CN104788423B - A kind of new cystic fibrosis transmembrane conductance regulator inhibitor - Google Patents

Abstract

The present invention relates to amidoimidazole-coupled dihydropyridone compounds shown in formula I and/or their pharmaceutically acceptable salts and preparation methods thereof, which can be used for the treatment and/or prevention of transmembrane conduction regulation due to cystic fibrosis Cystic fibrosis (CF)-related hereditary disease caused by factor (CFTR) gene mutation, and a pharmaceutical composition containing the compound. Wherein, R ¹ is a phenyl group substituted by at most 3 halogens, and R ² is a (C ₁ ‑C ₆ ) straight chain or branched chain alkyl group.

Description

A new inhibitor of cystic fibrosis transmembrane conductance regulator

technical field

The invention belongs to the field of medicinal chemistry, and specifically relates to a dihydropyridone imidazole compound and a physiologically acceptable salt thereof described in the claims, their preparation and their use in the treatment and/or prevention of cystic fibrosis Among the diseases related to male infertility caused by hypertrophy, abnormally increased intestinal secretion, secretory diarrhea, polycystic kidney disease, chronic obstructive pulmonary disease, chronic bronchitis, mucopolysaccharidosis, and congenital bilateral absence of the vas deferens (CBAVA) use.

technical background

Cystic fibrosis transmembrane conductance regulator (English for cystic fibrosis transmembrane conductance regulator, abbreviated as CFTR) is a member of the ATP binding box (English for ATP bindingcassette, abbreviated as ABC) transporter superfamily, and ABC transporter has functions such as absorbing nutrients Various important biological functions such as detoxifying substances, eliminating toxins, and mediating intercellular communication of eukaryotes and bacteria. More specifically, CFTR is a cAMP-activated ATP-gated epithelial chloride channel expressed in the apical plasma membrane of mammalian airway, digestive tract (gut, pancreas, etc.) Movement of ions across the apical membrane provides pathways and key sites involved in the activation of protein kinase A (PKA) responsible for salt and fluid transport in multiple organs, including the lung, and thereby regulates the ratio of salt and water transport across the epithelium , so, in epithelial cells, the normal function of CFTR is critical for maintaining electrolyte transport throughout the body, including respiratory and digestive tissues. Hormones such as beta-adrenergic agonists, cholera toxin, etc. can lead to an increase in cAMP, which is dependent on the activation of protein kinases and phosphorylation of the CFTR chloride channel and thus the opening of the chloride channel. CFTR chloride channel function is associated with many diseases, including cystic fibrosis (CF), abnormally increased intestinal secretion, secretory diarrhea, polycystic kidney disease, chronic obstructive pulmonary disease, chronic bronchitis, mucopolysaccharidosis, and congenital double Male infertility caused by lateral absence of the vas deferens (CBAVA), etc.

CFTR consists of approximately 1480 amino acids. These amino acids encode tandem repeats that form the transmembrane region and thus the protein, each repeat containing 6 transmembrane helices and 1 nucleotide binding domain. The two transmembrane domains are linked by a large polar, regulatory (R)-domain with multiple phosphorylation sites responsible for regulating channel activity and cellular trafficking.

Cystic fibrosis (cystic fibrosis, abbreviated as CF) is a fatal autosomal recessive disease and one of the most common genetic diseases in the human body, caused by mutations in the CFTR gene. Most CF mutations manifest as a decrease in the number of CFTR channels on the cell surface or impairment of channel function (such as gating or conductance mutations), or both. About 1 in 25 percent of children and tens of millions of adults in the United States have a CF-related disorder, and about the same number of people with the recessive genetic disorder live in Europe. Mutations in endogenously expressed CFTR in airway epithelial cells of CF patients lead to decreased anion secretion from the apical membrane, which unbalances ion and fluid transport. Reduced anion transport contributes to increased lung mucus accumulation in CF patients with subsequent microbial infection that can eventually lead to human death. In addition to respiratory problems, CF patients often suffer from typical gastrointestinal problems and pancreatic insufficiency, which can lead to death if left untreated. In addition, most men with CF are infertile, and women with CF are less fertile.

