CN109134431B - Aminoimidazole-coupled pyridone derivatives as cystic fibrosis transmembrane conductance regulator inhibitors - Google Patents

Abstract

The invention relates to an aminoimidazole-coupled pyridone compound shown in formula I and/or a medicinal salt thereof, a preparation method thereof, and a pharmaceutical composition containing the compound, wherein the aminoimidazole-coupled pyridone compound and/or the medicinal salt thereof can be used for treating and/or preventing Cystic Fibrosis (CF) related hereditary diseases caused by cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation.

Wherein R is C₄Or C₅Or C₆Or C₇Or C₈Cycloalkyl radical, C₃Or C₄Or C₅Or C₆Or C₇Or C₈Azacycloalkyl radical, C₃Or C₄Or C₅Or C₆Or C₇Or C₈Oxacycloalkyl, morpholinyl, piperazinyl substituted on the nitrogen by a straight or branched C1 or C2 or C3 or C4 or C5 alkyl.

Description

Aminoimidazole-coupled pyridone derivatives as cystic fibrosis transmembrane conductance regulator inhibitors

Technical Field

The invention belongs to the field of medicinal chemistry, and particularly relates to an aminoimidazole-coupled pyridone compound and physiologically acceptable salts thereof, preparation of the aminoimidazole-coupled pyridone compound and the physiologically acceptable salts thereof, and application of the aminoimidazole-coupled pyridone compound and the physiologically acceptable salts thereof in treating and/or preventing diseases related to cystic fibrosis, abnormally increased intestinal secretion, secretory diarrhea, polycystic kidney disease, chronic obstructive pulmonary disease, chronic bronchitis, mucopolysaccharidoses and male infertility caused by congenital bilateral vas deferens (CBAVA).

Technical Field

Cystic fibrosis transmembrane conductance regulator (CFTR) belongs to a member of the ATP-binding cassette (ABC) transporter superfamily, while ABC transporters have a variety of important biological functions such as absorbing nutrients, removing toxins, mediating intercellular communication in 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 (intestine, pancreas, etc.) and reproductive tract epithelial cells, which provides a pathway and key site for chloride movement across the apical membrane, and is involved in the activation of the protein kinase a (pka) responsible for salt and fluid transport in multiple organs, including the lung, and thus regulates the trans-epithelial salt and water transport ratio, so in epithelial cells, the normal function of CFTR is critical to maintain electrolyte transport throughout the body, including respiratory and digestive tissues. Hormones such as beta-adrenergic agonists, cholera toxin, etc., can cause an increase in cAMP, which is dependent on the activation of protein kinase and the phosphorylation of the CFTR chloride channel and thus causes the opening of the chloride channel. CFTR chloride channel function is associated with a number of diseases including Cystic Fibrosis (CF), abnormally increased intestinal secretion, secretory diarrhea, polycystic kidney disease, chronic obstructive pulmonary disease, chronic bronchitis, mucopolysaccharidoses, and male infertility due to congenital bilateral vasectomy (CBAVA).

CFTR consists of about 1480 amino acids. These amino acids encode tandem repeats forming transmembrane regions and thus constitute proteins, each repeat comprising 6 transmembrane helices and a 1 nucleotide binding domain. The 2 transmembrane domains are connected by a large polar and regulatable (R) -domain with multiple phosphorylation sites responsible for regulating channel activity and cellular trafficking.

Cystic fibrosis (CF for short) is a fatal autosomal recessive disease, one of the most common genetic diseases in humans, caused by mutations in the CFTR gene. Most CF mutations appear as a reduction in the number of CFTR channels on the cell surface or impairment of channel function (such as gating or conductance mutations), or both. In the united states, approximately one of two thousand five percent of children and tens of millions of adults suffer from CF-related disease, and in europe there are almost the same number of patients with this recessive genetic disorder. Mutations in endogenously expressed CFTR in the airway epithelial cells of CF patients result in reduced anion secretion from the apical membrane, imbalanced ion and fluid transport. The reduction in anion transport increases lung mucus accumulation in CF patients with microbial infections that ultimately can lead to human death. In addition to respiratory illness, CF patients typically suffer from typical gastrointestinal problems and pancreatic insufficiency, which if not treated in a timely manner, can lead to death. In addition, most CF patients in men are unable to give birth and CF patients in women have reduced fertility.

While modern medical efforts have progressed on the treatment of CF over the past decades, having greatly extended the life of CF patients, no safe and effective therapeutic approach has been found to date that directly targets CFTR. The interpretation of the CFTR gene helps one to further understand the pathogenesis of cystic fibrosis, while providing new clues for the diagnosis of the disease. While small molecule drugs that increase the likelihood of CFTR channel opening are a potential therapeutic strategy for treating CF, the development of many such drugs would help solve the CF problem.

