CN1739521A - Application of pyrimidine (thio) ketones in pharmaceuticals - Google Patents

Abstract

The present invention relates to the application of pyrimidone compounds in preparing medicine for treating secreted diarrhea and autosomal dominant inheritance polycystic kidney disease. Cystic fibrosis transmembrane conductance regulator (CFTR) factor is one kind of chloridion channel protein activated with cyclic adenosine monophosphate, and during designing model, several CFTR activating ways are considered and applied in the screening process, so as to screen out pyrimidone compounds to prepare medicine for treating secreted diarrhea and autosomal dominant inheritance polycystic kidney disease. The present invention can prevent and treat swine diarrhea effectively and inhibit the liquid secretion of epidermis cell of autosomal dominant inheritance polycystic kidney cyst lining, without obvious toxicity.

Description

Application of pyrimidine (thio) ketones in pharmacy

technical field

The invention relates to the application of pyrimidine (thio) ketone compounds in the preparation of medicines for treating secretory diarrhea and autosomal dominant hereditary polycystic kidney disease. The invention also relates to a method for establishing a cystic fibrosis model in a non-human mammal.

technical background

Cystic fibrosis transmembrane conductance regulator (cystic fibrosis transmembrane conductance regulator, CFTR) is a Cl ^- channel protein activated by cyclic adenosine monophosphate (adenosine 3'5'-cyclic monophosphate acidcAMP), the protein in mammals Expressed in the epithelial cells of airway, small intestine, kidney, pancreas and testis. Hormones (such as β-adrenaline) or toxins (such as cholera toxin) activate cAMP-dependent protein kinases by increasing cAMP levels in the body, thereby causing phosphorylation of CFTR, thereby opening CFTR Cl ^- channels. CFTR is abundantly expressed on epithelial cells, it provides a channel for Cl ^- across the apical membrane of epithelial cells, and it is also a regulatory point for the transport of salt and water across epithelial cells. The action of CFTR Cl ^- channel is related to many diseases such as cystic fibrosis (cystic fibrosis, CF), male infertility, polycystic kidney disease and secretory diarrhea.

1. CFTR and secretory diarrhea

Diarrhea can be triggered by exposure to a variety of pathogens or causative agents, including cholera toxin, pathogenic E. coli endotoxin, diarrhea due to AIDS, diarrhea due to ulcerative colitis, food poisoning, or intestinal Other factors of increased secretion. Secretory diarrhea is the leading cause of death among children in developing countries, with approximately 5 million children dying from this disease each year (Gabriel et al., 1994 Science 166:107-109)). In economically developed countries, the pathogenicity and lethality of diarrhea are also higher. Each year in the United States, 8 million people suffer from acute diarrhea, with 250,000 hospital admissions and more than 500 deaths. In addition, infectious secretory diarrhea is also one of the main diseases that harm many economic animals. Studies using mice have shown that CFTR Cl ^- channels are the final common pathway for various agonists to cause intestinal Cl ^- secretion (Snyder et al.1982 Bull.World Health Organ.60:605-613; Chao et al 1994 EMBO J.13 : 1065-1072; Kimberg et al. 1971 J. Clin. Invest. 50: 1218-1230).

Fluid secretion plays a crucial role in the physiology of the gastrointestinal tract. Under normal circumstances, the intestinal tract mainly absorbs the liquid, electrolytes and nutrients in the intestinal lumen, and at the same time, a small amount of basal secretion maintains the moistening of the intestinal mucosa and helps mix the food entering the intestinal lumen. Intestinal fluid secretion is a process of transepithelial fluid movement driven by intestinal glandular epithelial cells actively transporting Cl ^- from the basal side to the intestinal lumen (Thiagarajah JR., et al., 2003, Current Opinion in Pharmacology 3: 594-599) . The concentration difference between Na ⁺ and Cl ^- produced by Na ⁺ K ⁺ -ATPase and K ⁺ channels on the basement membrane of intestinal gland epithelial cells drives Cl ^- to enter the cell from the side of the basement membrane through the NKCC co-transporter; Cl ^- relies on The electrochemical action is secreted into the intestinal lumen through the CFTR chloride ion channel on the apical membrane; Na ⁺ and water enter the intestinal lumen through the intercellular pathway along with Cl ^- . Many physiological and pathological factors can activate the secretion of Cl ^- and intestinal fluid. For example, the neuromodulatory pathway of intestinal secretion releases serotonin through enterochromaffin cells, leading to the activation of cholinergic and vasoactive intestinal peptidergic neurons and increased cAMP and Ca ⁺ in the glandular epithelium, which in turn activates ^Cl- channels and Intestinal secretion process. Inflammatory mediators such as prostaglandins, histamine and interleukins can also activate intestinal Cl ^- secretion through different signaling pathways. In addition, nucleotide and purinergic signaling pathways can also stimulate intestinal Cl ^- secretion through Ca ⁺⁺ and cAMP signaling pathways.

The CFTR chloride channel plays a pivotal role in intestinal fluid secretion. CFTR knockout mice cause intestinal obstruction due to decreased intestinal secretion and increased absorption (Grubb BR., et al., 1997, Am J Physiol, 273:258-266); cholera toxin causes a large amount of small intestinal fluid in normal mice secreted, but not in CFTR knockout mice. In addition, in vitro experiments have also proved that the secretion of Cl ^- in intestinal mucosa and cultured intestinal epithelial cells induced by various agonists can be suppressed by anion channel blockers glibenclamide and 5-nitro-2-(3-phenylpropylamine) Benzoic acid [5-nitro2-(3-phenylpropylamino)benzoate, NPPB] inhibition.

Since diarrhea will eventually lead to dehydration, one of the goals of treatment is to correct dehydration, usually by means of rehydration and feeding. High-affinity CFTR inhibitors will play an important role in the treatment of secretory diarrhea. Although benzidine-2-carboxylic acid (diphenylamine-2-carboxylate, DPC), NPPB and glibenclamide can also inhibit the activity of CFTR under high concentration conditions, but their above-mentioned activities are non-specific (Cabantchik et al ., 1992, Am.J.Physiol.262:C803-C827; McDonough et al., 1994, Neuron 13:623-634; Schultz et al., 1999, Physiol.Rev.79:S109-S144.Edwards et al. , 1993, Br.J.Pharmacol.110:1280-1281; Rabe et al., 1995, Pflugers Arch.429:659-662; Yamazaki et al., 1997, Circ.Res.81:101-109). So far, there is no effective drug for the treatment of secretory diarrhea by specifically inhibiting CFTR Cl ^- channel function.

