KR19980078465A - 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten-1-one useful as an inhibitor of cyclooxygenase- - Google Patents

Abstract

The present invention relates to novel 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten-1-one of the formula 1, pharmaceutical compositions and cyclooxygenases Lt; RTI ID = 0.0 > (II) < / RTI > mediated diseases.

[Chemical Formula 1]

Description

2- (3,5-difluorophenyl) -3- (4-methylsulfonyl) phenyl) -2-cyclopenten-1-one useful as an inhibitor of cyclooxygenase-

The present invention relates to a method for the treatment of cyclooxygenase mediated diseases and to certain pharmaceutical compositions therefor.

Nasal steroidal anti-inflammatory agents exhibit most of their inflammatory, analgesic and antipyretic activity and inhibit hormone-induced uterine contractions and certain types of cancer growth through the inhibition of prostaglandin G / H synthase, known as cyclooxygenase. Initially, only one form of cyclooxygenase has been known, which corresponds to the cyclooxygenase-1 (COX-1) or constitutive enzyme, as originally identified in bovine hypocotyls. Recently, genes for the second inducible form of cyclooxygenase (cyclooxygenase-2; COX-2) have been cloned, sequenced and characterized from chickens, rats and humans. These enzymes are distinguished from COX-1 that has been cloned, sequenced and characterized from various sources including sheep, mice and humans. The second form of cyclooxygenase, COX-2, can be quickly and easily induced by a number of drugs including mitotic materials, endotoxins, hormones, cytokines and growth factors. Because prostaglandins have both physiological and pathological roles, we believe that the constitutive enzyme, COX-1, is predominantly responsible for the intrinsic basal release of prostaglandins, and thus is involved in physiological actions such as gastrointestinal integrity and maintenance of renal blood flow Concluded that it was important. Conversely, the present inventors conclude that COX-2 is mainly responsible for the pathological effects of prostaglandins, in which an inducible form of the enzyme is induced in response to drugs such as antiinflammatory agents, hormones, growth factors and cytokines I built it. Thus, selective inhibitors of COX-2 have similar anti-inflammatory, antipyretic and analgesic properties as conventional non-steroidal anti-inflammatory agents, inhibit hormone-induced uterine contractions and have potent anti-cancer effects, but some side effects based on the mechanism The ability to induce is reduced. In particular, the compounds reduce the potential for gastrointestinal toxicity, reduce the potential for kidney side effects, reduce the effect on bleeding times, and possibly reduce the ability to induce asthma attacks in aspirin-sensitive asthmatic patients .

Moreover, such compounds also inhibit prostaglandin-induced smooth muscle by preventing the synthesis of shrinking prostanoids, and thus can be used in the treatment of dysmenorrhea, premature labor, asthma and eosinophil-related diseases. It will also be useful in the treatment of Alzheimer's disease, particularly in the reduction of bone loss in postmenopausal women (e.g., in the treatment of osteoporosis) and in the treatment of glaucoma.

A brief description of the effective use of cyclooxygenase-2 inhibitors can be found in John Vane, Nature, Vol. 367, pp. 215-216, 1994 and Drug News and Perspectives, Vol. , 1994).

The present invention relates to novel compounds such as 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten-1-one, pharmaceutical compositions and cyclooxygenases -2 < / RTI > mediated diseases.

The first aspect of the present invention relates to a process for the preparation of the compound of formula (I), which comprises reacting the compound 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2- .

[Chemical Formula 1]

A second aspect of the present invention is a pharmaceutical composition comprising 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten-1-one and a pharmaceutically acceptable carrier Lt; RTI ID = 0.0 > COX-2 < / RTI > mediated diseases.

A third aspect of the present invention is a pharmaceutical composition comprising 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten-1-one and a pharmaceutically acceptable carrier ≪ / RTI >

A fourth aspect of the present invention relates to a method for the treatment of COX-2 mediated diseases, which method comprises administering 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) - Includes the use of on.

In a further aspect, compositions, methods of treatment and uses for oral administration are preferred.

In a further aspect, compositions, methods of treatment and uses for administration once a day or twice a day are preferred.

The following are the meanings of the abbreviations:

AA = arachidonic acid

CHO = Chinese hamster ovary

CMC = 1-cyclohexyl-3- (2-morpholinoethyl) -carbodiimide metho-p-toluenesulfonate

COX = cyclooxygenase

DMF = N, N-dimethylformamide

HBSS = Hans adjusted salt solution

HEPES = N- [2- hydroxyethyl] piperazine -N ¹ - [2- ethanesulfonic acid]

HWB = human whole blood

LPS = lipopolysaccharide

mCPBA = meta-chloroperbenzoic acid

MMPP = magnesium monoperoxyphthalate

NSAID = non-steroidal anti-inflammatory drug

PDC = pyridinium dichromate

r.t. = Room temperature

THF = tetrahydrofuran

Compound A can be used for the treatment of rheumatic fever, influenza or other viral infections, symptoms associated with cold, lower back and neck pain, dysmenorrhea, headache, toothache, sprains and constipation, myositis, neuralgia, synovitis, rheumatoid arthritis, Osteoarthritis), gout and ankylosing spondylitis, bursitis, burns, injuries followed by surgery and dental treatment, to relieve pain, heat and inflammation of various conditions. In addition, such compounds can inhibit cell neoplastic outgrowth and metastatic tumor growth, and can be used to treat cancers such as colon cancer. Compound A can also be used for the treatment and / or prevention of cyclooxygenase-mediated proliferative disorders that can occur in diabetic retinopathy and ontogeny.

Compound A also inhibits prostanoid-induced smooth muscle contraction by preventing the synthesis of shrinking prostanoids, and thus can be used for the treatment of dysmenorrhea, premature labor, asthma and eosinophil-related diseases. The compound A will also be useful in the treatment of Alzheimer's disease, particularly in the reduction of bone loss in postmenopausal women (e.g., treatment of osteoporosis) and glaucoma.

Due to the specificity of cyclooxygenase-2 over the high cyclooxygenase-2 (COX-2) activity and / or cyclooxygenase-1 (COX-1), Compound A is particularly effective against non- Patients with peptic ulcer, gastritis, gastroenteritis, ulcerative colitis, diverticulitis, gastrointestinal recurrence; Gleevec bleeding, coagulation disorders, including anemia such as hypoprothrombinemia, hemophilia or other bleeding problems; Patients with kidney disease; Will be demonstrated to be useful as a replacement for conventional non-steroidal anti-inflammatory agents (NSAID'S) when contraindicated to patients prior to surgical treatment or anticoagulant treatment.

Similarly, Compound A would be useful as a partial or complete replacement for conventional NSAID'S in the form of a formulation to be administered with other reagents or ingredients. Accordingly, in a further aspect, the present invention provides a non-toxic therapeutically effective amount of a compound of formula I as defined above and one or more components such as another pain reliever comprising acetaminophen or phenacetin; A synergist comprising caffeine; H2-antagonistic substances, aluminum hydroxide or magnesium, simethicone, decongestants such as phenyl ephrin, phenylpropanolamine, pseudophedrine, oxymetazoline, epinephrine, napalin, xyl methatholines, propylhexadrine or levo- Dexedephedrine); Codeine, hydrocodone, caramiphen, carbbetapentane, or dextramethorphan; Prostaglandins including misoprostol, noprostil, riofostil, ornoprostol or rosaprostol; diuretic; Comprising a pharmaceutical composition for treating a cyclooxygenase-2 mediated disease as defined above, comprising an antagonist, an antagonist, an antagonist, an antagonist, or an antagonist. The present invention also relates to a method of treating a cyclooxygenase mediated disease, optionally comprising administering to a patient to be treated a therapeutically effective amount of a non-toxic therapeutically effective amount of a non-toxic, .

