
uses involving oral ~lmini~tration is preferred.
In a further aspect, compositions, methods of treatment and
15 use involving once a day or twice a day ~lmini~tration is preferred.
The following abbreviations have the indicated meanings:
AA = arachidonic acid
CHO = chinese hamster ovary
CMC = l-cyclohexyl-3-(2-morpholinoethyl)
carbodiimidemetho-p-toluenesulfonate
COX = cyclooxygenase
DMF = N,N-dimethylform~mide
HBSS = Hanks balanced salt solution
HEPES = N-[2-Hydroxyethyl]piperazine-NI-[2-
ethanesulfonic acid]
HWB = human whole blood
LPS = lipopolysaccharide
mCPBA = meta-chloroperbenzoic acid
MMPP = magnesium monoperoxyphthalate
NSAID = non-steroidal anti-infl~mm~tory drug
PDC = pyridinium dichromate
r.t. = room temperature
THF = tetrahydrofuran
The Compound A is useful for the relief of pain, fever and
infl~mm~tion of a variety of conditions including rheumatic fever,
symptoms associated with influenza or other viral infections, common
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cold, low back and neck pain, dysmenorrhea, headache, toothache,
sprains and strains, myositis, neuralgia, synovitis, arthritis, including
rheumatoid arthritis, degenerative joint diseases (osteoarthritis), gout and
ankylosing spondylitis, bursitis, burns, injuries following surgical and
5 dental procedures. In addition, such a compound may inhibit cellular
neoplastic transformations and metastic tumour growth and hence can be
used in the treatment of cancer, such as cancer of the colon. Compound
A may also be of use in the treatment and/or prevention of
cyclooxygenase-mediated proliferative disorders such as may occur in
10 diabetic retinopathy and tumour angiogenesis.
Compound A will also inhibit prostanoid-induced smooth
muscle contraction by preventing the synthesis of contractile prostanoids
and hence may be of use in the treatment of dysmenorrhea, premature
labour, asthma and eosinophil related disorders. It will also be of use in
15 the treatment of Alzheimer's disease, for decreasing bone loss particularly
in postmenopausal women (i.e. treatment of osteoporosis), and for
treatment of glaucoma.
By virtue of its high cyclooxygenase-2 (COX-2) activity
and/or its specificity for cyclooxygenase-2 over cyclooxygenase-l (COX-
20 1), Compound A will prove useful as an alternative to conventional non-
steroidal antiinfl~mm~tory drugs (NSAID'S) particularly where such non-
steroidal antiinfl~mm~tory drugs may be contra-indicated such as in
patients with peptic ulcers, gastritis, regional enteritis, ulcerative colitis,
diverticulitis or with a recurrent history of gastrointestinal lesions; GI
25 bleeding, coagulation disorders including anemia such as
hypoprothrombinemia, haemophilia or other bleeding problems; kidney
disease; those prior to surgery or taking anticoagulants.
Similarly, Compound A, will be useful as a partial or
complete substitute for conventional NSAID'S in preparations wherein
30 NSAIDS are presently co-atlmini~tered with other agents or ingredients.
Thus in further aspects, the invention encompasses ph~rm~ceutical
compositions for treating cyclooxygenase-2 mediated diseases as defined
above comprising a non-toxic therapeutically effective amount of the
compound of Formula I as defined above and one or more ingredients
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such as another pain reliever including acetominophen or phenacetin; a
potentiator including caffeine; an H2-antagonist, aluminum or
magnesium hydroxide, simethicone, a decongestant including
phenylephrine, phenylpropanolamine, pseudoephedrine, oxymetazoline,
S ephinephrine, naphazoline, xylometazoline, propylhexedrine, or levo-
desoxyephedrine; an antiitussive including codeine, hydrocodone,
caramiphen, carbetapentane, or dextramethorphan; a prostaglandin
including misoprostol, enprostil, rioprostil, ornoprostol or rosaprostol; a
diuretic; a sedating or non-sedating antihistamine. In addition the
10 invention encompasses a method of treating cyclooxygenase mediated
diseases comprising: ~rlministration to a patient in need of such treatment
a non-toxic therapeutically effective amount of the compound A,
optionally co-~(1ministered with one or more of such ingredients as listed
irnmediately above.
For the treatment of any of these cyclooxygenase mediated
diseases Compound A may be ~(lmini.stered orally, topically, parenterally,
by inhalation spray or rectally in dosage unit formulations containing
conventional non-toxic pharmaceutically acceptable carriers, adjuvants
and vehicles. The term parenteral as used herein includes subcutaneous
20 injections, intravenous, intramuscular, intrasternal injection or infusion
techniques. In addition to the treatment of warm-blooded :~nim~ls such as
mice, rats, horses, cattle sheep, dogs, cats, etc., the compound of the
invention is effective in the treatment of humans.
As indicated above, pharmaceutical compositions for
25 treating cyclooxygenase-2 mediated diseases as defined may optionally
include one or more ingredients as listed above.
The ph~rm~ceutical compositions cont~ining the active
ingredients may be in a form suitable for oral use, for example, as tablets,
troches, lozenges, aqueous or oily suspensions, dispersible powders or
30 granules, emulsions, hard or soft capsules, or syrups or elixirs.
Compositions intended for oral use may be prepared according to any
method known to the art for the manufacture of ph~rm~ceutical
compositions and such compositions may contain one or more agents
selected from the group consisting of sweetening agents, flavoring agents,
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coloring agents and preseNing agents in order to provide
pharmaceutically elegant and palatable preparations. Tablets contain the
active ingredient in admixture with non-toxic pharmaceutically
acceptable excipients which are suitable for the manufacture of tablets.
5 These excipients may be, for example, inert diluents, such as calcium
carbonate, sodium carbonate, lactose, calcium phosphate or sodium
phosphate; granulating and disintegrating agents, for example, corn
starch, or alginic acid; binding agents, for example starch, gelatin or
acacia, and lubricating agents, for example, magnesium stearate, stearic
10 acid or talc. The tablets may be uncoated or they may be coated by
known techniques to delay disintegration and absorption in the
gastrointestinal tract and thereby provide a sustained action over a longer
period. For exarnple, a time delay material such as glyceN~ 1 monostearate
or glyceryl distearate may be employed. They may also be coated by the
15 technique described in the U.S. Patent 4,256,108; 4,166,452; and
4,265,874 to form osmotic therapeutic tablets for control release.
Formulations for oral use may also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an inert solid
diluent, for example, calcium carbonate, calcium phosphate or kaolin, or
20 as soft gelatin capsules wherein the active ingredients is mixed with water
or miscible solvents such as propylene glycol, PEGs and ethanol, or an oil
medium, for example peanut oil, liquid paraffin, or olive oil.
Aqueous suspensions contain the active materials in
admixture with excipients suitable for the manufacture of aqueous
25 suspensions. Such excipients are suspending agents, for example, sodium
carboxymethylcellulose, methylcellulose, hydroxy-propylmethycellulose,
sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;
dispersing or wetting agents may be a naturally-occurring phosphatide,
for example lecithin, or condensation products of an alkylene oxide with
30 fatty acids, for example polyoxyethylene stearate, or condensation
products of ethylene oxide with long chain aliphatic alcohols, for
example heptadecaethyleneoxycetanol, or condensation products of
ethylene oxide with partial esters derived from fatty acids and a hexitol
such as polyoxyethylene sorbitol monooleate, or condensation products
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of ethylene oxide with partial esters derived from fatty acids and hexitol
anhydrides, for ex~mple polyethylene sorbitan monooleate. The aqueous
suspensions may also contain one or more preservatives, for example
ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one
5 or more flavoring agents, and one or more sweetening agents, such as
sucrose, saccharin or aspartame.
