WO2019245010A1 - Therapeutic agent for cystic fibrosis - Google Patents

Abstract

The present invention addresses the problem of providing a medication which is for cystic fibroses that develop through the mutation of a cystic fibrosis transmembrane conductance regulator (CFTR) that is a type of chloride channel, and which is capable of expressing a therapeutic effect for any cystic fibrosis from Class I to VI, regardless of the mutation type of CFTR. The present invention provides a medication/treatment method characterized by administering a combination of: (a) a compound represented by formula (I) or a pharmaceutically acceptable salt thereof; and (b) one or more drugs individually selected from the group consisting of a CFTR function improving medicine and an ENaC inhibitor.

Description

Cystic fibrosis drug

The present invention has a G protein-coupled receptor 39 (G protein-coupled receptor 39, which may be abbreviated as GPR39 in the present specification) agonistic action, and activates a calcium-dependent chloride channel (GPR39) through activation of GPR39. Calcium-activated {Chloride} Channel, which may be abbreviated as CaCC in the present specification) and a compound having an effect of improving the function of Cyclic, Fibrosis, Transmembrane, conductance, and Regulator (which may be abbreviated as CFTR in this specification). A drug (preferably a drug) which is combined with a drug or a drug which inhibits epithelial sodium @ channel (may be abbreviated as ENaC in this specification). The pharmaceutical) or therapeutic methods for the treatment of cystic fibrosis.

Cystic fibrosis is a serious hereditary disease caused by a genetic mutation in CFTR, a type of chloride channel, and it is estimated that there are more than 70,000 patients worldwide. In recent years, although treatments have been developed, the average life expectancy is about 40 years, so that a satisfactory treatment has not yet been established.

CFTR is a major cAMP-dependent anion channel expressed on the luminal membrane of whole epithelial cells. When the function of this channel is reduced by gene mutation, the transport of ions and water through the epithelium / mucosa is impaired in the respiratory tract, intestinal tract, pancreatic duct, bile duct, sweat duct, etc., resulting in excessive mucus / secretion in the lumen. , Causing luminal obstruction and susceptibility to infection. Particularly severe pulmonary damage, most cystic fibrosis patients die from respiratory failure due to tracheal obstruction and respiratory infections. Since CFTR was identified as a causative gene for cystic fibrosis, studies on cystic fibrosis have progressed, and it has been reported that about 1900 or more mutations exist at present. Of these mutations, they are classified into six classes according to the CFTR dysfunction caused by the mutations (class I; nonsense mutation, class II; protein misfolding mutation, class III; mutation in abnormal channel opening, class IV; channel conductance). Mutations, class V; mutations that decrease the amount of CFTR produced, class VI; mutations that decrease the stability of CFTR), and particularly the class I, class II, and class III mutations, since the chloride ion is hardly secreted, and the symptoms are severe. The unmet medical needs are very high.

Conventional cystic fibrosis treatment has mainly been symptomatic treatments such as ibuprofen and antibacterial agents, but in recent years, Kalydeco (registered trademark) (Ivacaftor (CFTR potentiaor)), Orkambi (registered trademark) (Ivacaftor (CFTR potentiator)) Drugs that directly act on CFTR, such as a combination of Lumacaftor (CFTR collector) and Symdeko (registered trademark) (a combination of Ivacaftor (CFTR potentator) and Tezacaftor (CFTR collector)), have been launched to improve the function of CFTR. . (Note that, in this specification, Ivacaftor may be referred to as VX-770 and Lumacaftor may be referred to as VX-809.) However, Kalydeco is a class III and class IV mutation in some patients, and Orkambi is a class II. Among the mutations, Symdeko has an effect on the ΔF508 homozygous mutation and only on some patients with the ΔF508 homozygous mutation or the class III and class IV mutations. Orkambi and Symdeko were confirmed to have a statistically significant respiratory function-improving effect, but their efficacy was limited, and there were many patients who did not yet have a sufficient therapeutic drug, centering on the class I and class II mutations. . One of the methods for solving these problems is to open a chloride channel other than CFTR to substitute for a malfunction of CFTR. If a compound having such a profile can be obtained, it is considered that all cystic fibrosis patients can be treated theoretically without depending on the mutation of CFTR. In addition, since they open chloride channels other than CFTR, they can be used in combination with existing CFTR agents such as Kalydeco (registered trademark), Orkambi (registered trademark), and Symdeko (registered trademark) to further enhance the efficacy of the drug. There is expected. This idea is based on the concept that Denufosol, which is a P2Y ₂ (a type of G protein-coupled receptor (GPCR)) agonist, opens a chloride channel, Calcium-activated Chloride Channel (CaCC), which is different from CFTR. Although it was being developed, its development has been suspended for reasons such as Denfofosol being unstable in the lungs of patients (Non-Patent Document 1). In addition, a low-molecular compound that directly acts on CaCC and activates it has been screened and reported to be promising as a therapeutic agent for cystic fibrosis (Non-Patent Document 2). It is unknown. As described above, the preceding therapeutic drug for cystic fibrosis is only effective for patients with limited mutations, and the drug efficacy against Orkambi or Symdeko's ΔF508 homozygous mutation is not sufficient. Is eagerly awaited. Although there has been a therapeutic concept of opening a chloride channel other than CFTR and substituting a CFTR dysfunction, a compound showing a medicinal effect in clinical practice has not been obtained at present.

Most cystic fibrosis patients die from respiratory failure due to tracheal obstruction or infection of the respiratory tract, indicating that respiratory function may be improved. Become. On the other hand, since there is no animal model that reflects pulmonary dysfunction of cystic fibrosis, in-vitro drug efficacy evaluation is mainly performed. In-vitro drug efficacy evaluation is based on the point that a three-dimensional culture system using airway epithelial cells derived from a cystic fibrosis patient (Air-Liquid Interface Interfacial Assay; ALI assay) absorbs water quickly and hardly secretes water. Since it is close to the lung condition of cystic fibrosis patients, it is widely used as a drug efficacy evaluation system. In fact, Kalydeco moved water in the ALI assay (Non-patent Document 3), and was developed as a therapeutic agent for cystic fibrosis, and its clinical efficacy was confirmed (Non-patent Document 4).

On the other hand, GPR39 (G protein-coupled receptor 39) is a member of the ghrelin receptor family and has been reported to be expressed in the digestive tract, pancreas, liver, kidney, adipose tissue, thyroid, heart, lung, and the like. . The ligand for GPR39 has been unknown for a long time, but since it is a member of the ghrelin receptor family, the ligand was presumed to be a peptide, and there were times when obestatin was considered to be a natural ligand. However, in recent reports, obestatin is not a GPR39 ligand, but zinc (Zn ²⁺ ) is said to be a GPR39 ligand.

GPR39 is expressed in mouse @ intestinal @ fibroblast-like @ cells, and it has been reported that GPR39 activation is associated with CaCC activation (Non-Patent Document 5), but cells derived from cystic fibrosis patients have been reported. Is unknown, and there is no report that a compound capable of activating GPR39 and continuously opening CaCC has been obtained.

AZ7914, AZ4237, AZ1395 (Non-Patent Document 6), pyridylpyrimidine compound (Non-Patent Document 7) and the like have been reported as GPR39 agonists.

The present inventors have found that, in cystic fibrosis caused by CFTR mutation, if a chloride channel different from CFTR that causes the disease can be opened, the dysfunction of CFTR can be substituted, leading to treatment. As a result of intensive studies, they have found that a pyrimidine derivative having a specific chemical structure opens CaCC via GPR39 agonism and is effective in treating cystic fibrosis independently of CFTR. Then, they have found that when the pyrimidine derivative is administered in combination with a CFTR function improving agent or an ENaC inhibitor, the pyrimidine derivative exerts an excellent effect on the treatment of cystic fibrosis, thereby completing the present invention.

The present invention relates to the following.
[1] (a) Formula (I):

Or a pharmaceutically acceptable salt thereof and (b) at least one drug independently selected from the group consisting of CFTR function improving agents and ENaC inhibitors. Medicine.
[2] The medicament according to [1], for treating cystic fibrosis.
[3] at least one drug independently selected from the group consisting of (a) a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, and (b) a CFTR function improving agent and an ENaC inhibitor. The medicament according to [1] or [2], wherein the medicament is contained as active ingredients of different preparations, and is administered at the same time or at different times.
[4] (a) a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof and (b) at least one drug independently selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor; The medicament according to [1] or [2], which is contained in a single preparation.
[5] (a) a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof and (b) at least one drug independently selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor The medicament according to [1] or [2], which is a kit preparation containing the medicament.
[6] The method according to any one of [1] to [5], which is for a patient who has been treated with at least one drug independently selected from the group consisting of a CFTR function improving drug and an ENaC inhibitor. Medicine.

[7] At least one drug independently selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor is a CFTR function improving agent, wherein the CFTR function improving agent is CFTR potentiator, CFTR collector, or CFTR amplifier. The medicament according to any one of [1] to [6], which is at least one kind independently selected from the group consisting of RNA therapy and Gene therapy.
[8] At least one drug independently selected from the group consisting of a CFTR function improver and an ENaC inhibitor is a CFTR function improver, and the CFTR function improver is a group consisting of CFTR potentiator and CFTR collector The medicament according to any one of [1] to [6], which is at least one kind independently selected from:
[9] At least one drug independently selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor is a CFTR function improving agent, wherein the CFTR function improving agent is Ivacaftor, QBW251, VX-561. PTI-808, GLPG1837, GLPG2451, GLPG3067, Lumacaftor, Tezacaftor, VX-445, VX-440, VX-152, VX-659, FDL169, GLPG2222, GLPG2851, GLPG2737, PTI-801, PTI-801, PTI-801, PTI-801, PTI-801 The medicament according to any one of [1] to [6], which is at least one independently selected from the group consisting of:
[10] At least one drug independently selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor is a CFTR function improving agent, and the CFTR function improving agent is a group consisting of Ivacaftor, Lumacaftor and Tezacaftor. The medicament according to any one of [1] to [6], which is at least one kind independently selected from:
[11] At least one drug independently selected from the group consisting of CFTR function improving agents and ENaC inhibitors is an ENaC inhibitor, and the ENaC inhibitor is selected from the group consisting of QBW276, SPX-101 and AZD5634 The medicament according to any one of [1] to [6], which is at least one selected from the group consisting of:
[12] The medicament according to any one of [1] to [11], wherein the pharmaceutically acceptable salt is a magnesium salt, a calcium salt, a zinc salt, a sodium salt, a tert-butylamine salt or a diisopropylamine salt.
[13] The compound represented by the formula (I) or a pharmaceutically acceptable salt thereof is bis [5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid] magnesium salt. The medicament according to any one of [1] to [11].
[14] (b) Formula (I) for administration in combination with at least one drug independently selected from the group consisting of CFTR function improving agents and ENaC inhibitors:

