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WO2001022965A1 - Imizadoles substitues a activite inhibitrice de la cytokine - Google Patents

Imizadoles substitues a activite inhibitrice de la cytokine Download PDF

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Publication number
WO2001022965A1
WO2001022965A1 PCT/US2000/026358 US0026358W WO0122965A1 WO 2001022965 A1 WO2001022965 A1 WO 2001022965A1 US 0026358 W US0026358 W US 0026358W WO 0122965 A1 WO0122965 A1 WO 0122965A1
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Prior art keywords
alkyl
compound
optionally substituted
halogen
substituted
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PCT/US2000/026358
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English (en)
Inventor
Christopher F. Claiborne
David A. Claremon
Nigel J. Liverton
Kevin T. Nguyen
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Merck & Co., Inc.
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Publication date
Application filed by Merck & Co., Inc. filed Critical Merck & Co., Inc.
Priority to AU77154/00A priority Critical patent/AU7715400A/en
Publication of WO2001022965A1 publication Critical patent/WO2001022965A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond

Definitions

  • the present invention relates to substituted heterocyclic compounds which have cytokine inhibitory activity.
  • Cytokine mediated diseases and cytokine inhibition, suppression and antagonism are used in the context of diseases or conditions in which excessive or unregulated production or activity of one or more cytokines occurs.
  • Examples of cytokines which are effected typically include Interleukin-1 (IL-1), Interleukin-6 (IL-6), Interleukin-8 (IL-8) and Tumor Necrosis Factor (TNF).
  • IL-1 Interleukin-1
  • IL-6 Interleukin-6
  • IL-8 Interleukin-8
  • TNF Tumor Necrosis Factor
  • Interleukin-1 IL-1
  • Tumor Necrosis Factor TNF
  • IL-1 Interleukin-1
  • TNF Tumor Necrosis Factor
  • rheumatoid arthritis osteoarthritis
  • endotoxemia toxic shock syndrome
  • acute and chronic inflammatory diseases such as the inflammatory reaction induced by endotoxin or inflammatory bowel disease
  • tuberculosis atherosclerosis, muscle degeneration, cachexia, psoriatic arthritis, Reiter's syndrome, rheumatoid arthritis, gout, traumatic arthritis, rubella arthritis and acute synovitis.
  • IL-1 activity Recent evidence also links IL-1 activity to diabetes.
  • Interleukin-1 has been demonstrated to mediate a variety of biological activities thought to be important in immunoregulation and other physiological conditions. Dinarello et ah, Rev. Infect. Disease, 6, 51 (1984).
  • the known biological activities of IL-1 include the activation of T helper cells, induction of fever, stimulation of prostaglandin or collagenase production, neutrophil chemotaxis, induction of acute phase proteins and the suppression of plasma iron levels.
  • TNF tumor necrosis factor
  • TNF cytomegalovirus
  • Interleukin-6 is a cytokine effecting the immune system and hematopoiesis. It is produced by several mammalian cell types in response to agents such as IL-1, and is correlated with disease states such as angiofollicular lymphoid hyperplasia.
  • Interleukin-8 is a chemotactic factor first identified and characterized in 1987. Many different names have been applied to IL-8, such as neutrophil attractant/activation protein- 1 (NAP-1), monocyte derived neutrophil chemotactic factor (MDNCF), neutrophil activating factor (NAP), and T-cell lymphocyte chemotactic factor. Like IL-1, IL-8 is produced by several cell types, including mononuclear cells, fibroblasts, endothelial cells and ketainocytes. Its production is induced by IL-1, TNF and by lipopolysaccharide (LPS). IL-8 stimulates a number of cellular functions in vitro.
  • NAP-1 neutrophil attractant/activation protein- 1
  • MDNCF monocyte derived neutrophil chemotactic factor
  • NAP neutrophil activating factor
  • T-cell lymphocyte chemotactic factor T-cell lymphocyte chemotactic factor.