Oily suspensions may be formulated by suspending the
active ingredients in a vegetable oil, for example, arachis oil, olive oil,
sesame oil or coconut oil, or in mineral oil such as liquid paraffin. The
10 oily suspensions may contain a thickening agent, for example, beeswax,
hard paraffin or cetyl alcohol. Sweetening agents such as those set forth
above, and flavoring agents may be added to provide a palatable oral
preparation. These compositions may be preserved by the addition of an
anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredients in admixture with a dispersing or wetting agent, suspending
agent and one or more preservatives. Suitable dispersing or wetting
agents and suspending agents are exemplified by those already mentioned
20 above. Additional excipients, for example, sweetening, flavoring and
coloring agents, may also be present.
The pharmaceutical compositions of the invention may also
be in the form of an oil-in-water emulsions. The oily phase may be a
vegetable oil, for example, olive oil or arachis oil, or a mineral oil, for
25 example, liquid paraffin or mixtures of these. Suitable emulsifying
agents may be naturally-occurring phosphatides, for example, soy bean,
lecithin, and esters or partial esters derived from faKy acids and hexitol
anhydrides, for ex~mple, sorbitan monooleate, and condensation products
of the said partial esters with ethylene oxide, for example, polyoxy-
30 ethylene sorbitan monooleate. The emulsions may also containsweetening and flavouring agents.
Syrups and elixirs may be formulated with sweetening
agents, for example, glycerol, propylene glycol, sorbitol or sucrose. Such
formulations may also contain a demulcent, a preservative and flavoring
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and coloring agents. The pharmaceutical compositions may be in the
form of a sterile injectable aqueous or oleagenous suspension. This
suspension may be formulated according to the known art using those
suitable dispersing or wetting agents and suspending agents which have
5 been mentioned above. The sterile injectable preparation may also be a
sterile injectable solution or suspension in a non-toxic parenterally-
acceptable diluent or solvent, for example, as a solution in 1,3-butane
diol. Among the acceptable vehicles and solvents that may be employed
are water, Ringer's solution and isotonic sodium chloride solution.
10 Cosolvents such as ethanol, propylene glycol or polyethylene glycols may
also be used. In addition, sterile, fixed oils are conventionally employed
as a solvent or suspending medium. For this purpose any bland fixed oil
may be employed including synthetic mono- or diglycerides. In addition,
fatty acids such as oleic acid find use in the preparation of injectables.
Compound A may also be ~(lmini~tered in the form of a
suppositories for rectal ~clmini~tration of the drug. These compositions
can be prepared by mixing the drug with a suitable non-irritating
excipient which is solid at ordinary temperatures but liquid at the rectal
temperature and will therefore melt in the rectum to release the drug.
Such materials are cocoa butter and polyethylene glycols.
For topical use, creams, ointments, gels, solutions or
suspensions, etc., containing the compound A are employed. (For
purposes of this application, topical application shall include mouth
washes and gargles.) Topical formulations may generally be comprised
of a ph~ ceutical carrier, cosolvent, emulsifier, penetration enhancer,
preservative system, and emollient.
Other suitable formulations are set forth in U.S. Patent No.
5,474,995. We have found the following oral formulations to be of
particular value:
Rapidisc(~) - In view of the above mentioned characteristics,
2-(3 ,5-difluorophenyl)-3-(4-(methylsulfonyl)phenyl)-2-cyclopenten- 1-
one is particularly well suited for a rapid dissolving sublingual
formulation. For example, due to the lack of GI side-effects, the agent
need not be take with a large amount of water. Suitable Rapidisc(~)
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formulations and methods of making same are disclosed in US 4,305,502,
US 4,371,516, US 4,470,202, US 4,758,598, US 4,754,597, US 5,046,618
and US 5,188,882, all of which are hereby incorporated by reference.
As mentioned elsewhere in this specification, we have found
5 2-(3,5-difluorophenyl)-3-(4-(methylsulfonyl)phenyl)-2-cyclopenten- 1 -
one to possess a surprising combination of attributes. Not only is
compound A active, potent, safe and effective at modest oral dosages of
10 to 250 mg of agent per day, but in addition Compound A possesses a
half-life in humans of sufficient length that one or two oral doses of 10 to
10 250 mg of active agent per day will provide effective safe anti-
infl~mm~tory treatment over a 24 hour period. Such agents are
particularly useful in the treatment of chronic indications, such as
rheumatoid and osteo arthritis as well as Alzheimer's Disease.
Oral and intravenous dosage levels for agent 2-(3,5-
15 difluorophenyl)-3-(4-(methylsulfonyl)phenyl)-2-cyclopenten-1-one are of
the order of from about 10 to 250 mg per patient once or twice a day.
The amount of active agent that may be combined with the
carrier materials to produce a single dosage form will vary depending
upon the host treated and the particular mode of ~lministration. For
20 example, a formulation intended for the oral ~lministration of humans
may contain from 10 to 250 mg of agent compounded with an
appropriate and convenient amount of carrier material which may vary
from about 5 to about 95 percent of the total composition. Dosage unit
forms may typically contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125 or
25 250 mg of active agent.
It will be understood, however, that the specific dose level
for any particular patient will depend upon a variety of factors including
the age, body weight, general health, sex, diet, time of ~(lmini~tration,
rate of excretion, drug combination and the type and severity of the
30 particular disease undergoing therapy. For many patients, a dosage
range of 10 to 50 or 60 to 120 mg once or twice a day is preferred.
For long term therapy, such as in the treatment of chronic
diseases including rheumatoid arthritis, osteoarthritis or Alzheimer
disease, a dosage of 10 to 50 or 60 to 120 mg once or twice day is
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preferred. More particularly, for the treatment of osteoarthritis, a dosage
of 10, 20, 30, 40, 50, 60,70, 80, 90 or 100 mg once or twice a day is
preferred, whereas for the treatment of rheumatoid arthritis, 10, 20, 30,
40, S0, 60, 70, 80, 90 or 100 mg once or twice a day is preferred. For the
treatment of non-chronic indications such as headache or post-operative
swelling and pain, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg once or
twice a day is preferred.
Accordingly, in one aspect the invention is directed to a unit
dose oral form which comprises from 10 to 250 mg of the
10 cyclooxygenase inhibitor, for example, 10 to 50 or 60 to 120 mg.
In another aspect this invention is directed to a
pharmaceutical composition for the treatment of cyclooxygenase-2
mediated diseases, said composition suitable for once or twice a day oral
~lmini~tration, said composition comprising a 10 to 250 mg of 2-(3,5-
15 difluorophenyl)-3-(4-(methylsulfonyl)phenyl)-2-cyclopenten- 1 -one, and a
pharmaceutical carried therefor.
Within this aspect there is a first genus of compositions
comprising 10 to 50 mg of 2-(3,5-difluorophenyl)-3-(4-
(methylsulfonyl)phenyl)-2-cyclopenten- l-one.