Or a pharmaceutically acceptable salt thereof.
[15] The compound of [14] or a pharmaceutically acceptable salt thereof for the treatment of cystic fibrosis.
[16] The compound of [14] or [15] or a pharmaceutical thereof according to [14], which is intended for a patient who has been treated with at least one drug independently selected from the group consisting of a CFTR function improving drug and an ENaC inhibitor. Above acceptable salts.
[17] At least one drug independently selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor is a CFTR function improving agent, wherein the CFTR function improving agent is CFTR potentiator, CFTR collector, or CFTR amplifier. The compound according to any one of [14] to [16], which is at least one kind independently selected from the group consisting of RNA therapy and Gene therapy, or a pharmaceutically acceptable salt thereof.
[18] At least one drug independently selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor is a CFTR function improving agent, wherein the CFTR function improving agent is a group consisting of CFTR potentiator and CFTR collector. Or the pharmaceutically acceptable salt thereof according to any one of [14] to [16], which is at least one member independently selected from the group consisting of:
[19] At least one drug independently selected from the group consisting of CFTR function improvers and ENaC inhibitors is a CFTR function improver, wherein the CFTR function improver is Ivacaftor, QBW251, VX-561. PTI-808, GLPG1837, GLPG2451, GLPG3067, Lumacaftor, Tezacaftor, VX-445, VX-440, VX-152, VX-659, FDL169, GLPG2222, GLPG2851, GLPG2737, PTI-801, PTI-801, PTI-801, PTI-801, PTI-801 The compound or the pharmaceutically acceptable salt thereof according to any one of [14] to [16], which is at least one kind independently selected from the group consisting of:
[20] At least one drug independently selected from the group consisting of a CFTR function-improving agent and an ENaC inhibitor is a CFTR function-improving agent, wherein the CFTR function-improving agent is a group consisting of Ivacaftor, Lumacaftor, and Tezacaftor. Or the pharmaceutically acceptable salt thereof according to any one of [14] to [16], which is at least one member independently selected from the group consisting of:

[21] At least one drug independently selected from the group consisting of CFTR function improving agents and ENaC inhibitors is an ENaC inhibitor, and the ENaC inhibitor is selected from the group consisting of QBW276, SPX-101 and AZD5634. Or the pharmaceutically acceptable salt thereof according to any one of [14] to [16].
[22] The compound according to any one of [14] to [21], wherein the pharmaceutically acceptable salt is a magnesium salt, a calcium salt, a zinc salt, a sodium salt, a tert-butylamine salt or a diisopropylamine salt. A pharmaceutically acceptable salt thereof.
[23] The compound represented by the formula (I) or a pharmaceutically acceptable salt thereof is magnesium bis [5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid]. The compound according to any one of [14] to [21] or a pharmaceutically acceptable salt thereof.
[24] (a) Formula (I):

Or a pharmaceutically acceptable salt thereof and (b) at least one drug independently selected from the group consisting of CFTR function improving agents and ENaC inhibitors. How to treat the disease.
[25] The treatment method according to [24], wherein the disease is cystic fibrosis.
[26] A compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, and (b) at least one drug independently selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor: The therapeutic method according to [24] or [25], wherein the therapeutic agents are contained as active ingredients of different preparations, and are administered at the same time or at different times.
[27] at least one drug independently selected from the group consisting of (a) a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, and (b) a CFTR function improving agent and an ENaC inhibitor The therapeutic method according to [24] or [25], which is contained in a single preparation.
[28] At least one drug independently selected from the group consisting of (a) the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, and (b) a CFTR function improving agent and an ENaC inhibitor The therapeutic method according to [24] or [25], which is a kit preparation containing them.
[29] The method according to any one of [24] to [28], which is intended for a patient who has been treated with at least one drug independently selected from the group consisting of a CFTR function improving drug and an ENaC inhibitor. Treatment method.
[30] At least one drug independently selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor is a CFTR function improving agent, wherein the CFTR function improving agent is CFTR potentiator, CFTR collector, or CFTR amplifier. The treatment method according to any one of [24] to [29], which is at least one kind independently selected from the group consisting of RNA therapy and Gene therapy.
[31] At least one drug independently selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor is a CFTR function improving agent, wherein the CFTR function improving agent is a group consisting of CFTR potentiator and CFTR collector. The therapeutic method according to any one of [24] to [29], which is at least one kind independently selected from:

[32] At least one drug independently selected from the group consisting of CFTR function improvers and ENaC inhibitors is a CFTR function improver, wherein the CFTR function improver is Ivacaftor, QBW251, VX-561. PTI-808, GLPG1837, GLPG2451, GLPG3067, Lumacaftor, Tezacaftor, VX-445, VX-440, VX-152, VX-659, FDL169, GLPG2222, GLPG2851, GLPG2737, PTI-801, PTI-801, PTI-801, PTI-801, PTI-801 The treatment method according to any one of [24] to [29], which is at least one kind independently selected from the group consisting of:
[33] At least one drug independently selected from the group consisting of CFTR function improvers and ENaC inhibitors is a CFTR function improver, and the CFTR function improver is a group consisting of Ivacaftor, Lumacaftor and Tezacaftor. The therapeutic method according to any one of [24] to [29], which is at least one kind independently selected from:
[34] At least one drug independently selected from the group consisting of CFTR function improving agents and ENaC inhibitors is an ENaC inhibitor, and the ENaC inhibitor is selected from the group consisting of QBW276, SPX-101 and AZD5634 The therapeutic method according to any one of [24] to [29], which is at least one selected from the group consisting of:
[35] The method according to any one of [24] to [34], wherein the pharmaceutically acceptable salt is a magnesium salt, a calcium salt, a zinc salt, a sodium salt, a tert-butylamine salt or a diisopropylamine salt. .
[36] The compound represented by the formula (I) or a pharmaceutically acceptable salt thereof is bis [5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid] magnesium salt The therapeutic method according to any one of [24] to [34].
[37] (a) a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, and (b) at least one drug independently selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor The therapeutic method according to any one of [24] to [36], wherein the administered subject is a warm-blooded animal.
[38] The treatment method according to [37], wherein the warm-blooded animal is a human.

化学 The chemical name of the compound of the above formula (I) of the present invention is 5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid.

に お い て In the present invention, “a pharmaceutically acceptable salt” refers to a salt that has no significant toxicity and can be used as a medicament. Since the compound represented by the formula (I) of the present invention has an acidic group, it can be converted to a salt by reacting with a base.

Such salts include, for example, alkali metal salts such as sodium salt, potassium salt and lithium salt, alkaline earth metal salts such as calcium salt and magnesium salt, metal salts such as aluminum salt, iron salt and zinc salt. Inorganic salts such as ammonium salts, tert-butylamine salts, tert-octylamine salts, diisopropylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts Guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzylphenethylamine salt, piperazine salt, tetramethylammonium And organic amine salts such as tris (hydroxymethyl) aminomethane salt; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate and aspartate. It is not limited to these.

Examples of preferred salts include magnesium salt, calcium salt, zinc salt, sodium salt, tert-butylamine salt and diisopropylamine salt.

化合物 The compound represented by the formula (I) of the present invention can be combined with the above-mentioned acid or base in any ratio to form a salt. For example, hydrochloride includes salts that can be formed, such as monohydrochloride, dihydrochloride, trihydrochloride, and magnesium salt includes salts that can be formed, such as monomagnesium, マ グ ネ シ ウ ム magnesium. In the compound names of the present invention, "monoacid salt" and "monobasic salt" may be abbreviated as "acid salt" in which "one" is omitted. “Hydrochloride” and “monomagnesium salt” may be indicated as “magnesium salt”.

The compound represented by the formula (I) of the present invention or a pharmaceutically acceptable salt thereof may be left in the atmosphere or recrystallized to absorb adsorbed water or take in water molecules. , And hydrates, and such hydrates are also included in the compounds of the present invention or pharmaceutically acceptable salts.

The compound of the present invention represented by the formula (I) or a pharmaceutically acceptable salt thereof absorbs a certain solvent by being left in a solvent or recrystallized in the solvent, It may be possible to form a solvate, and such a solvate is also included in the compound of the present invention or a pharmaceutically acceptable salt. The solvent capable of forming a solvate is not particularly limited as long as it does not have remarkable toxicity and can be used as a medicine. For example, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, Butanol, acetone, methyl ethyl ketone, methyl isobutyl ketone, dimethyl sulfoxide, ethyl formate, ethyl acetate, propyl acetate, diethyl ether, tetrahydrofuran, formic acid, acetic acid, pentane, heptane, cumene, anisole and the like.

Each of the hydrates or solvates formed by combining the compound represented by the formula (I) of the present invention or a pharmaceutically acceptable salt thereof with water or a solvent in any ratio, or a mixture thereof, Included in the invention. For example, hydrates that can be formed, such as monohydrate, dihydrate, hemihydrate, 3/2 hydrate, and solvates, disolvates, and 1/2 solvents Solvates that can be formed, such as solvates, 3/2 solvates, are encompassed by the present invention. In the compound name of the present invention, when `` hydrate '' or `` solvate '' is described without specifying the hydration number or solvation number, any number of hydrates or solvates is included. It is.

More specific preferred examples of the compound represented by the formula (I) of the present invention or a pharmaceutically acceptable salt thereof include 5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2- Examples include carboxylic acid or its magnesium salt, calcium salt, zinc salt, sodium salt, tert-butylamine salt or diisopropylamine salt. These may be in the form of hydrates.

More specific one embodiment of the compound of the present invention includes 5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid hydrate, magnesium salt hydrate, calcium salt hydrate, Zinc salt hydrate, sodium salt anhydride, tert-butylamine salt anhydride or diisopropylamine salt anhydride, more preferably hydrate or magnesium salt hydrate.

More preferably, bis [5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid] monomagnesium salt dihydrate or 5- (2-chlorobenzyl) -4- Methoxy-6-methylpyrimidine-2-carboxylic acid 3/2 hydrate.

に お い て In the present specification, a CFTR function improving drug is a drug that directly acts on CFTR dysfunction caused by mutation of CFTR that is a causative gene of cystic fibrosis and improves the function thereof. CFTR function improving drugs include CFTR @ potentiator, CFTR @ corrector, CFTR @ amplifier, RNA therapy and Gene therapy.