Although the efforts of modern medicine have made progress in the treatment of CF in the past few decades, and the life span of CF patients has been greatly extended, so far no safe and effective treatment methods directly targeting CFTR have been found. Deciphering the CFTR gene will help people further understand the pathogenesis of cystic fibrosis, and provide new clues for the diagnosis of the disease. Small-molecule drugs that increase the possibility of CFTR channel opening are a potential therapeutic strategy for the treatment of CF, and the development of more such drugs will help solve the problem of CF.

Summary of the invention

The present invention describes compounds of formula I, and/or their pharmaceutically acceptable salts

Wherein, R ¹ is a phenyl group substituted by at most 3 halogens, and R ² is a (C ₁ -C ₆ ) linear or branched alkyl group.

Preferably, R is selected from ² -chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2,4-dichlorophenyl, 2,5-dichlorophenyl, 3,5-dichlorobenzene Base, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl or 3,5-difluorophenyl, while R ² is (C ₁ -C ₆ ⁾ linear or branched chain alkyl, that is, for R substituents, representative examples are as follows,

An asterisk (*) indicates that the bond is attached to the carbon atom at the 4-position of the imidazole ring.

The present invention also relates to the use of a compound of formula I and/or a pharmaceutically acceptable salt thereof as a medicine (or medicament) in the preparation of medicines for the prevention and/or treatment of the following diseases, i.e. cystic fibrosis, abnormally increased intestinal secretion, Secretory diarrhea, polycystic kidney disease, chronic obstructive pulmonary disease, chronic bronchitis, mucopolysaccharidosis, and male infertility due to congenital bilateral absence of the vas deferens (CBAVA).

The present invention also relates to pharmaceutical preparations (or pharmaceutical compositions) containing an effective amount of at least one compound of formula I and/or a pharmaceutically acceptable salt thereof, physiologically tolerated excipients and carriers, and where appropriate other additives and/or other active ingredients. The drug can be administered orally, for example in the form of pills, tablets, lacquered tablets, coated tablets, granules, hard and soft gelatin capsules, solutions, syrups, emulsions, suspensions or aerosol mixtures. However, administration can also be carried out rectally, for example in the form of suppositories; or parenterally, for example intravenously, intramuscularly or subcutaneously as injection or infusion solutions, microcapsules, implants or implants. or administered transdermally or topically, for example, as an ointment, solution or tincture; or by other routes, for example, as an aerosol or nasal spray.

The pharmaceutical formulations of the present invention are prepared in a manner known per se and familiar to those skilled in the art, using, in addition to the compounds of formula I and/or their pharmaceutically acceptable salts and/or their prodrugs, pharmaceutically acceptable inert inorganic and/or organic carrier substances and/or additives. For the preparation of pills, tablets, coated tablets and hard gelatine capsules it is possible to use, for example, lactose, corn starch or derivatives thereof, talc, stearic acid or its salts and the like. Carrier materials for soft gelatine capsules and suppositories are, for example, fats, waxes, semi-solid and liquid polyols, natural or hardened oils, and the like. Carrier substances suitable for the preparation of solutions, such as injectable solutions or emulsions or syrups, are, for example, water, saline, alcohols, glycerol, polyols, sucrose, invert sugar, glucose, vegetable oils and the like. Suitable carrier substances for microcapsules, implants or implant rods, for example copolymers of glycolic acid and lactic acid. Pharmaceutical preparations generally contain from about 0.5 to about 90% by weight of compounds of formula I and/or their pharmaceutically acceptable salts and/or their prodrugs. The amount of the active ingredient compound of formula I and/or their pharmaceutically acceptable salts and/or their prodrugs in the pharmaceutical preparation is usually about 0.5 to about 1000 mg, preferably about 1 to about 500 mg.

In addition to the active ingredients of formula I and/or their pharmaceutically acceptable salts and carrier substances, the pharmaceutical preparations may contain one or more additives, such as fillers, disintegrants, binders, lubricants, wetting agents, Stabilizers, emulsifiers, preservatives, sweeteners, colorants, flavoring agents, fragrances, thickeners, diluents, buffer substances, solvents, solubilizers, agents for depot effect, salts for changing osmotic pressure , coating agent or antioxidant. They may also contain two or more compounds of formula I and/or their pharmaceutically acceptable salts. When the pharmaceutical composition contains two or more compounds of formula I, the selection of individual compounds may depend on the particular overall pharmacological properties of the pharmaceutical formulation. For example, a highly potent compound with a shorter duration of action could be combined with a less potent, longer acting compound. The flexibility permitted with regard to the choice of substituents in the compounds of formula I enables a great deal of control over the biological and physicochemical properties of the compounds, thereby enabling the selection of such desired compounds. Furthermore, the pharmaceutical preparations may contain, in addition to at least one compound of the formula I and/or its pharmaceutically acceptable salts, one or more other therapeutically or prophylactically active ingredients.