Within this aspect there is a second genus of compositions
comprising 60 to 120 mg of 2-(3,5-difluorophenyl)-3-(4-
(methylsulfonyl)phenyl)-2-cyclopenten- l-one.
The amount of active ingredient that may be combined with
the carrier materials to produce a single dosage form will vary depending
25 upon the host treated and the particular mode of ~lmini~tration. For
example, a formulation intended for the oral ~lmini~tration of humans
may contain from 10 mg to 250 mg of active agent compounded with an
appropriate and convenient amount of carrier material which may vary
from about S to about 95 percent of the total composition. Dosage unit
30 forms will generally contain between from about 10 mg to about 250 mg
of an active ingredient, typically 10 mg, 25 mg, 50 mg, 100 mg, 125 mg,
or250mg.
It will be understood, however, that the specific dose level
for any particular patient will depend upon a variety of factors including
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the age, body weight, general health, sex, diet, time of ~flmini.stration,
route of ~lmini~tration, rate of excretion, drug combination and the
severity of the particular disease undergoing therapy.
S Assays for determining Biological Activity
The compound A can be tested using the following assays to
determine their cyclooxygenase-2 inhibiting activity.
INHIBITION OF CYCLOOXYGENASE ACTIVITY
Whole cell assays for COX-2 and COX-l using CHO transfected
cell lines
Chinese harnster ovary (CHO) cell lines which have been
stably transfected with an eukaryotic expression vector pCDNAIII
containing either the human COX- 1 or COX-2 cDNA's are used for the
assay. These cell lines are referred to as CHO [hCOX-l] and CHO
[hCOX-2], respectively. For cyclooxygenase assays, CHO[hCOX-l]
20 cells from suspension cultures and CHO[hCOX-2] cells prepared by
trypsinization of a&erent cultures are harvested by centrifugation (300 x
g, 10 min) and washed once in HBSS containing 15 mM HEPES, pH 7.4,
and resuspended in HBSS, 15 mM HEPES, pH 7.4, at a cell concentration
of 1.5 x 106 cells/rnl. Drugs to be tested are dissolved in DMSO to 66.7-
25 fold the highest test drug concentration. Compounds are typically testedat 8 concentrations in duplicate using serial 3-fold serial dilutions in
DMSO of the highest drug concentration. Cells (0.3 x 106 cells in 200
~11) are preincubated with 3 111 of the test drug or DMSO vehicle for 15
rnin at 37~C. Working solutions of peroxide-free AA (5.5 llM and 110
30 IlM AA for the CHO [hCOX-1] and CHO [COX-2] assays, respectively)
are prepared by a 10-fold dilution of a concentrated AA solution in
ethanol into HBSS containing 15 mM HEPES, pH 7.4. Cells are then
challenged in the presence or absence of drug with the AA/HBSS
solution to yield a final concentration of 0.5 ~M AA in the CHO[hCOX-
35 1] assay and a final concentration of 10 IlM AA in the CHO[hCOX-2]
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assay. The reaction is termin~ted by the addition of 10 111 1 N HCl
followed by neutralization with 20 ~11 of 0.5 N NaOH. The samples are
centrifuged at 300 x g at 4~C for 10 min, and an aliquot of the clarified
supernatant is appropriately diluted for the deterrnination of PGE2 levels
using an enzyme-linked imrnunoassay for PGE2 (Correlate PGE2 enzyme
imrnunoassay kit, Assay Designs, Inc.). Cyclooxygenase activity in the
absence of test compounds is determined as the difference in PGE2
levels of cells challenged with arachidonic acid versus the PGE2 levels in
cells mock-challenged with ethanol vehicle. Inhibition of PGE2 synthesis
10 by test compounds is calculated as a percentage of the activity in the
presence of drug versus the activity in the positive control sarnples.
Assay of COX-1 Activity from U937 cell microsomes
U 937 cells are pelleted by centrifugation at 500 x g for 5
15 min and washed once with phosphate-buffered saline and repelleted.
Cells are resuspended in homogenization buffer consisting of 0.1 M Tris-
HCl, pH 7.4, 10 mM EDTA, 2 llg/ml leupeptin, 2 llg/rnl soybean trypsin
inhibitor, 2 ~lg/ml aprotinin and 1 mM phenyl methyl sulfonyl fluoride.
The cell suspension is sonicated 4 times for 10 sec and is centrifuged at
20 10,000 x g for 10 min at 4~ C. The supernatant is centrifuged at 100,000
x g for 1 hr at 4~ C. The 100,000 x g microsomal pellet is resuspended in
0.1 M Tris-HCl, pH 7.4, 10 mM EDTA to approximately 7 mg protein/ml
and stored at -80~ C.
Microsomal preparations are thawed immediately prior to
25 use, subjected to a brief sonication, and then diluted to a protein
concentration of 125 llg/ml in 0.1 M Tris-HCl buffer, pH 7.4 containing
10 mM EDTA, 0.5 mM phenol, 1 mM reduced glutathione and 1 ~M
hematin. Assays are performed in duplicate in a final volume of 250 IlL
Initially, S ',11 of DMSO vehicle or drug in DMSO are added to 20 111 of
30 0.1 M Tris-HCl buffer, pH 7.4 containing 10 mM EDTA in wells of a 96-
deepwell polypropylene titre plate. 200 ~11 of the microsomal preparation
are then added and pre-incubated for 15 min at room temperature before
addition of 25 111 of 1 M arachidonic acid in 0.1 M Tris-HCl and 10 mM
EDTA, pH 7.4. Samples are incubated for 40 min at room temperature
35 and the reaction is stopped by the addition of 25 ~11 of 1 N HCl. Samples
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are neutralized with 25 111 of 1 N NaOH prior to quantitation of PGE2
content by radioimmunoassay (Dupont-NEN or Amersham assay kits).
Cyclooxygenase activity is defined as the difference between PGE2 levels
in samples incubated in the presence of arachidonic acid and ethanol
5 vehicle.
Assay of the activitv of purified human COX-2
The enzyme activity is measured using a chromogenic assay
based on the oxidation of N,N,N',N'-tetramethyl-p-phenylenedi~mine
10 (TMPD) during the reduction of PGG2 to PGH2 by COX-2 (Copeland et
al. (1994) Proc. Natl. Acad. Sci. 91, 11202-11206).
Recombinant human COX-2 is purified from Sf9 cells as
previously described (Percival et al (1994) Arch. Biochem. Biophys. 15,
111-118). The assay mixture (180 IlL) contains 100 mM sodium
15 phosphate, pH 6.5, 2 mM genapol X-100, 1 ,uM hematin, 1 mg/ml gelatin
, 80-100 units of purified enzyme (One unit of enzyme is defined as the
amount of enzyme required to produce an O.D. change of 0.001/min at
610 nm) and 4 IlL of the test compound in DMSO. The mixture is pre-
incubated at room temperature (22~C) for 15 minutes prior to initiation of
20 the enzymatic reaction by the addition of 20 ,uL of a sonicated solution of
1 mM arachidonic acid (AA) and 1 mM TMPD in assay buffer (without
enzyme or hematin). The enzymatic activity is measured by estimation of
the initial velocity of TMPD oxidation over the first 36 sec of the
reaction. A non-specific rate of oxidation is observed in the absence of
25 enzyme (0.007 - 0.010 O.D. /min) and is subtracted before the calculation
of the % inhibition. IC50 values are derived from 4-parameter least
squares non-linear regression analysis of the log-dose vs % inhibition
plot.