{For example, CFTR} potentiators act on and open CFTR with class III and class IV mutations, thereby improving its function. Representative examples include Ivacaftor (VX-770); QBW251 (Am \ J \ Respir \ Crit \ Care \ Med .; 193: A7789 (2016)); VX-561 (J \ Pharmacol \ Exp \ Ther .; 362: 359-67 (2017)); PTI-808 (Cystic Fibrosis Foundation 2017 ANNUAL REPORT); GLPG1837 (J Med Chem .; 61: 1425-1435 (2018)); GLPG2451 or GLPG 3067 (J Cyst Fibros.); Compounds described in International Publication WO2015 / 018823; compounds described in International Publication WO2018 / 073175.

ＣＦFurthermore, CFTR corrector normalizes the dysfunction by expressing CFTR having class II mutation on the cell surface. Representative examples include Lumacaftor (VX-809); Tezacaftor; VX-445, VX-440, VX-152 or VX-659 (J \ Cyst \ Fibros .; 17 (S2): S52-60 (2018)); FDL169 ( 27 (2018)); GLPG2222 (J Med Chem .; 61: 1436-1449 (2018)); GLPG2851 or GLPG2737 (JCyst Fibros .; 17 (S2): S52-60 (2018)); Compounds described in International Publication WO2015 / 018823; Compounds described in International Publication WO2018 / 073175; PTI-801 (J Cyst Fibros .; 17 (S2): S52-60 (2018), Cystic} ibrosis Foundation 2017 ANNUAL REPORT) and the like.

ＣＦCFTR amplifier normalizes dysfunction by enhancing the expression of CFTR protein. Representative examples include PTI-428 (J @ Cyst Fibros .; 17 (S3): S1-2 (2018), J @ Transl @ Med .; 15:84 (2017), Cystic @ Fibrosis \ Foundation 2017 2017 ANNUAL REPORT, N- [trans. -3- [5-[(1R) -1-hydroxyethyl] -1,3,4-oxadiazol-2-yl] cyclobutyl] -3-phenyl-1,2-oxazole-5-carboxamide, International Publication WO2016 / 105485, and the like.

ＲＮＡRNA therapy also restores the expression of mRNA encoding CFTR and normalizes its dysfunction by enhancing the expression of CFTR protein. Representative examples include Eluforsen (QR-010, J Transl Med .; 15:84 (2017), Cyclic Fibrosis Foundation 2017 ANNUAL REPORT), the antisense oligonucleotide described in International Publication WO2014 / 011053, MRT5005FyCy 17 (S3): S16 (2018), Cyclic Fibrosis Fundation 2017 2017 ANNUAL REPORT, and the like.

In addition, Gene therapy functions by introducing a gene encoding CFTR using a virus or a non-viral vector, or by repairing an abnormality of the gene encoding CFTR by a genome editing technique such as CRISPR / Cas9. Normalize the abnormalities (Front Pharmacol .; 9: 396 (2018), J @ Transl Med .; 15:84 (2017)).

In the present invention, particularly preferred examples of the CFTR function improving agent are at least one kind independently selected from the group consisting of CFTR @ potentiator and CFTR @ corrector. A preferred specific example is at least one independently selected from the group consisting of Ivacaftor, Lumacaftor and Tezacaftor.

Furthermore, two or more CFTR function-improving agents may be used in combination. For example, Orkambi (registered trademark) which is a combination of Ivacaftor and Lumacaftor, Symdeko (registered trademark) which is a combination of Tezacaftor and Ivacaftor, VX -445 and Tezacaftor and Ivacaftor the mixture (Cystic Fibrosis Foundation 2017 ANNUAL REPORT), mixture of VX-440 and Tezacaftor and Ivacaftor, VX-152 and Tezacaftor and Ivacaftor the mixture, VX-659 and Tezacaftor and Ivacaftor the mixture (J Cyst Fibros .; 17 (S3): S3 (2018), Cystic Fibrosis} compound, described in International Publication No. WO2017 / 060880, a combination of GLPG2451, GLPG2222 and GLPG2737, a combination of PTI-428, PTI-801 and PTI-808 (Expert Rev. MediGed.Dev. 3: 107-117 (2018)) is also preferably used.

In the present specification, the ENaC inhibitor inhibits the opening of ENaC (epithelial sodium channel) expressed on the surface of epithelial cells, or inhibits the absorption of sodium ions by internalizing ENaC expressed on the surface of epithelial cells. It is a drug. Representative examples include QBW276 (Cystic Fibrosis Foundation 2017 ANNUAL REPORT), Bioorg Med Chem Chem Lett. 22 (2): 929-932 (2012); Bioorg {Chem} Lett. 22 (8): 2877-2879 (2012), the compound described in SPX-101 (Am J Respir Crit Care Med .; 196: 671-672 (2017)), the peptide described in International Publication WO2016 / 057795, AZD5634 (J Transl Med .; 15:84 (2017), Cyclic Fibrosis Foundation 2017 ANNUAL REPORT), and the compounds described in International Publication No. WO 2015/140527.

Furthermore, CFTR function improving drugs and ENaC inhibitors can be used in combination with one or more drugs.

The compound of the present invention represented by the formula (I) or a pharmaceutically acceptable salt thereof has a strong chloride ion secretion action through GPR39 agonist action and transfers water. Since this compound can open a chloride channel different from CFTR which causes disease, it exhibits an excellent therapeutic effect when combined with a CFTR function improving agent or an ENaC inhibitor. Therefore, the medicament / therapeutic method for treating cystic fibrosis by the combination of the compound of the present invention or a pharmaceutically acceptable salt thereof and a CFTR function-improving agent can be performed from class I to VI regardless of the type of CFTR mutation. The therapeutic effect can be exhibited in any of the above. A preferred treatment target is a patient with a class II ΔF508５０homozygous mutation, a class III mutation, and a class IV mutation in which an existing CFTR improving drug is used.

FIG. 1 is a graph showing changes in the expression level of human GPR39 (mRNA expression analysis) in CuFi-1 cells, a bronchial epithelial cell line derived from a cystic fibrosis patient, by siRNA treatment. The vertical axis represents the relative expression level relative to the control @ siRNA treatment group. The average value and standard deviation of N = 3 are shown. FIG. 2 is a graph showing the influence of each compound on chloride secretion activity by treatment with siRNA in CuFi-1 cells. The vertical axis represents the rate of increase when comparing the average value of the five fluorescence values from 110 to 120 seconds during the measurement for 120 seconds with the average value of the fluorescence values at 17 points from 1 to 34 seconds. ing. The average value and standard deviation of N = 8 are shown. FIG. 3 is a graph showing the water transfer effect of each compound in MucilAir-CF ^TM cells (lot number MD048502) having ΔF508 homozygous, which is a Class II mutation of CFTR. The vertical axis indicates the moisture remaining ratio in the upper layer. N = 3-20 means and standard deviation. Dunnett's multiple comparison test was performed with the group to which no compound was added, and the group having a p value of 0.01 or less is indicated by **. # Indicates that the p-value by the Student's t-test was 0.032 for the group without the compound and the group with the combination of VX-809 (30 μM) / VX-770 (1 μM). FIG. 4 is a graph showing the water transfer effect of each compound in MucilAir-CF ^™ cells (lot number MD020802) having 2184ΔA + W1282X, which is a Class I mutation of CFTR. The vertical axis indicates the moisture remaining ratio in the upper layer. It is shown by the average value and the standard deviation of N = 4. Dunnett's multiple comparison test was performed with the group to which no compound was added, and the group having a p value of 0.01 or less is indicated by **. ## indicates that the p-value by the Student's t-test was 0.0017 for the group of Example 1 (100 μM) and the group of VX-809 (30 μM) / VX-770 (1 μM). It indicates that there is. FIG. 5 is a graph showing the water transfer effect of each compound in MucilAir-CF ^TM cells (Lot No. MD062011) having N1303K heterozygous, which is a Class II mutation of CFTR. The vertical axis indicates the moisture remaining ratio in the upper layer. It is shown by the average value and the standard deviation of N = 4. Dunnett's multiple comparison test was performed with the group to which no compound was added, and the group having a p value of 0.01 or less is indicated by **. FIG. 6 shows a mixture of VX-809 (30 μM) / VX-770 (1 μM) alone and the compound of Example 1 in MucilAir-CF ^™ cells (lot No. MD048502) having ΔF508 homozygous which is a Class II mutation of CFTR. 4 is a graph showing a water transfer effect in combination with the above. The vertical axis indicates the moisture remaining ratio in the upper layer. N = 4-20 means and standard deviation. Dunnett's multiple comparison test was performed with the combination of VX-809 (30 μM) / VX-770 (1 μM) and the group with a p-value of 0.01 or less is indicated by **. # Indicates that the p-value by the Student's t-test was 0.032 for the group without the compound and the group with the combination of VX-809 (30 μM) / VX-770 (1 μM).

Next, a typical method for producing the compound represented by the formula (I) will be described. The compound of the present invention can be produced by various production methods, and the production methods shown below are examples, and the present invention should not be construed as being limited thereto. The compound represented by the formula (I) and a production intermediate thereof can be produced using various known reactions described below. At that time, the functional group may be protected with a suitable protecting group at the stage of the raw material or the intermediate. Examples of such a functional group include a hydroxyl group, a carboxy group, an amino group, and the like. The kind of the protecting group and the conditions for introducing and removing the protecting group are, for example, Protective Groups, Organic, Synthesis, Third Edition. (TW Green @ and PGM Wuts, John Wiley & Sons, Inc., New York) can be referred to.

[Production method 1]
The compound represented by the formula (I) (represented as compound 1a in the following reaction formula) can be produced, for example, by the following reaction formula.
[Wherein, G is a 2-chlorophenyl group, and R ¹ is a methyl group. Q ¹ is represents a methylene ^{group, P a, P b, P} c represents a protecting group. R ^2a is a methoxy group. ]

(1) Conversion of Compound 2a to Compound 3a Conversion of Compound 2a to Compound 3a can be performed by using a suitable solvent that does not adversely affect the reaction (eg, benzene, toluene, diethyl ether, dichloromethane, tetrahydrofuran, or N, N-dimethylformamide). Or a mixed solvent thereof) in a suitable base (eg, sodium hydride, sodium methoxide, potassium tert-butoxide, etc.) at −30 ° C. to the boiling point of the solvent used in the reaction, preferably at 0 ° C. to 100 ° C. (A mixture thereof) in the presence of the ester compound 2a and the corresponding alkyl halide. The reaction time is preferably from 10 minutes to 72 hours, more preferably from 8 hours to 24 hours.