  • IL-8 is produced by several cell types, including mono
  • the present invention relates to compound I of the formula
  • Q is CH or N
  • R, and R ⁇ are independently hydrogen or C,-C 6 alkyl; said alkyl being optionally substituted by 1-3 groups selected from halogen, hydroxy, CF 3 N ⁇ and NO2; or
  • R, and R ⁇ taken together represent an optionally substituted 4 to 10 membered heterocyclic ring containing at least one N atom, and optionally containing 1-2 additional N atoms and 0-1 O atom; said ring optionally substituted by 1-3 groups selected from C j -C 4 alkyl, halogen, hydroxy,
  • R 3 is hydrogen, halogen, S(C ⁇ -C 6 alkyl), SO 2 (d-C 6 alkyl), NH(cycloalkyl), NH(C ⁇ -C 6 alkyl), said alkyl being optionally substituted by (C ⁇ -C 6 alkyl), or NH(C ⁇ -Cg alkyl) aryl; said aryl group being optionally substituted by 1-3 groups selected from halogen, hydroxy, CF 3 , NH2, and NO2;
  • R 4 , R 5 and R 6 independently represent a member selected from the group consisting of hydrogen, halo, hydroxy, CFo NH 2 NO 2 Ci-Cg alkyl, substituted Ci-Cg alkyl, Ci-Cg alkoxy, substituted C1-C6 alkoxy, C3-C8 cycloalkyl, substituted C3-C8 cycloalkyl, aryl or substituted aryl;
  • R 7 and R 8 independently represent a member selected from the group consisting of hydrogen or C j -C 8 alkyl, with the proviso that only one of the nitrogen atoms can be substituted; or
  • R ⁇ and R 7 taken together represent an optionally substituted 4 to 10 membered heterocyclic ring containing at least one N atom, and optionally containing 1-2 additional N atoms and 0-1 O atom; said ring optionally substituted by 1-3 groups selected from C,-C 4 alkyl, OH, O(C j -C 6 alkyl);
  • This invention also relates to a pharmaceutical composition which is comprised of a compound of formula I as defined above in combination with a pharmaceutically acceptable carrier. Additionally, the invention relates to a method of treating a cytokine mediated disease in a mammal, comprising administering to a mammalian patient in need of such treatment an amount of a compound of formula I which is effective for treating said cytokine mediated disease.
  • the present invention relates to compound I of the formula
  • Q is CH or N
  • R, and R 2 are independently hydrogen or C,-C 6 alkyl; said alkyl being optionally substituted by 1-3 groups selected from halogen, hydroxy, CF 3 NH 2j and NO 2 ; or
  • R, and R 2 taken together represent an optionally substituted 4 to 10 membered heterocyclic ring containing at least one N atom, and optionally containing 1-2 additional N atoms and 0-1 O atom; said ring optionally substituted by 1-3 groups selected from C j -C 4 alkyl, halogen, hydroxy,
  • R 3 is hydrogen, halogen, S(d-C 6 alkyl), SO 2 (d-C 6 alkyl), NH(cycloalkyl),
  • R 4 , R 5 and R 6 independently represent a member selected from the group consisting of hydrogen, halo, hydroxy, CFo NH 2 NO 2 Cj-Cg alkyl, substituted
  • R 7 and R 8 independently represent a member selected from the group consisting of hydrogen or C j -C 6 alkyl, with the proviso that only one of the nitrogen atoms can be substituted; or
  • R 2 and R 7 taken together represent an optionally substituted 4 to 10 membered heterocyclic ring containing at least one N atom, and optionally containing 1-2 additional N atoms and 0-1 O atom; said ring optionally substituted by 1-3 groups selected from C,-C 4 alkyl, OH, O(C,-C 6 alkyl);
  • This invention also relates to a pharmaceutical composition which is comprised of a compound of formula I as defined above in combination with a pharmaceutically acceptable carrier and to a method of treating a cytokine mediated disease in a mammal, comprising administering to a mammalian patient in need of such treatment an amount of a compound of formula I which is effective for treating said cytokine mediated disease.