30 HUMAN WHOLE BLOOD ASSAY
Rationale
Human whole blood provides a protein and cell-rich m ieu
appropriate for the study of biochernical efficacy of anti-infl~mm~tory
35 compounds such as selective COX-2 inhibitors. Studies have shown that
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normal human blood does not contain the COX-2 enzyme. This is
consistent with the obseNation that COX-2 inhibitors have no effect on
PGE2 production in normal blood. These inhibitors are active only after
incubation of human whole blood with LPS, which induces COX-2. This
5 assay can be used to evaluate the inhibitory effect of selective COX-2
inhibitors on PGE2 production. As well, platelets in whole blood contain
a large amount of the COX-1 enzyme. Immediately following blood
clotting, platelets are activated through a thrombin-mediated mechanism.
This reaction results in the production of thromboxane B2 (TXB2) via
10 activation of COX-1. Thus, the effect of test compounds on TxB2 levels
following blood clotting can be examined and used as an index for COX-
1 activity. Therefore, the degree of selectivity by the test compound can
be determined by measuring the levels of PGE2 after LPS induction
(COX-2) and TxB2 following blood clotting (COX-1) in the sarne assay.
Method
A. COX-2 (LPS-induced PGE2 production)
Fresh blood is collected in heparinized tubes by
venipuncture from both male and female volunteers. The subjects have
20 no apparent infl~mm~tory conditions and have not taken any NSAIDs for
at least 7 days prior to blood collection. Plasma is immediately obtained
from a 2mL blood aliquot to use as blank (basal levels of PGE2). The
rem~ining blood is incubated with LPS (100 llg/ml final concentration,
Sigma Chem, #L-2630 from E. coli; diluted in 0.1% BSA (Phosphate
25 buffered s~line) for 5 minutes at room temperature. Five hundred IIL
aliquots of blood are incubated with either 211L of vehicle (DMSO) or
2~L of a test compound at final concentrations varying from 10nM to
30,uM for 24 hours at 37~C. At the end of the incubation, the blood is
centrifuged at 12,000 x g for 5 minutes to obtain plasma. A 100~L
30 aliquot of plasma is n~ixed with 400~1L of methanol for protein
precipitation. The supernatant is obtained and is assayed for PGE2 using
a radioimmunoassay kit (Amersham, RPA#530) after conversion of
PGE2 to its methyl oximate derivative according to the manufacturer's
procedure.
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B. COX-1 (Clotting-induced TxB2 production)
Fresh blood is collected into vacutainers containing no
anticoagulants. Aliquots of 500~L are immediately transferred to
siliconized rnicrocentrifuge tubes preloaded with 2,uL of either DMSO or
S a test compound at final concentrations varying from 10nM to 3011M.
The tubes are vortexed and incubated at 37~C for 1 hour to allow blood to
clot. At the end of incubation, serum is obtained by centrifugation
(12,000 x g for 5 min.). A 100,uL aliquot of serum is mixed with 400~L
of methanol for protein precipitation. The supernatant is obtained and is
10 assayed for TxB2 using a enzyme immunoassay kit (Cayman, #519031)
according to the manufacturer's instruction.
RAT PAW EDEMA ASSAY
1 5 Protocol
Male Sprague-Dawley rats (150-200 g) are fasted overnight
and are given, po, either vehicle (1% methocel or 5% Tween 80) or a test
compound. One hr later, a line is drawn using a permanent marker at the
level above the ankle in one hind paw to define the area of the paw to be
20 monitored. The paw volume (Vo) is measured using a plethysmometer
(Ugo-Basile, Italy) based on the principle of water displacement. The
~nim~ls are then injected subplantarly with 50 ~11 of 1% carrageenan
solution in saline (FMC Corp, Maine) into the paw using an insulin
syringe with a 25-gauge needle (i.e. 500 ,ug carrageenan per paw). Three
25 hr later, the paw volume (V3) is measured and the increases in paw
volume (V3 - Vo) are calculated. The ~nim~l.s are sacrificed by CO2
asphyxiation and the absence or presence of stomach lesions scored.
Data is compared with the vehicle-control values and percent inhibition
calculated. All treatment groups are coded to elimin~te observer bias.
NSAID-INDUCED GASTROPATHY IN RATS
Rationale
The major side effect of conventional NSAIDs is their ability
35 to produce gastric lesions in man. This action is believed to be caused by
inhibition of Cox-1 in the gastrointestinal tract. Rats are particularly
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sensitive to the actions of NSAIDs. In fact, rat models have been used
commonly in the past to evaluate the gastrointestinal side effects of
current conventional NSAIDs. In the present assay, NSAID-induced
gastrointestinal damage is observed by measuring fecal 51Cr excretion
5 after systemic injection of 51Cr-labeled red blood cells. Fecal 51Cr
excretion is a well-established and sensitive technique to detect
gastrointestinal integrity in ~nim~l~ and man.
Methods
Male Sprague Dawley rats (150 - 200 g) are ~lmini.stered
orally a test compound either once (acute dosing) or b.i.d. for S days
(chronic dosing). Immediately after the ~lmini.stration of the last dose,
the rats are injected via a tail vein with 0.5 mL of 51Cr-labeled red blood
cells from a donor rat. The ~nim~l~ are placed individually in metabolism
15 cages with food and water ad lib. Feces are collected for a 48 h period
and 51Cr fecal excretion is calculated as a percent of total injected dose.
51Cr-labeled red blood cells are prepared using the following procedures.
Ten mL of blood is collected in heparinized tubes via the vena cava from
a donor rat. Plasma is removed by centrifugation and replenished with
20 equal volume of HBSS. The red blood cells are incubated with 400 Ci of
sodium Slchromate for 30 min. at 37~ C. At the end of the incubation,
the red blood cells are washed twice with 20 mL HBSS to remove free
sodium S lchromate. The red blood cells are finally reconstituted in 10
mL HBSS and 0.5 mL of the solution (about 20 Ci) is injected per rat.
PROTEIN-LOSING GASTROPATHY IN SOUIRREL MONKEYS
Rationale
Protein-losing gastropathy (manifested as appearance of
30 circulating cells and plasma proteins in the GI tract) is a significant and
dose-limiting adverse response to standard non-steroidal anti-
infl~mm~tory drugs (NSAIDs). This can be quantitatively assessed by
intravenous ~clmini~tration of 51CrCl3 solution. This isotopic ion can
avidly bind to cell and serum globins and cell endoplasmic reticulum.
35 Measurement of radioactivity appearing in feces collected for 24 h after
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~lministration of the isotope thus provides a sensitive and quantitative
index of protein-losing gastropathy.
Methods
Groups of male squirrel monkeys (0.8 to 1.4 kg) are treated
by gavage with either 1% methocell or 5% Tween 80 in H20 vehicles,
(3mL/kg b.i.d.) or test compounds at doses from 1 - 100 mg/kg b.i.d. for 5
days. Intravenous 5 lCr (SCi/kg in 1 ml/kg phosphate buffer saline
(PBS)) is ~mini.~tered 1 h after the last drug/vehicle dose, and feces
collected for 24 h in a metabolism cage and assessed for excreted 51Cr
by gamrna-counting. Venous blood is sampled 1 h and 8 h after the last
drug dose, and plasma concentrations of drug measured by RP-HPLC.
