Patent Application
20110039848
The present invention relates to a novel 5-membered ring compound or a salt thereof, and a pharmaceutical use thereof. Specifically, the present invention relates to a novel 5-membered ring compound, which is effective for the treatment of various inflammations by binding to a specific binding site of L-threo-3-(3,4-dihydroxyphenyl)-N-[3-(4-fluorophenyl)propyl]serine pyrrolidine amide in vivo and inhibiting infiltration of leukocytes including eosinophils, lymphocytes, etc., or a salt thereof and a pharmaceutical composition comprising the same.
A method for causing immediate asthmatic response (IAR) by administering inhaled allergens to atopic asthma patients has been carried out as an experimental model for dyspnea in bronchial asthma. Specifically, when inhaled allergens have been administered to atopic asthma patients, the patients have had asthmatic response (i.e., bronchial spasm) about 20 minutes after administration and recovered about two hours thereafter. After a continuation of observations of the patients, it has been found that bronchial spasm has been caused again after 6 to 10 hours in about half of the patients who have had the immediate asthmatic response, which has been referred to as Late asthmatic response (LAR). In late asthma, bronchial spasm response has long-continued in association with lung hyperinflation, which has been strongly suppressed by steroid drugs. Therefore, it has been recognized that the bronchial asthma caused by the allergens has been important as a clinical model for dyspnea in steroid-dependent severe bronchial asthma. It has been also recognized that an immediate response has been type I allergy caused as a result of the activation of mast cells by IgE antibody, and a late response has been T lymphocytic and eosinophilic allergy (i.e., eosinophilic inflammations). It has become evident that these immediate and late responses have been also caused by allergic rhinitis and dermatitis. It has been reported that when the late asthmatic response has been caused by allergens in bronchial asthma patients, eosinophils have accumulated in the lungs. Since eosinophilia have been found in blood and expectorated sputum of many bronchial asthma patients, significant numbers of eosinophil infiltration have been found in the lung tissues of patients who have died of asthma, a deposition of major basic protein (MBP) which has been tissue injurious protein derived from eosinophils has been found in bronchial walls and mucus plugs of patients, etc., it has been believed that products derived from eosinophils have played an important role in injuries of airway epithelium associated with late asthma attack.
Steroid drugs have been the only heroic drug against severe bronchial asthma and atopic dermatitis, which such drugs have had strong efficacies as well as adverse effects including hypertension, diabetes, obesity, immune suppression, cataract, mental diseases, atrophia cutis, etc. Although inhaled steroid drugs have been developed for the purpose of the reduction of the systemic adverse effects, concerns about the adverse effects inherent to inhaled steroid drugs have not been dispelled due to the difficulty in proving that the inhaled steroid drugs have not circulated throughout the body. Recently, since the adverse effects of the inhaled steroid drugs have been reported in Europe and the United States, FDA has instructed to weave letters of warning about the risk of the adverse effects into the packaging insertions of the inhaled steroid drugs for the treatment of bronchial asthma and nasal steroid drugs for the treatment of allergic rhinitis.
As mentioned above, infiltration of eosinophils into involved sites has played an important role in development and degradation of the late response of bronchial asthma as well as allergic dermatitis and rhinitis. However, a heroic drug for the treatment of allergy diseases including bronchial asthma by inhibiting infiltration and activation of eosinophils has been steroid drugs only, and orally-available anti-inflammatory drugs with fewer adverse effects which may be substituted for the steroid drugs have been desired in medical practice. For example, anti IL-5 neutralizing antibody which has been an antibody that neutralizes interleukin 5 causing proliferation and differentiation of eosinophil precursors and extension of survival of mature eosinophils, low-molecular inhibitors of eosinophil-specific adhesion factor Very Late Antigen 4 (VLA-4), and low-molecular antagonists to eosinophil-specific chemokine eotaxin receptor CCR3 causing eosinophil migration have been considered for trying to develop any drugs suppressing eosinophilic inflammations, but these have not been an alternative for steroid drugs.
On the other hand, it has been known that L-threo-3-(3,4-dihydroxyphenyl)-N-[3-(4-fluorophenyl)propyl]serine pyrrolidine amide has an inhibitory effect on eosinophil migration (Patent Document 1). A specific binding site of L-threo-3-(3,4-dihydroxyphenyl)-N-[3-(4-fluorophenyl)propyl]serine pyrrolidine amide in vivo has been a receptor-like membrane protein, which has been also referred to as SMBS protein (SMBP) (Patent Document 1).
Accordingly, it is possible to treat allergy diseases including asthma, chronic obstructive pulmonary diseases when eosinophil migration may be inhibited by binding to the SMBS protein.
It has been known that some low-molecular compounds have been useful as a therapeutic agent for allergy diseases including asthma by binding to the SMBS protein (Patent Documents 2, 3, 4).
[Patent Document 1] WO 98/26065 pamphlet
[Patent Document 2] WO 02/002542 pamphlet
[Patent Document 3] WO 2003/057693 pamphlet
[Patent Document 4] JP-A-2005-206515
The problem to be resolved by the present invention is to provide an effective compound as a therapeutic agent for various inflammations by inhibiting infiltration of leukocytes including eosinophils, lymphocytes, etc., which may be high safety in long-term administration, and a medicament comprising the same.
Since it has been believed that drugs for treating asthma, etc. are administered for long periods, the use of high safety drugs has been especially desired. It has been shown that compounds disclosed in Patent Document 2 have inhibitory effects on rat steroid hormone synthesis in significantly high compound concentrations.