  • R 1 and R 2 are independently hydrogen or C,-C 6 alkyl; said alkyl being optionally substituted by 1-3 groups selected from halogen, hydroxy, CF, NH 2 and NO 2 ; or
  • R, and R 2 taken together represent a piperazine, piperidine, pyridine or morpholine ring, each ring optionally substituted by 1-3 groups selected from C,-C 6 alkyl, halogen, hydroxy, CF 3 NH 2 and NO 2 ;
  • R 3 is hydrogen, NH(cycloalkyl) or NH(C ⁇ -C6 alkyl)phenyl; said phenyl group being optionally substituted by 1-3 groups selected from halogen, hydroxy, CF 3 , NH 2 , and NO 2 ;
  • R., Rr and R 6 are independently hydrogen, halogen, alkyl or CF 3 ;
  • R 7 and R 8 are independently hydrogen or CH 3 ;
  • Representative species falling within the present invention include the following:
  • the dotted bond in the imidazole ring designates that there is a double bond at one of the two positions.
  • alkyl refers to a monovalent alkane (hydrocarbon) derived radical containing from 1 to 15 carbon atoms unless otherwise defined. It may be straight or branched, and when of sufficient size, e.g., C -15 may be cyclic. Preferred straight or branched alkyl groups include methyl, ethyl, propyl, isopropyl, butyl and t-butyl. Preferred cycloalkyl groups include cyclopropyl, cyclopentyl and cyclohexyl.
  • Alkyl also includes an alkyl group substituted with a cycloalkyl group, such as cyclopropylmethyl.
  • alkylene and monovalent alkyl portion(s) of the alkyl group can be attached at any available point of attachment to the cycloalkylene portion.
  • substituted alkyl this refers to a straight, branched or cyclic alkyl group as defined above, substituted with 1-3 groups as defined with respect to each va ⁇ able
  • aryl refers to aromatic ⁇ ngs, e g., phenyl, substituted phenyl and like groups as well as ⁇ ngs which are fused, e g , naphthyl and the like
  • Aryl thus contains at least one ⁇ ng having at least 6 atoms, with up to two such ⁇ ngs being present, containing up to 10 atoms therein, with alternating (resonating) double bonds between adjacent carbon atoms
  • the prefe ⁇ ed aryl groups are phenyl and naphthyl
  • Aryl groups may likewise be substituted as defined below
  • Preferred substituted aryls include phenyl or naphthyl substituted with one or two groups
  • heterocycloalkyl and “heterocyclyl” refer to a cycloalkyl group (nonaromatic) in which one of the carbon atoms in the ⁇ ng is replaced by a heteroatom selected from O, S(O) y or N, and m which up to three additional carbon atoms may be replaced by said heteroatoms When three heteroatoms are present m the heterocycle, they are not all linked together
  • heterocyclyls are pipe ⁇ dinyl, morphohnyl, azetidinyl, pyrrolidinyl, tetrahydrofuranyl, imidazolmyl, piperazinyl, pyrol ⁇ d ⁇ n-2-one, pipe ⁇ dm-
  • halogen or "halo" is intended to include fluo ⁇ ne, chlo ⁇ ne, bromine and iodine.
  • composition is intended to encompass a product comp ⁇ sing the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts
  • optical isome ⁇ c forms that is mixtures of enantiomers, e.g , racemates, or diastereomers as well as individual enantiomers or diastereomers of the instant compound are included.
  • These individual enantiomers are commonly designated according to the optical rotation they effect by the symbols (+) and (-), (L) and (D), (1) and (d) or combinations thereof.
  • These isomers may also be designated according to their absolute spatial configuration by (S) and (R), which stands for sinister and rectus, respectively.
  • the individual optical isomers may be prepared using conventional resolution procedures, e.g., treatment with an appropriate optically active acid, separating the diastereomers and then recovering the desired isomer.
  • the individual optical isomers may be prepared by asymmetric synthesis.
  • a given chemical formula or name shall encompass pharmaceutically acceptable addition salts thereof and solvates thereof, such as hydrates.
  • the compounds of the present invention while effective themselves, may be formulated and administered in the form of their pharmaceutically acceptable addition salts for purposes of stability, convenience of crystallization, increased solubility and other desirable properties.
  • the compounds of the present invention may be administered in the form of pharmaceutically acceptable salts.
  • pharmaceutically acceptable salt is intended to include all acceptable salts.