LPS-Induced Pyrexia in Conscious Rats
Male Sprague-Dawley rats (150 - 200 g) were fasted for 16 -
1~ h before use. At approximately 9:30 a.m., the ~nim~l~ were placed
temporarily in plexiglass restrainers and their baseline rectal temperature
was recorded using a flexible temperature probe (YSI series 400)
connected to a digital thermometer (Model 08502, Cole Parmer). The
same probe and thermometer were used for all ~nim~l~ to reduce
experimental error. The ~nim~l~ were returned to their cages after the
temperature measurements. At time zero, the rats were injected
intraperitoneally with either saline or LPS (2 mg/kg, Sigma Chem) and
the rectal temperature was remeasured at 5, 6 and 7 h following LPS
injection. After the measurement at 5 h, when the increase in temperature
had reached a plateau, the LPS-injected rats were given either the vehicle
(1% methocel) or a test compound orally to deterrnine whether the
compound could reverse the pyrexia. Percent reversal of the pyrexia was
calculated using the rectal temperature obtained at 7 h in the control
(vehicle-treated) group as the reference (zero reversal) point. Complete
reversal of pyrexia to the pre-LPS baseline value is taken as 100%.
LPS-Induced Pyrexia in Conscious Squirrel Monkeys
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Temperature probes were surgically implanted under the
abdominal skin in a group of squirrel monkeys (Saimiri sciureus) (1.0 -
1.7 kg). This allows for the monitoring of body temperature in conscious,
unrestrained monkeys by a telemetric sensing system (Data Sciences
5 International, Minnesota). The ~nim~l.s were fasted and were placed in
individual cages for acclimati7~tion 13 - 14 h before use. Electronic
receivers were installed on the side of the cages which pick up signals
from the implanted temperature probes. At approximately 9:00 a.m. on
the day of the experiment, the monkeys were restrained temporarily in
10 training chairs and were given a bolus I.V. injection of LPS, (6 mg/kg,
dissolved in sterile saline). The ~nim~ls were returned to their cages and
body temperature was recorded continuously every 5 min. Two h after
injection of LPS, when the body temperature had increased by 1.5 - 2~ C,
the monkeys were dosed orally with either vehicle (1% methocel) or a
15 test compound (3 mg/kg). One hundred minutes later, the difference
between the body temperature and the baseline value was determined.
Percent inhibition was calculated taking the value in the control group as
0% inhibition.
20 Acute Inflammatory Hyperalgesia Induced by Carrageenan in Rats
Experiments were performed using male Sprague Dawley
rats (90-110g). Hyperalgesia to mechanical compression of the hind paw
was induced by intraplantar injection of carrageenan (4.5 mg into one
25 hind paw) 3 h previously. Control anim~l~ received an equivalent volume
of saline (0.15 ml intraplantar). A test compound (0.3-30 mg/kg,
suspended in 0.5% methocel in distilled water) or vehicle (0.5%
methocel) was ~clmini~tered orally (2ml/kg) 2 h after carrageenan. The
vocalisation response to compression of the hind paw was measured 1 h
30 later using a Ugo Basile algesiometer.
Statistical analysis for carrageenan-induced hyperalgesia was
performed using one-way ANOVA (BMDP Statistical Software Inc.).
Hyperalgesia was determined by subtracting the vocalisation threshold in
saline injected rats from that obtained in ~nim~ injected with
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carrageenan. Hyperalgesia scores for drug-treated rats were expressed as
a percentage of this response. IDso values (the dose producing 50% of
the maximum observed response) were then calculated by nonlinear least
squares regression analysis of mean data using GraFit (Erithacus
5 Software).
Adjuvant-Induced Arthritis in Rats
Seventy, 6.5-7.5 week old, female Lewis rats (body weight
~146-170 g) were weighed, ear marked, and assigned to groups (a
10 negative control group in which arthritis was not induced, a vehicle
control group, a positive control group ~lmini.stered indomethacin at a
total daily dose of 1 mg/kg and four groups ~tlmini~tered with a test
compound at total daily doses of 0.10-3.0 mg/kg) such that the body
weights were equivalent within each group. Six groups of 10 rats each
15 were injected into a hind paw with 0.5 mg of Mycobacterium butyricum
in 0.1 ml of light mineral oil (adjuvant), and a negative control group of
10 rats was not injected with adjuvant. Body weights, contralateral paw
volumes (determined by mercury displacement plethysmography) and
lateral radiographs (obtained under Ketamine and Xylazine anesthesia)
20 were determined before (day -1) and 21 days following adjuvant
injection, and primary paw volumes were deterrnined before (day -1) and
on days 4 and 21 following adjuvant injection. The rats were
anesthetized with an intramuscular injection of 0.03 - 0.1 ml of a
combination of Ketamine (87 mg/kg) and Xylazine (13 mg/kg) for
25 radiographs and injection of adjuvant. The radiographs were made of
both hind paws on day 0 and day 21 using the Faxitron (45 kVp, 30
seconds) and Kodak X-OMAT TL film, and were developed in an
automatic processor. Radiographs were evaluated for changes in the soft
and hard tissues by an investigator who was blinded to experimental
30 treatment. The following radiographic changes were graded numerically
according to severity: increased soft issue volume (0-4), narrowing or
widening of joint spaces (0-5) subchondral erosion (0-3), periosteal
reaction (0-4), osteolysis (0-4) subluxation (0-3), and degenerative joint
changes (0-3). Specific criteria were used to establish the numerical
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grade of severity for each radiographic change. The maximum possible
score per foot was 26. A test compound at total daily doses of 0.1, 0.3, 1,
and 3 mg/kg/day, Indomethacin at a total daily dose of 1 mg/kg/day, or
vehicle (0.5% methocel in sterile water) were ~-lmini~tered per os b.i.d.
5 beginning post injection of adjuvant and continuing for 21 days. The
compounds were prepared weekly, refrigerated in the dark until used, and
vortex mixed immediately prior to ~clmini~tration.
Two-factor ('treatment' and 'time') analysis of variance with
repeated measures on 'time' were applied to the % changes for body
10 weight and foot volumes and to the rank-transformed radiographic total
scores. A post hoc Dunnett' s test was conducted to compare the effect of
treatments to vehicle. A one-way analysis of variance was applied to the
thymic and spleen weights followed by the Dunnett' s test to compare the
effect of treatments to vehicle. Dose-response curves for % inhibition in
15 foot volumes on days 4, 14 and 21 were fitted by a 4-parameter logistic
function using a nonlinear least squares' regression. IDso was defined as
the dose corresponding to a 50% reduction from the vehicle and was
derived by interpolation from the fitted 4-parameter equation.