In order to solve the problem, the present inventors have found compounds which are effective as a therapeutic agent for various inflammations by inhibiting infiltration of leukocytes including eosinophils, lymphocytes, etc. via binding to SMBS and significantly reduce inhibitory effects on the steroid hormone synthesis by screening using pharmacological tests and assessment tests for rat steroid hormone synthesis inhibition disclosed in Patent Document 2 in compounds with a structural feature that pyrazinyl, pyrimidinyl or pyridazinyl which is substituted by morpholino or dialkylamino is attached on 4-position of thiazolinone, and have achieved the present invention.
The present invention of the present application is as follows:
[1] A 5-membered ring compound of formula (I):
wherein R1 is phenyl optionally substituted by a halogen atom, C1-C3 alkyl, C1-C3 alkoxy or trifluoromethyl; or pyridyl optionally substituted by a halogen atom, C1-C3 alkyl, C1-C3 alkoxy or trifluoromethyl;
R2 is pyrazinediyl optionally substituted by a halogen atom or C1-C3 alkyl; pyrimidinediyl optionally substituted by a halogen atom or C1-C3 alkyl; or pyridazinediyl optionally substituted by a halogen atom or C1-C3 alkyl;
R3 is C1-C3 alkyl;
R4 and R5 are each independently C1-C3 alkyl; or —N(R4)R5 may be morpholino;
Y2 is C2-C4 alkylene; or a pharmaceutically acceptable salt thereof.
[2] The 5-membered ring compound of [1], wherein R2 is pyridazinediyl optionally substituted by a halogen atom or C1-C3 alkyl, or a pharmaceutically acceptable salt thereof.
[3] The 5-membered ring compound of [1], wherein R2 is pyridazinediyl, or a pharmaceutically acceptable salt thereof.
[4] The 5-membered ring compound of [1], wherein —N(R4)R5 is morpholino, or a pharmaceutically acceptable salt thereof.
[5] The 5-membered ring compound of [1], wherein R1 is phenyl optionally substituted by a halogen atom, or a pharmaceutically acceptable salt thereof.
[6] The 5-membered ring compound of [1] to [5], wherein R3 is methyl or ethyl, and Y2 is C2-C3 alkylene, or a pharmaceutically acceptable salt thereof.
[7] The 5-membered ring compound of any one of [1] to [6], wherein the wavy line represents (Z)-coordination, or a pharmaceutically acceptable salt thereof.
[8] A therapeutic agent for inflammations, comprising the 5-membered ring compound of any one of [1] to [7] or a pharmaceutically acceptable salt thereof.
[9] A therapeutic agent for autoimmune inflammations or allergic inflammations, comprising the 5-membered ring compound of any one of [1] to [7] or a pharmaceutically acceptable salt thereof.
[10] A therapeutic agent for chronic obstructive pulmonary diseases, comprising the 5-membered ring compound of any one of [1] to [7] or a pharmaceutically acceptable salt thereof.
[11] A therapeutic agent for bronchial asthma, comprising the 5-membered ring compound of any one of [1] to [7] or a pharmaceutically acceptable salt thereof.
[12] A therapeutic agent for rhinitis, comprising the 5-membered ring compound of any one of [1] to [7] or a pharmaceutically acceptable salt thereof.
[13] A method for treating inflammations, comprising administering the 5-membered ring compound of any one of [1] to [7] or a pharmaceutically acceptable salt thereof to a patient in need thereof.
[14] Use of the 5-membered ring compound of any one of [1] to [7] or a pharmaceutically acceptable salt thereof in the manufacture of a therapeutic agent for inflammations.
The 5-membered ring compound or a salt thereof of the present invention inhibits infiltration of leukocytes including eosinophils, lymphocytes, etc., and therefore, it has been possible to provide a therapeutic agent for various inflammations.
Each substituent has the following meaning throughout the specification.
The “C1-C3 alkyl” includes straight- or branched-chain C1-C3 alkyl, particularly methyl, ethyl, n-propyl, 2-propyl. A preferable one includes methyl, ethyl, more preferably methyl.
The “C1-C3 alkoxy” includes straight- or branched-chain C1-C3 alkoxy, particularly methoxy, ethoxy, n-propoxy, 2-propoxy. A preferable one includes methoxy, ethoxy, more preferably methoxy.
The “halogen atom” includes fluorine, chlorine, bromine, iodine, preferably fluorine, chlorine, bromine, more preferably fluorine, chlorine.
The “C2-C4 alkylene” includes straight- or branched-chain C2-C4 alkylene, particularly ethylene, trimethylene, tetramethylene, methylethylene, 2-methyltrimethylene, etc. A preferable one includes ethylene, trimethylene.
When R1 is substituted by substituents, the substituents of R1 are the same or different and the number of the substituents includes 1 to 3.
The 5-membered ring compound of formula (I) of the present invention may be a pharmaceutically acceptable salt. The pharmaceutically acceptable salt includes an acid addition salt and a base addition salt. The acid addition salt includes an inorganic acid salt such as hydrochloride, hydrobromide, sulfate, and an organic acid salt such as citrate, oxalate, malate, tartrate, fumarate, maleate, etc.
The compound encompassed in the present invention may have asymmetric centers or any substituents with asymmetric carbons, and may have any optical isomers and geometric isomers. The present invention includes a mixture of each isomer and an isolated one. The present invention also includes a solvate including hydrate of the 5-membered ring compound or a pharmaceutically acceptable salt thereof.