2. CFTR and autosomal dominant polycystic kidney disease

Polycystic kidney disease (Polycystic kidney disease, PKD) is a common genetic disease characterized by the formation of multiple progressively enlarged cysts in the renal tubules, and is one of the main causes of end-stage renal failure (Wilson PD.2004, NEngl J Med. 350:151-164). Polycystic kidney disease can be divided into two types: autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD). The incidence rate of ARPKD is low, about 1/20000, and it is more common in infants and young children, and most of the children die in the early stage. The incidence rate of ADPKD population is relatively high, about 1/400～1/1000. About 50% of ADPKD patients enter end-stage renal failure before the age of 60, and 8-10% of patients receiving kidney transplantation and dialysis clinically are end-stage renal failure patients with ADPKD (Hateboer N., et al., 1999, Lancet , 353:103-107). In addition to renal manifestations, ADPKD also involves multiple organs, such as cyst formation in the liver, pancreas, and spleen, as well as intracranial aneurysms and mitral valve prolapse. Three pathogenic genes of ADPKD have been found: PKD1 located at 16p13.3, PKD2 and PKD3 located at 4q21-23 (Ariza M., et al., 1997, J Med Genet, 34:587-589). ADPKD genes are relatively large and the mutation spectrum is relatively complex, there is no obvious mutation hotspot, and various mutations are distributed throughout the gene. The pathogenesis of ADPKD is still unclear at the cellular and molecular levels, resulting in the lack of effective treatments for ADPKD.

The pathological changes of ADPKD include abnormal proliferation and apoptosis of renal tubular epithelial cells, renal tubular dilatation caused by abnormal secretion of fluid in the renal tubules, and abnormal remodeling of the extracellular matrix (GRANTHAM, JJ1993, J. Am. Soc. Nephrol, 3: 1841- 1857). Studies have proved that the fluid secretion of cyst lining epithelial cells is related to the abnormally increased cAMP level in polycystic kidney tissue, and plays a key role in the formation and development of ADPKD cysts. Accumulating evidence shows that Cl ^- secretion mediated by cAMP-activated CFTR chloride channel plays an important role in the process of fluid accumulation in ADPKD cysts (Perso A., et al., 2000, J Am Soc Nephrol.11: 2285 -2296; Murcia NS., et al., 1999, Kidney Int.55: 1187-1197; Hanaoka K., et al., 1998, J Am Soc Nephrol.9: 903-916), suggesting that by inhibiting CFTR ^Cl- Transport activity to block the fluid accumulation in ADPKD cysts to prevent cyst formation and progression may become a new strategy for the treatment of ADPKD.

3. CFTR and cystic fibrosis

Cystic fibrosis (CF) is a fatal genetic disease caused by recessive mutations in CFTR. The results of studies on cystic fibrosis patients and CF mouse models show that CFTR plays an important role in the fluid transport of the small intestine and pancreas and male fertility (Grubb et al., 1999. Physiol. Rev. 79: S193-S214; Wong, P.Y. , 1997, Mol. Hum. Report. 4:107-110). Although progressive lung function lesions are a common cause of morbidity and mortality in CF patients, the mechanism by which CFTR mutations cause airway lesions in patients with cystic fibrosis is still unclear (Pilewski et al., 1999, Physiol. Rev. 79: S215 -S255). It is of great significance to establish cystic fibrosis models by using human tissues or animals to clarify the lung injury and pathogenic mechanism caused by cystic fibrosis. More and more studies have shown that the cause of pulmonary cystic fibrosis is the loss of lung villi clearance function and bacterial infection due to insufficient secretion of airway submucosal gland fluid and excessive secretion of macromolecules. However, research has been severely limited by the fact that airway material from lung transplantation is severely diseased. Using CFTR inhibitors to establish large animal cystic fibrosis models will be of great significance for studying the role of submucosal gland CFTR in the process of water, salt and macromolecule secretion. The discovery of high-affinity CFTR inhibitors will play an important role in establishing large animal models of CF, and it is also of great significance for studying the pathogenesis of CF and finding effective therapeutic approaches.

Contents of the invention

The purpose of the present invention is to provide the application of pyrimidine (thio) ketone compounds in the preparation of medicines for treating secretory diarrhea and autosomal dominant hereditary polycystic kidney disease. Another object of the present invention is to provide a method for establishing a cystic fibrosis model in a non-human mammal.

The present invention provides high affinity pyrimidine(thio)one CFTR inhibitors and methods of using them to treat symptoms or conditions associated with cystic fibrosis transmembrane conductance regulator (CFTR) ion transport dysfunction in a subject. Pyrimidin(thio)ones were discovered on the basis of screening a large number of compounds using a model for screening inhibitors of direct binding to CFTR. Since multiple activation pathways of CFTR are considered in the design process of the model, and multiple activation pathways are jointly applied to the screening process, the pyrimidine (thio) ketone compounds in the present invention are considered to be bound at the Cl ^- transport channel . By screening compounds in a combinatorial chemical library containing 100,000 structurally diverse compounds, we obtained several pyrimidin(thio)one compounds that can effectively inhibit CFTR Cl ^-transport function. These compounds are structurally distinct from known CFTR inhibitors or activators. Among them, the most active CFTR inhibitor can effectively inhibit the Cl ^- current of human airway cells when the concentration is 10M. When the concentration is 100 μg/kg, it can effectively inhibit the accumulation of fluid in the small intestine of mice caused by cholera toxin. When given to piglets with diarrhea at 0.25mg/kg once a day for 7 consecutive days, it can effectively prevent the occurrence of diarrhea in piglets without showing obvious toxic effects in vivo. When the concentration is 500μg/kg, it can effectively inhibit the fluid secretion of epithelial cells lining autosomal dominant polycystic kidney cysts. In addition, the CFTR inhibitors we found have rapid, reversible and CFTR-specific inhibitory effects on CFTR.