20 Representative Biological Data
Compounds of the present invention are inhibitors of COX-2
and are thereby useful in the treatment of COX-2 mediated diseases as
enumerated above. The activities of compound A against
cyclooxygenase may be seen in the representative results shown below in
25 the Table. In the assays, inhibition is determined by measuring the
amount of a prostaglandin synthesized in the presence and absence of a
putative inhibitor. The IC50 values represent the concentration of putative
inhibitor required to lower prostaglandin synthesis to 50% of that
obtained as compared to the uninhibited control. The ED50 values in the
30 rat paw edema assay represent the dose of compound required to reduce
edema formation by 50% as compared to the vehicle control. Shown also
are the corresponding biological activities of Compound B, which
compound is disclosed in WO 95/00501, Example 7 and US 5,536,752.
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Table
Assay ,1~ SO2CH3 ~ ~0, SO2CH3
b ~,F
F Compound B
Compound A
COX-2:
CHO Cells .03 ~M .02 ~M
Whole Blood .45 IlM .12 ,LM
COX- 1
CHO cells ~50 ~M 17
Whole Blood 77 ,uM 4.4 ~lM
U937 microsomes 11 ~,IM 0.45 ',lM
Rat Paw edema (ED50) 2.6 mg/kg 3.0 mg/kg
The invention will now be illustrated by the following non-
5 limiting examples in which, unless stated otherwise:
(i) all operations were carried out at room or ambient
temperature, that is, at a temperature in the range 18-25~C;
(ii) evaporation of solvent was carried out using a rotary
evaporator under reduced pressure (600-4000 pascals: 4.5-
30 mm Hg) with a bath temperature of up to 60~C;
(iii) the course of reactions was followed by thin layer
chromatography (TLC) and reaction times are given for
illustration only;
(iv) melting points are uncorrected and 'd' indicates
decomposition; the melting points given are those obtained
for the materials prepared as described; polymorphism may
result in isolation of materials with different melting points
m some preparatlons;
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(v) the structure and purity of all final products were assured by
at least one of the following techniques: TLC, mass
spectrometry, nuclear magnetic resonance (NMR)
spectrometry or microanalytical data;
(vi) yields are given for illustration only;
(vii) when given, NMR data is in the form of delta (o) values for
major diagnostic protons, given in parts per million (ppm)
relative to tetramethylsilane (TMS) as internal standard,
determined at 300 MHz or 400 MHz using the indicated
solvent; conventional abbreviations used for signal shape
are: s. singlet; d. doublet; t. triplet; m. multiplet; br. broad;
etc.: in addition "Ar" signifies an aromatic signal;
(viii) chemical symbols have their usual meanings; the following
abbreviations have also been used v (volume), w (weight),
b.p. (boiling point), M.P. (melting point), L (liter(s)), mL
(milliliters), g (gram(s)), mg (milligrams(s)), mol (moles),
mrnol (millimoles), eq (equivalent(s)).
Compound A can be prepared according to the following methods
Methods of Synthesis
Method A
Cyclopentenone (II) may be halogenated with bromine or
iodine followed by treatment with a base to give III (see Organic
Syntheses 6165). 3,5-Difluorophenylboronic acid may then be added via
a palladium-catalyzed coupling reaction to forrn IV. 4-Bromothioanisole
may be metalated with nBuLi or magnesium, and then treated with IV to
give the alcohol V. Oxidation with allylic transposition may then be
accomplished using an oxidant such as PDC to give cyclopentenone VI.
Sulfide oxidation using an oxidant such as H2O2 in the presence of a
tungstate catalyst, MMPP, or mCPBA then provides sulfone Ia.
Alternatively, VI may be converted to sulfonamide Ib as described in
U.S. Patent 5,474,995, Method A.
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(HO)2B,~ F
<~<~x
III base IV F M=Li, Mg
X = Br, I
F
ref: US Patent 5,474,995
Method A
~, SO2NH2
~F
O ~
Ib F
Method B
To bromocyclopentenone IIIa is added lithio or magnesium
S thioanisole to give tertiary alcohol VII. Oxidation with a reagent such as
PDC then provides the ketone vm. The sulfide may be oxidized at this
point using an oxidant such as H202 in the presence of a tungstate
catalyst, MMPP, or mCPBA to give the sulfone IX. Palladium-catalyzed
coupling of 3,5-difluorophenylboronic acid then provides Ia.
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<~~ M~ ~ PDC ~SMe
M = Li, Mg Br O VIII
VII
(HO)2B,~:~F SO2Me
base IX
Ia F
Method C
3-Ethoxycyclopentenone (X) may be bromin~ted followed by treatment
5 with a base to give XI (see Organic Syntheses 61 65). To
bromocyclopentenone XI is added lithio or magnesium thioanisole
followed by acid rearrangement of the intermediate addition product to
give VIII. The sulfide may be oxidized at this point using an oxidant
such as H202 in the presence of a tungstate catalyst, MMPP, or mCPBA
10 to give the sulfone IX. Palladium-catalyzed coupling of 3,5-
difluorophenylboronic acid then provides Ia.
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¢~ G~Br M <~SMe
OEt EtO ' Br
XI M=Li, Mg O VIII
(H~)2B ~ F
SOzMe ~J ~ ,~, SO2Me
~, Pd~ Br
O I~J base O
F
EXAMPLE 1
5 2-(3~5-DIFLUOROPHENYL)-3-(4-(METHYLSULFONYL)PHENYL)-
2-CYCLOPENTEN-l-ONE (COMPOUND A)
Step 1 3-(4-(Methylthio)phenyl)-2-cyclopenten- 1 -one
To a -65~C solution of 4-bromothioanisole (61 g, 0.3 mol) in
10 THF (800 mL) was added a solution of n-BuLi (2.3 M in hexanes, 120
mL) at a rate so as to m:~int~in an internal temperature below -55~C. The
resulting slush was stirred at -65~C for 2 h, then treated with a solution of
3-ethoxy-2-cyclopenten-1-one (35 g, 0.28 mmol) in THF (50 mL). After
30 min at -65~C, the solution was warmed to 0~C and quenched with sat.
15 aq. NH4Cl (400 mL). The product was extracted with 3 x lL of EtOAc
and the combined extracts were dried over MgSO4 and concentrated.
The resulting material was swished in 200 mL of 1:1 EtOAc/hexanes to
provide 45 g of the title compound as a white solid.
20Step 2 2-Bromo-3-(4-(methylthio)phenyl)-2-cvclopenten-1-one
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To a suspension of 3-(4-(methylthiol)phenyl)-2-cyclopenten-
l-one (9.64 g, 47.2 mmol) in CC14 (200 mL) was added a solution of
bromine (5 mL) in CCl4 (30 mL) over 20 min. The resulting orange
suspension was stirred for 1.5 h, then cooled in an ice-bath, and
5 triethylamine (14 mL, 100 mmol) was added. After 30 min, the mixture
was quenched with lM HCl (300 mL) and extracted with CH2Cl2. The
organic phase was washed with brine, filtered through cotton and
evaporated. Purification by silica gel chromatography, eluting with 90%
CH2Cl2/hexanes gave the title compound (7.13 g).
Step 3 2-Bromo-3-(4-(methylsulfonyl)phenyl)-2-cyclopenten- 1 -one
To a 0 ~C solution of 2-bromo-3-(4-(methylthio)phenyl)-2-
cyclopenten-l-one (5.73 g, 20.2 mmol) in CH2C12 (100 mL) and MeOH
(50 mL) was added MMPP (19 g, 30.7 mmol). The mixture was stirred 5
15 h at r.t., then concentrated. The residue was partitioned between sat. aq.