Compound 2a which is a raw material for production can be synthesized according to a commercially available or known method.
(2) Conversion of Compound 3a to Compound 5a Conversion of Compound 3a to Compound 5a is performed using a suitable solvent that does not adversely influence the reaction (eg, N, N-dimethylformamide, acetone, or the like, or a mixed solvent thereof). A suitable base (eg, triethylamine, N, N-diisopropylethylamine, 4-dimethylaminopyridine, N-methylmorpholine, pyridine, 2,6- Compound 4 in the presence of lutidine, diazabicyclo [5.4.0] undec-7-ene, or the like, or a mixture thereof). An excess amount can be used as the amount of the base. The reaction time is preferably 1 hour to 72 hours, more preferably 8 hours to 24 hours.

Compound 4 as a raw material for production can be synthesized according to the method described in Reference Example.
(3) Conversion of Compound 5a to Compound 6a Conversion of Compound 5a to Compound 6a can be performed by using a suitable solvent that does not adversely affect the reaction (eg, toluene, 1,4-dioxane, 1,2-dichloroethane, tetrahydrofuran, or the like, or In a mixed solvent thereof), a chlorinating agent such as carbon tetrachloride, trichloroacetonitrile, or N-chlorosuccinimide is used in the presence of triphenylphosphine at −30 ° C. to the boiling point of the solvent used in the reaction, preferably at room temperature to 120 ° C. The reaction can be carried out. The reaction time is preferably from 10 minutes to 12 hours, more preferably from 30 minutes to 2 hours. Further, when ^Pb is a methyl group, the compound is dissolved in a suitable solvent (for example, chloroform, dichloromethane, tetrahydrofuran, 1,4-dioxane or the like, or a mixed solvent thereof) which does not adversely affect the reaction, at a temperature of -30 ° C to 100 ° C. Alternatively, the reaction can be carried out by treating a suitable chlorinating agent (eg, oxalyl chloride, thionyl chloride, phosphorus oxychloride, etc.) at a temperature up to the boiling point of the solvent used in the reaction, preferably from room temperature to 100 ° C. The reaction time is preferably from 10 minutes to 24 hours, more preferably from 30 minutes to 12 hours. If necessary, a base such as triethylamine, N, N-dimethylaniline, N, N-diethylaniline can be added. Further, N, N-dimethylformamide or the like can be added as a reaction accelerator.
(4) conversion of converting compound 6a from compound 6a into compounds 7a into compounds 7a, the reaction conditions for deprotection varies depending on the type of P ^b. When ^Pb is a methyl group, it is added in a suitable solvent that does not adversely influence the reaction (eg, dichloromethane, chloroform, etc., or a mixed solvent thereof) from −78 ° C. to the boiling point of the solvent used in the reaction, preferably −40. It can be carried out by treating a deprotecting agent such as boron tribromide at a temperature from ℃ to room temperature. The reaction time is preferably from 1 hour to 72 hours, more preferably from 2 hours to 24 hours. When ^Pb is a tert-butyl group, the reaction can be carried out by treating with trifluoroacetic acid, hydrochloric acid, formic acid or the like at -30 ° C to the boiling point of the solvent used in the reaction, preferably at -20 ° C to room temperature. The reaction time is preferably from 10 minutes to 72 hours, more preferably from 30 minutes to 24 hours.
(5) Conversion of Compound 7a to Compound 8a Conversion of Compound 7a to Compound 8a can be carried out by a general oxidation reaction for converting a primary alcohol to a carboxylic acid. Typical oxidizing agents include potassium permanganate, chromium trioxide and dilute sulfuric acid (Jones oxidation), or (2,2,6,6-tetramethyl-1-piperidinyl) oxyl (TEMPO) and a co-oxidizing agent ( Hypochlorite, bromite, N-chlorosuccinimide, etc.). Examples of the solvent used in the reaction include acetone, acetonitrile, water and the like, or a mixed solvent thereof. The reaction temperature is from −78 ° C. to 100 ° C. or the boiling point of the solvent, preferably from room temperature to 80 ° C. The time can be from 1 hour to 48 hours, preferably from 1 hour to 24 hours.

Further, the aldehyde compound obtained by oxidizing the compound 7a can be oxidized again to obtain the compound 8a. Examples of the oxidation reaction for obtaining an aldehyde compound include chromic acid [pyridinium chlorochromate (PCC), pyridinium dichromate (PDC), etc.], dimethylsulfoxide and oxalyl chloride (Swern oxidation), dimethylsulfoxide and acetic anhydride, dimethylsulfoxide and trioxide. A reaction using a sulfur pyridine complex, 1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3 (1H) -one (Dess-Martin reagent) or the like is known. As an oxidation reaction for obtaining a carboxylic acid from an aldehyde compound, Pinnick oxidation using sodium chlorite in the presence of 2-methyl-2-butene is known.
(6) Conversion from Compound 8a to Compound 9a Conversion from Compound 8a to Compound 9a can be carried out by a general carboxy group protection reaction. For example, in a lower alcohol corresponding to ^Pc such as methanol or ethanol, at room temperature to the boiling point of the solvent used for the reaction, preferably at room temperature to 100 ° C., a suitable acid catalyst (eg, hydrogen chloride, sulfuric acid, or thionyl chloride) Can be implemented. The reaction time is preferably from 10 minutes to 72 hours, more preferably from 30 minutes to 24 hours.

In a suitable solvent that does not adversely influence the reaction (toluene, dichloromethane, or the like, or a mixed solvent thereof), a suitable esterifying agent (for example, from room temperature to the boiling point of the solvent used in the reaction, preferably from room temperature to 100 ° C.) A tert-butyl ester can be obtained by treating N, N-dimethylformamide di-tert-butyl acetal, O-tert-butyl-N, N′-diisopropylisourea, or the like. The reaction time is preferably from 10 minutes to 72 hours, more preferably from 30 minutes to 24 hours.
(7) Conversion of Compound 9a to Compound 10a Conversion of Compound 9a to Compound 10a is performed by a nucleophilic substitution reaction of Compound 9a with an alcohol, amine, or thiol. For example, when the above substitution reaction is performed using an alcohol, the substitution reaction is performed in an appropriate solvent (tetrahydrofuran, acetone, acetonitrile, 1,4-dioxane, dimethyl sulfoxide, or the like, or a mixed solvent thereof) that does not adversely affect the reaction. The reaction can be carried out by treating an appropriate base (eg, sodium hydride, potassium carbonate, cesium carbonate, etc.) at a temperature from room temperature to the boiling point of the solvent used in the reaction, preferably from room temperature to 100 ° C. The reaction time is preferably from 6 hours to 72 hours, more preferably from 12 hours to 24 hours. The amount of the base to be used may be 1 to excess molar equivalent relative to compound 9a, and more preferably 1 to 5 molar equivalents. The amount of the alcohol to be used may be 1 to an excess molar equivalent based on compound 10a, and the reaction may be carried out using alcohol as a solvent. It is also possible to carry out the reaction using a metal alkoxide. Further, a catalytic amount of crown ether may be added.

For example, when the above-mentioned reaction is carried out using an amine, the solvent used in the reaction may be heated from room temperature in a suitable solvent (tetrahydrofuran, 1,4-dioxane, or the like, or a mixed solvent thereof) which does not adversely influence the reaction. By treating with a suitable base (eg, an inorganic base such as potassium carbonate or cesium carbonate, an organic base such as triethylamine, N, N-diisopropylethylamine) at the boiling point, preferably at room temperature to 100 ° C., or without using a base It can be carried out by using an excess amount of the amine. The reaction time is preferably from 6 hours to 72 hours, more preferably from 12 hours to 24 hours. The amount of the base to be used may be 1 to excess molar equivalent relative to compound 9a, and more preferably 1 to 2 molar equivalents. The use amount of the amine may be 1 to 2 molar equivalents when a base is used, and preferably 2 to 30 molar equivalents to compound 9a when a base is not used. The above reaction can also be performed by treating in a sealed tube or under microwave irradiation.

When the above substitution reaction is carried out using thiol, it can be carried out by basically performing the same operation as in the case of using alcohol.
(8) Conversion of Compound 10a to Compound 1a In the conversion of Compound 10a to Compound 1a, the deprotection reaction conditions differ depending on the type of ^Pc . When P ^c is a methyl group, a suitable solvent that does not adversely affect the reaction (eg, methanol, ethanol, water, tetrahydrofuran, 1,4-dioxane, etc., or a mixture thereof; A suitable base (eg, sodium hydroxide, potassium hydroxide, lithium hydroxide, or the like) at −30 ° C. to the boiling point of the solvent used in the reaction, preferably at room temperature to 100 ° C. Potassium tert-butoxide). The reaction time is preferably from 10 minutes to 72 hours, more preferably from 30 minutes to 24 hours. When P ^c is a tert-butyl group, it is treated with trifluoroacetic acid, hydrochloric acid, formic acid or the like at −30 ° C. to the boiling point of the solvent used in the reaction, preferably at −20 ° C. to room temperature, in addition to the above deprotection reaction. This can also be implemented. The reaction time is preferably from 10 minutes to 72 hours, more preferably from 30 minutes to 24 hours.

[Production method 2]
Compound 1a can also be obtained by subjecting compound 8a of [Production Method 1] to the substitution reaction described in (7) of [Production Method 1].

(1) Conversion of compound 8a to compound 1a Conversion of compound 8a to compound 1a can be carried out by the same substitution reaction as described in the above-mentioned [Production method 1] (7).

[Production method 3]
The compound represented by the formula (I) (represented as compound 1c in the following reaction formula) can be produced, for example, by the following reaction formula using 6c that can be produced by the above [Production method 1] as a starting material.
[In the formula, G, R ¹ and P ^b have the same meaning as described above, but P ^b is preferably a tert-butyl group. Q ² represents a methylene group. M ^1c represents a methyl group. ]

(1) Conversion of Compound 6c to Compound 11c The conversion of Compound 6c to Compound 11c can be carried out by the same substitution reaction as in the case of using the alcohol described in (7) of [Production Method 1].
(2) Conversion of Compound 11c to Compound 12c Conversion of Compound 11c to Compound 12c can be carried out by the same general deprotection reaction as described in the above-mentioned [Production Method 1] (4).
(3) Conversion of compound 12c to compound 1c Conversion of compound 12c to compound 1c can be carried out by the same general oxidation reaction as described in the above-mentioned [Production method 1] (5).

In the present invention, at least one drug selected from the group consisting of (a) a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, and (b) a CFTR function improving agent and an ENaC inhibitor, The “medicament characterized by being administered in combination (preferably, a medicament for treating cystic fibrosis) / therapeutic method (preferably, a method for treating cystic fibrosis)” includes the formula (a) Or a pharmaceutically acceptable salt thereof, and (b) at least one drug selected from the group consisting of CFTR function improving agents and ENaC inhibitors, preferably for the treatment of cystic fibrosis. And pharmaceutical / therapeutic methods intended to be administered in combination.