For treatment of cyclooxygenase mediated diseases, Compound A may be administered 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, intravenous, intramuscular, intrasternal injection or infusion techniques. In addition to the treatment of warm-blooded animals such as mice, rats, horses, cows, sheep, dogs, cats and the like, the compounds of the present invention are effective in treating humans.

As indicated above, the pharmaceutical compositions for the treatment of diseases mediated by cyclooxygenase-2 may optionally comprise one or more components as described above.

Pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules or syrups or elixirs have. Oral compositions may be prepared according to methods known in the art for the manufacture of pharmaceutical compositions, which may be formulated with sweetening, flavoring, coloring and preservative agents to provide pharmaceutically elegant, high-quality, One or more reagents selected from the group. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. Such excipients include, for example, inert diluents (such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate); Granules and disintegrants such as corn starch, or alginic acid; (E.g. starch, gelatin or acacia) and lubricants (e.g. magnesium stearate, stearic acid or talc). They may be coated with a known technique so as not to coat the tablets or delay disintegration and absorption in the gastrointestinal tract so that long-term sustained action is exhibited. For example, time delay materials such as glyceryl monostearate or glyceryl distearate may be used. The tablets may also be coated with the techniques described in U.S. Patent Nos. 4,256,108, 4,166,452 and 4,265,874 to form osmotic therapeutic tablets for controlled release.

Oral formulations are prepared by mixing the active ingredient in a hard gelatine capsule, in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate or kaolin, or water, a miscible solvent such as propylene glycol, PEGs and ethanol, (E.g. peanut oil, liquid paraffin or olive oil).

Aqueous suspensions contain the active material mixed with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth and acacia rubbers; The dispersing or wetting agent may be a natural phosphatide such as lecithin or a condensation product of a fatty acid with an alkylene oxide such as polyoxyethylene stearate or a condensation product of an ethylene oxide with a long chain aliphatic alcohol such as heptadecaethyleneoxyethanol, Or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol such as polyoxyethylene sorbitol monooleate or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides such as polyethylene sorbitan Monooleate). The aqueous suspension may contain one or more preservatives (e.g., ethyl or n-propyl, P-hydroxybenzoate), one or more coloring agents, one 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 ingredient in vegetable oils (such as peanut oil, olive oil, sesame oil or coconut oil) or mineral oil (such as liquid paraffin). Oily suspensions may contain thickening agents (e.g. beeswax, hard paraffin or cetyl alcohol). Sweeteners and flavors such as those described above may be added to provide a tasty oral preparation. These compositions can be preserved by the addition of antioxidants such as ascorbic acid.

Dispersible powders and granules suitable for the manufacture of aqueous suspensions by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Examples of suitable dispersing or wetting agents and suspending agents are already mentioned above. Additional excipients such as sweetening, flavoring and coloring agents may also be present.

The pharmaceutical composition of the present invention may be in the form of a water-in-oil emulsion. The oily phase may be vegetable oil (e.g., olive oil or peanut oil) or mineral oil (e.g. liquid paraffin) or a mixture thereof. Suitable emulsifiers include esters or partial esters (e.g., sorbitan monooleate) derived from natural phosphatides (e.g. soybean, lecithin) and fatty acids and hexitol anhydrides, and condensation products of such partial esters with ethylene oxide : Polyoxyethylene sorbitan monooleate). The emulsion may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated using sweetening agents such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain spotting agents, preservatives and flavoring agents and coloring agents. The pharmaceutical composition may be in the form of a sterile injectable aqueous or oleaginous suspension. Such suspensions may be formulated according to the known art using the above-mentioned suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may be a non-toxic orally acceptable diluent or a sterile injectable solution or suspension in a solvent such as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. Co-solvents such as ethanol, propylene glycol or polyethylene glycol may also be used. In addition, sterile, nonvolatile oils are conventionally employed as a solvent or suspending medium. For this purpose, non-volatile stationary oils including synthetic mono- or diglycerides may be used. In addition, fatty acids such as oleic acid are used in the preparation of injectable formulations.

Compound A may also be administered in the form of suppositories for rectal administration of the drug. Such a composition may be prepared by mixing the drug with a suitable non-polar excipient which is solid at ambient temperature but liquid at rectal temperature and melts in the rectum to release the drug. These materials are cocoa butter and polyethylene glycol.

For topical use, creams, ointments, gels, solutions or suspensions, etc., containing the compound A are used (for such use, topical application includes mouthwash and mouthwash). Topical formulations may generally comprise a pharmaceutical carrier, co-solvent, emulsifier, penetration enhancer, preservative system and softener.

Other suitable agents are described in U.S. Patent No. 5,474,995. The inventors have found that the following oral preparations are particularly effective:

Rapidisc ^R - In terms of the properties mentioned above, 2- (3,5-difluorophenyl) -3- (4-methylsulfonyl) phenyl) -2-cyclopenten- Lt; RTI ID = 0.0 > rapidly < / RTI > For example, because there is no GI side effect, this formulation does not need to be eaten with large amounts of water. Suitable raffidisc preparations and methods for their preparation are described 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, the disclosures of which are incorporated herein by reference.

As mentioned herein, the present inventors have found that the surprising combination properties of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) . Compound A is active at an appropriate oral dose of 10 to 250 mg per day and is not only safe and effective but also has a sufficiently long half life in the body so that oral administration of 10 to 250 mg of active agent once or twice a day It also provides an effective and stable anti-inflammatory treatment over 24 hours. Such agents are particularly useful for the treatment of chronic diseases such as Alzheimer's disease as well as rheumatism and osteoarthritis.

Oral and intravenous dose levels of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten-1-one drug are about 10 to 250 mg per patient It is administered once or twice a day.

The amount of active agent that can be mixed with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation for oral administration for humans may contain from 10 mg to 250 mg of a formulation mixed with a suitable and convenient amount of carrier material, which may vary from about 5% to about 95% of the total composition. Dosage unit forms typically contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125 or 250 mg of active agent.

However, the specific dose level for a particular patient will depend on a variety of factors including age, weight, general health status, sex, diet, time of administration, rate of excretion, drug mix and the severity of the particular disease to be treated. For many patients, it is preferable to administer the dose in the range of 10 to 50 or 60 to 120 mg once or twice a day.

In the case of long-term treatment regimens such as in the treatment of chronic diseases including rheumatoid arthritis, osteoarthritis or Alzheimer's disease, it is preferable to administer a dose of 10 to 50 or 60 to 120 mg once or twice a day. More particularly, it is desirable to administer a dose of 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg once or twice a day in the treatment of osteoarthritis, while in the treatment of rheumatoid arthritis 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg is preferably administered once or twice a day. For the treatment of non-chronic diseases such as headache or postoperative swelling or pain, it is preferable to administer a dose of 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg once or twice a day Do.

Thus, one aspect of the invention relates to unit oral dosage forms comprising 10 to 250 mg, for example 10 to 50 or 60 to 120 mg of a cyclooxygenase inhibitor.