  • acid salts are hydro- chloric, nitric, sulfuric, phosphoric, formic, acetic, trifluoroacetic, propionic, maleic, succinic, malonic, methane sulfonic and the like which can be used as a dosage form for modifying the solubility or hydrolysis characteristics or can be used in sustained release or prodrug formulations.
  • pharmaceutically acceptable salts of the compounds of this invention include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, omithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl) aminomethane, and tetramethyl-ammonium hydroxide.
  • bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, omithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl) amino
  • esters can be employed, e.g. methyl, ethyl, butyl, acetate, maleate, pivaloyloxymethyl, and the like, and those esters known in the art for modifying solubility or hydrolysis characteristics for use as sustained release or prodrug formulations.
  • the compounds of the present invention may have chiral centers other than those centers whose stereochemistry is depicted in formula I, and therefore may occur as racemates, racemic mixtures and as individual enantiomers or diastereomers, with all such isomeric forms being included in the present invention as well as mixtures thereof.
  • some of the crystalline forms for compounds of the present invention may exist as polymorphs and as such are intended to be included in the present invention.
  • some of the compounds of the instant invention may form solvates with water or common organic solvents. Such solvates are encompassed within the scope of this invention.
  • TNF mediated disease or disease state refers to disease states in which TNF plays a role, either by production or increased activity levels of TNF itself, or by causing another monokine to be released, such as but not limited to IL-1 or IL-6.
  • cytokine as used herein means any secreted polypeptide that affects the functions of cells and is a molecule which modulates interactions between cells in the immune, inflammatory or hematopoietic response.
  • a cytokine includes, but is not limited to, monokines and lymphokines regardless of which cells produce them. Examples of cytokines include, but are not limited to, Interleukin-1 (IL-1), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Tumor Necrosis Factor-alpha (TNF- ⁇ ) and Tumor Necrosis Factor-beta (TNF- ⁇ ).
  • IL-1 Interleukin-1
  • IL-6 Interleukin-6
  • IL-8 Interleukin-8
  • TNF- ⁇ Tumor Necrosis Factor-alpha
  • TNF- ⁇ Tumor Necrosis Factor-beta
  • cytokine interfering or cytokine suppressive amount an effective amount of a compound of formula I which will cause a decrease in the in vivo activity or level of the cytokine to normal or sub-normal levels, when given to the patient for the prophylaxis or therapeutic treatment of a disease state which is exacerbated by, or caused by, excessive or unregulated cytokine production or activity.
  • Compound 1 is deprotonated by treatment with a strong base followed by quenching with an amide 2 to yield the ketone 3.
  • Compound 3 is reacted with an oxidizing agent such as selenium dioxide to prepare diketone 4.
  • Alkyl guanidine 5 is condensed with the diketone 4, followed by reduction to afford the imidazole 6.
  • Reaction of Compound 6 with alkylating agents leads to a mixture of imidazole regioisomers represented by 8 and 9. These products are separated and reacted with a nucleophile R 3 to provide compounds 10 and 11.
  • Fluoropyridine 12 is treated with lithium disopropylamine, followed by an amide 13 to yield ketone 14.
  • Compound 14 is reacted with selenium dioxide to prepare the diketone 15.
  • Substituted guanidine 16 is readily condensed with Compound 15 to afford a hydroxy imidazole intermediate which is reduced in situ by Pd on carbon in the presence of hydrogen to afford Compound 17.
  • the imidazole is alkylated with methyl iodide resulting in a mixture of regioisomeric products. This mixture is purified by silica gel chromatography, and the desired compound 18 is reacted with alpha-methyl benzylamine to yield the final compound.
  • the compounds of formula 1 can be used in the prophylactic or therapeutic treatment of disease states in mammals which are exacerbated or caused by excessive or unregulated cytokines, e.g., IL-1, IL-6, LL-8 or TNF. Because the compounds of formula I inhibit cytokines, the compounds are useful for treating diseases in which cytokine presence or activity is implicated, such as rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions.
  • cytokines e.g., IL-1, IL-6, LL-8 or TNF.
  • the compounds of formula I are useful to treat disease states mediated by excessive or unregulated TNF production or activity.