The structure of the pyrimidine (thio) ketone compound in the present invention includes a heterocyclic ring consisting of six atoms, a carbonyl or thiourea group is attached to the heterocyclic ring, and it is a pyrimidinone when a carbonyl group is attached to the heterocyclic ring Compounds, when there is a thiourea group attached to the heterocycle, it is a pyrimidinethione compound. Especially in the molecular formula of pyrimidine (thio) ketone compounds in the present invention, wherein X is hydrogen, any organic group, any halogen atom, nitro group, azo group, hydroxyl group or mercapto group; Y ₁ and Y ₂ are respectively Hydrogen, any organic group, any halogen atom, nitro, azo group, hydroxyl or mercapto; A ₁ is an oxygen atom or a sulfur atom; A ₂ is hydrogen, any organic group, any halogen atom, nitric acid group, azo group, hydroxyl group or mercapto group.

In some specific cases pyrimidin(thio)ones have the formula:

X is methyl, ethyl, propyl, isopropyl or allyl, Y ₁ is C1-C6 alkyl or hydrogen, Y ₂ is C1-C6 alkyl or hydrogen, Y ₁ is the same as Y ₂ or Different, A ₁ is an oxygen atom or a sulfur atom, and A ₂ is a C1-C6 alkyl group or hydrogen.

Some specific forms of the compounds of the invention are:

(1) N-(2,3-dimethylphenyl)-6-methyl-2-sulfur-4-(4-methylphenyl)-1,2,3,4-tetrahydropyrimidine-5 -amide

(2) N-(2,4-dimethylphenyl)-6-methyl-2-sulfur-4-(4-methylphenyl)-1,2,3,4-tetrahydropyrimidine-5 -amide

(3) N-(2,3-dimethylphenyl)-4-(4-ethylphenyl)-6-methyl-2-sulfur-1,2,3,4-tetrahydropyrimidine-5 -amide

(4) N-(2,4-dimethylphenyl)-4-(4-ethylphenyl)-6-methyl-2-thio-1,2,3,4-tetrahydropyrimidine-5 -amide

(5) N-(2,3-dimethylphenyl)-4-(4-isopropylphenyl)-6-methyl-2-sulfur-1,2,3,4-tetrahydropyrimidine- 5-amide

(6) N-(2,4-dimethylphenyl)-4-(4-isopropylphenyl)-6-methyl-2-sulfur-1,2,3,4-tetrahydropyrimidine- 5-amide

(7) 4-(4-allylphenyl)-N-(2,3-diethylphenyl)-6-methyl-2-sulfur-1,2,3,4-tetrahydropyrimidine- 5-amide

(8) 4-(4-allylphenyl)-N-(2,4-diethylphenyl)-6-methyl-2-thio-1,2,3,4-tetrahydropyrimidine- 5-amide

(9) N-(2,6-dimethylphenyl)-6-methyl-2-sulfur-4-(4-methylphenyl)-1,2,3,4-tetrahydropyrimidine-5 -amide

(10) N-(2,6-dimethylphenyl)-4-(4-ethylphenyl)-6-methyl-2-thio-1,2,3,4-tetrahydropyrimidine-5 -amide

(11) N-(2,6-dimethylphenyl)-4-(4-isopropylphenyl)-6-methyl-2-sulfur-1,2,3,4-tetrahydropyrimidine- 5-amide

(12) 4-(4-allylphenyl)-N-(2,6-diethylphenyl)-6-methyl-2-sulfur-1,2,3,4-tetrahydropyrimidine- 5-amide

The present invention also relates to the dosage form of the medicine for treating secretory diarrhea and autosomal dominant hereditary polycystic kidney disease prepared by the pyrimidine (thio) ketone compound. The compounds of the present invention can be prepared in the form of solid, semi-solid, liquid or gas together with suitable pharmaceutical carriers, diluents, excipients or adjuvants. For example tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. The compounds of the present invention can be administered by a variety of routes. These routes include oral, buccal, rectal, intravenous, subperitoneal, subcutaneous, airway, and the like.

In dosage forms the compounds of the present invention may be in the form of their pharmaceutically acceptable salts, and they may also be administered alone or in combination or together with other pharmaceutically active compounds.

When made into oral medicine, the compounds of the present invention can be made into tablets, powders, granules or capsules alone or with appropriate additives. For example, conventional additives such as lactose, mannitol, corn starch, potato starch, or binders of cellulose, cellulose derivatives, acacia gum, corn starch, or gelatin, lubricants such as talc, magnesium stearate, etc. , or mixed with diluents, buffers, wetting agents, preservatives and flavoring agents as needed to make oral medicine.

The injection form of the compounds of the present invention can be dissolved, suspended, emulsified in water or non-aqueous solvents (such as vegetable oils, synthetic fatty acid glycerides, esters, higher fatty acids or propylene glycol). Conventional co-solvents, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives can be added when necessary.

The compounds of the present invention can be administered by inhalation in the form of aerosol formulations. The compounds of the present invention can be compressed with dichlorodifluoromethane, propane, nitrogen, etc. to make aerosol.

The compounds of the present invention can also be mixed with different bases such as emulsifying bases and water-soluble bases to make suppositories. Suppositories made of the compounds of the present invention can be administered rectally. The carrier of the suppository can be cocoa butter, carbowax, polyvinyl alcohol and the like which are solid at room temperature but melt at body temperature.

When administered orally or rectally, the compound of the present invention can be prepared in the form of a unit of measurement with syrup, formulation and suspension. The unit of measure may be teaspoon, tablespoon, tablet or peg. Each measurement unit contains a certain amount of one or more inhibitors. For intramuscular or intravenous injection, the compound of the present invention can be dissolved in sterile water, normal saline or other pharmaceutically acceptable carriers.

Dosages vary depending on the subject to be treated or the mode or route of administration. Since the doses required by different mammals vary greatly, for example, the dose required by humans per unit weight is 20, 30 or even 40 times lower than that of rats, so the dose range of the compounds of the present invention is very wide, for example, it can be 0.1-10 mg /kg/individual/day. Different administration methods also have an impact on the dosage. For example, a single intraperitoneal injection of 10-20 mg of the compound of the present invention can effectively block diarrhea in mice induced by cholera toxin, but the same effect can be achieved only when the dose is increased by dozens of times orally.