When using a compound of the formula I, the dosage may vary within wide limits and as customary and known to the physician, the dosage being suitable for each individual case. It depends, for example, on the particular compound employed, the nature and severity of the disease being treated, the mode and schedule of administration or whether acute or chronic conditions are being treated or prophylaxis. Suitable dosages can be established using clinical methods known in the medical arts. In general, the daily dose to obtain the desired result in an adult weighing about 75 kg is about 0.01 to about 100 mg/kg, preferably about 0.1 to about 50 mg/kg, especially about 0.1 to about 10 mg/kg (in each case In mg/kg body weight). Especially in the case of administration of relatively large amounts, the daily dose may be administered in fractions, for example in 2, 3 or 4 fractions. In general, depending on individual behaviour, it may be necessary to deviate upwards or downwards from the stated daily doses.

Furthermore, the compounds of formula I can be used as synthetic intermediates for the preparation of other compounds, especially other pharmaceutically active ingredients, which can be obtained from the compounds of formula I, for example by introducing substituents or modifying functional groups.

In most cases, reaction mixtures containing final compounds of the formula I or intermediates are worked up and, if necessary, the products are purified by conventional methods known to those skilled in the art. For example, the synthesized compounds can be purified using well known methods such as crystallization, chromatography or reverse phase high performance liquid chromatography (RP-HPLC) or other separation methods based on, for example, compound size, charge or hydrophobicity. Similarly, well known methods such as amino acid sequence analysis, NMR, IR and mass spectrometry (MS) can be used to characterize the compounds of the invention.

Accordingly, the following examples form a part of the present invention and serve to illustrate but not limit the invention.

It should be noted that modifications that do not substantially affect the activity of the various embodiments of the invention are included within the scope of the invention disclosed herein.

detailed description

Example: N-(5-(1-sec-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,4-difluorophenyl)-1H-imidazole- 2-yl) preparation of cyclopropyl formamide

The first step: 2-bromo-1-(2,4-difluorophenyl)ethanone

Copper bromide (28.6 g, 128.0 mmol) was added to a mixed solution of 1-(2,4-difluorophenyl)ethanone (10.0 g, 64.0 mmol) in chloroform (100 mL) and ethanol (80 mL). The oil bath was heated to 80° C., and the reaction was kept for about 10 hours. Insoluble matter was removed by filtration, ethyl acetate (200 mL) was added, and the resulting mixed solution was washed successively with saturated brine (200 mL*2) and saturated sodium sulfite solution (200 mL*2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain 13.2 g of a yellow oily substance, namely 2-bromo-1-(2,4-difluorophenyl)ethanone, with a yield of 87.8%. MS: m/z=235.0, 237.0 (M+H ⁺ ).

The second step: 2-(2,4-difluorophenyl)imidazo[1,2-a]pyridine

Add pyridin-2-amine (9.5g, 0.1mol) to a solution of 2-bromo-1-(2,4-difluorophenyl)ethanone (23.5g, 0.1mol) in ethylene glycol dimethyl ether (200mL) middle. The reaction solution was heated to 100° C. for about 8 hours, and the solvent was distilled off under reduced pressure. The resulting crude product was recrystallized from a mixed solvent of methanol and methyl tert-butyl ether (methanol:methyl tert-butyl ether=1:10), separated out, filtered and dried in vacuo to obtain 19.7 g of a white solid, namely 2-(2, 4-Difluorophenyl)imidazo[1,2-a]pyridine, the yield is 85.2%. MS: m/z = 232.1 (M+H ⁺ ).

The third step: 3-bromo-2-(2,4-difluorophenyl)imidazo[1,2-a]pyridine

N-bromosuccinimide (11.6 g, 64.9 mol) was added to 2-(2,4-difluorophenyl) imidazo[1,2-a]pyridine (15.0 g, 64.9 mmol) in chloroform ( 200mL) solution. The reaction solution was heated to 80° C. for about 3 hours, then cooled to room temperature, and dichloromethane (200 mL) was added. The resulting reaction solution was washed with saturated sodium bicarbonate solution (200 mL*3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain 18.9 g of a white solid, which was 3-bromo-2-(2,4-difluorophenyl)imidazo[1,2-a ] Pyridine, yield 93.9%. MS: m/z = 310.0, 312.0 (M+H ⁺ ).