Another aspect of the present invention is a pharmaceutical composition comprising 10 to 250 mg of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten- Or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a disease mediated by cyclooxygenase-2, which is suitable for oral administration once or twice daily.

A first example of a composition comprising 10-50 mg of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten- / RTI >

According to this aspect, a second example of a composition comprising 60 to 120 mg of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten- / RTI >

The amount of active ingredient that can be combined with the carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. For example, a formulation for oral administration to a human may comprise from 10 mg to 250 mg of active agent to be combined with a suitable and convenient amount of carrier which may vary from about 5 to about 95% per total composition. The dosage unit form will generally comprise from about 10 mg to about 250 mg, usually 10 mg, 25 mg, 50 mg, 100 mg, 125 mg or 250 mg of the active ingredient.

It will be understood, however, that the specific dose level for a particular patient will vary depending upon various factors including age, weight, general health status, sex, diet, time of administration, route of administration, rate of excretion, drug mix, It will be understood.

Test for determination of biological activity

Compound A can be tested using the following assay to determine cyclooxygenase-2 inhibitory activity.

Inhibition of cyclooxygenase activity

All cell assays for COX-2 and COX-1 using CHO transfected cell lines

A Chinese hamster ovary (CHO) cell line stably transfected with the eukaryotic expression vector pCDNAIII containing human COX-1 or COX-2 cDNA is used in the assay. These cell lines are referred to respectively as CHO [hCOX-1] and CHO [hCOX-2], respectively. CHO [hCOX-2] cells prepared by trypsinization of CHO [hCOX-1] cells from the suspension culture and adhesion cultures for the cyclooxygenase assay were collected by centrifugation (300xg, 10 minutes) and washed with 15 mM HEPES , pH 7.4 and resuspended at a cell concentration of 1.5 x 10 ⁶ cells / ml in HBSS, 15 mM HEPES, pH 7.4. The drug to be tested is dissolved in DMSO to a maximum test drug concentration of 66.7-fold. Compounds are tested in duplicate at 8 concentrations using a series of triplicate dilutions of the highest drug concentration in DMSO. Cells (0.3 x 10 ⁶ cells in 200 μl) are pre-incubated with 3 μl of test drug or DMSO vehicle for 15 min at 37 ° C. The working solution of AA without peroxide (5.5 [mu] M and 110 [mu] M AA for CHO [hCOX-1] and CHO [COX-2] assay, respectively) was diluted to 10 in 10% HBSS containing 15 mM HEPES, pH 7.4 ≪ / RTI > The following cells are challenged with AA / HBSS solution in the presence or absence of the drug to give a final concentration of 0.5 μM AA for the CHO [hCOX-1] assay and a final concentration of 10 μM AA for the CHO [hCOX-2] assay. This reaction is terminated by adding 10 μl of 1N HCl and neutralizing with 20 μl of 0.5 N NaOH. Removing 10 minutes and centrifuged at 4 ℃ samples to 300xg and suitably diluted aliquots of the supernatant Clarification enzyme for PGE ₂ - coupled immunoassay (associated with PGE ₂ enzyme immunoassay kit, Assay Designs, Inc. manufactured) such that the measured level of PGE ₂ used in the. The level of PGE ₂ in cells challenged with arachidonic acid for cyclooxygenase activity in the absence of the test compound is determined by the difference in PGE ₂ levels in cells simulated-challenged with an ethanol vehicle. The inhibition of PGE ₂ synthesis by the test compound is calculated as a percentage of the activity in the presence of the drug relative to the activity in the positive control sample.

Assay of COX-1 activity from U937 cell overlaid

U 937 cells are pelleted by centrifugation at 500 xg for 5 minutes, washed once with phosphate-buffered saline and repelleted. Cells were resuspended in homogenization buffer consisting of 0.1 M Tris-HCl, pH 7.4, 10 mM EDTA, 2 ug / ml leupeptin, 2 ug / ml soybean trypsin inhibitor, 2 ug / ml aprotinin and 1 mM phenylmethylsulfonyl fluoride Throw away. The cell suspension is sonicated four times for 10 seconds and centrifuged at 10,000 xg for 10 minutes at 4 ° C. The supernatant is centrifuged at 100,000 xg for 1 hour at 4 ° C. The 100,000 x g granular pellet is resuspended at approximately 7 mg protein / ml in 0.1 M Tris-HCl, pH 7.4, 10 mM EDTA and stored at -80 캜.

The granulation preparation was thawed immediately before use, slightly sonicated, and then lysed in a 0.1 M Tris-HCl buffer containing 10 mM EDTA, 0.5 mM phenol, 1 mM reduced glutathione and 1 μM hematin, a protein concentration of 125 μg / ml in pH 7.4 Lt; / RTI > Two rounds of assay are performed in a final volume of 250 [mu] l. Initially, 5 μl of drug in DMSO vehicle or DMSO is added to 20 μl of 0.1 M Tris-HCl buffer, pH 7.4, containing 10 mM EDTA in the wells of a 96-dip polypropylene titration plate. 200 μl of the following granular preparation is added and preliminary incubated at room temperature for 15 minutes before adding 25 μl of 1 M arachidonic acid in 0.1 M Tris-HCl and 10 mM EDTA, pH 7.4. The sample is incubated at room temperature for 40 minutes and 25 μl of 1 N HCl is added to stop the reaction. A sample before an appropriate amount of PGE ₂ content by radioimmunoassay black (Dupont-NEN or Amersham assay kits) and neutralized with 1N NaOH 25㎕. Cyclooxygenase activity is defined as the difference in PGE ₂ levels in the sample incubated in the presence of the incubated sample and the ethanol vehicle in the presence of arachidonic acid.

Assay of activity of purified human COX-2

Enzyme activity was measured using a colorimetric assay based on the oxidation of N, N, N ', N'-tetramethyl-p-phenylenediamine (TMPD) during the reduction of PGG ₂ to PGH ₂ by COX-2 (See Copeland et al. (1994) Proc. Natl. Acad. Sci. 91, 11202-11206].

Recombinant human COX-2 is purified from Sf9 cells as described above (Percival et al (1994) Arch. Biochem. Biophys. 15, 111-118. The black mixture (180 μL) was diluted with 100 mM sodium phosphate, pH 6.5, 2 mM GenePol X-100, 1 μM hematin, 1 mg / ml gelatin, 80-100 units of purified enzyme (1 unit of enzyme at 0.001 Lt; / RTI > of the test compound) and 4 [mu] L of the test compound. The mixture is pre-incubated at room temperature (22 DEG C) for 15 minutes before initiating the enzymatic reaction by adding 20 [mu] L of 1 mM arachidonic acid (AA) and 1 mM TMPD in assay buffer (enzyme or hematin) . Enzyme activity is measured by evaluating the initial rate of TMPD oxidation over the first 36 seconds of the reaction. The non-specific rate of oxidation is measured in the absence of enzyme (0.007-0.010 OD / min) and is subtracted before calculating% inhibition. IC ₅₀ values are obtained from a 4-parameter minimum angle non-linear regression analysis of the logarithm to inhibition plot percent of the dose.