The 5-membered ring compound of formula (I) of the present invention may be prepared according to the following methods and modifications thereof.
Compound (I) is prepared by the following method.
[In the formula, R1, R2, R3, R4, R5 and Y2 have the same meanings as defined above. X1 is a halogen atom such as chlorine, bromine.]
Thiourea compound (3) is reacted with α-haloketone compound (4) in a solvent in the presence or absence of a base to give compound (I). The solvent includes an alcohol solvent such as methanol, ethanol, 2-propanol, an ether solvent such as diethylether, tetrahydrofuran (THF), a halogenated hydrocarbon solvent such as dichloromethane, dichloroethane, chloroform, an aprotic solvent such as dimethylformamide, an aromatic solvent such as toluene, etc. The base includes an organic amine such as triethylamine, pyridine, 4-dimethylaminopyridine, an inorganic base such as potassium carbonate, sodium carbonate, etc. The reaction temperature is selected in the range of room temperature to a boiling point of a solvent.
Compound (I) is also prepared according to the following method. The preparation is useful when a protecting group is needed in the introduction of R3. A conventional protecting group of amino group may be used as the protecting group, and the following preparations are illustrated by using 2-methyl-2-propyloxycarbonyl, which is referred to as Boc hereinafter, as the protecting group.
[In formula, R1, R2, R3, R4, R5, X1 and Y2 have the same meanings as defined above.]
Thiourea compound (5) is reacted with α-haloketone compound (4) in a solvent in the presence or absence of a base to give compound (6). The solvent includes an alcohol solvent such as methanol, ethanol, 2-propanol, an ether solvent such as diethylether, THF, a halogenated hydrocarbon solvent such as dichloromethane, dichloroethane, chloroform, an aprotic solvent such as dimethylformamide, an aromatic solvent such as toluene, etc. The base includes an organic amine such as triethylamine, pyridine, 4-dimethylaminopyridine, and an inorganic base such as potassium carbonate, sodium carbonate. The reaction temperature is selected in the range of room temperature to a boiling point of a solvent.
Then, compound (6) is deprotected in the presence of an acid in a solvent to give compound (7). The acid includes an inorganic acid such as hydrochloric acid, and an organic acid such as trifluoroacetic acid. The solvent includes an ether solvent such as diethylether, THF, dioxane, a halogenated hydrocarbon solvent such as dichloromethane, dichloroethane, chloroform, etc. The reaction temperature is selected in the range of about 0° C. to a boiling point of a solvent.
Compound (7) is reacted with a compound such as a corresponding isocyanate or carbamic acid ester in the presence or absence of a base in a solvent to give compound (I). A concrete example of the compound includes alkyl isocyanate, ethyl alkyl carbamate, etc. The solvent includes an ether solvent such as diethylether, THF, a halogenated hydrocarbon solvent such as dichloromethane, dichloroethane, chloroform, an aprotic solvent such as dimethylformamide, an aromatic solvent such as toluene, etc. The base includes an organic amine such as triethylamine, pyridine. 4-dimethylaminopyridine, an inorganic base such as potassium carbonate, sodium carbonate, etc. The reaction temperature is selected in the range of room temperature to a boiling point of a solvent.
Compound (I) is also prepared according to the following method.
[In formula, R1, R2, R3, R4, R5 and Y2 have the same meanings as defined above. R10 is alkyl, or phenyl substituted by a halogen atom or nitro.]
Compound (7) is reacted with chloroformate (e.g., R10OCOCl wherein R10 is the same as defined above) in a solvent in the presence or absence of a base to give compound (8). The solvent includes an ether solvent such as diethylether, THF, a halogenated hydrocarbon solvent such as dichloromethane, dichloroethane, chloroform, an aprotic solvent such as dimethylformamide, an aromatic solvent such as toluene, etc. The base includes an organic amine such as triethylamine, pyridine, 4-dimethylaminopyridine, and an inorganic base such as potassium carbonate, sodium carbonate. The reaction temperature is selected in the range of room temperature to a boiling point of a solvent.
Compound (8) is reacted with an amine of R3NH2 in a solvent in the presence or absence of a base to give compound (I). The solvent includes an ether solvent such as diethylether, THF, dioxane, a halogenated hydrocarbon solvent such as dichloromethane, dichloroethane, chloroform, etc. The base includes the base as described above. The reaction temperature is selected in the range of about 0° C. to a boiling point of a solvent.
Starting materials used in the above Preparations 1 to 3 are prepared according to the following methods.
[In formula, R1, R3, R10 and Y2 have the same meanings as defined above.]
Amine compound (12) may be reacted with isocyanate compound (13) or dithiocarbamic acid ester (14) in a solvent to give thiourea compound (3). The solvent includes an alcohol solvent such as methanol, ethanol, 2-propanol, an ether solvent such as diethylether, THF, a halogenated hydrocarbon solvent such as dichloromethane, dichloroethane, chloroform, an aprotic solvent such as dimethylformamide, an aromatic solvent such as toluene, etc. The reaction temperature is selected in the range of room temperature to a boiling point of a solvent.
[In formula, R1, R3, R10 and Y2 have the same meanings as defined above.]
Amine compound (12) may be reacted with isocyanate compound (13) or dithiocarbamic acid ester (14) in a solvent to give thiourea compound (3). The solvent includes an alcohol solvent such as methanol, ethanol, 2-propanol, an ether solvent such as diethylether, THF, a halogenated hydrocarbon solvent such as dichloromethane, dichloroethane, chloroform, an aprotic solvent such as dimethylformamide, an aromatic solvent such as toluene, etc. The reaction temperature is selected in the range of room temperature to a boiling point of a solvent.