Summary of The Invention

ZL201510111497.9 describes some of our initial research efforts to develop aminoimidazole-coupled pyridone cystic fibrosis transmembrane conductance regulator inhibitors. We initially synthesized and screened only groups at the 4-position of imidazole (mainly substituted phenyl) and substituents at the 5-position of the pyridone nitrogen atom (mainly straight or linear alkyl). Activity measurement shows that when the 4-position of imidazole is fluorophenyl and the 5-position of pyridone is isopropyl, the biological activity is higher, so that subsequent work begins to synthesize and screen a group connected with an amido carbon atom at the 2-position of imidazole, and only 3-8-membered alicyclic groups are considered to be screened at present, and part of aza-alicyclic groups and oxa-alicyclic groups are included. The details are as follows.

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

Wherein R is C₃Or C₄Or C₅Or C₆Or C₇Or C₈Cycloalkyl radical, C₃Or C₄Or C₅Or C₆Or C₇Or C₈Azacycloalkyl radical, C₃Or C₄Or C₅Or C₆Or C₇Or C₈Oxacycloalkyl, morpholinyl, piperazinyl substituted at the nitrogen by a straight or branched C1 or C2 or C3 or C4 or C5 alkyl.

That is, for the R substituent, a representative structural formula is as follows,

the asterisk indicates that the bond is attached to the amido carbon atom at position 2 of the imidazole ring.

The invention also relates to the use of a compound of formula I and/or a pharmaceutically acceptable salt thereof as a medicament (or pharmaceutical) for the manufacture of a medicament for the prevention and/or treatment of cystic fibrosis, abnormally increased intestinal secretion, secretory diarrhoea, polycystic kidney disease, chronic obstructive pulmonary disease, chronic bronchitis, mucopolysaccharidoses and male infertility that is caused by congenital bilateral absence of vas deferens (CBAVA).

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

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

In addition to the active ingredients of the formula I and/or their pharmaceutically acceptable salts and carrier substances, the pharmaceutical preparations can contain one or more additives, such as fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, colorants, flavorants, aromatics, thickeners, diluents, buffer substances, solvents, solubilizers, agents for achieving a depot effect, salts for altering the osmotic pressure, coating agents or antioxidants. They may also contain two or more compounds of formula I and/or their pharmaceutically acceptable salts. Where a 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, highly potent compounds with shorter duration of action may be combined with long acting compounds with lower efficacy. The flexibility allowed with respect to the choice of substituents in the compounds of formula I allows for 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 a pharmaceutically acceptable salt thereof, one or more further therapeutically or prophylactically active ingredients.

When using the compounds of the formula I, the dosage can vary within wide limits and as is conventional and known to the skilled worker, the dosage being adapted to the individual case in each case. Depending on, for example, the particular compound employed, the nature and severity of the disease being treated, the mode and regimen of administration, or whether an acute or chronic condition is being treated or prevented. Suitable dosages may be established using clinical methods known in the medical arts. In general, the daily dose to achieve the desired result in an adult human weighing about 75kg is from about 0.01 to about 100mg/kg, preferably from about 0.1 to about 50mg/kg, in particular from about 0.1 to about 10mg/kg (in each case in mg/kg body weight). Especially in case of administration of relatively large amounts, the daily dose may be divided into several parts, e.g. 2, 3 or 4 parts. In general, depending on the individual behaviour, it may be necessary to deviate upwards or downwards from the stated daily dose.

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

In most cases, the reaction mixture containing the final compound of formula I or intermediates is worked up and, if necessary, the product is 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.

The following examples are, therefore, part of the present invention and are intended to illustrate, but not to limit, the invention.

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

Detailed Description

Example (b): preparation of 4-sec-butyl-N- (4- (2, 4-difluorophenyl) -5- (1-isopropyl-6-oxo-1, 6-dihydropyridin-3-yl) -1H-imidazol-2-yl) piperazine-1-carboxamide (Compound No. 1)

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

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

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

Pyridin-2-amine (9.5g, 0.1mol) was added to a solution of 2-bromo-1- (2, 4-difluorophenyl) ethanone (23.5g, 0.1mol) in ethylene glycol dimethyl ether (200 mL). The reaction mixture was heated to 100 ℃ and reacted for about 8 hours, and then the solvent was distilled off under reduced pressure. The crude product was recrystallized from a mixed solvent of methanol and methyl t-butyl ether (methanol: methyl t-butyl ether ═ 1:10), precipitated, filtered and vacuum-dried to obtain 19.7g of a white solid, i.e., 2- (2, 4-difluorophenyl) imidazo [1,2-a ═ 2, 4-difluorophenyl)]Pyrimidine, yield 85.2%. MS M/z 232.1(M + H)⁺)。