  • diseases include, but are not limited to, sepsis, septic shock, endotoxic shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcoidosis, bone resorption diseases, such as osteoporosis, reperfusion injury, graft v.
  • the compounds of formula I are also useful topically in the treatment of inflammation such as in the treatment of rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions, inflamed joints, eczema, psoriasis or other inflammatory skin conditions such as sunburn, inflammatory eye conditions including conjunctivitis, pyresis, pain and other conditions associated with inflammation.
  • the compounds of formula I are also useful in treating diseases characterized by excessive IL-8 activity. These disease states include psoriasis, inflammatory bowel disease, asthma, cardiac and renal reperfusion injury, adult respiratory distress syndrome, thrombosis and glomerulonephritis.
  • the invention thus includes a method of treating psoriasis, inflammatory bowel disease, asthma, cardiac and renal reperfusion injury, adult respiratory distress syndrome, thrombosis and glomerulo-nephritis, in a mammal in need of such treatment, which comprises administering to said mammal a compound of formula I in an amount which is effective for treating said disease or condition.
  • the dosage used can be varied within wide limits, depending upon the type of disease, the age and general condition of the patient, the particular compound administered, the presence or level of toxicity or adverse effects experienced with the drug and other factors.
  • a representative example of a suitable dosage range is from as low as about 0.01 mg/kg to as high as about 100 mg/kg. However, the dosage administered is generally left to the discretion of the physician.
  • the methods of treatment can be carried out by delivering the compound of formula I parenterally.
  • parenteral as used herein includes intravenous, intramuscular, or intraperitoneal administration. The subcutaneous and intramuscular forms of parenteral administration are generally preferred.
  • the instant invention can also be carried out by delivering the compound of formula I subcutaneously, intranasally, intrarectally, transdermally or intravaginally.
  • the compounds of formula I may also be administered by inhalation.
  • inhalation is meant intranasal and oral inhalation administration.
  • Appropriate dosage forms for such administration such as an aerosol formulation or a metered dose inhaler, may be prepared by convention techniques.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula I and a pharmaceutically acceptable carrier.
  • the compounds of formula I may also be included in pharmaceutical compositions in combination with a second therapeutically active compound.
  • the pharmaceutical carrier employed may be, for example, either a solid, liquid or gas.
  • solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, and the like.
  • liquid carriers are syrup, peanut oil, olive oil, water and the like.
  • gaseous carriers include carbon dioxide and nitrogen.
  • the carrier or diluent may include time delay material well known in the art, such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • a wide variety of pharmaceutical dosage forms can be employed. If a solid dosage is used for oral administration, the preparation can be in the form of a tablet, hard gelatin capsule, troche or lozenge. The amount of solid carrier will vary widely, but generally will be from about 0.025 mg to about 1 g. When a liquid dosage form is desired for oral administration, the preparation is typically in the form of a syrup, emulsion, soft gelatin capsule, suspension or solution. When a parenteral dosage form is to be employed, the drug may be in solid or liquid form, and may be formulated for administration directly or may be suitable for reconstitution. Topical dosage forms are also included. Examples of topical dosage forms are solids, liquids and semi-solids. Solids would include dusting powders, poultices and the like.
  • Liquids include solutions, suspensions and emulsions.
  • Semi- solids include creams, ointments, gels and the like.
  • the amount of a compound of formula I used topically will, of course, vary with the compound chosen, the nature and severity of the condition, and can be varied in accordance with the discretion of the physician.
  • a representative, topical, dose of a compound of formula I is from as low as about 0.01 mg to as high as about 2.0 g, administered one to four, preferably one to two times daily.
  • the active ingredient may comprise, for topical administration, from about 0.001% to about 10% w/w.
  • Drops according to the present invention may comprise sterile or non- sterile aqueous or oil solutions or suspensions, and may be prepared by dissolving the active ingredient in a suitable aqueous solution, optionally including a bactericidal and/or fungicidal agent and/or any other suitable preservative, and optionally including a surface active agent.
  • a suitable aqueous solution optionally including a bactericidal and/or fungicidal agent and/or any other suitable preservative, and optionally including a surface active agent.