Human whole blood test

principle

Human whole blood provides a cell-rich environment suitable for studying the biochemical potency of anti-inflammatory compounds such as selective COX-2 inhibitors. This study shows that normal human blood does not contain COX-2 enzyme. This is consistent with observations that COX-2 inhibitors do not affect PGE ₂ in normal blood. These inhibitors are active only after incubating human whole blood with COX-2 inducing LPS. This assay can be used to evaluate the inhibitory effect of selective COX-2 inhibitors on PGE ₂ production. Similarly, platelets in whole blood also contain large amounts of COX-1 enzyme. Immediately after blood coagulates, platelets are activated through a thrombin-mediated mechanism. This reaction produces thromboxane B ₂ (TxB ₂ ) through activation of COX-1. Thus, the effect of test compounds on TxB ₂ levels with blood clotting can be tested and used as an indicator of COX-1 activity. That is, the selectivity by the test compound is determined by evaluating the level of PGE ₂ after LPS induction (COX-2) and TxB ₂ followed by blood coagulation (COX-1) in the same assay.

Way

A. COX-2 (LPS-induced PGE ₂ production)

Fresh blood by venipuncture from male and female volunteers is collected into heparinized tubes. The subjects were not clearly inflamed and were not receiving NSAIDs for more than 7 days prior to blood collection. Plasma is immediately collected from 2 mL of blood aliquots and used as blink (basal value of PGE ₂ ). The remaining blood is incubated for 5 minutes at room temperature with LPS (100 μg / ml final concentration, Sigma Chem, # L-2630, available from E. coli; 0.1% BSA (phosphate buffered saline)). An aliquot of 500 μL of blood is incubated at 37 ° C. for 24 hours at 2 μL of vehicle (DMSO) or 2 μL of test compound at a final concentration varying from 10 nM to 30 μM. At the end of the incubation, the blood is centrifuged at 12,000 xg for 5 minutes to obtain plasma. An aliquot of 100 μL of plasma is mixed with 400 μL of methanol for protein precipitation. The supernatant is obtained and the PGE ₂ is converted to its methyloxyate derivative according to the manufacturer's method and then assayed against PGE ₂ using an immunoassay kit (Amersham, RPA # 530).

B. COX-1 (Coagulation-Induced TxB ₂ Generation)

Fresh blood is collected into a bacillus container containing no coagulation inhibitor. An aliquot of 500 μL is immediately transferred to a microcentrifuge tube made of 2 μL of DMSO or silicon pre-loaded with the test compound at a final concentration varying from 10 nM to 30 μM. The tube is vortexed and incubated at 37 ° C for 1 hour to allow blood to clot. At the end of the incubation, sera are obtained by centrifugation (12,000 xg for 5 minutes). An aliquot of 100 μL of serum is mixed with 400 μL of methanol for protein precipitation. The supernatant is obtained and assayed for TxB ₂ using an enzyme immunoassay kit (Cayman, # 519031) according to the manufacturer's instructions.

Rat swelling test

protocol

Male Sprague-Dawley rats (150-200 g) are fasted overnight and orally administered with vehicle (1% Methocel or 5% Tween 80) or test compound. One hour later, a line is drawn using a permanent marker on the ankle of one hind paw. The foot volume (V ₀ ) is measured using an intravascular anemometer (Ugo-Basile, Italy) according to the principle of water replacement. 50 μl of 1% carrageenan solution in saline (FMC Corp, Maine) is injected into the hind paw (ie, 500 μg of carrageenan per plant) using an insulin syringe with a 25-gauge needle on the next animal. After 3 hours, the plantar volume (V ₃ ) is measured and the increase of the plantar volume (V ₃ -V ₀ ) is calculated. Animals are choked to CO ₂ and beheaded and the absence or presence of gastric lesions is noted. Data are compared to vehicle-control values and% inhibition calculated. Encrypt all treatment groups to remove observer bias.

NSAID-induced < RTI ID = 0.0 >

principle

A major side effect of conventional NSAIDs is its ability to cause gastric lesions in humans. This action is believed to be caused by inhibiting Cox-1 in the gastrointestinal tract. Rats are particularly sensitive to the action of NSAIDs. In fact, rat models are now commonly used in the past to assess the gastrointestinal side effects of conventional NSAIDs. In this assay, NSAID-induced gastrointestinal damage is observed by measuring the release of the ⁵¹ Cr feces after systemic injection of ⁵¹ Cr-labeled red blood cells. ⁵¹ Cr fecal discharge is a well-established and precise technique for detecting the integrity of the stomach in animals and humans.

Way

Male Sprague Dawley rats (150 to 200 g) are orally administered the test compound at once (acutely induced dose) or twice daily (chronic induced dose) for 5 days. Immediately after the last dose, 0.5 mL of ⁵¹ Cr-labeled red blood cells from the donor rat is injected through the tail vein of the rat. These animals are individually placed in a metabolism that can be freely consumed with feed and water. The feces are collected for 48 hours and the ⁵¹ Cr fecal discharge is calculated as the total injection volume%. ⁵¹ Cr-labeled red blood cells are prepared using the following method. 10 mL of blood is collected from a donor rat through a vein into a heparinized tube. Plasma is removed by centrifugation and supplemented with equivalent volume of HBSS. The red blood cells are incubated with 400 Ci of sodium ⁵¹ chromate for 30 minutes at < RTI ID = 0.0 > 37 C. < / RTI > At the end of the incubation period, the red blood cells are washed twice with 20 mL of HBSS to remove the liberated sodium ⁵¹ chromate. The red blood cells are finally reconstituted in 10 mL of HBSS and 0.5 mL of solution (approximately 20 Ci) per rat is injected.

Protein-Lossy Sickness in Blue-headed Monkeys

principle

Protein-losing disease (proving the appearance of circulating cells and plasma proteins in the GI tract) is a significant and dose-limiting adverse reaction to nonsteroidal anti-inflammatory standard drugs (NSAIDs). This is assessed suitably by intravenous administration of ⁵¹ CrCl ₃ solution. These isotonic ions bind very well to cells and serum globulins and cell endoplasmic reticulum. Measurement of radioactivity in the feces collected 24 hours after administration of the isotope provides a precise and quantitative indicator of protein-deficient disease.

Way

(0.8-1.4 kg) groups were given 1% methotrexate or 5% Tween 80 (3 mL / kg, twice a day) in H ₂ O vehicle, or test compound at a dose of 1 to 100 mg / It is administered by feeding five times a day. ⁵¹ Cr ( ⁵ Ci / kg in phosphate buffer saline (PBS) at 1 ml / kg) was administered one hour after administration of the final drug / vehicle and the feces were collected for 24 hours in the metabolic cage to obtain ⁵¹ Cr is evaluated. Venous blood is sampled 1 and 8 hours after the last drug administration and the plasma concentration of the drug is assessed by RP-HPLC.

LPS-induced fever in conscious rats

Male Sprague-Dawley rats (150-200 g) are desensitized for 16-18 hours before use. At approximately 9:30 am, the animals are temporarily placed in a resinous glass confinement unit and their basal rectal temperature is recorded using a resilient temperature probe (YSI series 400) connected to a digital thermometer (Model 08502, Cole Parmer). The same probe and thermometer are used for all animals to reduce experimental error. After measuring the temperature, put the animal back in our room. At the beginning (0 h), rats are intraperitoneally injected with saline or LPS (2 mg / kg, Sigma Chem) and rectal temperature is re-measured after 5, 6 and 7 hours after LPS injection. Five hours after measurement, when the temperature increase is constant, the vehicle (1% methotrexate) or test compound is orally administered to the LPS-injected rats to determine whether the compound causes recovery of fever. The percent recovery of fever is calculated using the rectal temperature measured after 7 hours in the reference group (vehicle-treated group) as the reference point (heat point at which the fever is reduced: 0). Takes 100% of full recovery of fever for pre-LPS base value.