Thiourea compound (5) which is protected by 2-methyl-2-propyloxycarbonyl, etc. is obtained in the following method.
[In formula, R1, R10, Y2 and Boc have the same meanings as defined above.]
Amine compound (15) may be reacted with isocyanate compound (10) or dithiocarbamic acid ester (11) in a solvent to give thiourea compound (5). The solvent includes an alcohol solvent such as methanol, ethanol, 2-propanol, an ether solvent such as diethylether, THF, a halogenated hydrocarbon solvent such as dichloromethane, dichloroethane, chloroform, an aprotic solvent such as dimethylformamide, an aromatic solvent such as toluene, etc. The reaction temperature is selected in the range of room temperature to a boiling point of a solvent.
[In formula, R1, R10, Y2 and Boc have the same meanings as defined above.]
Amine compound (12) may be reacted with isocyanate compound (16) or dithiocarbamic acid ester (17) in a solvent to give thiourea compound (5). The solvent includes an alcohol solvent such as methanol, ethanol, 2-propanol, an ether solvent such as diethylether, THF, a halogenated hydrocarbon solvent such as dichloromethane, dichloroethane, chloroform, an aprotic solvent such as dimethylformamide, an aromatic solvent such as toluene, etc. The reaction temperature is selected in the range of room temperature to a boiling point of a solvent.
Isothiocyanate compounds (10), (13) and (16) are commercially available, or may be synthesized from corresponding amino compounds according to the methods of Synlett. 1997, 773-774, J. Org. Chem., 1997, 62, 4539-4540, or J. Med. Chem., 1984, 27, 1570-1574, for example, and may be also synthesized from corresponding carboxylic acid compounds according to the methods of Synth. Commun. 1997, 27, 751-756, or Indian, J. Chem., 1998, 1153-1156, for example.
Dithiocarbamic acid ester compounds (11), (14) and (17) are commercially available, or may be synthesized from corresponding amino compounds according to the methods of J. Chem. Soc. 1956, 1644-1649, or Syn. Commun. 1984, 537-546, for example.
α-Haloketone compound (4) is commercially available, or may be prepared according to the method of Bioorganic & Medicinal Chemistry, 2005, 13, 3705, for example.
The 5-membered ring compound (I) or an intermediate for preparing the same of the present invention may be purified in a conventional manner. The 5-membered ring compound (I) or an intermediate for preparing the same of the present invention with several isomers may be also purified in a similar manner. For example, the compound may be purified by column chromatography, recrystallization, etc. A solvent for recrystallization includes an alcoholic solvent such as methanol, ethanol, 2-propanol, an ether solvent such as diethylether, an ester solvent such as ethyl acetate, an aromatic hydrocarbon solvent such as toluene, a ketone solvent such as acetone, a hydrocarbon solvent such as hexane, or a mixed solvent thereof, etc.
A method for obtaining a pure optical isomer includes an optical resolution method. The optical resolution method includes a method for treating the present compound or an intermediate thereof which has a basic substituent such as amino group with an optically active acid (e.g., monocarboxylic acids such as mandelic acid, N-benzyloxyalanine, lactic acid, dicarboxylic acids such as tartaric acid, O-diisopropylidenetartaric acid, malic acid, sulfonic acids such as camphersulfonic acid, bromocamphersulfonic acid) in an inactive solvent (e.g., an alcoholic solvent such as methanol, ethanol, 2-propanol, an ether solvent such as diethylether, an ester solvent such as ethyl acetate, an aromatic hydrocarbon solvent such as toluene, acetonitrile, and a mixed solvent thereof) to form a salt. A method for treating the present compound or an intermediate thereof which has an acidic substituent such as carboxyl group with an optically active amine (e.g., organic amines such as α-phenethylamine, kinin, quinidine, cinchonidine, cinchonine, strychnine) to form a salt may be adopted. The reaction temperature in forming a salt in the optical resolution method includes the range of room temperature to a boiling point of a solvent. In order to improve the optical purity, the reaction temperature is preferably raised to around a boiling point of a solvent once. The yields may be optionally improved by cooling before the filtration of the precipitated salt, if necessary. The usage of the optically active acid or amine is appropriately in the range of about 0.5 to about 2.0 equivalents, preferably around 1 equivalent, to the substrate. The resulting crystal may be optionally recrystallized in an inactive solvent (e.g., an alcoholic solvent such as methanol, ethanol, 2-propanol, an ether solvent such as diethylether, an ester solvent such as ethyl acetate, an aromatic hydrocarbon solvent such as toluene, acetonitrile, and a mixed solvent thereof) to give an optically active salt in high yields. The resulting salt may be also optionally treated with an acid or a base in a conventional manner to give a free form.