Typical dosage forms may be solutions suitable for administration by injection; tablets to be taken 2-6 times a day; sustained release capsules or tablets to be taken once a day, and the like. The sustained release effect can be achieved by using capsule materials that dissolve under different pH conditions, or capsules that release slowly under different osmotic pressures, or other known controllable release materials.

The compounds of the present invention may be used together with other pharmaceutically active agents including CFTR inhibitors.

Pharmaceutical excipients such as diluents, carriers, and adjuvants are commonly available, and other pharmaceutically acceptable accessories such as pH regulators, buffers, osmotic pressure regulators, stabilizers, and wetting agents are commonly available.

Dosages will vary depending on the particular compound of the invention, the severity of disease symptoms, and the tolerance of the subject to side effects. For a certain compound in the present invention, the dosage is determined by different methods.

The CFTR inhibitor in the present invention can be administered 1-3 times a day by intraperitoneal, subcutaneous or other administration routes at a dose of 50-500 μg/kg body weight to create a non-human cystic fibrosis animal model.

The CFTR inhibitor in the present invention can effectively treat secretory diarrhea and autosomal dominant polycystic kidney disease, the action is rapid, reversible and does not show obvious toxicity in vivo. The CFTR inhibitor of the present invention can cause pigs, cattle, sheep and other non-human mammals to appear phenotypes similar to cystic fibrosis when administered in large quantities for a long period of time.

The CFTR inhibitors of the present invention are useful in the treatment of CFTR-related symptoms or conditions, that is, any condition, disorder, disease, or symptom associated with CFTR activity (eg, CFTR ion transport activity). These CFTR-associated conditions, disorders, diseases, symptoms can be treated by inhibiting CFTR activity, such as CFTR ion transport activity.

The CFTR inhibitors of the invention can be used to treat symptoms or conditions associated with abnormal increases in small intestinal fluid secretion, especially acute abnormal increases in small intestinal fluid. CFTR activity has been shown to induce increased intestinal secretion under the action of various agonists including cholera toxin, and CFTR inhibitors at doses effective to inhibit CFTR ion transport can reduce intestinal fluid secretion. Experiments on small intestinal fluid secretion in mice have shown that intraperitoneal injection of non-toxic doses of CFTR inhibitors to mice can effectively inhibit the excessive secretion of small intestinal fluid caused by cholera toxin, so CFTR inhibitors can be used to treat intestinal inflammatory disorders and diarrhea, especially secretory diarrhea.

CFTR inhibitors are also potentially useful in the treatment of diarrhea caused by AIDs (AIDs-associated diarrhea) and inflammatory gastrointestinal disorders such as ulcerative colitis, inflammatory bowel disease (IBD), Crohn's disease and similar conditions.

The CFTR inhibitor of the present invention can also be used to treat diseases such as polycystic kidney disease, and can be further developed as a male contraceptive by inhibiting the activity of CFTR in the testis.

Of particular interest is that the CFTR inhibitors of the present invention can be used to induce cystic fibrosis lesions in non-human animals. For example, CFTR inhibitors at doses effective to inhibit CFTR channel activity effectively mimic the functional deficits in CFTR found in cystic fibrosis in the lung. Routes of administration of CFTR inhibitors have been discussed in detail above.

Animals suitable for the application of the inhibitor-induced animal model in the present invention include humans and animals, especially mammals, such as non-human primates (monkeys, orangutans, gorillas, chimpanzees, etc.), rodents (rats, mice, etc.) , gerbils, hamsters, rabbits, ferrets, etc.), rabbits, pigs, horses, dogs, cats, etc. Of greatest interest are large animal models.

CFTR inhibitors can also be contacted with isolated human tissues to create in vitro disease models. These tissues can be exposed to a dose of a CFTR inhibitor effective to reduce CFTR activity in these tissues to create an in vitro human disease model within 15 minutes to 2 hours. Human tissues of interest include, but are not limited to, lungs (including bronchi and airways), liver, pancreas, testis, and the like. Physiological, biochemical, genomic or other studies using tissue treated with a CFTR inhibitor can identify novel therapeutic target molecules that play important roles in disease pathophysiology. For example, ex vivo tissues isolated from humans without cystic fibrosis can be exposed to inhibitors sufficient to induce the cystic fibrosis condition, and such studies can identify novel therapeutic agents that play an important role in the pathophysiology of cystic fibrosis. target molecule.

Description of drawings

Fig. 1 is a schematic diagram of the principle of the CFTR inhibitor screening technology of the present invention. A variety of activators were used to activate CFTR expressed on epithelial cells stably co-transfected with porcine CFTR and a Cl ^- /I ^- sensitive yellow fluorescent protein (YFP). After the test compound was added, the I ^- containing solvent was added to the cell culture wells to measure the influx velocity of ^I- .

Fig. 2 is a representative fluorescence change curve measured in a single cell culture well obtained by screening. The figure shows the fluorescence change curves of the control group without activator and test compound, the test compound group with no activity and the test compound group with the activity of inhibiting the opening of CFTR chloride ion channel.

Fig. 3a is the chemical structure of pyrimidinethione CFTR chloride channel inhibitors obtained by screening.

Figure 3b shows the complete chemical structure of CFTR chloride channel inhibitors, with relative activities of 0.9 (CFTRinh-1), 0.9 (CFTRinh-2), 0.9 (CFTRinh-3), 0.9 (CFTRinh-4), 1 (CFTRinh- 5), 0.8(CFTRinh-6), 0.9(CFTRinh-7), 0.1(CFTRinh-8), 0.6(CFTRinh-9), 0.9(CFTRinh-10), 0.8(CFTRinh-11), 0.2(CFTRinh-12 ).