LPS-induced fever in conscious blue-green monkeys

The temperature probe is implanted in the abdominal skin of a group of Saimiri sciureus (1.0 to 1.7 kg). This can be measured by a remote sensing system (Data Sciences International, Minnesota) to measure the body temperature of a conscious, unrestrained monkey. Animals are fasted and placed in individual cages for 13-14 hours to adapt before use. An electronic receiver is installed on one side to collect signals from the implanted temperature probe. At about 9 am on the day of the experiment, the monkeys were temporarily restrained in a training chair and infused intravenously with LPS (dissolved in sterile saline 6 mg / kg). Place this animal back in our room and record body temperature every 5 minutes. Two hours after the injection of LPS, monkeys are orally administered vehicle (1% methotrexate) or test compound (3 mg / kg) when the temperature rises by 1.5 to 2 ° C. After 100 minutes, measure the difference between body temperature and baseline. The inhibition% is calculated by taking the value of the control group as 0 inhibition%.

Acute inflammatory hyperalgesia induced by carrageenan in rats

The test is carried out using male Sprague Dawley rats (90-110 g). Hyperalgesia for mechanical compression of the sole is induced by injecting Karaghenan (4.5 mg on one foot) into the soles 3 hours before. A control group is injected with the same volume of saline (0.15 ml injected into the soles). The test compound (0.3 to 30 mg / kg, suspended in 0.5% methocel in distilled water) or vehicle (0.5% methocel) is orally administered (2 ml / kg) within 2 hours after the carrageenan injection. The loudness response to pressing of the sole is measured with a Ugo Basile algesiometer.

Statistical analysis of the carrageenan-induced hyperalgesia is performed using a one-way ANOVA (BMDP Statistical Software Inc.). The hyperalgesia is measured by subtracting the maximum loudness in the rat injected with saline from the loudness obtained from the animal injected with carrageenan. The hyperalgesia value for drug-treated rats is expressed as a percentage of this response. The ID ₅₀ value (the volume that causes 50% of the maximum measured response) is calculated by a nonlinear minimum angle regression analysis of the mean data using Graffit (Erithacus Software).

In rats, excipient-induced arthritis

A group of 70, 6.5 to 7.5 week old female levis rats (body weight about 146-170 g) were weighed and labeled on the ear and then induced with arthritis, vehicle control group, and indomethacin in a daily dose of 1 mg / The groups are divided into four groups administered with the test compound at a daily dose of 0.10 to 3.0 mg / kg, and the positive control group administered, with the body weights being such that each group is the same. Six groups of 10 rats were injected with 0.5 mg of Mycobacterium butyricum in 0.1 ml of light mineral oil (adjuvant) and no supplement was injected into groups of 10 negative control groups. Body weight, both foot volume (measured with a mercury gallometer) and lateral radiographs (obtained under ketamine and xylazine anesthesia) were measured on day -1 and on day 21 after adjuvant injection, and paw volume on day -1 and after supplementation 4 and 21 days. For radiography, rats are injected intramuscularly with 0.03-0.1 ml of a combination of ketamine (87 mg / kg) and xylazine (13 mg / kg) and anesthetized by injection of adjuvant. Using a paxitron (Faxitron, 45 kVp, 30 sec.) And Kodak X-OMAT TL film, radiographs of both the soles are taken on days 0 and 21 and developed with an autofocuser. Evaluate changes in soft tissue and soft tissues to a tester who is not familiar with the experimental treatment of radiographs. Changes in the following radiographs are numerical grades according to severity: increase in soft tissue volume (0-4), narrowing or widening of joint space (0-5), vocal fold erosion (0-3), periosteal reaction (0-4 ), Bone disruption (0-4), dislocation (0-3), and degenerative joint changes (0-3). Use specific criteria to assess the numerical severity of severity for each radiographic change. The maximum possible value per foot is 26. Test compounds at total daily doses of 0.1, 0.3, 1 and 3 mg / kg / day, indomethacin or vehicle (0.5% methocel in sterile water) at a total daily dose of 1 mg / kg / It is administered orally twice a day for 21 days continuously. Compounds are prepared weekly, refrigerated until used in a dark room, vortexed immediately before administration.

Apply two-factor ('treatment' and 'time') analysis of changes with repeated measurements in 'time' to the% change in body weight and foot volume and the total number of radiographs modified in sequence. A post hoc Dunnett's test is performed to compare the effect of treatment on the vehicle. A one-way analysis of changes is applied to the thymus and spleen body weights, followed by a cut-off test to compare the treatment effects on the vehicle. The dose-response curve for% inhibition on foot volume on days 4, 14 and 21 is applied to a 4-parameter algebraic function using nonlinear least-squares regression. ID ₅₀ is defined as the dose corresponding to a 50% reduction from the vehicle and is obtained by interpolation from the applied 4-parameter equation.

Representative biological data

Since the compounds of the present invention are inhibitors of COX-2, they are useful for the treatment of diseases mediated by COX-2 as described above. The activity of Compound A on cyclooxygenase is shown by the representative results shown in Table 1 below. In this assay, the inhibition is measured by measuring the amount of prostaglandin synthesized in the presence or absence of the putative inhibitor. IC ₅₀ represents the estimated inhibitor concentration required to reduce the prostaglandin synthesis obtained by 50% compared to the uninhibited control. The ED ₅₀ value in rat vet sweat analysis indicates the amount of compound required to reduce edema formation by up to 50% when compared to the vehicle control. The corresponding biological activities of the compounds B, which are the compounds described in documents WO 95/00501, Example 7 and US 5,536,752, are also shown.

[Table 1]

The invention is illustrated by the following non-limiting Examples, in which the following conditions are used, unless otherwise indicated:

(i) all processes are carried out at room or ambient temperature, i.e., at a temperature in the range of 18 to 25 ° C;

(ii) the solvent is evaporated using a rotary evaporator at a bath temperature of 60 DEG C or less under reduced pressure (600 to 4000 pascals: 4.5 to 30 mmHg);

(iii) the reaction is carried out by thin layer chromatography (TLC) and the reaction time is provided as an example only;

(iv) the melting point is not corrected and 'd' indicates decomposition; The melting points described are those obtained for the material prepared as described; Polymorphism can result in the separation of substances with different melting points in some formulations;

(v) The structure and purity of all final products are confirmed by one or more of the following techniques: TLC, mass spectrometer, nuclear magnetic resonance (NMR) photometer or microanalytical data;

(vi) yield is provided by way of example only;

(vii) The proposed NMR data is in the form of a delta (δ) value for the main diagnostic protons and is provided in ppm relative to tetramethylsilane (TMS) as internal standard and can be determined at 300 MHz or 400 MHz Measuring; Common abbreviations used for signal types are: s. Single line; d. Double line; t. Triple line; m. Multiple lines; br. Wide range; Etc. Also, Ar represents an aromatic signal;

(viii) The abbreviations of the formulas have their usual meanings; The following abbreviations are also used: v (volume), w (weight), bp (boiling point), MP (melting point), L (liter), mL (milliliters), g (grams) ), mmol (millimole), eq (equivalent).

Compound A can be prepared according to the following method.