The fourth step: 1-sec-butyl-5-(2-(2,4-difluorophenyl)imidazo[1,2-a]pyridin-3-yl)pyridin-2(1H)-one

Sodium carbonate (21.2g, 0.20mol), 1-sec-butyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) Pyridine-2(1H)-one (15.2g, 0.055mol), tetrakistriphenylphosphine palladium (2.9g, 2.5mmol) were sequentially added to 3-bromo-2-(2,4-difluorophenyl)imidazo[ 1,2-a]pyridine (15.5g, 0.05mol) in THF (200mL). The reaction solution was heated to 85° C. for about 10 hours and then cooled to room temperature. The reaction solution was diluted with ethyl acetate (200 mL). The resulting mixture was washed with saturated brine (200 mL*3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (gradient elution, eluent was petroleum ether:ethyl acetate=40:1 to 4: 1) 12.4g of yellow solid was obtained, which was 1-sec-butyl-5-(2-(2,4-difluorophenyl)imidazo[1,2-a]pyridin-3-yl)pyridine-2( 1H)-ketone, yield 65.2%. MS: m/z = 381.2 (M+H ⁺ ).

The fifth step: 5-(2-amino-4-(2,4-difluorophenyl)-1H-imidazol-5-yl)-1-sec-butylpyridin-2(1H)-one

Add hydrazine hydrate (55% aqueous solution, 9.8 g, 0.1 mol) to 1-sec-butyl-5-(2-(2,4-difluorophenyl)imidazo[1,2-a]pyridin-3-yl ) in a solution of pyridin-2(1H)-one (7.6g, 20.0mmol) in ethanol (100mL). The oil bath of the reaction solution was heated to reflux and reacted for about 10 hours, then cooled to room temperature. The solvent was evaporated under reduced pressure, the residue was dissolved in ethyl acetate (150 mL), and the resulting solution was washed with saturated brine (100 mL*2). After the organic phase was dried over anhydrous sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane:methanol=40:1) to obtain 5.2 g of a yellow solid, namely 5 -(2-Amino-4-(2,4-difluorophenyl)-1H-imidazol-5-yl)-1-sec-butylpyridin-2(1H)-one, yield 75.5%. MS: m/z = 345.1 (M+H ⁺ ). The sixth step: N-(5-(1-sec-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,4-difluorophenyl)-1H-imidazole -2-yl) cyclopropyl formamide

In 5-(2-amino-4-(2,4-difluorophenyl)-1H-imidazol-5-yl)-1-sec-butylpyridin-2(1H)-one (4.0g, 11.6mmol) 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (ie EDCI·HCl, 4.45g, 23.2mmol), N,N-lutidine-4-amine (ie DMAP, 0.71g, 5.8mmol), anhydrous potassium carbonate (3.2g, 23.2mmol) and cyclopropylformic acid (1.5g, 17.4mmol) . The reaction was carried out overnight at room temperature, monitored by TLC (petroleum ether: ethyl acetate = 1:1) until the reaction was complete. The reaction solution was diluted with ethyl acetate (200 mL). The resulting mixture was washed with saturated brine (100 mL*3). The organic phase was dried over anhydrous sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (gradient elution, eluent: dichloromethane:methanol=100:1 to 6:1) to obtain 2.4g light yellow solid, namely N-(5-(1-sec-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,4-difluorophenyl) -1H-imidazol-2-yl)cyclopropylformamide, yield 50.2%. MS: m/z = 413.2 (M+H ⁺ ).

¹ H NMR (300MHz, DMSO-d ₆ )δ: 11.70(d, J=18.6Hz, 1H), 11.48(s, 1H), 7.77(d, 1H), 7.46(d, J=5.8Hz, 2H) ,7.38-7.14(m,2H),6.37(d,J=9.2Hz,1H),5.08(t,J=6.8Hz,1H),1.90(t,J=6.3Hz,1H),1.39-1.34( m,2H),1.30-1.18(m,3H),1.05-0.97(m,3H),0.85(t,J＝4.6Hz,4H).