  • the resulting solution may then be clarified by filtration, transferred to a suitable container which is then sealed and sterilized by autoclaving or maintaining at 98-100°C for half an hour.
  • the solution may be sterilized by filtration and transfe ⁇ ed to the container aseptically.
  • bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%).
  • Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.
  • Lotions according to the present invention include those suitable for application to the skin or eye.
  • An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops.
  • Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or arachis oil.
  • Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application.
  • the base may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or macrogels.
  • hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap
  • a mucilage an oil of natural origin such as almond, corn, arachis, castor or olive oil
  • wool fat or its derivatives or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or macrogels.
  • the formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surfactant such as sorbitan esters or polyoxy- ethylene derivatives thereof.
  • suitable surface active agent such as an anionic, cationic or non-ionic surfactant such as sorbitan esters or polyoxy- ethylene derivatives thereof.
  • Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicas, and other ingredients such as lanolin may also be included.
  • Step 1A N-Methyl-N-methoxy-(3-trifluoromethyl)-phenyl-carboxamide To a suspension of N,O-dimethylhydroxylamine hydrochloride (58.2 g,
  • Step IB (2-Fluoro-pyridin-4-vI)-l-(3-trifluoromethyl-phenyl)-ethanone
  • n-butyllithium 54.0 mL, 2.5M in hexane, 0.135 mol
  • 2-fluoro-4- methylpyridine 10 g, 0.090 mol
  • Step 1C (2-Fluoro-pyridin-4-yl)-2-(3-trifluoromethyl-phenyl)-ethane-1.2-dione
  • Step ID l-[5-(2-Fluoro-pyridin-4-yl)-4-(3-trifluoromethyl-phenyl)-lH- imidazol-2-yll-piperazine
  • Step IE 4-[5-(2-Fluoro-pyridin-4-yl)-4-(3-trifluoromethyl-phenyl)-lH- imidazol-2-v ⁇ -piperazine- 1-carboxylic acid benzyl ester
  • Step IF 4-[5-[2-(l-(S)-Phenyl-ethylamino)-pyridin-4-yl]-4-(3-trifluoromethyl- phenyl)-lH-imidazol-2-yl1-piperazine-l-carboxylic acid benzyl ester
  • 4-[5-(2-fluoro-pyridin-4-yl)-4-(3-trifluoromethyl- phenyl)-lH-imidazol-2-yl]-piperazine-l-carboxylic acid benzyl ester (1.04 g, 1.979 mmol) in (S)-(-)-( ⁇ ) methyl benzylamine (5.10 mL, 39.581 mmol) was heated at 150°C for 47 hrs.
  • the reaction was cooled to room temperature, diluted with ethyl acetate (500 ml), washed with a solution of pH 4.5 (5 x 25 mL) (10% citric acid/10 N sodium hydroxide), 1:1 wate ⁇ sodium bicarbonate solution (20 mL), water (50 mL), brine (30 mL), dried over sodium sulfate, filtered and concentrated .
  • the residue was chromatographed on 300 g silica gel, eluting with 70% ethyl acetate in hexane to give 0.629 g (51%) of the title compound.
  • Step G 4- ⁇ l-Methyl-5-[2-(l-(S)-phenylethylamino)-pyridin-4-yl]-4-[3-
  • This reaction mixture was slowly warmed to RT over 3 hrs, diluted with EtOAc (300 mL), washed with water (5 x 10 mL), brine (20 mL), dried over sodium sulfate, filtered and concentrated to an oil. This oil was chromatographed on 200 g silica gel, eluting with 50% ethyl acetate in hexane to give the title compound 0.235 g (48%).