The 5-membered ring compound of formula (I) or a salt thereof of the present invention is useful as a medicament, especially with an inhibitory effect on infiltration of leukocytes including eosinophils, lymphocytes, etc. Due to the effect, the present invention is useful as a therapeutic agent for autoimmune inflammations, allergic inflammations, acute inflammations, other cell infiltrative inflammatory diseases, etc. The autoimmune inflammations include rheumatism, multiple sclerosis, inflammatory bowel diseases, type I diabetes, etc. The allergic inflammations include bronchial asthma, inflammatory bowel diseases, allergic rhinitis, atopic dermatitis, hives, allergic conjunctivitis, etc. The present 5-membered ring compound is especially useful for late asthma in bronchial asthma. The acute inflammations include inflammatory lung diseases, etc. The other inflammatory diseases include eosinophilia, eosinophilic angiitis, eosinophilic granuloma, transplantation rejection, tumor metastasis, etc. The present compound used as anti-inflammatory drugs may be administered in combination with steroid drugs as a therapeutic agent for inflammatory diseases, and the combination use may enhance the therapeutic effects of the present compound and allow for the reduction or elimination of steroid drugs with strong adverse effects. The present compound used as a therapeutic agent for allergy diseases may be administered in combination with an antiallergic agent (including an inhibitor of liberation of chemical messengers, antihistamine agent, antileukotriene agent, antithromboxane agent, etc.), and in bronchial asthma, a bronchodilator agent (including xanthine preparation including theophylline, β-stimulant), anticholinergic agents, steroid drugs. The present compound used as a therapeutic agent for autoimmune diseases including rheumatism may be administered in combination with nonsteroidal anti-inflammatory drugs including cyclooxygenase (COX) inhibitors.
The present 5-membered ring compound or a salt thereof may be administered orally or parenterally. The oral administration may be carried out in the conventional dosage form. The parenteral administration may be carried out in the form of topical administration, injection, transdermal administration, transnasal administration, etc. A preparation for oral or rectal administration includes a capsule, a tablet, a pill, a powder, a cachet, a suppository, a solution, etc. The injection includes a sterile solution or a suspension, etc. The topical preparation includes a cream, an ointment, a lotion, a transdermal preparation (including a conventional patch, matrix), etc.
The above dosage form is formulated using a pharmaceutically acceptable excipient and additive in a conventional manner. The pharmaceutically acceptable excipient and additive include a carrier, a binder, a perfume, a buffer, a thickener, a colorant, a stabilizer, an emulsifier, a dispersant, a suspending agent, a preservative agent, etc.
The pharmaceutically acceptable carrier includes magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethyl cellulose, low-melting wax, cocoa butter, etc.
The capsule may be formulated by setting the present compound in a capsule together with a pharmaceutically acceptable carrier. The present compound may be set in a capsule by mixing with a pharmaceutically acceptable excipient, or without any excipient. The cachet may be also formulated in a similar manner.
The powder is formulated together with a pharmaceutically acceptable powder base. The base includes talc, lactose, starch, etc. The drop may be formulated together with aqueous or nonaqueous base and one or more of a pharmaceutically acceptable diffusing agent, suspending agent, solubilizer, etc.
The injectable solution includes a solution, a suspension, an emulsion, for example aqueous solution, water-propylene glycol solution, etc. The solution may contain water, and may be also prepared in the form of a solution of polyethylene glycol or/and propylene glycol. The appropriate solution for oral administration may be prepared by adding the present compound to water together with a colorant, a perfume, a stabilizing agent, a sweetening agent, a solubilizer, a thickener, etc., if necessary. The appropriate solution for oral administration may be also prepared by adding the present compound to water together with a dispersant to thicken. The thickener includes a pharmaceutically acceptable natural or synthetic gum, resin, methylcellulose, sodium carboxymethyl cellulose, or known suspending agent.
The topical formulation includes the solution, and a cream, an aerosol, a spray, a powder, a lotion, an ointment, etc. The topical formulation may be prepared by mixing the present compound with a conventional pharmaceutically acceptable diluent and carrier. The ointment and cream may be formulated by adding a thickener and/or a gelator to an aqueous or oily base. The base includes water, liquid paraffin, vegetable oil (including peanut oil, castor oil). The thickener includes soft paraffin, aluminum stearate, cetostearyl alcohol, propyleneglycol, polyethylene glycol, lanolin, hydrogenated lanolin, bees wax, etc.
For the lotion, one or more of a pharmaceutically acceptable stabilizer, suspending agent, emulsifier, diffusing agent, thickener, colorant, perfume, etc. may be added to an aqueous or oily base.
The topical formulation may optionally contain an antiseptic, a cell-proliferation preventive agent including methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chloride.
The present compound may be also nasally administered in the form of a solution spray, a powder or a drop formulation.
The dosage amounts and the number of doses may vary depending on conditions, ages, weights, dosage forms, and may be orally administered in the range of about 1 to about 1000 mg, preferably about 2 to about 500 mg, particularly preferably about 5 to about 100 mg, once or in several divided doses per day to an adult. The administration by injection may be in the range of about 0.1 to about 300 mg, preferably about 1 to about 200 mg, once or in several divided doses.
The present invention is illustrated with Examples more specifically, but is not limited thereto.
To a solution of 3,6-dichloropyridazine (1.49 g) and tetrakis(triphenylphosphine)palladium (115 mg) in toluene (50 ml) was added (1-ethoxyvinyl)tributyltin (4.33 g), and the mixture was heated to reflux under nitrogen for 5 hours. To the reaction mixture was added 1N hydrochloric acid (20 ml), and the mixture was stirred at 60° C. for 1 hour, then extracted with ethyl acetate. The organic layer was dried over sodium sulfate, and then the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography [hexane:ethyl acetate=4:1] to give the titled compound (653 mg).
1H-NMR (CDCl3): δ 2.88 (3H, s), 7.67 (1H, d, J=8.8 Hz), 8.10 (1H, d, J=8.8 Hz).