In the present invention, at least one drug selected from the group consisting of (a) a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, and (b) a CFTR function-improving agent and an ENaC inhibitor is used. The term "administered in combination" means that, during a certain period of time, the administered subject is (a) a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, (b) a CFTR function improving agent and It means that at least one drug selected from the group consisting of ENaC inhibitors is taken into the body. (A) a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof and (b) at least one drug selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor in a single preparation The included formulations may be administered, or each may be separately formulated and administered separately. When formulated separately, the timing of administration is not particularly limited, and they may be administered simultaneously, or may be administered at different times at different times or on different days. (A) a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, and (b) at least one drug selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor, at different times or When administered daily, the order of administration is not particularly limited. Usually, each preparation is administered according to each administration method, so that the administration may be the same or different. Further, when each is formulated separately, the administration method (administration route) of each formulation may be the same or may be administered by different administration methods (administration routes). Further, (a) a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof and (b) at least one drug selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor are simultaneously administered in the body. It is not necessary to be present in the body for a certain period of time (for example, one month, preferably one week, more preferably several days, even more preferably one day). Sometimes the other active ingredient may have disappeared from the body.

Examples of the dosage form of the medicament of the present invention include, for example, 1) (a) a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, and (b) a CFTR function improving agent and an ENaC inhibitor. Administration of a single preparation containing at least one drug selected from the group, 2) (a) a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, and (b) a CFTR function improving agent and Simultaneous administration by the same administration route of two formulations obtained by separately formulating at least one drug selected from the group consisting of ENaC inhibitors, 3) (a) a compound represented by the formula (I) or Time difference in the same administration route between two formulations obtained by separately formulating the pharmaceutically acceptable salt thereof and (b) at least one drug selected from the group consisting of CFTR function improving agents and ENaC inhibitors Administration in the ) (A) separately formulating a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof and (b) at least one drug selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor. Administration of the two preparations obtained by different administration routes, 5) (a) a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, and (b) a CFTR function-improving agent and ENaC inhibition Examples include administration of two formulations obtained by separately formulating at least one drug selected from the group consisting of drugs with different administration routes at different times.

に お い て In the present invention, when two different preparations are used, a kit containing them may be used.

When the CFTR function-improving drug is RNA therapy or Gene therapy, “administer the drug” means that the gene encoding the antisense oligonucleotide or CFTR is introduced directly or by using a viral or non-viral vector. Or repairing the abnormality of the gene encoding CFTR by a genome editing technique such as CRISPR / Cas9.

Further, one embodiment of the present invention provides (a) a compound represented by the formula (I) which is administered in combination with at least one drug independently selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor. The compound or a pharmaceutically acceptable salt thereof. Here, “(b) administered in combination with at least one drug selected from the group consisting of CFTR function improving agents and ENaC inhibitors” means (b) CFTR function improving agents and ENaC inhibitors. And administering to a patient who has been treated with at least one drug selected from the group consisting of:

The medicament according to the present invention is (a) at least one selected from the group consisting of a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof and / or (b) a CFTR function improving agent and an ENaC inhibitor. Can be administered in various forms. Examples of the administration form include oral administration by tablets, capsules, granules, emulsions, pills, powders, syrups (solutions) and the like, or injections (intravenous, intramuscular, subcutaneous or intraperitoneal administration), Parenteral administration by drops, suppositories (rectal administration) and the like can be mentioned. These various preparations are commonly used in the pharmaceutical preparation technical field such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizing agents, suspending agents, coating agents, and the like, in accordance with the conventional method. It can be formulated with the aid obtained.

When used as tablets, carriers include excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, etc .; water, ethanol, propanol, simple syrup, glucose Liquid, starch liquid, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, and other binders; dried starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid Disintegrators such as esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose; disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil; quaternary ammonium salts, sodium lauryl sulfate Absorbents such as glycerin and starch; Adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid; Lubricants such as purified talc, stearate, boric acid powder, polyethylene glycol, etc. can do. If necessary, tablets coated with a conventional coating, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets or double tablets or multilayer tablets, can be prepared.

When used as a pill, as a carrier, for example, excipients such as glucose, lactose, cocoa butter, starch, hydrogenated vegetable oil, kaolin, talc; binders such as gum arabic powder, tragacanth powder, gelatin, ethanol; laminaran; Disintegrators such as agar can be used.

(4) When used as a suppository, those conventionally known in the art can be widely used as the carrier, and examples thereof include polyethylene glycol, cocoa butter, higher alcohols, higher alcohol esters, gelatin, and semi-synthetic glycerides.

場合 When used as an injection, it can be used as a liquid, emulsion or suspension. These solutions, emulsions or suspensions are preferably sterilized and isotonic with blood. The solvent used in the production of these liquid preparations, emulsions or suspensions is not particularly limited as long as it can be used as a diluent for medical use. For example, water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol Examples thereof include stearyl alcohol and polyoxyethylene sorbitan fatty acid esters. In this case, the preparation may contain a sufficient amount of salt, glucose or glycerin to prepare an isotonic solution, and may also contain ordinary solubilizing agents, buffers, soothing agents and the like. You may go out.

製 剤 In addition, the above-mentioned preparations may contain a coloring agent, a preservative, a flavor, a flavoring agent, a sweetening agent, and the like, if necessary, and may further contain other pharmaceuticals.

量 The amount of the active ingredient compound contained in the above preparation is not particularly limited and may be appropriately selected in a wide range, but usually contains 0.5 to 70% by weight, preferably 1 to 30% by weight of the whole composition.

The amount used depends on the symptoms, age, etc. of the patient (warm-blooded animal, especially human), but in the case of oral administration, the lower limit is 0.01 mg / kg body weight (preferably 0.1 mg / kg) per dose. / Kg body weight), 500 mg / kg body weight (preferably 100 mg / kg body weight) as an upper limit, and 0.001 mg / kg body weight (preferably, once per intravenous administration) 0.01 mg / kg body weight), and, as an upper limit, 50 mg / kg body weight (preferably 10 mg / kg body weight) is desirably administered once or several times a day depending on the symptoms.

In the case of Kalydeco (registered trademark) (Ivacaftor), usually 150 mg for children and adults aged 6 years or older, 75 mg for children aged 2 to 6 years older than 14 kg, and 2 to 6 years for a body weight of 14 kg or less. A preferred example is oral administration of a child of less than 50 mg every 12 hours, but it may be adjusted as appropriate according to the patient's condition.

In the case of Orkambi (registered trademark) (a combination of Ivacaftor and Lumacaftor), adult Ivacaftor125 mg and Lumacaftor200 mg for 12-year-old and older, and child Ivacaftor125 mg and Lumacaftor100 小 児 mg for 60-year-old and 12-year-old for every 12 hours. Is a preferable example, but may be appropriately increased or decreased depending on the condition of the patient.

In the case of Symdeko (registered trademark) (a combination of Tezacaftor and Ivacaftor), oral intake of Ivacaftor 150 mg and Tezafactor 100 mg in the morning for adults 12 years of age and older, and Ivacaftor 150 mg in the evening 12 hours later may be mentioned as preferred examples. It may be appropriately increased or decreased according to the condition of the patient.

The type of cystic fibrosis to be treated is not limited, but is particularly suitable for patients with class III and class IV mutations of CFTR when used in combination with CFTR potentiator, and has class II mutation when used in combination with CFTR corrector. Particularly suitable for patients.

Hereinafter, the present invention will be described in more detail with reference to Reference Examples, Examples, and Test Examples, but the scope of the present invention is not limited thereto.

Elution in column chromatography in Reference Examples and Examples was performed under observation by TLC (Thin Layer Chromatography, thin-layer chromatography). In the TLC observation, silica gel 60F254 manufactured by Merck was used as a TLC plate, a solvent used as an elution solvent in column chromatography was used as a developing solvent, and a UV detector was used as a detection method. For column chromatography, an automatic purifying device of Yamazen Corporation or an automatic purifying device of Shokosha was used as appropriate. As the elution solvent, the solvent specified in each Reference Example and Example was used. The abbreviations used in Reference Examples and Examples have the following meanings.

Mg: milligram, g: gram, μl: microliter, ml: milliliter, L: liter, M: molarity, MHz: megahertz.

In the following Examples, nuclear magnetic resonance (hereinafter, ¹ H-NMR: 400 MHz) spectra were described using chemical values of δ (ppm) with tetramethylsilane as a standard substance. The fission pattern was indicated by s for singlet, d for doublet, t for triplet, q for quadruple, spt for sevenfold, m for multiplet, and br for broad.

The powder X-ray diffraction measurement was performed using a reflection type powder X-ray diffractometer (RINT-TTRIII) manufactured by Rigaku Corporation, wavelength: CuKα, λ = 1.54 angstroms, and the sample was measured using a non-reflective sample holder (tube). (Voltage: 50 kV, tube current: 300 mA, scanning range: 2 to 40 °, scanning speed: 20 ° / min, sampling width: 0.02 °, rotation speed: 120 rpm).

The moisture was measured using a Karl Fischer moisture meter (coulometric titration method MKC-610) manufactured by Kyoto Electronics Industry Co., Ltd. (Anolyte: Hydranal Coulomat AG (Sigma Aldrich), Catholyte: Hydranal Coulomat CG (Sigma Aldrich) )).

For thermal analysis (simultaneous measurement of differential heat and thermal mass @ TG-DTA), TG / DTA6200 manufactured by Hitachi High-Tech Science Corp. was used (heating rate: 10 ° C / min, atmosphere gas: nitrogen, nitrogen gas flow rate: 200 ml / min).