Detailed ways

Example 1

This example describes the establishment of a high-throughput cell-based screening model for small molecule inhibitors of CFTR

The basis for studying the opening of CFTR Cl ^- ion channel in the cell membrane and screening its inhibitors is to establish a cell model with high sensitivity, strong specificity, stability and reliability, and suitable for high-throughput screening. First, we stably co-transfected Fischer cells with a porcine CFTR expression plasmid (SV40 promoter, zeocin as a selection marker) and a halogen-sensitive fluorescent green protein mutant YFP-H148Q expression plasmid (CMV promoter, G418 as a selection marker). Mouse thyroid (fischer rat thyroid, FRT) epithelial cell line. The clone FRT/pCFTR/YFP-H148Q/I152L with high fluorescence intensity, rapid growth, basic chloride ion permeability and stable and high expression on the plasma membrane was selected. When performing high-throughput screening, FTR cells were suspended in F-12Coon's culture medium (containing 10% fetal bovine serum, 2mM L-Gln, 500u/mL penicillin and streptomycin, 500ug/ml Zeocin), and the above cell suspension Added to a 96-well plate with black walls and transparent bottom (Corning-Costar 3904), the number of cells in each well was 20,000. After culturing for 16-24 hours, the cells were grown into a monolayer, and the chloride ion channel function was measured on a FluoStar Galaxy fluorescence instrument. For short-circuit current measurement mediated by chloride ion channels, cells were cultured on Snapwell support carriers (Corning-Costar), and the number per well was about 500,000. After growing for 8-10 days, the Ussing Chamber was used to measure the chloride ion transmembrane current after the intercellular tight junction (transcellular electrical impedance) was formed.

Example 2

This example describes the high-throughput cell screening method and results of small molecule inhibitors of CFTR

1. Compound

A library of 100,000 structurally diverse drug-like small molecules (molecular weight range 350-550 Daltons) was purchased from ChemBridge Co. (ChemBridge Co. San Diego). The concentration of the compound is 10 mM DMSO solution, which is dispensed on a 96-well plate. In the initial screening, the above-mentioned small molecule compounds were screened. Secondary screening and dose-effect analysis were performed on the screened positive compounds. Conduct structural analysis on the exact high-affinity positive compounds, and purchase or synthesize their analogs for further testing.

2. High-throughput screening system for CFTR inhibitors

Primary screening was performed on a Beckman high-throughput cell assay screening system. The system mainly consists of an automatic manipulator, a 3-meter-long slideway, a carbon dioxide incubator, a multi-functional stage, a code recognition machine, a plate washer, a cell culture plate removing device, an oscillator, a plate sealing machine, a liquid transfer device and Fluorescence detector and other components. The fluorescence detector is FLUOstar (Galaxy, BMGLab Technologies), which contains two syringe pumps, the excitation wavelength is HQ500/20X (500±10nm), and the emission wavelength is HQ535/30M (535±15nm). The system is controlled by a central host, and the control software is provided by Beckman.

3. High-throughput cell-based screening method for CFTR small molecule inhibitors

Spread FTR/pCFTR/YFP-H148Q/I152L cells in a 96-well plate at a density of 20,000 per well, in a carbon dioxide incubator (37°C, humidity 90%, CO _{concentration} 5%, air concentration ) were cultured to form a monolayer. All of the following screening procedures are automated processes operated by a robotic arm: take the cell culture plate out of the carbon dioxide incubator, remove the cover, wash the cells three times with phosphate buffered solution (PBS), 300 μl each time, keep 50 μl of PBS for the last time, send to On a pipetting device, 10 μl of a mixture containing an activator (containing 5 μM FSK, 100 μM IBMX, and 25 μM APG) was added to each well, and 5 minutes later the small molecule compounds to be tested were added to a final concentration of 10 μM for the individual compounds. After 15 minutes, it was sent to a fluorescence measuring instrument to measure the fluorescence quenching kinetics of YFP in the cell by iodide ion flowing through the chloride ion channel. The method of fluorescence analysis is as follows: measure continuously for 14 seconds at a speed of 5 points/second, and the first 2 seconds are the baseline. After 2 seconds, 160 μl of PBS containing 137 mM NaI (replacing NaCl) was quickly injected into the cell culture well, and the measurement was continued for 12 seconds. Calculate the initial velocity of fluorescence change using a custom cubic equation.

4. Primary Screening Results

The purpose of this study was to find inhibitors that directly block the chloride channel of wild-type CFTR, so first we used a cocktail of activators (containing 5 μM FSK, 100 μM IBMX and 25 μM APG) to keep the CFTR chloride channel in a persistent open state . Compounds were screened at a concentration of 10 μM. 15 minutes after the compound was added, I ^- was rapidly injected into the system, and the intracellular fluorescence dynamics change (ie, the internal flow velocity of I -) was measured (Fig. 1).

We used a PBS saline solution containing a mixture of activators as a negative control, and the smaller the slope of the curve, the greater the activity of the compound at the measured concentration ( FIG. 2 ). Among the 100,000 compounds screened, 99,995 compounds had no significant effect on the influx velocity of ^I- at a concentration of 10 μM (the slope of the curve decreased by <10%), and 5 compounds reduced the influx velocity of ^I- 10-50% off. The screened positive compounds all have the core structure of pyrimidine (thio) ketones ( FIG. 3 a ).

We further analyzed the fluorescence activity of 196 compounds with the core structure of pyrimidine (thio) ketones, and the one with the highest activity is shown in Figure 3b.

Example 3

This example describes the method and results of the determination of the properties of CFTR small molecule inhibitors

1. Compound

FSK, IBMX, APG, purchased from Sigma Company (Sigma Chemical Co.St.Louis, Mo.) CFTRinh-1, CFTRinh-5, CFTRinh-7 self-synthesized

2. Kinetic analysis of CFTR inhibitors inhibiting CFTR ion transport activity

The FRT/pCFTR/EYFP-H148Q cells were plated in a 96-well plate (Corning-Costar 3904) with a black-walled transparent bottom at a density of 30,000 cells per well, and the culture components were F-12Coon's culture medium (containing 10% fetal bovine serum, 2mM glutamine, 500u/mL penicillin streptomycin), cultured in a carbon dioxide incubator (37°C, humidity 90%, _CO2 concentration 5%, air concentration 95%) for 24 hours to form a single layer before use .

Take the cell culture plate out of the carbon dioxide incubator, wash the cells three times with phosphate buffered saline (PBS), 300 μl each time, keep 40 μl of PBS for the last time, add 10 μl of CFTR activator mixture (5 μM forskolin, 100 μM IBMX) to each well , 50 μM genistein) in PBS solution, after 5 minutes, add 10 μl of the CFTR inhibitor to be tested, respectively at the 2nd, 4th, 6th, 8th, 10th, 15th, 20th, 30th, and 60th minute after adding the CFTR inhibitor, carry out Fluorescence analysis. The method of fluorescence analysis is as follows: measure continuously for 14 seconds at a speed of 5 points/second, and the first 2 seconds are the baseline. After 2 seconds, quickly inject 160μl of PBS containing 137mM NaI (replacing NaCl) into the cell culture well, and continue to measure for 12 seconds. The functional experiment of cell chloride ion transport is directly reflected by the fluorescence value and time change curve. Calculate the initial velocity of the fluorescence change.