To 3-acetyl-6-chloropyridazine obtained in Reference Example 1 was added morpholine (652 mg), and the mixture was stirred at 110° C. for 6 hours. The reaction mixture was concentrated under reduced pressure and azeotroped with toluene twice. Then, thereto was added water, and the mixture was extracted with ethyl acetate. The organic layer was dried over sodium sulfate, and then the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography [hexane:ethyl acetate=2:1]. The solvent was evaporated under reduced pressure. To the residue was added ethanol (8 ml) at 70° C., and the precipitated crystal was filtered to give the titled compound (432 mg).
1H-NMR (CDCl3): δ 2.78 (3H, s), 3.79-3.81 (4H, m), 3.85-3.87 (4H, m), 6.90 (1H, d, J=9.6 Hz), 7.92 (1H, d, J=9.6 Hz).
To a 48% aqueous hydrogen bromide solution (4 ml) containing 4-(3-acetylpyridazin-6-yl)morpholine (249 mg) obtained in Reference Example 2 was added dropwise bromine (182 mg)-48% aqueous hydrogen bromide solution (0.5 ml) at room temperature, and the mixture was stirred at 45° C. for 1.5 hours. The reaction solution was poured into aqueous saturated sodium bicarbonate solution to be neutralized, and extracted with chloroform. The organic layer was dried over sodium sulfate, and the solvent was evaporated under reduced pressure. Then, thereto were added N-[2-({[(3-fluorophenyl)amino]carbonothioyl}amino)ethyl]-N′-methylurea (260 mg) and ethanol (6 ml), and the mixture was stirred at 70° C. for 2 hours, then stirred at room temperature overnight. The mixture was neutralized by aqueous saturated sodium bicarbonate solution, and extracted with 10% chloroform-methanol solution. The organic layer was dried over sodium sulfate; and then the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography [3% chloroform-methanol] to give the titled compound (356 mg).
MS (ESI-MS): 458 [M+1]+, rt=2.6 min.
1H-NMR (CDCl3): δ 2.76 (3H, d, J=4.6 Hz), 3.64 (2H, bs), 3.70-3.72 (4H, m), 3.87-3.89 (4H, m), 4.24 (2H, t, J=6.7 Hz), 6.07 (1h, s), 6.76-6.83 (1H, m), 6.86-6.89 (1H, m), 6.97 (1H, d, J=9.5 Hz), 7.30 (1H, m), 7.43 (1H, d, J=9.5 Hz).
The acetophenone obtained in Reference Example 2 was reacted with various thioureas in a similar manner to Example 1 to give compounds shown in Table 1.
MS detector Perkin-Elmer Sciex API 150EX Mass spectrometer, HPLC Shimadzu LC 8A, Column YMC CombiScreen ODS-A-300-CC 4.6 mm×50 mm, Gradient condition; A: 0.35% TFA/CH3CN, B: 0.05% TFA/H2O, 0.0-0.5 min A 10% B 90% 0.5-4.2 min Linear gradient from A 10% B 90% to A 99% B 1% 4.2-6.3 min A 99% B 1%.
To a solution of 3-amino-5-chloropyridine (51 mg) in tetrahydrofuran (2 ml) were added triethylamine (277 μl) and thiophosgene (46 μl) under ice-cooling, and the mixture was stirred at room temperature for 50 minutes. Thereto was added N-(2-amino)ethyl-N′-methylurea methanesulfonate (231 mg), and the mixture was stirred at room temperature for 1 hour. To the reaction mixture was added aqueous saturated sodium bicarbonate solution, and the mixture was extracted with ethyl acetate. The organic layer was dried over sodium sulfate, and then the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography [3% chloroform-methanol+1% triethylamine] to give the titled compound (88 mg).
1H-NMR (CDCl3): δ 2.75 (3H, d, J=4.9 Hz), 3.41-3.45 (2H, m), 3.73 (2H, m), 4.57-4.48 (1H, m), 5.0 (1H, brs), 8.13 (1H, brs), 8.37-8.41 (2H, m).
The acetophenone (16 mg) obtained in Reference Example 2 was reacted with N-[2-({[(3-chloropyridin-5-yl)amino]carbonothioyl}amino)ethyl]-N′-methylurea (16 mg) obtained in Reference Example 3 to give the titled compound (9.5 mg) in a similar manner to Example 1.
MS (ESI-MS): 475 [M+1]+, rt=2.0 min.
The acetophenone obtained in Reference Example 2 was reacted with various thioureas in a similar manner to Example 13 to give compounds shown in Table 2.
Analytical conditions in the above Table: MS detector Waters micromass ZQ, HPLC waters 2790 separations module, Column Impact Cadenza CD-C18 2.0 mm×20 mm, Gradient condition; A: H2O, B: CH3CN, C: 2% HCOOH/CH3CN, 0.0-0.1 min A 95% B 2% C3% 0.1-3.1 min Linear gradient from A 95% B 2% C 3% to A 1% B 96% C 3% 3.1-3.5 min A 1% B 96% C 3%.
The 3-acetyl-6-chloropyridazine obtained in Reference Example 1 was reacted with diethylamine in a similar manner to Reference Example 2 to give the titled compound.
1H-NMR (CDCl3): δ 1.27 (6H, t, J=7.1 Hz), 2.77 (3H, s), 3.66-3.71 (4H, q, J=7.1 Hz), 6.75 (1H, d, J=9.6 Hz), 7.85 (1H, d, J=9.6 Hz).
The 3-acetyl-6-N,N-diethylaminopyridazine obtained in Reference Example 4 was reacted with various thioureas in a similar manner to Example 1 to give compounds shown in Table 3.