NaHCO3 and CH2Cl2. The organic phase was washed with brine,
filtered through cotton and evaporated. The resulting solid was swished
in CH2Cl2/hexanes to provide the title compound (5.57 g).
20 Step 4 2-(3,5-Difluorophenyl)-3-(4-(methylsulfonyl)phenyl)-2-
cyclopenten- l-one
To a mixture of 2-bromo-3-(4-(methylsulfonyl)phenyl)-2-
cyclopenten-l-one (664 mg, 2.1 1 mmol), 3,5-difluorophenylboronic acid
(832 mg, 5.27 mmol), tris(dibenzylideneacetone)dipalladium(0) (83 mg,
25 0.09 mmol), and triphenylphosphine (96 mg, 0.36 mmol) was added 40
mL of 3:1:1 toluene:n-propanol:water and the mixture was purged with
nitrogen. After stirring 10 min, diethyl~mine (1.1 mL, 10.6 rnmol) was
added and the solution was heated to reflux for 4 h. The mixture was
cooled and partitioned bet~,veen lM NaOH and EtOAc. The organic
30 phase was washed with water, lM HCl and brine, and dried over MgSO4.
Purification by silica gel chromatography, eluting with 50%
CH2C12/hexanes, followed by cryst~lli7~tion from CH2C12/ether/hexanes
gave the title compound (223 mg).
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.
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1H NMR (CD3COCD3) o 7.95 (2H, d), 7.65 (2H, d), 6.98 (lH, m), 6.82
(2H, m), 3.19 (2H, m), 3.15 (3H, s), 2.68 (2H, m).
EXAMPLE lA
Step 1 2-Bromo-2-cyclopenten-1-one
To a 0 ~C solution of 2-cyclopenten-1-one (125 g, 1.52 mol)
in CCl4 (1.2 L) in a three-neck flask equipped with an overhead stirrer
was added a solution of bromine (269 g, 1.68 mol) in CCl4 (400 rnL)
10 dropwise over 4 h, m~int~ining an internal temperature <2 ~C. A solution
of Et3N (310 mL, 2.22 mol) in CCl4 (200 mL) was then added dropwise
over 1.5 h, maintaining an internal temperature <10 ~C. The resulting
suspension was warmed to r.t. for 1 h, then cooled to 0 ~C and filtered.
The filtrate was washed with two 700 mL portions of 3M HCl and 500
15 mL of brine, then filtered through cotton. Concentration provided 228 g
of an orange oil which was crystalized from 150 mL of 2: 1 hexane: ether
to provide 191 g of the title compound.
1H NMR (CD3COCD3) o 7.94 (lH, t), 2.72 (2H, m), 2.46 (2H, m).
Step 2 2-Bromo-3-(4-(methylthio)phenyl)-2-cyclopenten-1-one
To a -78 ~C solution of 4-bromothioanisole (35.1 g, 173
mmol) in THF (500 mL) was added nBuLi (1.6 M in hexanes, 107.5 mL,
172 mmol). The solution was stirred for 45 min, then a solution of 2-
25 bromo-2-cyclopenten-1-one (25.4 g, 158 mmol) in THF (150 rnL) was
added and the mixture was allowed to warm to 0 ~C and was quenched
with saturated aqueous NH4Cl. The majority of the solvent was removed
in vacuo and the residue was suspended in water and extracted with two
portions of EtOAc. The organic layers were washed with brine, dried
30 over MgSO4, filtered and concentrated. This material was dissolved in
DMF (300 rnL), cooled to 0 ~C and treated with PDC (72.4 g, 192 mmol).
The resulting mixture was warmed to r.t. and stirred for 2 h, then poured
into water (1.2 L) and extracted with two 500 mL portions of EtOAc.
The organic layers were washed with brine, dried over MgSO4, filtered
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and concentrated to give the title compound as a light brown solid which
was used directly in the next step.
Step 3 2-Bromo-3-(4-(methylsulfonyl)phenyl)-2-cyclopenten-1-one
To a 0 ~C solution of 2-bromo-3-(4-(thiomethyl)phenyl)-2-
cyclopenten-l-one in 2:1 CH2C12/MeOH (500 mL) was added MMPP
(100 g). The mixture was stirred at r.t. overnight, then concentrated and
partitioned between saturated NaHCO3, lM Na2S2O3 and CH2C12. The
aqueous layer was extracted with CH2C12, and the combined organics
10 were washed with brine, filtered through cotton and evaporated. The
resulting solid was swished in CH2C12/ether to provide 23 g of the title
compound.
lH NMR (CD3COCD3) o 8.12 (4H, m), 3.22 (2H, m), 3.20 (3H, s), 2.69
15 (2H, m).
Step 4 3,5-Difluorophenylboronic acid
To a -78 ~C solution of l-bromo-3,5-difluorobenzene (50 g,
0.26 mol) in ether (860 mL) was added nBuLi (2.4 M in hexanes, 108
20 mL, 0.26 mol) dropwise over 20 min. The resulting solution was stirred
for 10 min, then treated with triisopropylborate (61 mL, 0.27 mol). The
reaction mixture was warmed to 0 ~C for 30 min, then quenched with 1 M
HCl. After stirring 30 min, the mixture was partitioned between ethyl
acetate and water. The organic layers were washed with brine, dried over
25 MgSO4, filtered, and evaporated. The resulting product was dried under
high vacuum overnight to give 38 g of the title compound.
lH NMR (CD3COCD3) ~ 7.50 (2H, br s), 7.40 (2H, m), 7.04 (lH, m).
30 Step 5 2-(3,5-Difluorophenyl)-3-(4-(methylsulfonyl)phenyl)-2-
cyclopenten- 1 -one
A mixture of 2-bromo-3-(4- (methylsulfonyl)phenyl)-2-
cyclopenten-l-one (52.2 g, 166 rnmol), 3,5-difluorophenylboronic acid
(31.9 g, 202 mmol), tris(dibenzylideneacetone)dipalladium(0) (3.3 g, 3.6
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mmol), and triphenylphosphine (1.89 g, 7.2 mmol) were dissolved in
toluene (800 mL) and nPrOH (250 mL) and degassed. After stirring for
10 min, diethylamine (21 mL, 203 mmol) and water (250 rnL) was added.
The mixture degassed again, heated to reflux for lh, then cooled and
poured into EtOAc (2L). The aqueous layer was separated, and the
organic layer was washed with 0.2N NaOH (500 mL), 0.5N HCl (500
mL) and brine. The organic phase was then dried over MgSO4, filtered
through celite and evaporated. The resulting light brown solid was
swished in hot EtOAc (250 mL), cooled and filtered to give 45.8 g of the
10 title compound, m.p. 174-175 ~C.
H NMR (CD3COCD3) â 7.95 (2H, m), 7.66 (2H, m), 6.98 (lH, m), 6.80
(2H, m), 3.17 (2H, m), 3.12 (3H, s), 2.68 (2H, m).