Synthesis method

Method A

Halogenation of the cyclopentenone (II) with bromine or iodine and subsequent treatment with a base gives compound (III) [see Organic Syntheses 61 65]. The 3,5-difluorophenylboronic acid may then be added via palladium catalyzed coupling reaction to form compound (IV). The 4-bromothioanisole can be metallized with nBuLi or magnesium and then treated with compound (IV) to give alcohol V. Oxidation by allyl substitution can then be carried out using an oxidizing agent such as PDC to yield cyclopentenone (VI). Subsequently, sulfone oxidation to give sulfone (Ia) using an oxidizing agent such as H ₂ O _{2 in} the presence of a tungstate catalyst, MMPP, or mCPBA. Alternatively, compound (VI) can be converted to sulfonamide (Ib) as described in U.S. Patent No. 5,474,995, Method A.

[Reaction Scheme 1]

Method B

Litorio or magnesium thioanisole is added to bromocyclopentenone (IIIa) to obtain a tertiary alcohol (VII). The ketone (VIII) is then provided by oxidation with a reagent such as PDC. At this point, the sulfide can be oxidized using an oxidizing agent such as H ₂ O _{2 in} the presence of a tungstate catalyst, MMPP or mCPBA. Compound (Ia) is then provided by coupling of 3,5-difluorophenylboronic acid catalyzed by palladium.

[Reaction Scheme 2]

Method C

3-ethoxycyclopentenone (X) is brominated and then treated with a base to give compound (XI) (reference: Orgonic Synetheses 61 65). Compound (VIII) is obtained by adding lithio or magnesium thioanisole to the bromocyclopentenone (XI) followed by acid disposition of the intermediate addition product. At this point, sulfone (IX) can be obtained by oxidizing the sulfide using an oxidizing agent such as H ₂ O _{2 in} the presence of a tungstate catalyst, MMPP or mCPBA. Compound (Ia) is then provided by coupling of 3,5-difluorophenylboronic acid catalyzed by palladium.

[Reaction Scheme 3]

Example 1

2- (3,5-difluorophenyl) -3- (4-methylsulfonyl) phenyl) -2-cyclopenten-

Step 1

3- (4- (methylthio) phenyl) -2-cyclopenten-l-one

To a solution of 4-bromothioanisole (-65 ° C) in THF (800 mL) is added n-BuLi solution (2.3 M in hexane, 120 mL) at such a rate as to keep the internal temperature below -55 ° C . The resulting slush was stirred at -65 ° C for 2 hours and then treated with a solution of 3-ethoxy-2-cyclopenten-l-one (35 g, 0.28 mmol) in THF (50 mL). After 30 minutes at -65 ℃ past, thereby heating the solution to 0 ℃ to inhibit the reaction with NH ₄ Cl saturated aqueous solution (400mL). The product was extracted three times with 1L EtOAc and dry the combined extracts over MgSO ₄ and concentrated. The resulting material was swished with 200 mL of 1: 1 EtOAc / hexanes to give 45 g of the title compound as a white solid.

Step 2

2-bromo-3- (4- (methylthio) phenyl) -2-cyclopenten-

CCl ₄ (200mL) solution of 3 (4- (methyl thiol) phenyl) -2-cyclopenten-1-one Bromine (5mL) solution of _{(9.64g, 47.2mmol) CCl 4 (} 30mL) for 20 minutes to a suspension . The resulting orange suspension is stirred for 1.5 hours, then cooled in a freeze-bath and triethylamine (14 mL, 100 mmol) is added. After 30 minutes, the mixture is quenched with 1 M HCl (300 mL) and extracted with CH ₂ Cl ₂ . The organic phase is washed with brine, filtered through a cotton pad and evaporated. Purification by silica gel chromatography eluting with 90% CH ₂ Cl ₂ / hexane gave the title compound (7.13 g).

Step 3

2-bromo-3- (4-methylsulfonyl) phenyl) -2-cyclopenten-

A solution of 2-bromo-3- (4- (methylthio) phenyl) -2-cyclopenten-l-one (5.73 g, 20.2 mmol) in CH ₂ Cl ₂ (100 mL) and MeOH Is added MMPP (19 g, 30.7 mmol). The mixture is stirred at room temperature for 5 hours and then concentrated. The residue is partitioned between saturated aqueous NaHCO ₃ and CH ₂ Cl ₂ . The organic phase is washed with brine, filtered through a cotton pad and evaporated. The resulting solid was swished with CH ₂ Cl ₂ / hexane to give the title compound (5.57 g).

Step 4

2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) -phenyl) -2-cyclopenten-

(664 mg, 2.11 mmol), 3,5-difluorophenylboronic acid (832 mg, 5.27 mmol), tris (4-methylphenyl) 1: 1 toluene: n-propanol: water was added to a mixture of tetrahydrofuran (dibenzylideneacetone) dipalladium (O) (83 mg, 0.09 mmol) and triphenylphosphine (96 mg, 0.36 mmol) Purge with nitrogen. After stirring for 10 minutes, diethylamine (1.1 mL, 10.6 mmol) is added and the solution is heated to reflux for 4 hours. The mixture was cooled and partitioned between 1 M NaOH and EtOAc. The organic phase is washed with water, 1 M HCl and brine, and dried over MgSO ₄ . Purify by silica gel chromatography, eluting with 50% CH ₂ Cl ₂ / hexanes, followed by crystallization from CH ₂ Cl ₂ / ether / hexanes to give the title compound (223 mg).

^{_{1 H NMR (CD 3 COCD 3}} ) δ7.95 (2H, d), 7.65 (2H, d), 6.98 (1H, m), 6.82 (2H, m), 3.19 (2H, m), 3.15 (3H, s), 2.68 (2 H, m).

Example 1A

Step 1

2-Bromo-2-cyclopenten-l-one

To a solution of 2-cyclopenten-l-one (125 g, 1.52 mol) in CCl ₄ (1.2 L) in a 3-necked flask equipped with an overhead stirrer was added bromine (269 g, 1.68 mol) in CCl ₄ ) Solution is added dropwise over 4 hours while maintaining the internal temperature below 2 < 0 > C. A solution of Et ₃ N (310 mL, 2.22 mol) in CCl ₄ (200 mL) is then added dropwise over 1.5 hours, keeping the internal temperature below 10 ° C. The resulting suspension was warmed to room temperature for 1 hour, then cooled to 0 < 0 > C and filtered. The filtrate is washed twice with 700 mL of 3M HCl and 500 mL of brine and then filtered through a cotton wool pad. And concentrated to give 228 g of an orange oil which was crystallized from 150 mL of 2: 1 hexane: ether to give 191 g of the title compound.

¹ H NMR (CD ₃ COCD ₃ )? 7.94 (1H, t), 2.72 (2H, m), 2.46 (2H, m).

Step 2

2-bromo-3- (4-methylthio) phenyl) -2-cyclopenten-

NBuLi (1.6M in hexane, 107.5 mL, 172 mmol) is added to a solution of 4-bromothioanisole (35.1 g, 173 mmol) in THF (500 mL) at -78 < The solution to the 2-bromo-2-cyclopenten-1-one (25.4g, 158mmol) was added and the mixture was warmed to 0 ℃ and NH ₄ Cl saturated aqueous solution in 45 minutes and then stirred, THF (150mL) It inhibits the reaction. Most of the solvent is removed in vacuo and the residue is suspended in water and extracted twice with EtOAc. The organic layer is suspended in water and extracted twice with EtOAc. The organic layer was washed with brine, dried over MgSO ₄ and, filtered and concentrated. This material is dissolved in DMF (300 mL), cooled to 0 &lt; 0 &gt; C and treated with PDC (72.4 g, 192 mmol). The resulting mixture was allowed to warm to room temperature and stirred for 2 h, then poured into water (1.2 L) and extracted twice with 500 mL EtOAc. The organic layer was washed with brine, dried over MgSO _4, filtered and concentrated to give the title compound as a light brown solid state, and directly used in the next step.