MS detector Perkin-Elmer Sciex API 150EX Mass spectrometer, HPLC Shimadzu LC 8A, Column YMC CombiScreen ODS-A-300-CC 4.6 mm×50 mm, Gradient condition; A: 0.35% TFA/CH3CN, B: 0.05% TFA/H2O, 0.0-0.5 min A 10% B 90% 0.5-4.2 min Linear gradient from A 10° A, B 90% to A 99% B 1% 4.2-6.3 min A 99% B 1%.
To 3,6-dichloro-4-methylpyridazine (815 mg) was added morpholine (5 ml), and the mixture was stirred at 50 to 60° C. for 6 hours. The reaction mixture was cooled to room temperature, and thereto was added aqueous saturated sodium bicarbonate solution (10 ml). The mixture was extracted with ethyl acetate. The organic layer was washed with water, then saturated saline, dried over sodium sulfate, and then the solvent was evaporated under reduced pressure. To the residue was added ethanol at 50° C., and the mixture was cooled to room temperature and stirred overnight. The precipitated crystal was filtered to give the titled compound (570 mg).
1H-NMR (CDCl3): δ 2.32 (3H, s), 3.58-3.60 (4H, m), 3.82-3.84 (4H, m), 6.77 (1H, s).
To a solution of 4-(3-chloro-4-methylpyridazin-6-yl)morpholine (570 mg) obtained in Reference Example 5 and tetrakis(triphenylphosphine)palladium (309 mg) in toluene (14 ml) was added (1-ethoxyvinyl)tributyltin (1.94 g), and the mixture was stirred under nitrogen at 110° C. for 9.5 hours. The reaction mixture was filtered through Celite, and then the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography [hexane:ethyl acetate=1:1] to give the titled compound (476 mg).
1H-NMR (CDCl3): δ 2.55 (3H, s), 2.77 (3H, s), 3.75-3.79 (4H, m), 3.83-3.86 (4H, m), 6.61 (1H, s).
The 4-(3-acetyl-4-methylpyridazin-6-yl)morpholine obtained in Reference Example 6 was reacted with various thioureas in a similar manner to Example 1 to give compounds shown in Table 4.
MS detector Perkin-Elmer Sciex API 150EX Mass spectrometer, HPLC Shimadzu LC 8A, Column YMC CombiScreen ODS-A-300-CC 4.6 mm×50 mm, Gradient condition; A: 0.35% TFA/CH3CN, B: 0.05% TFA/H2O, 0.0-0.5 min A 10% B 90% 0.5-4.2 min Linear gradient from A 10% B 90% to A 99% B 1% 4.2-6.3 min A 99% B 1%.
The assessment test was carried out according to the method of Sugasawa, T. et al., J. Biol. Chem., 272, 21244-21252 (1997).
Lung taken out from SD male rat (7-week old under test, Charles River Laboratories Japan, Inc.) was removed trachea and blood vessels to shred and washed with iced tris-saline buffer (10 mM tris hydrochloric acid-154 mM sodium chloride, pH7.4). The resultant was homogenized by Hiscotron with ice-cold tris-saline buffer containing a homogenization buffer (1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), 5 μg/ml aprotinin, 5 μg/ml leupeptine) (maximum rate: 1 minute). The supernatant after low-speed centrifugation (1500×g, 20 minutes, 4° C.) was ultracentrifuged (100000×g, 20 minutes, 4° C.), and the pellet was suspended in tris-saline buffer to store at −80° C. The protein concentrations were determined by Bio-Rad Protein Assay Kit using bovine serum albumin (BSA) as a standard.
To each well in protein-nonabsorptive round-bottom 96-well assay plate (commercially available from Iwaki Glass Co., Ltd.) were added tris-saline buffer (200 μl) containing 1 nM [125I]-iodocyanopindolol (commercially available from Amersham plc), 10 μM serotonine, 20 μm dl-propranolol, 10 μM phentolamine, 1.1 mM ascorbic acid and 100 μg lung membrane, and the mixture was pipetted to combine and then incubated at 37° C. for 30 minutes. The test compounds were dissolved in 100% dimethyl sulfoxide solution, and thereto was added 2 μl (final DMSO concentration: 1%). In order to calculate nonspecific binding amounts, to the mixture was added L-threo-3-(3,4-dihydroxyphenyl)-N-[3-(4-fluorophenyl)propyl]serine pyrrolidine amide at 100 μM of the final concentration as an alternative to the test compounds. During this time, 100 μl of 0.3% polyethyleneimine (PEI)/tris-saline buffer was added to MultiScreen plate (96-well B glass fiber, Millipore Cat. No. MAFB NOB10), and incubated for over 30 minutes. The resultant was filtered with aspiration (aspiration by the addition of 200 μl iced tris-saline buffer) to wash, and the reaction solutions on 96-well assay plate were filtered with aspiration to wash four times on MultiScreen plate. B glass fiber filter paper at the bottom of MultiScreen plate was punched out, and γ-ray doses of [125I]-iodocyanopindolol trapped on the filter paper were determined as binding amounts. The binding amounts in the presence of DMSO (final concentration: 1%) were total binding amounts, and the binding amounts in the presence of 100 μM L-threo-3-(3,4-dihydroxyphenyl)-N-[3-(4-fluorophenyl)propyl]serine pyrrolidine amide were nonspecific binding amounts. The values subtracted the nonspecific binding amounts from the total binding amounts were the specific binding amounts. The binding activity of a compound was calculated according to the following formula, which was shown in the ratio of inhibiting the specific binding of [125I]-iodocyanopindolol to rat lung membrane SMBS by the test compound.