The results showed that when the concentration of CFTRinh-1 was 0.4-0.7 μM, it had a significant inhibitory effect on wild-type CFTR. The activity can reach the maximum within 10 min (t _1/2 =5 min). When the concentration of CFTRinh-5 is 0.3-0.6μM, it has a significant inhibitory effect on wild-type CFTR. The activity can reach the maximum within 10 minutes (t _1/2 =4 minutes). When the concentration of CFTRinh-7 is 0.3-0.6μM, it has a significant inhibitory effect on wild-type CFTR. The activity can reach the maximum within 10 min (t _1/2 =5 min).

3. Analysis of the reversibility of CFTR inhibitors inhibiting the ion transport activity of CFTR

The FRT/CFTR/EYFP-H148Q cells were plated in a 96-well plate (Corning-Costar 3904) with a black-walled transparent bottom at a density of 30,000 cells per well, and the culture ingredients were F-12Coon's medium (containing 10% fetal calf serum, 2mM glutamine, 500u/mL penicillin streptomycin), cultured in a carbon dioxide incubator (37°C, humidity 90%, _CO2 concentration 5%, air concentration 95%) for 24 hours to form a single layer before use .

Take the cell culture plate out of the carbon dioxide incubator, wash the cells three times with phosphate buffered saline (PBS), 300 μl each time, keep 40 μl of PBS for the last time, add 40 μl of CFTR activator mixture (5 μM forskolin, 100 μM IBMX) to each well , 50 μM genistein) in PBS solution, after 5 minutes, add 10 μl of the CFTR inhibitor to be tested, after 15 minutes of action, wash the cells three times with phosphate buffer solution (PBS), 300 μl each time, keep 50 μl PBS for the last time, add to each 10 μl of PBS solution containing CFTR activator mixture (5 μM forskolin, 100 μM IBMX, 50 μM genistein) was added to the wells, and fluorescence analysis was performed at 5, 10, 15, 20, 30, and 60 minutes after adding the activator mixture. The method of fluorescence analysis is as follows: measure continuously for 14 seconds at a speed of 5 points/second, and the first 2 seconds are the baseline. After 2 seconds, quickly inject 160μl of PBS containing 137mM NaI (replacing NaCl) into the cell culture well, and continue to measure for 12 seconds. The functional experiment of cell chloride ion transport is directly reflected by the fluorescence value and time change curve. Calculate the initial velocity of the fluorescence change.

The results showed that the activity of CFTR recovered more than 1/2 after removing CFTRinh-1 in the system for 5 minutes; the activity of CFTR recovered more than 1/2 after removing CFTRinh-5 in the system for 5 minutes; the activity of CFTR recovered 1 /2 or more

4. CFTR inhibitors exert their functions through direct interaction with CFTR

CFTRinh-1, CFTRinh-5 and CFTRinh-7 inhibitors can all inhibit the activation of CFTR by FSK, IBMX and APG, and can also inhibit the activation of CFTR including GEN, CPT-cAMP, CPX, 8-MPO, UCCF-029 and Activity of UCCF-853. Since the CFTR activators mentioned above are almost unrelated in structure, we believe that CFTRinh-1, CFTRinh-5 and CFTRinh-7 exert their functions through direct interaction with CFTR.

5. Electrophysiological assay results of CFTR inhibitors inhibiting CFTR ion transport activity

Cells were cultured on 6-well Snapwell support carriers (Corning-Costar) at 500,000 per well. After growing for 8-10 days, after the formation of intercellular tight junctions (transcellular electrical impedance), use the Ussing Chamber to measure the chloride ion transmembrane current. , Inc). Then add the solution containing 130mM NaCl, 2.7mM KCl, 1.5mM KH ₂ PO ₄ , 1mM CaCl ₂ , 0.5mM MgCl ₂ , 10mM glucose, 10mM Na-Hepes (pH7.3) to the side of the basement membrane, and add the solution containing A solution of 65mM sodium gluconate, 65mM NaCl, 2.7mM KCl, 1.5mM KH ₂ PO ₄ , 2mM CaCl ₂ , 0.5mM MgCl ₂ , 10mM glucose, 10mM Na-Hepes (pH 7.3). The system was continuously ventilated with air at 37°C, and then the basement membrane was permeabilized with 250 μg/ml amphotericin B. During the measurement, the Ussing cell was connected to a DVC-1000 voltage clamp (World Precision Instruments), and the short-circuit current between the Ag/AgCl electrode and the 1M KCl agarose bridge was measured.

We used electrophysiological methods to study the specificity of CFTRinh-1, CFTRinh-5 and CFTRinh-7 in inhibiting CFTR activity. We added CFTRinh-1 (or CFTRinh-5, or CFTRinh-7) to the epithelial membrane side of FRT cells expressing CFTR, and determined its effect on short-circuit current. The study found that CFTRinh-1, CFTRinh-5 and CFTRinh-7 can rapidly and effectively inhibit the short-circuit current of FRT cells, and the inhibitory activity is positively correlated with its concentration.

6. Toxicity analysis of CFTR inhibitors

In this experiment, the toxicity of CFTRinh-1, CFTRinh-5 and CFTRinh-7 was analyzed in vitro and in vivo, respectively. The cytotoxicity of CFTRinh-1, CFTRinh-5 and CFTRinh-7 was determined by dihydrorhodamine method. The results showed that the CFTRinh-1, CFTRinh-5 and CFTRinh-7 we found had no obvious toxicity to FRT cells when they were co-incubated for 24 hours at a concentration of 100 μM.

Give mice intraperitoneal injection of 1 mg/kg body weight of the above-mentioned compound, inject once a day, continuously inject 7 days or a one-time high dose (10mg/kg) intraperitoneal injection, the mouse diet and water intake are normal, serum electrolytes, glucose concentration, Liver function index, serum creatine, amylase, hematocrit, etc. had no significant difference with normal control mice.