Step 3

2-Bromo-3- (4- (methylsulfonyl) phenyl) -2-cyclopenten-

MMPP (100 g) is added to a solution of 2-bromo-3- (4-thiomethyl) phenyl) -2-cyclopenten-1-one in 2: 1 CH ₂ Cl ₂ / MeOH . The mixture is stirred at room temperature overnight, then concentrated and partitioned with saturated NaHCO ₃ , 1M Na ₂ S ₂ O ₃ and CH ₂ Cl ₂ . The aqueous layer is extracted with CH ₂ Cl ₂ , the combined organics are washed with brine, filtered through a cotton wool pad and evaporated. The resulting solid was swapped with CH ₂ Cl ₂ / ether to give 23 g of the title compound.

¹ H NMR (CD ₃ COCD ₃ )? 8.12 (4H, m), 3.22 (2H, m), 3.20 (3H, s), 2.69 (2H, m).

Step 4

3,5-Difluorophenylboronic acid

NBuLi (2.4 M in hexane, 108 mL, 0.26 mol) is added dropwise over 20 minutes to a solution of 1-bromo-3,5-difluorobenzene (50 g, 0.26 mol) in ether (860 mL) . The resulting solution was stirred for 10 minutes and then treated with triisopropyl borate (61 mL, 0.27 mol). The reaction mixture is allowed to warm to 0 &lt; 0 &gt; C for 30 minutes and then the reaction is quenched with 1M HCl. After stirring for 30 minutes, the mixture is partitioned between ethyl acetate and water. The organic layer was washed with brine, dried over MgSO ₄ and, filtered and evaporated. The resulting product was dried under high vacuum overnight to give 38 g of the title compound.

¹ H NMR (CD ₃ COCD ₃ )? 7.50 (2H, br s), 7.40 (2H, m), 7.04 (1H, m).

Step 5

2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten-

(52.2 g, 166 mmol), 3,5-difluorophenylboronic acid (31.9 g, 202 mmol), 2-bromo-3- (4- (methylsulfonyl) A mixture of tris (dibenzylideneacetone) dipalladium (O) (3.3 g, 3.6 mmol) and triphenylphosphine (1.89 g, 7.2 mmol) was dissolved in toluene (800 mL) and nPrOH (250 mL) do. After stirring for 10 minutes, diethylamine (21 mL, 203 mmol) and water (250 mL) are added. The gas is again removed from the mixture and heated to reflux for 1 hour, then cooled and added to EtOAc (2 L). The aqueous layer is separated and the organic layer is washed with 0.2N NaOH (500 mL), 0.5N HCl (500 mL) and brine. The organic phase is then dried over MgSO ₄ , filtered through celite and evaporated. The resulting pale brown solid was swished in hot EtOAc (250 mL), cooled and filtered to give the title compound (45.8 g, mp 174-175 ° C).

^{_{1 H NMR (CD 3 COCD 3}} ) δ7.95 (2H, m), 7.66 (2H, m), 6.98 (1H, m), 6.80 (2H, m), 3.17 (2H, m), 3.12 (3H, s), 2.68 (2 H, m).

B. Another method for the preparation of 2-bromo-3- (4- (methylsulfonyl) phenyl) -2-cyclopenten-l-one (Example 1, step 3 is developed as follows)

Step 1

2-Bromo-3-ethoxy-2-cyclopenten-l-one

Of bromine (219g, 1.37mol) in a solution of 3 in CHCl ₃ 1.8L-2-cyclopenten-1-one CHCl ₃ (200mL) to (165g, 1.30mol) solution (-15 ℃) through the dropping funnel 1.5 hours to give a thick yellow slurry. After further stirring for 30 minutes, a solution of Et ₃ N (210 mL, 1.5 mol) in CHCl ₃ (300 mL) is added via a dropping funnel over 30 minutes. The resulting mixture is concentrated, suspended in EtOAc and filtered through a pad of 2 cm celite on top of 1 cm silica gel. The filtrate is concentrated and the resulting material is recrystallized from 4: 1 ether / hexanes to give 245 g of the title compound.

¹ H NMR (CD ₃ COCD ₃ )? 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-

NBuLi (2.5 M in hexane, 500 mL, 1.25 mol) was added to a solution of 4-bromothioanisole (253 g, 1.25 mol) in THF (5 L) at -78 ° C over 20 minutes to give a thick slurry Stir at -78 &lt; 0 &gt; C for 2 h. A solution of 2-bromo-2-cyclopenten-l-one (242 g, 1.18 mol) in THF (1 L) was added via cannula and the mixture was warmed to -10 <0> C. 6N HCl (430 mL) is then added and the resulting mixture is stirred for 20 minutes. Add EtOAc (3 L) and separate the aqueous phase. The organic layer is concentrated. The residue is suspended in ether and filtered to give 287 g of the title compound.

¹ H NMR (CD ₃ COCD ₃ )? 8.12 (4H, m), 3.22 (2H, m), 3.20 (3H, s), 2.69 (2H, m).

The following examples of pharmaceutical formulation are for the purpose of illustrating the present invention.

Step 3

2-Bromo-3- (4- (methylsulfonyl) phenyl) -2-cyclopenten-

EtOAc (500mL) and CH ₂ Cl ₂ (100mL) NaWO to a solution of 2-bromo-3- (4-methylthio) phenyl) -2-cyclopenten-1-one (67.4g, 238mmol) suspension (at room temperature) ₄ 2H ₂ O (3.1 g, 9 mmol) and Aliquat 336 (12 g, 30 mmol). 10 mL of 30% H ₂ O ₂ is added and the mixture is warmed to 40 ° C to initiate oxidation. Then 85 mL of H ₂ O _{2 is} added dropwise for 20 minutes while cooling the outside to maintain an internal temperature of approximately 50 ° C. The dark reaction mixture is then maintained at 40 占 폚 for 1.5 hours. The aqueous layer is separated and the organic phase is 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 granule type tablet composition

Refined sugar component

25 mg COX-2 inhibitor

79.7 mg Crystalline cellulose

79.7 mg Lactose monohydrate

6 mg hydroxypropylcellulose

8 mg croscarmellose sodium

1 mg magnesium stearate

Tablet dose concentrations of 5 to 125 mg can be adjusted by varying the total tablet weight and the first three component ratios. In general, it is desirable to maintain the ratio of microcrystalline cellulose: lactose monohydrate at 1: 1. If yellow or orange is desired, iron oxide may be added to the or other composition in varying amounts of 5 mg or less.