Compounds of formula I meeting the claims of the patent can be obtained by a synthetic method similar to that of the above-mentioned examples, and only need to change different starting materials. Representative compounds are as follows:

N-(5-(1-sec-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2-chlorophenyl)-1H-imidazol-2-yl)cyclopropyl methyl formamide;

N-(5-(1-sec-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3-chlorophenyl)-1H-imidazol-2-yl)cyclopropyl methyl formamide;

N-(5-(1-sec-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(4-chlorophenyl)-1H-imidazol-2-yl)cyclopropane methyl formamide;

N-(5-(1-sec-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,4-dichlorophenyl)-1H-imidazol-2-yl ) cyclopropyl formamide;

N-(5-(1-sec-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,5-dichlorophenyl)-1H-imidazol-2-yl ) cyclopropyl formamide;

N-(5-(1-sec-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3,5-dichlorophenyl)-1H-imidazol-2-yl ) cyclopropyl formamide;

N-(5-(1-sec-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2-fluorophenyl)-1H-imidazol-2-yl)cyclopropane methyl formamide;

N-(5-(1-sec-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3-fluorophenyl)-1H-imidazol-2-yl)cyclopropyl methyl formamide;

N-(5-(1-sec-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(4-fluorophenyl)-1H-imidazol-2-yl)cyclopropane methyl formamide;

N-(5-(1-sec-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,5-difluorophenyl)-1H-imidazol-2-yl ) cyclopropyl formamide;

N-(5-(1-sec-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3,5-difluorophenyl)-1H-imidazol-2-yl ) cyclopropyl formamide;

N-(5-(1-n-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2-chlorophenyl)-1H-imidazol-2-yl)cyclopropane methyl formamide;

N-(5-(1-n-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3-chlorophenyl)-1H-imidazol-2-yl)cyclopropyl methyl formamide;

N-(5-(1-n-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(4-chlorophenyl)-1H-imidazol-2-yl)cyclopropane methyl formamide;

N-(5-(1-n-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,4-dichlorophenyl)-1H-imidazol-2-yl ) cyclopropyl formamide;

N-(5-(1-n-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,5-dichlorophenyl)-1H-imidazol-2-yl ) cyclopropyl formamide;

N-(5-(1-n-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3,5-dichlorophenyl)-1H-imidazol-2-yl ) cyclopropyl formamide;

N-(5-(1-n-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2-fluorophenyl)-1H-imidazol-2-yl)cyclopropane methyl formamide;

N-(5-(1-n-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3-fluorophenyl)-1H-imidazol-2-yl)cyclopropane methyl formamide;

N-(5-(1-n-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(4-fluorophenyl)-1H-imidazol-2-yl)cyclopropane methyl formamide;

N-(5-(1-n-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,4-difluorophenyl)-1H-imidazol-2-yl ) cyclopropyl formamide;

N-(5-(1-n-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,5-difluorophenyl)-1H-imidazol-2-yl ) cyclopropyl formamide;

N-(5-(1-n-butyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3,5-difluorophenyl)-1H-imidazol-2-yl ) cyclopropyl formamide;

N-(5-(1-isopropyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2-chlorophenyl)-1H-imidazol-2-yl)cyclopropyl methyl formamide;

N-(5-(1-isopropyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3-chlorophenyl)-1H-imidazol-2-yl)cyclopropyl methyl formamide;

N-(5-(1-isopropyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(4-chlorophenyl)-1H-imidazol-2-yl)cyclopropyl methyl formamide;

N-(5-(1-isopropyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,4-dichlorophenyl)-1H-imidazol-2-yl ) cyclopropyl formamide;

N-(5-(1-isopropyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,5-dichlorophenyl)-1H-imidazol-2-yl ) cyclopropyl formamide;

N-(5-(1-isopropyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3,5-dichlorophenyl)-1H-imidazol-2-yl ) cyclopropyl formamide;

N-(5-(1-isopropyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2-fluorophenyl)-1H-imidazol-2-yl)cyclopropyl methyl formamide;

N-(5-(1-isopropyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3-fluorophenyl)-1H-imidazol-2-yl)cyclopropyl methyl formamide;

N-(5-(1-isopropyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(4-fluorophenyl)-1H-imidazol-2-yl)cyclopropyl methyl formamide;

N-(5-(1-isopropyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,4-difluorophenyl)-1H-imidazol-2-yl ) cyclopropyl formamide;