[Example 1]
5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid

[Step 1]
Methyl 2- (2-chlorobenzyl) -3-oxobutanoate Methyl 3-oxobutanoate was added to a suspension of sodium hydride (60% oil, 0.68 g) in tetrahydrofuran (25 ml) under ice-cooling. A solution of (3.0 g) in tetrahydrofuran (5 ml) was added and the mixture was stirred at room temperature for 30 minutes. A solution of 2-chlorobenzyl bromide (5.3 g) in tetrahydrofuran (5 ml) was added to the reaction solution, and the mixture was stirred at room temperature for 2 days. Ice water and 1 M hydrochloric acid were added to the reaction solution, and the mixture was extracted with ethyl acetate. The extract was washed with saturated saline and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the obtained residue was purified by silica gel column chromatography (n-hexane / ethyl acetate) to obtain the title compound (5.6 g).
¹ H-NMR (CDCl ₃ ) δ: 2.22 (3H, s), 3.24 (1H, dd, J = 14.1, 8.0 Hz), 3.30 (1H, dd, J = 14.1, 6.8 Hz), 3.69 (3H, s ), 3.97 (1H, dd, J = 8.2, 6.7 Hz), 7.14-7.19 (2H, m), 7.22-7.25 (1H, m), 7.33-7.36 (1H, m).
[Step 2]
2- (tert-butoxymethyl) -5- (2-chlorobenzyl) -6-methylpyrimidin-4 (3H) -one N, N-dimethylformamide of the compound (5.6 g) obtained in the above step 1 To the (30 ml) solution were added the compound obtained in Step 1 of Reference Example 1 (5.8 g) and 1,8-diazabicyclo [5.4.0] undec-7-ene (10.5 ml). The mixture was stirred at 75 ° C. for 26 hours. After cooling, a saturated aqueous solution of sodium hydrogen carbonate was added to the reaction solution, and the mixture was extracted with ethyl acetate. The extract was washed sequentially with water and saturated saline, and then dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and diethyl ether was added to the obtained residue, followed by filtration to obtain the title compound (2.8 g).
¹ H-NMR (CDCl ₃ ) δ: 1.29 (9H, s), 2.20 (3H, s), 4.01 (2H, s), 4.40 (2H, s), 6.98-7.02 (1H, m), 7.09-7.14 (2H, m), 7.34-7.38 (1H, m).
[Step 3]
2- (tert-butoxymethyl) -4-chloro-5- (2-chlorobenzyl) -6-methylpyrimidine A suspension of the compound (2.8 g) obtained in Step 2 above in toluene (10 ml) was added. Triphenylphosphine (6.9 g) and trichloroacetonitrile (0.88 ml) were added and the mixture was stirred at 120 ° C. for 1 hour. After cooling, a saturated aqueous sodium hydrogen carbonate solution was added to the reaction solution, and the mixture was extracted with chloroform. The extract was washed with saturated saline and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the obtained residue was purified by silica gel column chromatography (n-hexane / ethyl acetate) to obtain the title compound (2.7 g).
¹ H-NMR (CDCl ₃ ) δ: 1.33 (9H, s), 2.44 (3H, s), 4.24 (2H, s), 4.63 (2H, s), 6.65 (1H, dd, J = 7.5, 1.8 Hz ), 7.12 (1H, td, J = 7.5, 1.4 Hz), 7.19 (1H, td, J = 7.7, 1.7 Hz), 7.43 (1H, dd, J = 7.9, 1.4 Hz).

[Step 4]
2- (tert-butoxymethyl) -5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine A solution of the compound (1.5 g) obtained in Step 3 above in methanol (4 ml) was added with carbonic acid. Cesium (1.44 g) was added and the mixture was stirred at room temperature for 2 days. Water was added to the reaction solution, which was extracted with ethyl acetate. The extract was washed with saturated saline and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the obtained residue was purified by silica gel column chromatography (n-hexane / ethyl acetate) to obtain the title compound (1.15 g).
¹ H-NMR (CDCl ₃ ) δ: 1.33 (9H, s), 2.34 (3H, s), 3.95 (3H, s), 4.03 (2H, s), 4.55 (2H, s), 6.68-6.71 (1H , m), 7.07 (1H, td, J = 7.5, 1.4 Hz), 7.14 (1H, td, J = 7.7, 1.8 Hz), 7.38 (1H, dd, J = 7.9, 1.4 Hz).
[Step 5]
(5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidin-2-yl) methanol To the compound (1.15 g) obtained in the above step 4 was added trifluoroacetic acid (2 ml), and a mixture was obtained. Was stirred at room temperature for 24 hours and a half. Water was added to the reaction solution, which was extracted with ethyl acetate. The extract was washed successively with a saturated aqueous solution of sodium hydrogen carbonate and a saturated saline solution, and then dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the obtained residue was purified by silica gel column chromatography (n-hexane / ethyl acetate) to obtain the title compound (0.81 g).
¹ H-NMR (CDCl ₃ ) δ: 2.36 (3H, s), 3.76 (1H, t, J = 4.9 Hz), 3.95 (3H, s), 4.05 (2H, s), 4.69 (2H, d, J = 4.8 Hz), 6.70 (1H, dd, J = 7.3, 1.5 Hz), 7.10 (1H, td, J = 7.5, 1.4 Hz), 7.15 (1H, td, J = 7.5, 1.8 Hz), 7.40 (1H , dd, J = 7.8, 1.5 Hz).
[Step 6]
5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid To a solution of the compound obtained in the above step 5 (0.8 g) in acetonitrile (6 ml) was added sodium phosphate buffer. (0.67 M, pH 6.7, 4 ml) and (2,2,6,6-tetramethylpiperidin-1-yl) oxyl (31 mg) were added, and the mixture was stirred at 35 ° C. An aqueous solution of sodium chlorite (2.0 M, 3.3 ml) and an aqueous solution of sodium hypochlorite (0.26%, 2.9 ml) were simultaneously added dropwise to the mixture, and the mixture was stirred at 35 ° C. for 23 hours and a half. did. After cooling, the reaction solution was diluted with a 2 M aqueous sodium hydroxide solution (4 ml), a 10% aqueous sodium thiosulfate solution (6 ml) was added under ice cooling, and the mixture was stirred at the same temperature for 30 minutes. Ethyl acetate was added to the reaction solution, and after separating two layers, 2M hydrochloric acid (10 ml) was added to the aqueous layer, and the mixture was extracted with ethyl acetate. The extract was washed sequentially with water and saturated saline, and then dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and n-hexane / ethyl acetate was added to the obtained residue, followed by filtration to obtain the title compound (0.71 g).
¹ H-NMR (CDCl ₃ ) δ: 2.45 (3H, s), 4.08 (3H, s), 4.13 (2H, s), 6.67 (1H, dd, J = 7.3, 2.0 Hz), 7.11 (1H, td , J = 7.5, 1.3 Hz), 7.18 (1H, td, J = 7.7, 1.7 Hz), 7.41 (1H, dd, J = 7.9, 1.4 Hz).
MS (m / z): 293 (M + H) ⁺ .

[Example 2]
5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid 3/2 hydrate

[Step 1]
5- (2-Chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid 3/2 hydrate To the compound (200 mg) obtained in Step 6 of Example 1 was added 2-propanol (1. 8 ml) and water (0.2 ml) were added and the mixture was stirred at 95 ° C. for 15 minutes. After cooling to room temperature, the mixture was stirred at room temperature overnight. The precipitate was collected by filtration, washed with water, and dried to give the title compound (188 mg).
¹ H-NMR (CDCl ₃ ) δ: 2.46 (3H, s), 4.09 (3H, s), 4.14 (2H, s), 6.66-6.69 (1H, m), 7.12 (1H, td, J = 7.5, 1.5 Hz), 7.19 (1H, td, J = 7.7, 1.8 Hz), 7.42 (1H, dd, J = 7.9, 1.4 Hz).
Anal.Calcd for C ₁₄ H ₁₃ ClN ₂ O ₃・ 3 / 2H ₂ O: C, 52.59; H, 5.04; Cl, 11.09; N, 8.76.
Found: C, 52.40; H, 5.07; Cl, 11.17; N, 8.70.

[Reference Example 1]
2- (tert-butoxy) acetimidamide hydrochloride

[Step 1]
2- (tert-butoxy) acetimidamide hydrochloride To a solution of 2- (tert-butoxy) acetonitrile (Nature Chemistry, 2010, 937-943) (69.89 g) in methanol (400 ml) was added sodium methoxide (3.08 g). ) Was added and the mixture was stirred at room temperature for 8 hours. Ammonium chloride (34.69 g) was added to the reaction solution, and the mixture was stirred at 40 ° C. overnight, and then at room temperature overnight. The precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. Ethanol (400 ml) was added to the obtained residue, and the mixture was stirred at 80 ° C for 30 minutes. The precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. Ethyl acetate (300 ml) was added to the obtained residue, n-hexane (300 ml) was added dropwise at 40 ° C. with stirring, and the mixture was stirred at room temperature overnight. The suspension was filtered to give the title compound (68.57 g).
¹ H-NMR (DMSO-d ₆ ) δ: 1.19 (9H, s), 4.21 (2H, s), 8.61 (4H, br s).

[Example 3]
Bis [5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid] magnesium salt To a compound (301.89 mg) obtained in the same manner as in Example 2 was added 2-propanol (1208). μl) and water (3302 μl). To this solution was added a 1.0 mol / l aqueous solution of potassium hydroxide (989 μl), and then a 1.0 mol / l aqueous solution of magnesium chloride (539 μl) was added. The mixture was stirred at 40 ° C. for about 24 hours, then left at room temperature for about 0.5 hour. The solid was collected by filtration, dried overnight at room temperature, added with water (6.0 ml), and stirred at room temperature for about 2 hours. The solid was collected by filtration and dried overnight at room temperature to give the title compound (274.93 mg, 88% recovery).

Elemental analysis _{C 28 H 24 Cl 2 MgN 4} O 6 · 3.0H 2 O Calculated: C, 50.82; H, 4.57 ; N, 8.47; Cl, 10.71; Mg, 3.67.
Found: C, 50.63; H, 4.69; N, 8.41; Cl, 10.88; Mg, 3.63.
From the above results, it was estimated to be bis [5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid] magnesium salt hydrate.

[Example 4]
Bis [5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid] magnesium salt To a compound (1501 mg) obtained in the same manner as in Example 2 were added 2-propanol (6005 μl), Water (16.42 ml) was added. To this solution was added a 1.0 mol / l aqueous potassium hydroxide solution (4920 μl), and then a 1.0 mol / l aqueous magnesium chloride solution (2679 μl). After stirring the mixture at 40 ° C. for about 2 days, a small amount of a seed crystal obtained by the same method as described below was added, and the mixture was allowed to stand at 40 ° C. for about 1 day and then at room temperature for about 0.5 hour. The solid was collected by filtration, water (30.0 ml) was added, and the mixture was stirred at room temperature for about 2 hours. The solid was collected by filtration and dried overnight at room temperature to give the title compound (1416 mg, 93% recovery).

Method for Obtaining Seed Crystal To a compound (701.13 mg) obtained in the same manner as in Example 3, a 1,4-dioxane / dimethylsulfoxide (1/1) solution (30 ml) was added and dissolved. 428 μl of this solution was dispensed, and the solvent was distilled off by freeze-drying. To the obtained freeze-dried product was added 20% aqueous 2-propanol (100 μl), and the mixture was stirred at 40 ° C. for about 24 hours. The solid was collected by filtration and dried at room temperature overnight to obtain a seed crystal.