Example 2a

Wet granule type tablet composition

Refined sugar component

125 mg COX-2 inhibitor

425 mg microcrystalline cellulose

425 mg lactose monohydrate

10 mg hydroxypropylcellulose

14 mg croscarmellose sodium

1 mg magnesium stearate

Example 2b

Wet granule type tablet composition

Refined sugar component

10 mg COX-2 inhibitor

87.2 mg Crystalline cellulose

87.2 mg Lactose monohydrate

6 mg hydroxypropylcellulose

8 mg croscarmellose sodium

1 mg magnesium stearate

Example 2c

Wet granule type tablet composition

Refined sugar component

5 mg COX-2 inhibitor

89.7 mg microcrystalline cellulose

89.7 mg Lactose monohydrate

6 mg hydroxypropylcellulose

8 mg croscarmellose sodium

1 mg magnesium stearate

Example 3

Directly compressed tablet composition

Refined sugar component

25 mg COX-2 inhibitor

106.9 mg microcrystalline cellulose

106.9 mg Lactose anhydrous

7.5 mg croscarmellose sodium

3.7 mg magnesium stearate

Tablet dose concentrations of 5 to 125 mg can be adjusted by varying the total tablet weight and the first three component ratios. It is generally desirable to maintain the ratio of microcrystalline cellulose: lactose monohydrate at 1: 1.

Example 3a

Directly compressed tablet composition

Refined sugar component

125 mg COX-2 inhibitor

425 mg microcrystalline cellulose

425 mg lactose monohydrate

15 mg croscarmellose sodium

10 mg magnesium stearate

Example 3b

Directly compressed tablet composition

Refined sugar component

10 mg COX-2 inhibitor

42.5 mg Crystalline cellulose

42.5 mg lactose monohydrate

4 mg croscarmellose sodium

1 mg magnesium stearate

Example 3c

Directly compressed tablet composition

Refined sugar component

5 mg COX-2 inhibitor

45 mg microcrystalline cellulose

45 mg lactose monohydrate

4 mg croscarmellose sodium

1 mg magnesium stearate

Example 4

Hard capsule composition

Refined sugar component

25 mg COX-2 inhibitor

37 mg microcrystalline cellulose

37 mg lactose monohydrate

1 mg croscarmellose sodium

1 capsule hard gelatin capsule

Capsule dose concentrations from 1 to 50 mg can be adjusted by varying the total fill weight and the first three component ratios. In general, it is desirable to maintain the ratio of microcrystalline cellulose: lactose monohydrate at 1: 1.

Example 5

Oral solution

Both components per 5 mL dose

50 mg COX-2 inhibitor

5 mL polyethylene oxide 400 &lt; RTI ID = 0.0 &gt;

A solution dose concentration of 1 to 50 mg / 5 mL can be adjusted by varying the ratio of the two components.

Example 6

Oral Suspension

Both components per 5 mL dose

100 mg COX-2 inhibitor

150 mg polyvinylpyrrolidone

2.5 mg polyoxyethylene sorbitan monolaurate

10 mg benzoic acid

5 mL of sorbitol solution (70%) containing the above components,

Suspension dose concentrations of 1 to 50 mg / 5 ml can be adjusted by varying the ratio of the first two components.

Example 7

Intravenous infusion

Both components per 200 mL dose

1 mg COX-2 inhibitor

0.2 mg Polyethylene Oxide 400

1.8 mg sodium chloride

200 mL &lt; / RTI &gt; of purified water

2- (3,5-Difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten-l-one is effective in treating cyclooxygenase-2 mediated diseases.

Claims (30)

A pharmaceutical composition comprising a therapeutically effective amount of a 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten- A pharmaceutical composition for the treatment of diseases mediated by cyclooxygenase-2. 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten-1-one and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 1, which comprises 10 to 250 mg of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten- &Lt; / RTI &gt; 3. The process according to claim 1 or 2, wherein at least one of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) 40 or 50 mg. 3. The compound according to claim 1 or 2, wherein the compound is 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2- 100, 110, or 120 mg. 3. The pharmaceutical composition according to claim 1 or 2 comprising 60 to 120 mg of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten- A pharmaceutical composition. The pharmaceutical composition according to claim 1 or 2, which comprises 10 to 50 mg of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten- A pharmaceutical composition. Comprising administering to a patient in need of treatment from 10 to 250 mg of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten- A method of treating an inflammatory disease susceptible to treatment with a non-steroidal anti-inflammatory agent. 9. The method of claim 8, further comprising administering a therapeutically effective amount of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) , &Lt; / RTI &gt; 40 or 50 mg. 9. The method of claim 8, wherein the patient is treated with a therapeutically effective amount of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) , 90, 100, 110, or 120 mg per day. 9. The method according to claim 8, wherein 60 to 120 mg of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten- &Lt; / RTI &gt; 9. The method according to claim 8, wherein 10 to 50 mg of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten- &Lt; / RTI &gt; 9. The method of claim 8, wherein the non-chronic headache, pain or swelling is treated. 9. The method of claim 8, wherein the osteoarthritis is treated. 9. The method of claim 8, wherein the method is for treating rheumatoid arthritis. The use of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2- cyclohexane in the preparation of a medicament for the treatment of inflammatory diseases susceptible to treatment with non- Penten-1-one. 17. A method according to claim 16 for the preparation of a dosage form for the treatment of inflammatory diseases susceptible to treatment with a non-steroidal anti-inflammatory agent, comprising administering a therapeutically effective amount of 2- (3,5-difluorophenyl) -3- (4- Sulfonyl) phenyl) -2-cyclopenten-l-one from 10 to 250 mg. 17. A method according to claim 16 for the preparation of a dosage form for the treatment of inflammatory diseases susceptible to treatment with a non-steroidal anti-inflammatory agent, comprising administering a therapeutically effective amount of 2- (3,5-difluorophenyl) -3- (4- Sulfonyl) phenyl) -2-cyclopenten-l-one 10, 20, 30, 40 or 50 mg. 17. A method according to claim 16 for the preparation of a dosage form for the treatment of inflammatory diseases susceptible to treatment with a non-steroidal anti-inflammatory agent, comprising administering a therapeutically effective amount of 2- (3,5-difluorophenyl) -3- (4- Sulfonyl) phenyl) -2-cyclopenten-l-one 60, 70, 80, 90, 100, 110, or 120 mg. 17. A method according to claim 16 for the preparation of a dosage form for the treatment of inflammatory diseases susceptible to treatment with a non-steroidal anti-inflammatory agent, comprising administering a therapeutically effective amount of 2- (3,5-difluorophenyl) -3- (4- Sulfonyl) phenyl) -2-cyclopenten-l-one from 10 to 50 mg. 17. A method according to claim 16 for the preparation of a dosage form for the treatment of inflammatory diseases susceptible to treatment with a non-steroidal anti-inflammatory agent, comprising administering a therapeutically effective amount of 2- (3,5-difluorophenyl) -3- (4- Sulfonyl) phenyl) -2-cyclopenten-l-one 60-120 mg. 17. The use of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopentene- 1-on 10 to 50 mg use. 17. The use of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopentene- 1-on 60 to 120 mg use. 17. The use of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopentene in the preparation of a dosage form of a medicament for the treatment of rheumatoid arthritis, -1-one &lt; / RTI &gt; 17. The use of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopentene in the preparation of a dosage form for the treatment of rheumatoid arthritis, -1-one &lt; / RTI &gt; 17. The use of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -pyrrolidine for the preparation of a dosage form for the treatment of non-chronic headache, pain or swelling. ) -2-cyclopenten-l-one &lt; / RTI &gt; 17. The use of 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -pyrrolidine for the preparation of a dosage form for the treatment of non-chronic headache, pain or swelling. ) -2-cyclopenten-l-one &lt; / RTI &gt; 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten-1-one. Therapeutic 2- (3,5-difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten-1-one. 2- (3,5-Difluorophenyl) -3- (4- (methylsulfonyl) phenyl) -2-cyclopenten-l-one crystals.