Collagenase from Clostridium histolyticum (Sigma-Aldrich)
Dimethylsulfoxide (referred to as DMSO hereinafter) (nacalai tesque)
Fetal Bovine Serums (referred to as FBS hereinafter) (Bioflud)
Multiple well plates for adhesive cell culture, 96 wells (IWAKI)
N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (referred to as cAMP hereinafter) (Sigma)
Control examples 1 to 4: Control example 1 was Example 350 of Patent Document 2. Control examples 2 to 4 were synthesized according to Patent Document 2.
Adrenal glands were taken out from rats, and removed excess connective tissues, adiposes and membranes in D-MEM. The adrenal glands were transferred to polytubes, and quickly segmentalized with scissors. Thereto was added D-MEM containing 1 mg/mL collagenase and 0.25 μL DNase I, and then the mixture was shaken at 37° C. for 10 minutes in 80 times/minute. Then, the mixture was pipetted by 1 mL truncated pipette tip, and shaken again under the same condition. After 10 minutes, the mixture was pipetted again by 1 mL pipette tip, and the cell suspension was filtered through metal mesh. The filtrate was centrifuged at 4° C. for 5 minutes in 500 g. The supernatant was removed, and thereto was added a mixed solution of 0.16 M NH4Cl and 0.17 M Tris-HCl in 3 mL. The mixture was let stand on ice for 5 minutes, and then centrifuged under the same condition. Then, the supernatant was removed and then thereto was added D-MEM. The cell suspension was prepared and centrifuged under the same condition. Again, the supernatant was removed and then thereto was added D-MEM to give a cell suspension. The cell suspension was filtered by Cell Strainer (BD Falcon®). The filtrate was centrifuged under the same condition. After the completion of the centrifugation, the supernatant was removed, and thereto was added D-MEM containing 1% Penicillin Streptomycin Mixed Solution and 10% FBS. The mixture was seeded in Multiple well plate for adhesive cell culture, 96 wells (IWAKI) in 10000 cells/well, and incubated at 37° C. overnight in CO2 incubator.
Test article concentrations were prepared in DMSO to 2000 μM, and D-MEM containing 20 μM test article in D-MEM was prepared.
Cell-seeded plate was incubated overnight, and then the medium was removed and thereto was added D-MEM containing 20 μM test article in 50 μL/well. Then, to the mixture was promptly added D-MEM containing 20 μM cAMP in 50 μL/well so that the final concentration of the test article was 10 μM. After the addition of 2 types of D-MEM, the mixture was incubated at 37° C. for 2 hours in CO2 incubator. After 2 hours, the supernatant (50 μL) of the medium was collected. For cells, the cell toxicity of the test article was assessed according to a conventional method using Cell Titer-Glo® (Promega). For the collected supernatant, the Corticosterone concentration in the supernatant was determined according to a conventional method using Corticosterone EIA kit (Cayman).
The inhibition rate of 10 μM test article to Corticosterone generation was calculated according to the following formula confirming that the cell toxicity by the test article was not recognized.
The results of the above Tests 1 and 2 are shown in Table 5.
Binding Activities (Effects) and Steroid Synthesis Inhibition Rates (by-Effects) of Example Compounds
The assessment was carried out using a compound obtained in Example 1.
Hartley male guinea pigs (commercially available from Japan SLC, Inc.) were pre-bred about 1 week after arrival, and then exposed by inhalation to 2% (w/v) ovalbumin (OA) saline in a plastic box (4 guinea pigs/box) for 5 minutes using a ultrasonic nebulizer (OMRON NE-U12, condition: maximum nebulizing amounts, maximum air volume), and sensitized (day 0). The same operation was carried out on day 7. On day 14 or 15, each guinea pig was administered 2% OA by inhalation for 5 minutes to initiate a response (challenge). One hour before the challenge, an antihistamine agent pyrilamine maleate (dissolved in saline, 10 mg/2 ml/kg) was intraperitoneally administered. A test compound was suspended in 0.5% methylcellulose (MC), and was orally administered one hour before the antigen challenge in 3 mg/5 ml/kg. 0.5% MC was administered to a control group in a similar manner.
A determination and analysis of respiratory function were carried out according to the method of Penny A. Hutson et al. Am Rev Respir Dis 1988 137, 548-557. The respiratory function was determined before the antigen challenge (before drug administration) and 5 minutes and 1, 2, 3, 4, 5, 6, 7 and 8 hours after the antigen challenge, and wave profiles were imported using MacLab Chart v3.4 (AD Instruments) and analyzed later. The assessment was carried out on the basis of sRaw/TV by subtracting the former sRaw/TV value from sRaw/TV value for each animal at each point of time. The sRaw/TV values subtracting the former values were plotted against measured times, and each area under the curve (AUC4-8hr) of each animal was calculated to compare each other in the range of 4 to 8 hours after the antigen challenge. The statistical analysis was carried out using SAS preclinical package Version 5.0. It was evaluated by Dunnett-type multiple comparison whether or not there were any significant differences between a control group and each drug administration group.
The results are shown in Table 6.
Example 1 compound had an improvement rate of 47%, and a significant difference was shown compared to the control group (p<0.01; Dunnett's test).
The 5-membered ring compound or a salt thereof of the present invention inhibits infiltration of leukocytes including eosinophils, lymphocytes, etc., and hence, is useful for the treatment of various inflammations.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2008-115988 | Apr 2008 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2009/058053 | 4/23/2009 | WO | 00 | 10/25/2010 |