Elemental analysis _{C 28 H 24 Cl 2 MgN 4} O 6 · 2H 2 O Calculated: C, 52.24; H, 4.38 ; N, 8.70; Cl, 11.01; Mg, 3.78.
Found: C, 51.82; H, 4.52; N, 8.64; Cl, 10.90; Mg, 3.70.
Water content (Karl Fischer _{method) C 28 H 24 Cl 2 MgN} 4 O 6 · 2H 2 O Calculated: 5.58%
Measured: 6.10%
Weight reduction rate (thermal analysis _{TG-DTA) C 28 H 24} Cl 2 MgN 4 O 6 · 2H 2 O Calculated: 5.58%
Measured: 5.61%
From the above measurement results, the product was identified to be bis [5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid] monomagnesium salt dihydrate.

[Example 5]
Bis [5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid] calcium salt To a compound (300.93 mg) obtained in the same manner as in Example 2 was added 2-propanol (1202). μl) and water (3284 μl). A 1.0 mol / l aqueous solution of potassium hydroxide (985 μl) was added to this solution, and then a 1.0 mol / l aqueous solution of calcium chloride (538 μl) was added. The mixture was stirred at 40 ° C. for about 24 hours, then left at room temperature for about 0.5 hour. The solid was collected by filtration, dried overnight at room temperature, added with water (6.0 ml), and stirred at room temperature for about 2 hours. The solid was collected by filtration and dried overnight at room temperature to give the title compound (301.22 mg, 92% recovery).

Elemental analysis calculated for C ₂₈ H ₂₄ Cl ₂ CaN ₄ O ₆・ 4.0 H ₂ O: C, 48.35; H, 4.64; N, 8.05; Cl, 10.19; Ca, 5.76.
Found: C, 48.10; H, 4.74; N, 7.98; Cl, 10.26; Ca, 4.28.
From the above results, it was presumed to be bis [5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid] calcium salt hydrate.

[Example 6]
Bis [5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid] zinc salt To a compound (300.10 mg) obtained in the same manner as in Example 2 was added 2-propanol (1200 μl) and water (3280 μl). A 1.0 mol / l aqueous solution of potassium hydroxide (984 μl) was added to this solution, and then a 1.0 mol / l aqueous solution of zinc bromide (538 μl) was added. The mixture was stirred at 40 ° C. for about 24 hours, then left at room temperature for about 0.5 hour. The solid was collected by filtration, dried overnight at room temperature, added with water (6.0 ml), and stirred at room temperature for about 2 hours. The solid was collected by filtration and dried overnight at room temperature to give the title compound (293.65 mg, 92% recovery).

Elemental analysis _{C 28 H 24 Cl 2 ZnN 4} O 6 · 1.5H 2 O Calculated: C, 49.76; H, 4.03 ; N, 8.29; Cl, 10.49.
Found: C, 49.94; H, 4.12; N, 8.28; Cl, 10.61.
From the above results, it was presumed to be bis [5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid] zinc salt hydrate.

[Example 7]
5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid sodium salt To a compound (301.24 mg) obtained in the same manner as in Example 2, ethanol (5042 μl) was added. A 1.0 mol / l sodium hydroxide ethanol solution (982 μl) was added. The mixture was stirred at 40 ° C. for about 24 hours, then left at room temperature for about 0.5 hour. The solid was collected by filtration and dried at room temperature overnight to give the title compound (289.98 mg, recovery 97%).

Example 8
5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid tert-butylamine salt To a compound (300.90 mg) obtained in the same manner as in Example 2 was added acetone (5913 μl). In addition, tert-butylamine (105 μl) was added. The mixture was stirred at 40 ° C. for about 24 hours, then left at room temperature for about 0.5 hour. The solid was collected by filtration and dried overnight at room temperature to give the title compound (332.74 mg, 97% recovery).

Elemental analysis _{C 14 H 13 ClN 2 O 3} · 1.0C 4 H 11 N Calculated: C, 59.09; H, 6.61 ; N, 11.49; Cl, 9.69.
Found: C, 58.69; H, 6.53; N, 11.34; Cl, 9.74.

[Example 9]
5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid diisopropylamine salt To a compound (300.05 mg) obtained in the same manner as in Example 2 was added acetone (5863 μl). , Diisopropylamine (138 μl). The mixture was stirred at 40 ° C. for about 24 hours, then left at room temperature for about 0.5 hour. The solid was collected by filtration and dried overnight at room temperature to give the title compound (353.95 mg, 96% recovery).

Elemental analysis _{C 14 H 13 ClN 2 O 3} · 1.0C 6 H calcd _{15 N: C, 60.98; H} , 7.16; N, 10.67; Cl, 9.00.
Found: C, 60.89; H, 7.17; N, 10.65; Cl, 9.05.

[Test Example 1] Examination of chloride ion secretion activity of test compound Measurement of chloride ion secretion activity was carried out according to a previously reported report (West and Molloy, 1996). That is, CuFi-1 cells, a bronchial epithelial cell line derived from a cystic fibrosis patient, were seeded on a 96-well plate at 3 × 10 ⁴ cells / well and cultured overnight. After removing the medium, 100 μl / well of N- (Ethoxycarbonylmethyl) -6-Methoxyquinolinium Bromide (MQAE) diluted with the medium and adjusted to a concentration of 5 mM was added. After adding MQAE, the cells were cultured overnight and passively loaded. After loading, the plate was washed twice with Assay buffer, and 100 μl / well of Assay buffer was added. The chloride ion secretion ability was measured using Flexstation 1 (Ex: 355 nm / Em: 460 nm fluorescence wavelength). That is, in order to measure a basal value, nothing is added for 34 seconds immediately after the start of the measurement, and a test compound diluted with a chloride ion-free assay buffer and zinc (final concentration: 10 μM) are added at 100 μl / well at 34 seconds. And then for 86 seconds (total of 120 seconds). The chloride ion secretion ability was calculated by dividing the average of the RFU values for 10 seconds from 110 seconds to 120 seconds from the start of measurement by the average value from 0 seconds to 34 seconds (basal value). The chloride ion secretion activity (EC ₅₀ value) of the test compound was calculated from the chloride ion secretion ability at each concentration of the serially diluted test compound at each concentration. The concentration was calculated as the concentration of the test compound giving a% secretory ability.

The chloride ion secretion activity (EC ₅₀ value) of the compound of Example 1 was 10.0 nM.

[Test Example 2] Effect of GPR39 knockdown on chloride secretion by test compound It was confirmed by gene knockdown that chloride secretion by the test compound observed in Test Example 1 was a GPR39-mediated reaction. That is, CuFi-1 cells cultured in Pneumacult-EX (STEMCELL Technologies) were seeded on a 96-well plate at 3 × 10 ⁴ cells / 100 μl / well, and Lipofectamine RNAiMAX (ThermoFisherFisherFisherFisherFisherFisherFisherFisherFisherFisher.com) using Lipofectamine RNAiMAX (MISSION siRNA SASI_Hs02_00332000, SASI_Hs02_00332001, Sigma-Aldrich) or control siRNA (Ambion Silencer Select, Thermo Fisher Scientific) was added at 1 pmol / 10 μl / w, followed by 1 pmol / 10 l culture. N- (Ethoxycarbonylmethyl) -6-methoxyquinolinium bromide (MQAE) diluted with the medium was added at 10 μl / well to a final concentration of 5 mM. After adding MQAE, the cells were cultured overnight and passively loaded. Thereafter, washing and measurement of chloride ion secretion ability were performed in the same manner as in Test Example 1. GPR39 gene expression was analyzed using RNA extracted from the well in which the same operation was performed. That is, total RNA is extracted from the cells using RNAiso Plus (Takara Bio Inc.), purified using RNeasy Micro Kit (Qiagen), and then purified using High Capacity cDNA Reverse Transcription Kit (Thermo Fisher cDNA Inc.). The synthesis was performed, and TaqMan Gene Expression Assays (human GPR39: Hs00230762_m1, human GAPDH: Hs02758891_g1, Thermo Fisher Scientific) and THUNDERBIRD Probe were used for quantitative PCR (Tokyo, Inc.). The relative expression level of GPR39 corrected by GAPDH was calculated by the standard curve method. The results are shown in FIGS.

It was confirmed that GPR39 was knocked down by siRNA treatment (see FIG. 1. siGPR39-1: 17%, siGPR39-2: 15% vs control siRNA). Under the conditions where GPR39 was knocked down, chloride ion secretion in CuFi-1 cells by the test compound (the compound of Example 2) was significantly suppressed as compared with the control siRNA-treated group (see FIG. 2. siGPR39-). 1: 17%, siGPR39-2: 37% {vs \ control \ siRNA). Chloride ion secretion by UTP was not affected by GPR39 knockdown (see FIG. 2. siGPR39-1: 111%, siGPR39-2: 89% {vs @ control} siRNA). Therefore, it was shown that chloride secretion by the test compound in CuFi-1 cells was induced through GPR39.

[Test Example 3] Effect of test compound in ALI assay using water transfer as an index ΔF508 homozygous (class II mutation), 2184ΔA + W1282X (class I mutation) and N1303K heterozygous primary bronchial epithelial cells having heterozygous (class II mutation). Air-Liquid Interface (ALI) cultured cells (MucilAir-CF ^™ ) were purchased from Epithelix. After obtaining, after confirming the resistance value, mucin production, and ciliary movement, 200 μl of HBSS (+) was added to the upper layer, and the mucin was washed. A medium in which a test compound and zinc (final concentration: 10 μM) were dissolved was added to the upper layer at a volume of 100 μl / well, and a medium in which a test compound and zinc (final concentration: 10 μM) were dissolved was added to the lower layer at a volume of 500 μl / well. The weight of the upper layer medium 72 hours after the addition of the test compound was measured with an electronic balance to confirm the water transfer effect of the test compound. As controls, the effects of VX-809 (lumacaftor) alone (Selleck Chemicals) and VX-809 (lumacaftor) / VX-770 (ivacaftor) combination (Selleck Chemicals) were also examined. The results for the above three types of cells using the compound of Example 1 as the test compound are shown in FIGS.

In the ALI culture system using the ΔF508 homozygous mutation, the compound of Example 1 moved water in a concentration-dependent manner. The effect was similar to that of VX-809 alone or the combination of VX-809 / VX-770. In addition, the compound of Example 1 also exhibited an activity equivalent to that of the ΔF508ｚhomozygus mutation in mutations other than the ΔF508 homozygus mutation (2184ΔA + W1282X (class I mutation)) and N1303K heterozygos, and could be effective without depending on the mutation. Was done. However, the combination of VX-809 / VX-770 had a weak effect on 2184ΔA + W1282X (class I mutation), and it was considered that the effect was dependent on the mutation. In particular, the effect of the compound of Example 1 on 2184ΔA + W1282X (class I mutation) was significant as compared with the combination of VX-809 / VX-770.