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

N-bromosuccinimide (11.6g, 64.9mol) was added to 2- (2, 4-difluorophenyl) imidazo [1,2-a]Pyridine (15.0g, 64.9mmol) in chloroform (200 mL). The reaction mixture was heated to 80 ℃ and reacted for about 3 hours, then cooled to room temperature, and methylene chloride (200mL) was added. The resulting reaction was washed with saturated sodium bicarbonate solution (200mL × 3). Drying the organic phase with anhydrous sodium sulfate, filtering, and evaporating under reduced pressure to remove solvent to obtain 18.9g white solid, i.e. 3-bromo-2- (2, 4-difluorophenyl) imidazo [1,2-a]Pyrimidine, yield 93.9%. MS M/z 310.0, 312.0(M + H)⁺)。

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

Sodium carbonate (21.2g, 0.20mol), 1-isopropyl-5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2 (1H) -one (14.5g, 0)055mol), Tetratriphenylphosphine Palladium (2.9g, 2.5mmol) 3-bromo-2- (2, 4-difluorophenyl) imidazo [1,2-a ] is added successively]Pyrimidine (15.5g, 0.05mol) in tetrahydrofuran (200 mL). The reaction mixture was heated to 85 ℃ and reacted for about 10 hours, and then cooled to room temperature. The reaction was diluted with ethyl acetate (200 mL). The resulting mixture was washed with saturated brine (200mL × 3). Mixing organic phases, drying with anhydrous sodium sulfate, filtering, evaporating under reduced pressure to remove solvent, purifying the residue by silica gel column chromatography (gradient elution with petroleum ether as eluent: ethyl acetate: 40:1 to 4:1) to obtain 12.7g of yellow solid, i.e. 5- (2- (2, 4-difluorophenyl) imidazo [1,2-a ] solid]Pyrimidin-3-yl) -1-isopropylpyridin-2 (1H) -one in 69.3% yield. MS M/z 367.1(M + H)⁺)。

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

Hydrazine hydrate (55% aqueous solution, 9.8g, 0.1mol) was added to 5- (2- (2, 4-difluorophenyl) imidazo [1,2-a ]]Pyrimidin-3-yl) -1-isopropylpyridin-2 (1H) -one (7.3g, 20.0mmol) in ethanol (100 mL). The reaction was heated to reflux in an oil bath and allowed to react for about 10 hours, then cooled to room temperature. The solvent was evaporated under reduced pressure, the residue was dissolved in ethyl acetate (150mL), and the resulting solution was washed with saturated brine (100 mL. multidot.2). 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 (dichloromethane: methanol 40:1 as eluent) to obtain 5.5g of a yellow solid, i.e., 5- (2-amino-4- (2, 4-difluorophenyl) -1H-imidazol-5-yl) -1-isopropylpyridin-2 (1H) -one, yield 83.2%. MS M/z 331.1(M + H)⁺). And a sixth step: 4-sec-butyl-N- (4- (2, 4-difluorophenyl) -5- (1-isopropyl-6-oxo-1, 6-dihydropyridin-3-yl) -1H-imidazol-2-yl) piperazine-1-carboxamide (Compound No. 1)

At 5-To a solution of (2-amino-4- (2, 4-difluorophenyl) -1H-imidazol-5-yl) -1-isopropylpyridin-2 (1H) -one (3.8g, 11.6mmol) in N, N-dimethylformamide (60mL) was added 1-ethyl- (3-dimethylaminopropyl) carbodiimides hydrochloride (i.e., EDCI. HCl, 4.45g, 23.2mmol), N-dimethylpyridin-4-amine (i.e., DMAP, 0.71g, 5.8mmol), anhydrous potassium carbonate (3.2g, 23.2mmol), and 4-sec-butylpiperazine-1-carboxylic acid (3.2g, 17.4mmol) in succession. The reaction was allowed to proceed overnight at room temperature and monitored by TLC (petroleum ether: ethyl acetate 1:1) until the reaction was complete. The reaction was diluted with ethyl acetate (200 mL). The resulting mixture was washed with saturated brine (100mL × 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 with dichloromethane: methanol: 50:1 to 5:1 as eluent) to obtain 3.1g of a yellow solid, which was 4-sec-butyl-N- (4- (2, 4-difluorophenyl) -5- (1-isopropyl-6-oxo-1, 6-dihydropyridin-3-yl) -1H-imidazol-2-yl) piperazine-1-carboxamide in 53.6% yield. MS M/z 499.3 (M + H)⁺)。