N-(5-(1-isopropyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,5-difluorophenyl)-1H-imidazol-2-yl ) cyclopropyl formamide;

N-(5-(1-isopropyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3,5-difluorophenyl)-1H-imidazol-2-yl ) cyclopropyl formamide;

N-(5-(1-propyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2-chlorophenyl)-1H-imidazol-2-yl)cyclopropyl Formamide;

N-(5-(1-propyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3-chlorophenyl)-1H-imidazol-2-yl)cyclopropyl Formamide;

N-(5-(1-propyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(4-chlorophenyl)-1H-imidazol-2-yl)cyclopropyl Formamide;

N-(5-(1-Propyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,4-dichlorophenyl)-1H-imidazol-2-yl) Cyclopropyl formamide;

N-(5-(1-Propyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,5-dichlorophenyl)-1H-imidazol-2-yl) Cyclopropyl formamide;

N-(5-(1-Propyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3,5-dichlorophenyl)-1H-imidazol-2-yl) Cyclopropyl formamide;

N-(5-(1-propyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2-fluorophenyl)-1H-imidazol-2-yl)cyclopropyl Formamide;

N-(5-(1-propyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3-fluorophenyl)-1H-imidazol-2-yl)cyclopropyl Formamide;

N-(5-(1-propyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(4-fluorophenyl)-1H-imidazol-2-yl)cyclopropyl Formamide;

N-(5-(1-Propyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,4-difluorophenyl)-1H-imidazol-2-yl) Cyclopropyl formamide;

N-(5-(1-Propyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) Cyclopropyl formamide;

N-(5-(1-Propyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3,5-difluorophenyl)-1H-imidazol-2-yl) Cyclopropyl formamide;

N-(5-(1-ethyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2-chlorophenyl)-1H-imidazol-2-yl)cyclopropyl Formamide;

N-(5-(1-ethyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3-chlorophenyl)-1H-imidazol-2-yl)cyclopropyl Formamide;

N-(5-(1-ethyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(4-chlorophenyl)-1H-imidazol-2-yl)cyclopropyl Formamide;

N-(5-(1-ethyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,4-dichlorophenyl)-1H-imidazol-2-yl) Cyclopropyl formamide;

N-(5-(1-Ethyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,5-dichlorophenyl)-1H-imidazol-2-yl) Cyclopropyl formamide;

N-(5-(1-Ethyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3,5-dichlorophenyl)-1H-imidazol-2-yl) Cyclopropyl formamide;

N-(5-(1-ethyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2-fluorophenyl)-1H-imidazol-2-yl)cyclopropyl Formamide;

N-(5-(1-ethyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3-fluorophenyl)-1H-imidazol-2-yl)cyclopropyl Formamide;

N-(5-(1-ethyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(4-fluorophenyl)-1H-imidazol-2-yl)cyclopropyl Formamide;

N-(5-(1-ethyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,4-difluorophenyl)-1H-imidazol-2-yl) Cyclopropyl formamide;

N-(5-(1-Ethyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) Cyclopropyl formamide;

N-(5-(1-ethyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3,5-difluorophenyl)-1H-imidazol-2-yl) Cyclopropyl formamide;

N-(5-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2-chlorophenyl)-1H-imidazol-2-yl)cyclopropyl Formamide;

N-(5-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3-chlorophenyl)-1H-imidazol-2-yl)cyclopropyl Formamide;

N-(5-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(4-chlorophenyl)-1H-imidazol-2-yl)cyclopropyl Formamide;

N-(5-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,4-dichlorophenyl)-1H-imidazol-2-yl) Cyclopropyl formamide;

N-(5-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,5-dichlorophenyl)-1H-imidazol-2-yl) Cyclopropyl formamide;

N-(5-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3,5-dichlorophenyl)-1H-imidazol-2-yl) Cyclopropyl formamide;

N-(5-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2-fluorophenyl)-1H-imidazol-2-yl)cyclopropyl Formamide;

N-(5-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3-fluorophenyl)-1H-imidazol-2-yl)cyclopropyl Formamide;

N-(5-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(4-fluorophenyl)-1H-imidazol-2-yl)cyclopropyl Formamide;

N-(5-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,4-difluorophenyl)-1H-imidazol-2-yl) Cyclopropyl formamide;

N-(5-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(2,5-difluorophenyl)-1H-imidazol-2-yl) Cyclopropyl formamide;

N-(5-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-(3,5-difluorophenyl)-1H-imidazol-2-yl) Cyclopropylformamide.