  • Step 1H 4- ⁇ l-Methyl-5-[2-(l-(S)-phenylethylamino)- ⁇ yridin-4-yl]-4-[3-
  • Step 10A 2-Methanesulfanyl-4-methylpyrimidine
  • Step 10B 2-(2-Methanesulfanylpyrimidine-4-yl)-l-(3- trifluoromethylphenvDethanone
  • Step 10C 2-Bromo-2-(2-methanesulfanylpyrimidine-4-yl)-l-(3- tri fl uoromethylphen yl )-eth an one
  • Step 10D 3-(2-Methylsulfanylpyrimidin-4-yl)-2-(3- trifluoromethylphenyl)imidazo[L2-a]-pyrimidine
  • Step 10E 3-(2-Methylsulfonylpyrimidin-4-yl)-2-(3- trifluoromethylphenyl)imidazori,2-a1-pyrimidine
  • step D The product from step D (2.32g, 5.99mmol), sodium tungstate (200mg, 0.60mmol), 30% hydrogen peroxide (2.7 lmL, 24mmol), ethyl acetate (200mL) and methanol (20mL) were combined under Argon then heated at a gentle reflux for 18h. The contents of the reaction flask were cooled and sodium bisulfite (aq.) was added to quench excess peroxides. Methanol was removed in vacuo. Sodium bicarbonate (aq., sat.) was added and the mixture was extracted with ethyl acetate (3 X lOOmL).
  • Step 10F 2-Amino-5-(2-cyclopropylaminopyrimidin-4-yl)-4-(3- trifluoromethylphenyl) imidazole
  • PBMC Human peripheral blood mononuclear cells
  • the PBMC's are washed two times in Hanks Balanced Salt Solution and then resuspended to a final concentration of 2 x 10 cell/mL in RPMI containing 5% fresh autologous human serum, penicillin streptomycin (10 U/mL) and 0.05% DMSO.
  • Lipopoly- saccharide (Salmonella type Re545; Sigma Chemicals) is added to the cells to a final concentration of 100 ng/mL.
  • An aliquot (0.1 mL) of the cells is quickly dispensed into each well of a 96 well plate containing 0.1 mL of the test compound, at the appropriate dilution, and are incubated for 24 hours at 37°C in 5% CO 2 .
  • cell culture supematants are assayed for IL-l ⁇ , TNF- ⁇ , IL-6 and PGE production using specific ELISA.
  • Human peripheral blood mononuclear cells are isolated from fresh human blood according to the procedure of Chin and Kostura, 7. Immunol. 151, 5574-5585 (1993).
  • Whole blood is collected by sterile venipuncture into 60 mL syringes coated with 1.0 mL of sodium heparin (Upjohn, 1000 U/mL) and diluted 1:1 in Hanks Balanced Salt Solution (Gibco).
  • the erythrocytes are separated from the PBMC's by centrifugation on a Ficoll-Hypaque lymphocyte separation media.
  • the PBMC's are washed three times in Hanks Balanced Salt Solution and then resuspended to a final concentration of 2 x 10 cell/mL in RPMI containing 10% fresh autologous human serum, penicillin streptomycin (10 U/mL) and 0.05% DMSO. Endotoxin free recombinant human IL-l ⁇ is then added to a final concentration of 50 pMolar. An aliquot (0.1 mL) of the cells is quickly dispensed into each well of a 96 well plate containing 0.1 mL of the compound at the appropriate dilution, and are incubated for 24 hours at 37°C in 5% CO 2 . At the end of the culture period, cell culture supematants are assayed for TNF- ⁇ , IL-6 and PGE 2 synthesis using specific ELISA.
  • Human EL-l ⁇ can be detected in cell-culture supematants or whole blood with the following specific trapping ELISA.
  • Ninety-six well plastic plates (Immulon 4; Dynatech) are coated for 12 hours at 4°C with 1 mg/mL protein-A affinity chromatography purified mouse anti-human IL-l ⁇ monoclonal antibody (purchased as an ascites preparation from LAO Enterprise, Gaithersburg Maryland.) diluted in Dulbecco's phosphate -buffered saline (-MgCl2, -CaCl2).
  • IL-l ⁇ standards are prepared from purified recombinant IL-l ⁇ produced from E. coli. The highest concentration begins at 10 ng/mL followed by 11 two-fold serial dilutions. For detection of IL-l ⁇ from cell culture supematants or blood plasma, 10 - 25 mL of supernatant is added to each test well with 75-90 mL of PBS Tween.