7. CFTR inhibitor multidrug resistance activity (MDR-1) analysis results

Since CFTR is structurally homologous to the ABC (ATP-binding cassette, ABC) transporter MDR-1, we studied the inhibitory effects of CFTRinh-1, CFTRinh-5 and CFTRinh-7 on MDR-1. We determined the activity of multidrug resistance protein-1 (MDR-1) by measuring the accumulation degree of ³ H-vincristine in 9HTEo-/Dx of human bronchial cells. The specific method is to induce 9HTEo-/Dx cells to express MDR-1 with doxorubicin (Rasola et al.1994 J.Biol.Chem.269:1432-1436), and then spread the cells on a 24-well plate at an amount of 200,000/well After 48 hours, the above cells were washed with washing solution (containing 130mM NaCl, 2mM KCl, 1mM KH ₂ PO ₄ , 2mM CaCl ₂ , 2mM MgCl ₂ , 10mM Na-HEPES pH7.3) and mixed with 200μl ³ H-vinca A solution of neobase (0.7 μM; 1 μCi/ml) and the sample to be tested was incubated at 37° C. for 1 hour. Finally, the above-mentioned cells were washed three times with pre-cooled washing solution, and the radiation dose was measured after the cells were lysed with 0.25M NaOH. When CFTRinh-1, CFTRinh-5 and CFTRinh-7 were at 5 μM, they had no inhibitory effect on the translocation activity of MDR-1 to vincristine.

Example 4

This example describes the experimental method and results of small-molecule inhibitors of CFTR inhibitors inhibiting cholera toxin-induced secretion in the small intestine of mice.

The experimental animals were fasted (without water) for 12 hours and then anesthetized. Three 2-10 cm long nodes were ligated in the ileum during the operation. During enteral administration, inject 0.2-2 milliliters of phosphate buffer solution into each segment of the intestinal cavity, containing the following components: the first segment (without other components); the second segment (containing 10-100 micrograms of CFTRinh-1 or CFTRinh- 5 or CFTRinh-7); the third paragraph (containing 1-10 micrograms of cholera toxin and 10-100 micrograms of CFTRinh-1 or CFTRinh-5 or CFTRinh-7); the fourth paragraph (containing 1-10 micrograms of cholera toxin and 10- 100 micrograms of CFTRinh-1 or CFTRinh-5 or CFTRinh-7 inactive analog); fifth paragraph (containing 1-10 micrograms of cholera toxin). After the operation, the abdominal cavity was closed, the animals were woken up, and allowed to move freely. Animals were sacrificed at 6 hours, and each section of small intestine was taken out, weighed and measured for length, and the weight/length ratio was calculated. It was found that after 6 hours, the fluid accumulated in the small intestine injected with cholera toxin, causing the intestinal segment to be extremely distended, and the fluid accumulation in the small intestine of the experimental groups injected with cholera toxin and CFTRinh-1, CFTRinh-5 and CFTRinh-7 was reduced More than 80%.

For intravenous administration, the experimental animals were divided into two groups, and two 2-10 cm long knots were respectively ligated in the ileum of all animals. Inject 0.2-2 ml of phosphate buffer solution containing 1-10 micrograms of cholera toxin into each intestinal cavity respectively, and close the abdominal cavity after the operation to maintain the anesthesia state of the animals. From 2 hours after the operation, the animals in the first group were intravenously infused with CFTRinh-1 or CFTRinh-5 or CFTRinh-7 saline solution at 0.25 mg/kg body weight, and the animals in the second group were infused with inactive CFTRinh-1 or CFTRinh -5 or CFTRinh-7 analog saline solution was dripped over 4 hours. Each section of small intestine was removed, weighed and measured for length, and the weight/length ratio was calculated. It was found that fluid accumulation in the small intestine was reduced by more than 60% in the group given intravenous CFTRinh-1 or CFTRinh-5 or CFTRinh-7.

Example 5

This example describes the experimental method and results of the therapeutic effect of CFTR inhibitor small molecule inhibitor on porcine infectious diarrhea

This topic carried out a field anti-diarrhea study in Chengxi breeding pig farm in Changchun City. At that time, there was a severe epidemic of yellow scour in piglets in this kind of pig farm, which mainly occurred in newborn piglets 3-10 days after birth, and most of them showed the disease in the whole litter. Sick pigs showed watery diarrhea, rapid emaciation and severe dehydration, and died 3-7 days after onset. Litter mortality is as high as 50-100%. The study showed that the intramuscular injection of synthetic CFTRinh-1 or CFTRinh-5 or CFTRinh-7 (0.5 mg/kg) alone in 10 newborn piglets with infectious diarrhea and severe dehydration resulted in 8 sick pigs passing the critical period And recovery, the curative effect is extremely remarkable. Only 2 out of 10 piglets injected with DMSO survived.

In addition, during the high-incidence period of piglet infectious diarrhea, 33 randomly selected litters of 33 piglets were given preventive medication at 0.25 mg/kg once a day for 7 consecutive days. As a result, none of the piglets developed diarrhea within 30 days. Blood biochemical analysis showed no significant difference in liver function, kidney function, electrolytes, blood sugar, and total plasma protein between the medication group and the DMSO control group, indicating that there was no obvious toxic effect in vivo.

Example 6

This example describes the experimental method and results of CFTR inhibitor small molecule inhibitor induced porcine cystic fibrosis model

Give newborn piglets intramuscular injection of CFTRinh-1 or CFTRinh-5 or CFTRinh-7 at a dose of 0.25 mg/kg body weight, twice a day (12 hours apart), continuous injection administration. Continuous administration for 90 days. Lung and bronchial submucosal glands present pathological changes similar to cystic fibrosis symptoms.

Example 7

This example describes the experimental methods and results of the effects of small molecule inhibitors of CFTR inhibitors on the cAMP-stimulated fluid secretion rate of primary cultured ADPKD cyst lining epithelial cells.