15 B. An alternative method of preparing 2-bromo-3-(4-
(methylsulfonyl)phenyl)-2-cyclopenten-1-one (Exarnple 1, Step 3 has
been developed as follows:
Step 1 2-Bromo-3-ethoxy-2-cyclopenten-1-one
To a -15 ~C solution of 3-ethoxy-2-cyclopenten-1-one (165g,
1.30 mol) in 1.8L of CHCl3 was added a solution of bromine (219 g, 1.37
mol) in CHCl3 (200 mL) via dropping funnel over 1.5h to give a thick
yellow slurry. After 30 min additional stirring, a solution of Et3N (210
mL, 1.5 mol) in CHCl3 (300 mL) was added via dropping funnel over 30
25 min. The resulting mixture was concentrated, suspended in EtOAc and
filtered through a pad of 2 cm celite over 1 cm silica gel. The filtrate was
concentrated, and the resulting material recrystalized from 4: 1
ether/hexanes to provide 245 g of the title compound.
30 H NMR (CD3COCD3) ~ 4.43 (2H, q), 2.95 (2H, m), 2.47 (2H, m), 1.39
(3H, t).
Step 2 2-Bromo-3-(4-(methylthio)phenyl)-2-cyclopenten-1-one
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To a -78 ~C solution of 4-bromothioanisole (253 g, 1.25
mol) in THF (5 L) was added nBuLi (2.5 M in hexanes, 500 mL, 1.25
mol) over 20 min to give a thick slurry which was stirred 2h at -78 ~C. A
solution of 2-bromo-2-cyclopenten-1-one (242 g, 1.18 mol) in THF (1 L)
S was then added via cannula and the mixture was allowed to warm to -10
~C. 6N HCl (430 mL) was then added and the resulting mixture stirred
20 min. EtOAc (3L) was added and the aqueous phase was separated.
The organic layer was concentrated. The residue was suspended in ether
and filtered to give 287 g of the title compound.
1H NMR (CD3COCD3) o 8.12 (4H, m), 3.22 (2H, m), 3.20 (3H, s), 2.69
(2H, m).
The following example pharmaceutical formulation are
lS illustrative of the invention:
Step 3 2-Bromo-3-(4-(methylsulfonyl)phenyl)-2-cyclopenten-1-one
To a room temperature suspension of 2-bromo-3-(4-
(methylthio)phenyl)-2-cyclopenten-1-one (67.4 g, 238 mmol) in EtOAc
20 (500 mL) and CH2C12 (100 mL) was added NaWO4 2H20 (3.1 g, 9
mmol) and Aliquat 336 (12 g, 30 mmol). 10 mL of 30% H202 was
added and the mixture warmed to 40~C to initiate the oxidation. 85 mL
of H202 was then added dropwise over 20 min with external cooling to
maintain an internal temperature of approximately 50~C. The dark
25 reaction mixture was then maintained at 40~C for l.S h. The aqueous
layer was separated and the organic phase was washed twice with warm
water. The organic phase was then concentrated to 500 mL and the
precipitate was filtered and washed with ether to give 52.2 g of the title
compound as a white solid.
EXAMPLE 2
Wet ~ranulated tablet composition
Amount per tablet Ingredient
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25 mg COX-2 Inhibitor
79.7 mg Microcrystalline cellulose
79.7 mg Lactose monohydrate
6 mg Hydroxypropyl cellulose
8 mg Croscarmellose sodium
1 mg Magnesium stearate
Tablet dose strengths of between S and 125 mg can be accomodated by
10 varying total tablet weight, and the ratio of the first three ingredients.
Generally it is preferable to maintain a 1:1 ratio for microcrystalline
cellulose: lactose monohydrate. Varying amounts of up to S mg. of iron
oxide may be added to this or the other compositions if a yellow or
orange color is desired.
EXAMPLE 2a
Wet ~ranulated tablet composition
Amount per tablet Ingredient
125 mg COX-2 Inhibitor
425 mg Microcrystalline cellulose
425 mg Lactose monohydrate
10 mg Hydroxypropyl cellulose
14 mg Croscarmellose sodium
1 mg Magnesium stearate
EXAMPLE 2b
Wet granulated tablet composition
Amount per tablet Ingredient
10 mg COX-2 Inhibitor
87.2 mg Microcrystalline cellulose
87.2 mg Lactose monohydrate
6 mg Hydroxypropyl cellulose
8 mg Croscarmellose sodium
1 mg Magnesiumstearate
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EXAMPLE 2c
Wet granulated tablet composition
S Amount per tablet Ingredient
5 mg COX-2 Inhibitor
89.7 mg Microcrystalline cellulose
89.7 mg Lactose monohydrate
6 mg Hydroxypropyl cellulose
8 mg Croscarmellose sodium
1 mg Magnesium stearate
EXAMPLE 3
Directly compressed tablet composition
Amount per tablet Ingredient
25 mg COX-2 Inhibitor
106.9 mg Microcrystalline cellulose
106.9 mg Lactose anhydrate
7.5 mg Croscarmellose sodium
3.7 mg Magnesium stearate
Tablet dose strengths of between 5 and 125 mg can be accomodated by
varying total tablet weight, and the ratio of the first three ingredients.
Generally it is preferable to maintain a 1: 1 ratio for microcrystalline
cellulose: lactose monohydrate.
EXAMPLE 3a
Directly compressed tablet composition
Amount per tablet Ingredient
125 mg COX-2 Inhibitor
425 mg Microcrystalline cellulose
425 mg Lactose anhydrate
15 mg Croscarmellose sodium
10 mg Magnesium stearate
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EXAMPLE 3b
Directly compressed tablet composition
Amount per tablet Ingredient
10 mg COX-2 Inhibitor
42.5 mg Microcrystalline cellulose
1042.5 mg Lactose anhydrate
4 mg Croscarmellose sodium
1 mg Magnesium stearate
EXAMPLE 3c
Directly compressed tablet composition
Amount per tablet Ingredient
20 5 mg COX-2 Inhibitor
45 mg Microcrystalline cellulose
45 mg Lactose anhydrate
4 mg Croscarmellose sodium
1 mg Magnesium stearate
EXAMPLE 4
Hard ~elatin capsule composition
30Amount per capsule Ingredient
25 mg COX-2 Inhibitor
37 mg Microcrystalline cellulose
37 mg Lactose anhydrate
35 1 mg Magnesiumstearate
1 capsule Hard gelatin capsule
Capsule dose strengths of between 1 and S0 mg can be accomodated by
varying total fill weight, and the ratio of the first three ingredients.
40 Generally it is preferable to maintain a 1:1 ratio for microcrystalline
cellulose: lactose monohydrate.
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EXAMPLE 5
Oral solution
s
Amount per 5 mL dose Ingredient
50 mg COX-2 Inhibitor
to 5 mL with Polyethylene oxide 400
Solution dose strengths of between 1 and 50 mg/SmL can be
accomodated by varying the ratio of the two ingredients.
EXAMPLE 6
Oral suspension
Amount per 5 mL dose Ingredient
100 mg COX-2 Inhibitor
150 mg Polyvinylpyrrolidone
2.5 mg Poly oxyethylene sorbitan monolaurate
10 mg Benzoic acid
to 5 mL with sorbitol solution (70%)
Suspension dose strengths of between 1 and 50 mg/Sml can be
accomodated by varying the ratio of the first two ingredients.
EXAMPLE 7
Intravenous infusion
Amount per 200mL dose Ingredient
1 mg COX-2 inhibitor
0.2 mg Polyethylene oxide 400
1.8 mg Sodium chloride
to 200mL Purified water