  • Peroxidase activity was determined using TMB peroxidase substrate kit (Kirkegaard and Pe ⁇ y) with quantitation of color intensity on a 96-well plate Molecular Devices spectrophotometer set to determine absorbance at 450 nM. Samples are evaluated using a standard curve of absorbance versus concentration. Four-parameter logistics analysis generally is used to fit data and obtain concentrations of unknown compounds.
  • Immulon 4 (Dynatech) 96-well plastic plates are coated with a 0.5 mg/mL solution of mouse anti-human TNF- ⁇ monoclonal antibody.
  • the secondary antibody is a 1:2500 dilution of a rabbit anti-human TNF- ⁇ polyclonal serum purchased from Genzyme. All other operations are identical to those described above for IL-l ⁇ .
  • the standards are prepared in PBS-Tween + 10% FBS or HS. Eleven twofold dilutions are made beginning at 20 ng/mL TNF- ⁇ .
  • ELISA as described previously in Chin and Kostura, 7. Immunol. 151, 5574-5585 (1993). (Dynatech) ELISA plates are coated with mouse anti-human IL-6 monoclonal antibody diluted to 0.5 mg/mL in PBS. The secondary antibody, a rabbit anti-human IL-6 polyclonal antiserum, is diluted 1:5000 with PBS-Tween. All other operations are identical to those described above for IL-lb. The standards are prepared in PBS- Tween + 10% FBS or HS. Eleven twofold dilutions are made beginning at 50 ng/mL IL-6.
  • Prostaglandin E2 is detected in cell culture supematants from LPS or
  • IL-1 stimulated PBMC's using a commercially available enzyme immunoassay .
  • the assay purchased from the Cayman Chemical (Catalogue number 514010) and is run according to the manufacturer's instructions.
  • Interleukin8 (IL-8)
  • the present compounds can also be assayed for IL-8 inhibitory activity as discussed below.
  • Primary human umbilical cord endothelial cells (HUVEC) (Cell Systems, Kirkland, Wa) are maintained in culture medium supplemented with 15% fetal bovine serum and 1% CS-HBGF consisting of aFGF and heparin. The cells are then diluted 20-fold before being plated (250 ⁇ l) into gelatin coated 96-well plates. Prior to use, culture medium is replaced with fresh medium (200 ⁇ l).
  • Buffer or test compound (25 ⁇ l, at appropriate concentrations) is then added to each well in quadruplicate wells and the plates incubated for 6h in a humidified incubator at 37°C in an atmosphere of 5% CO2- At the end of the incubation period, supernatant is removed and assayed for IL-8 concentration using an IL-8 ELISA kit obtained from R&D Systems (Minneapolis, MN). All data is presented as mean value (ng/mL) of multiple samples based on the standard curve. IC50 values where appropriate are generated by non-linear regression analysis.

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Abstract

L'invention concerne des composés représentés par la formule (I) et leurs sels pharmaceutiquement acceptables qui conviennent pour le traitement de pathologies à médiation par la cytokine telles que l'arthrite.
PCT/US2000/026358 1999-09-28 2000-09-25 Imizadoles substitues a activite inhibitrice de la cytokine WO2001022965A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU77154/00A AU7715400A (en) 1999-09-28 2000-09-25 Substituted imidazoles having cytokine inhibitory activity

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US15650999P 1999-09-28 1999-09-28
US60/156,509 1999-09-28

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7612094B2 (en) 2002-04-04 2009-11-03 Biogen Idec Ma Inc. Tri-substituted heteroaryls and methods of making and using the same

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09124640A (ja) * 1995-08-25 1997-05-13 Nippon Soda Co Ltd ピリジルイミダゾール化合物、製法および農園芸用殺菌剤

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09124640A (ja) * 1995-08-25 1997-05-13 Nippon Soda Co Ltd ピリジルイミダゾール化合物、製法および農園芸用殺菌剤

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE CAPLUS [online] CHEM. ABSTR. (COLUMBUS, OHIO, USA); 1997, HAGIWARA ET AL.: "The preparation of pyridylimidazole compounds as agrochemical fungicides", XP002937654, Database accession no. 1997:361716 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7612094B2 (en) 2002-04-04 2009-11-03 Biogen Idec Ma Inc. Tri-substituted heteroaryls and methods of making and using the same

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