¹HNMR(400MHz,DMSO-d₆)δ:10.06(s,1H),8.76(s,1H),7.78(d,1H),7.19(d,1H), 7.05(d,1H),6.92(s,1H),6.51(d,1H),4.49-4.43(m,1H),3.33-3.24(m,4H),2.94-2.85 (m,4H),2.69-2.63(m,1H),1.52-1.44(m,8H),1.11(d,3H),0.89(t,3H).

The compounds of formula I in accordance with the patent claims can be obtained by a synthesis similar to the above examples, with only a change in the starting materials. Representative compounds are shown in the following table:

more representative compounds are not listed.

Activity assay

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

The determination principle is as follows: the principle of transmembrane potentiometry is to pre-treat cells with a test compound using a negatively charged fluorescent voltage sensing dye (such as a FLIPR membrane potential dye) and then load the voltage sensing dye, which binds the quencher extracellularly, and following depolarization of the cell, the negatively charged dye redistributes to the intracellular compartment, thereby releasing from the membrane non-osmotic quencher, resulting in increased fluorescence. Changes in transmembrane potential on FLIPR iii were measured as an increased readout (conductance) of functional af 508-CFTR gating in NIH3T3 cells with a fluorescent plate reader. 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 suitably equipped fluorescence detectors such as a FLIPR (fluorescence imaging plate reader). CFTR activity can be quantitatively detected in this manner by measuring transmembrane potential.

Cell culture: membrane potential experiments were performed with NIH3T3 Chinese Hamster Ovary (CHO) cells stably expressing the Δ F508-CFTR channel. Cells were incubated at 37 ℃ with 5% v/vCO₂And maintained in Modified Eagle Medium (MEM) at 100% humidity. The medium was supplemented with 8% v/v fetal bovine serum, 100. mu.g/mL methotrexate and 100U/mL penicillin/streptomycin. The cells were grown at 225cm²In tissue culture flasks. For membrane potential determination, cells were seeded at 40,000 cells/well in 96-well matrigel coated culture plates, allowed to adhere, and cultured at 26 ℃ for 48 hours for enhancer determination.

And (3) determination of an enhancer: the membrane potential screening assay utilizes low chloride ion (5mM) with extracellular solution and a double-addition HTS assay protocol. The first addition was a buffer with or without test compound, and 5 minutes later forskolin (1-20. mu.M) was added. This protocol favors maximum chlorine flux in response to activation of Δ F508-CFTR. The af 508-CFTR mediated chloride efflux leads to membrane depolarization, which is optionally monitored by FMP dyes. Solution: the low-chlorine extracellular solution (with concentration of the order of magnitude of mM) consists of 120 parts of sodium gluconate and 1.2 parts of CaCl₂、3.3KH₂PO₄、 1.2MgCl₂10.0D-glucose, 20.0HEPES, pH adjusted to 7.4 with NaOH.

FMP dye: the low chloride extracellular solution was prepared according to the instructions for use, 10 fold final concentration, stored in 1mL aliquots at-20 ℃.

And (3) measuring results: measurement results (EC) of activity of part of representative Compounds measured by the above-described method₅₀Values) are shown in the following table.

。

Claims (5)

1. A compound of formula I, and/or a pharmaceutically acceptable salt thereof

Wherein R is C₃Or C₄Or C₅Or C₆Or C₇Or C₈Azacycloalkyl radical, C₄Or C₅Or C₆Or C₇Or C₈Oxacycloalkyl, morpholinyl, nitrogen-substituted straight or branched C₁Or C₂Or C₃Or C₄Or C₅Alkyl-substituted piperazinyl.

2. The use of a compound of formula I as claimed in claim 1 and/or a pharmaceutically acceptable salt thereof in the manufacture of a medicament of the cystic fibrosis transmembrane conductance regulator class.

3. The use of a compound of the formula I as claimed in claim 1 and/or of a pharmaceutically acceptable salt thereof for producing a medicament for the prophylaxis and/or treatment of the following diseases: 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 vasectomy (CBAVA).

4. The use as claimed in claim 3 of a compound of formula I and/or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prevention and/or treatment of cystic fibrosis.

5. A medicament comprising an effective amount of a compound of formula I as claimed in claim 1 and/or its pharmaceutically acceptable salts, physiologically tolerated excipients and carriers.