Primary culture of ADPKD cyst lining epithelial cells: The isolated ADPKD mouse (Pkd2 ^WS25/-type ) and human renal cyst tissues (polycystic kidney tissue and isolated single cysts were obtained from clinically accepted kidney transplantation or due to complications) Partially or totally resected diseased kidneys, the patient’s consent should be obtained in advance. Generally, specimens can be obtained 1-2 times a month) The cyst cavity was washed 3 times with PBS, enzymatically digested with XIV protease at 4°C overnight, and then treated with serum-free DME- The cyst cavity was washed with F12 culture solution, and the cyst lining epithelial cells were collected. The above-mentioned epithelial cells were continued in serum-free DME-F12 medium (containing 2% fetal bovine serum, insulin, transferrin, ethanolamine, phosphoethanolamine, hydrocortisone, triiodothyronine, retinoic acid, bovine pituitary extract) for culture and expansion.

Measurement of CFTR-mediated Cl ^- current in ADPKD cyst lining epithelial cells: The above cells were trypsinized, plated on a permeable carrier (Transwell-COL, Costar) at a high density and cultured for 24 hours, and then the bottom culture medium was replaced with DME -F12 mixed culture medium (containing 2% fetal bovine serum, insulin, transferrin, ethanolamine, phosphoethanolamine, hydrocortisone, triiodothyronine, retinoic acid, bovine pituitary extract), top surface Continue culturing in air for about 10 days to obtain a polar monolayer columnar epithelial cell layer. The CFTR-mediated Cl ^- current was measured using a Ussing chamber by placing the Snapwell of a monolayer of ADPKD epithelial cells in a polar growth state into a Ussing chamber (Physiologic Instruments, Inc). Then add 130mM NaCl, 2.7mM KCl, 1.5mM KH ₂ PO4, 1mM CaCl ₂ , 0.5mM MgCl ₂ , 10mM glucose, 10mM Na-Hepes (pH7.3) and different concentrations of CFTRinh-1 or CFTRinh to one side of the basement membrane -5 or CFTRinh-7 solution, and the epithelial membrane side was added containing 65mM sodium gluconate, 65mM NaCl, 2.7mM KCl, 1.5mM KH ₂ PO ₄ , 2mM CaCl ₂ , 0.5mM MgCl ₂ , 10mM glucose, 10mM Na-Hepes ( pH7.3) solution. The system was continuously ventilated with air at 37°C, and then the basement membrane was permeabilized with 250 μg/ml amphotericin B. During the measurement, the Ussing cell was connected to the DVC-1000 voltage clamp (WorldPrecision Instruments), and the short-circuit current between the Ag/AgCl electrode and the 1M KCl agarose bridge was measured. It was found that CFTRinh-1, CFTRinh-5 and CFTRinh-7 can all rapidly Effectively inhibit the short-circuit current of FRT cells, and the inhibitory activity is positively correlated with its concentration.

Measurement of fluid secretion of epithelial cells lining ADPKD cysts: When measuring transepithelial fluid secretion by filter paper method, cAMP and different concentrations of CFTR-specific inhibitor CaCl ₂ were added to the culture medium at the bottom of the polar single-layer columnar epithelial cell layer obtained above. , 0.5mM MgCl ₂ , 10mM glucose, 10mM Na-Hepes (pH7.3) and different concentrations of CFTRinh-1 or CFTRinh-5 or CFTRinh-7. The study found that CFTRinh-1, CFTRinh-5 and CFTRinh-7 can rapidly and effectively inhibit the fluid secretion of ADPKD cyst lining epithelial cells.

Example 8

This example describes the experimental method and results of the effect of small molecule inhibitors of CFTR inhibitors on inhibiting the fluid secretion rate of renal cyst tissue of ADPKD patients cultured in vitro.

Fresh ADPKD kidneys were preserved in pre-cooled DME-F12 tissue culture medium, and cystic tissue with a volume weight of 5-50 g on the surface of the kidney was isolated. The cyst cavity fluid was completely sucked out with a sterile syringe, the fluid volume in each cyst was accurately recorded, and the cysts were divided into A, B, and C groups. In group A, 1/3 volume of mixed cyst cavity fluid was reinjected; in group B, 1/3 volume of mixed cyst cavity fluid containing 10mol/L CFTRinh-1 or CFTRinh-5 or CFTRinh-7 was injected; group C cyst Inject 1/3 volume of mixed cyst cavity fluid containing 10mol/L CFTRact-09 (a CFTR specific activator). The cysts of the three groups A, B, and C above were respectively placed in DME-F12 tissue culture medium containing 10mol/L forskolin (containing 5% fetal bovine serum, insulin, transferrin, selenium, cortisol, triiodothyronine acid, penicillin and streptomycin), in a carbon dioxide incubator (37 ° C, humidity of 90%, _CO2 concentration of 5%, air concentration of 95%) to measure the change of cyst weight in each group after culturing for 24 hours. The change in cyst weight was divided by the surface area of the cyst to obtain the rate of fluid secretion per unit surface area of the cyst. Taking group A as the control, it was found that CFTRinh-1, CFTRinh-5 and CFTRinh-7 could all significantly inhibit the fluid secretion rate of the cyst.

Claims (3)

1. The application of pyrimidine (thio) ketones in pharmacy, which is characterized in that the pyrimidine (thio) ketones are used in the preparation of drugs for the treatment of secretory diarrhea and autosomal dominant polycystic kidney disease. 2. Preparation of pyrimidine (thio) ketone compounds in dosage forms for treating secretory diarrhea and autosomal dominant polycystic kidney disease, characterized in that pyrimidine (thio) ketone compounds can be mixed with suitable pharmaceutical carriers, diluents, Excipients or adjuvants in solid, semi-solid, liquid or gaseous form: i.e. tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols; pyrimidine (thio) ketones Can be administered through a variety of routes: including oral, oral, rectal, intravenous, subperitoneal, subcutaneous, airway; pyrimidine (thio) ketone compounds can also be in the form of their pharmaceutically acceptable salts, which can be used alone or in combination or with other drugs The active compounds are co-administered. 3. A method for establishing a porcine cystic fibrosis model, which is characterized by intramuscularly injecting CFTRinh-1 or CFTRinh-5 or CFTRinh-7 into newborn piglets at a dose of 0.25 mg/kg body weight, twice a day with an interval of 12 hours, continuously After 90 days of injection, the lungs and bronchial submucosal glands showed pathological changes similar to those of cystic fibrosis.

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