More representative compounds are not listed one by one.

activity assay

The compounds of formula I and/or their pharmaceutically acceptable salts as CFTR inhibitors can be initially tested for their activity by optical fluorescence transmembrane potential assay.

Measuring principle: The principle of transmembrane potential assay is to use negatively charged fluorescent voltage to transduce dyes (such as FLIPR membrane potential dyes), pre-treat cells with the compound to be tested, and then load the voltage to transduce the dye, and the transfected dye is extracellular When the quencher is bound, upon cell depolarization, the negatively charged dye redistributes to the intracellular compartment, where it is released from the membrane-impermeable quencher, resulting in an increase in fluorescence. Changes in transmembrane potential on FLIPRIII were measured with a fluorescent plate reader as a readout (conductance) for increased gating of functional ΔF508-CFTR in NIH3T3 cells. This change in fluorescence is proportional to the change in transmembrane potential due to CFTR activity. Changes in fluorescence can be monitored in real time in 96 or 384-well microtiter plates by an appropriately equipped fluorescence detector such as FLIPR (Fluorescence Imaging Plate Reader). CFTR activity can be quantitatively detected in this way by measuring the transmembrane potential.

Cell culture: NIH3T3 Chinese hamster ovary (CHO) cells stably expressing the ΔF508-CFTR channel were used for membrane potential experiments. Cells were maintained in Modified Eagle Medium (MEM) at 37°C, 5% v/v CO ₂ and 100% humidity. The medium was supplemented with 8% v/v fetal bovine serum, 100 μg/mL methotrexate and 100 U/mL penicillin/streptomycin. Cells were grown in ²²⁵ cm tissue culture flasks. For membrane potential assays, cells were seeded at 40,000 cells/well in 96-well Matrigel-coated culture plates, allowed to adhere, and incubated at 26°C for 48 hours for enhancer assays.

Enhancer Assay: Membrane Potential Screening Assay utilizes a low chloride ion (5 mM) containing extracellular solution and a dual addition HTS assay protocol. The first addition was buffer with or without the compound to be tested, followed by the addition of forskolin (1-20 [mu]M) 5 minutes later. This protocol favors maximal chlorine efflux in response to ΔF508-CFTR activation. ΔF508-CFTR-mediated chloride efflux results in membrane depolarization, which is optionally monitored with an FMP dye.

Solution: Low-chloride extracellular solution (concentration level is mM) is composed of 120 sodium gluconate, 1.2CaCl ₂ , 3.3KH ₂ PO ₄ , 1.2MgCl ₂ , 10.0D-glucose, 20.0HEPES, adjust the pH value to 7.4. FMP Dye: Prepare the above low-chloride extracellular solution according to the instruction manual at 10-fold final concentration and store in 1 mL aliquots at -20°C.

Measurement results: the detection results (EC ₅₀ values) of several representative compounds using the above method for activity determination are shown in the table below.

Claims (5)

1. compound of formula I, and/or their officinal salt

Wherein, R¹It is at most by the phenyl of 3 halogen substiuted, R²For (C₁-C₆) straight or branched alkyl.

2. according to the compound of formula I of claim 1, wherein R¹Selected from 2-chlorphenyl, 3-chlorphenyl, 4-chlorphenyl, 2,4-dichloro Phenyl, 2,5-Dichlorobenzene base, 3,5-Dichlorobenzene base, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2,4 difluorobenzene base, 2,5- Difluorophenyl or 3,5-difluorophenyl, simultaneously R²For (C₁-C₆) straight or branched alkyl.

3. in claim 1 and 2 at least one compound of formula I described in any one and/or its officinal salt preparation prevention and/ Or treat the purposes in the medicine of following disease: cystic fibrosis, abnormal increase intestinal secretion, secretory diarrhea, polycystic kidney Dirty disease, chronic obstructive pulmonary disease, chronic bronchitis, mucopolysaccharide disease and congenital bilateral deferent duct disappearance (CBAVA) The male infertility caused.

4. the purposes described in claim 3, disease therein refers to cystic fibrosis.

5. medicine, it comprises in the claim 1 and 2 of effective dose at least one compound of formula I described in any one and/or it can Pharmaceutical salts, the excipient of physiology's tolerance and carrier, and also have other additives and/or other active component in due course.