[Test Example 4] Effect of Combination of Test Compound with VX-809 / VX-770 Combination in ALI Assay Using Water Movement as Index ΔF508 homozygous Mutant ALI culture system using the compound of Example 1 and VX- The effect of the combination of 809 (lumacaftor) / VX-770 (ivacaftor) with the combination was examined. The combined effect was examined in the same manner as in Test Example 3. That is, cells obtained by ALI-cultured primary bronchial epithelial cells derived from a cystic fibrosis patient having a ΔF508 homozygous mutation (MucilAir-CF ^™ ) were purchased from Epithelix. After obtaining, after confirming the resistance value, mucin production, and ciliary movement, 200 μl of HBSS (+) was added to the upper layer, and the mucin was washed. The medium was added to the upper layer at a volume of 100 μl / well, and the medium containing a mixture of VX-809 / VX-770, a test compound and zinc (final concentration: 10 μM) was added to the lower layer at a volume of 500 μl / well. . 72 hours after the addition of the VX-809 / VX-770 mixture alone or the addition of the VX-809 / VX-770 mixture and the test compound, the weight of the upper medium was measured with an electronic balance, and the water transfer of the test compound was measured. The effect was confirmed. FIG. 6 shows the results of using the compound of Example 1 as a test compound.

In the ALI culture system using the ΔF508 homozygous mutation, the mixture of VX-809 / VX-770 significantly moved water, similarly to the result of Test Example 3. As a result of adding the compound of Example 1 to the mixture of VX-809 / VX-770, the compound of Example 1 was concentration-dependent on the water transfer amount of the mixture of VX-809 / VX-770, and Moisture was added additively. Therefore, the combined effect of the test compound with the combination of VX-809 / VX-770 was confirmed.

In this test, the combined effect of VX-809 / VX-770 with the combination drug was examined as a representative example. As shown in Test Example 3, the compound of Example 1 was not dependent on mutation in the ALI culture system. Because of the possibility of being effective, it is not limited to the combination of VX-809 / VX-770, but a drug having an action mechanism different from that of a GPR39 agonist, for example, other than the combination of VX-809 / VX-770 It is thought that a combination effect can be expected even in combination with a CFTR function improving drug or an ENaC inhibitor.

The compound represented by the formula (I) of the present invention or a pharmaceutically acceptable salt thereof has a strong chloride ion secretion action through GPR39 agonist action and transfers water. Since this compound can open a chloride channel different from CFTR which causes disease, it exhibits an excellent therapeutic effect when combined with a CFTR function improving agent or an ENaC inhibitor. Therefore, the medicament / therapeutic method using the compound of the present invention or a pharmaceutically acceptable salt thereof in combination with a CFTR function improving agent or an ENaC inhibitor can be carried out by any of class I to VI irrespective of the type of CFTR mutation. It is useful because it can also exert a therapeutic effect on fibrosis.

Claims (36)

1. (A) Formula (I):
   

   

   
   Or a pharmaceutically acceptable salt thereof and (b) at least one drug independently selected from the group consisting of CFTR function improving agents and ENaC inhibitors. Medicine.
2. The medicament according to claim 1, for treating cystic fibrosis.
3. Formulations in which (a) the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof and (b) at least one drug independently selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor are different from each other. The medicament according to claim 1, wherein the medicament is contained as an active ingredient and is administered simultaneously or at different times.
4. A single preparation comprising at least one drug independently selected from the group consisting of (a) the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, and (b) a CFTR function improving agent and an ENaC inhibitor. The medicament according to claim 1, wherein the medicament is contained therein.
5. A kit preparation comprising (a) the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, and (b) at least one drug independently selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor. The medicament according to claim 1 or 2, wherein
6. The medicament according to any one of claims 1 to 5, wherein the medicament is for a patient who has been treated with at least one drug independently selected from the group consisting of a CFTR function improving drug and an ENaC inhibitor.
7. At least one drug independently selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor is a CFTR function improving agent, wherein the CFTR function improving agent is CFTR @ potentiator, CFTR @ corrector, CFTR @ amplifier, RNA therapy 7. The medicament according to any one of claims 1 to 6, wherein the medicament is at least one kind independently selected from the group consisting of Gene therapy and Gene therapy.
8. At least one drug independently selected from the group consisting of a CFTR function improver and an ENaC inhibitor is a CFTR function improver, wherein the CFTR function improver is independently a group consisting of CFTR 薬 potentiator and CFTR collector. The medicament according to any one of claims 1 to 6, which is at least one selected from the group consisting of:
9. At least one drug independently selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor is a CFTR function improving agent, wherein the CFTR function improving agent is Ivacaftor, QBW251, VX-561, or PTI-808. GLPG1837, GLPG2451, GLPG3067, Lumacaftor, Tezacaftor, VX-445, VX-440, VX-152, VX-659, FDL169, GLPG2222, GLPG2851, GLPG2737, PTI-801, PTI-801, PTI-801, PTI-801, PTI-801, MTI The medicament according to any one of claims 1 to 6, which is at least one independently selected.
10. At least one drug independently selected from the group consisting of a CFTR function improver and an ENaC inhibitor is a CFTR function improver, wherein the CFTR function improver is independently from the group consisting of Ivacaftor, Lumacaftor and Tezacaftor. The medicament according to any one of claims 1 to 6, which is at least one selected from the group consisting of:
11. At least one drug independently selected from the group consisting of CFTR function improving agents and ENaC inhibitors is an ENaC inhibitor, and the ENaC inhibitor is at least one selected from the group consisting of QBW276, SPX-101 and AZD5634. The medicament according to any one of claims 1 to 6, which is a species.
12. The medicament according to any one of claims 1 to 11, wherein the pharmaceutically acceptable salt is a magnesium salt, a calcium salt, a zinc salt, a sodium salt, a tert-butylamine salt or a diisopropylamine salt.
13. The compound represented by the formula (I) or a pharmaceutically acceptable salt thereof is bis [5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid] magnesium salt. 12. The medicament according to any one of 1 to 11.
14. (B) Formula (I) for administration in combination with at least one drug independently selected from the group consisting of CFTR function improving agents and ENaC inhibitors:
    

    

    
    Or a pharmaceutically acceptable salt thereof.
15. 15. The compound according to claim 14, or a pharmaceutically acceptable salt thereof, for the treatment of cystic fibrosis.
16. The compound according to claim 14 or 15, or a pharmaceutically acceptable salt thereof, which is intended for a patient who has been treated with at least one drug independently selected from the group consisting of a CFTR function improving drug and an ENaC inhibitor. .
17. At least one drug independently selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor is a CFTR function improving agent, wherein the CFTR function improving agent is CFTR @ potentiator, CFTR @ corrector, CFTR @ amplifier, RNA therapy 17. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 14 to 16, which is at least one member independently selected from the group consisting of Gene therapy and Gene therapy.
18. At least one drug independently selected from the group consisting of a CFTR function improver and an ENaC inhibitor is a CFTR function improver, wherein the CFTR function improver is independently a group consisting of CFTR 薬 potentiator and CFTR collector. The compound according to any one of claims 14 to 16, or a pharmaceutically acceptable salt thereof, which is at least one member selected from the group consisting of:
19. At least one drug independently selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor is a CFTR function improving agent, wherein the CFTR function improving agent is Ivacaftor, QBW251, VX-561, or PTI-808. GLPG1837, GLPG2451, GLPG3067, Lumacaftor, Tezacaftor, VX-445, VX-440, VX-152, VX-659, FDL169, GLPG2222, GLPG2851, GLPG2737, PTI-801, PTI-801, PTI-801, PTI-801, PTI-801, MTI The compound according to any one of claims 14 to 16, or a pharmaceutically acceptable salt thereof, which is at least one independently selected.
20. At least one drug independently selected from the group consisting of a CFTR function improver and an ENaC inhibitor is a CFTR function improver, wherein the CFTR function improver is independently from the group consisting of Ivacaftor, Lumacaftor, and Tezacaftor. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 14 to 16, which is at least one member selected from the group consisting of:
21. At least one drug independently selected from the group consisting of CFTR function improving agents and ENaC inhibitors is an ENaC inhibitor, and the ENaC inhibitor is at least one selected from the group consisting of QBW276, SPX-101 and AZD5634. 17. The compound according to any one of claims 14 to 16, or a pharmaceutically acceptable salt thereof, which is a species.
22. The compound according to any one of claims 14 to 21, or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is a magnesium salt, a calcium salt, a zinc salt, a sodium salt, a tert-butylamine salt or a diisopropylamine salt. Salt.
23. The compound represented by the formula (I) or a pharmaceutically acceptable salt thereof is bis [5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid] magnesium salt. 22. The compound according to any one of 14 to 21 or a pharmaceutically acceptable salt thereof.
24. (A) Formula (I):
    

    

    
    Or a pharmaceutically acceptable salt thereof and (b) at least one drug independently selected from the group consisting of CFTR function improving agents and ENaC inhibitors. How to treat the disease.
25. The method according to claim 24, wherein the disease is cystic fibrosis.
26. Formulations in which (a) the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof and (b) at least one drug independently selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor are different from each other. The method according to claim 24 or 25, wherein the therapeutic agent is contained as an active ingredient and is administered simultaneously or at different times.
27. A single preparation comprising at least one drug independently selected from the group consisting of (a) the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, and (b) a CFTR function improving agent and an ENaC inhibitor. The treatment method according to claim 24 or 25, wherein the method is contained therein.
28. At least one drug independently selected from the group consisting of (a) the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, and (b) a CFTR function improving agent and an ENaC inhibitor includes them. The therapeutic method according to claim 24 or 25, which is a kit preparation.
29. 29. The treatment method according to any one of claims 24 to 28, wherein the method is for a patient who has been treated with at least one drug independently selected from the group consisting of a CFTR function improving drug and an ENaC inhibitor.
30. At least one drug independently selected from the group consisting of a CFTR function improving agent and an ENaC inhibitor is a CFTR function improving agent, wherein the CFTR function improving agent is CFTR @ potentiator, CFTR @ corrector, CFTR @ amplifier, RNA therapy 30. The treatment method according to any one of claims 24 to 29, wherein the treatment method is at least one kind independently selected from the group consisting of Gene therapy and Gene therapy.
31. At least one drug independently selected from the group consisting of a CFTR function improver and an ENaC inhibitor is a CFTR function improver, wherein the CFTR function improver is independently a group consisting of CFTR @ potentiator and CFTR @ corrector. 30. The method according to any one of claims 24 to 29, wherein the method is at least one selected from the group consisting of:
32. At least one drug independently selected from the group consisting of CFTR function improving agents and ENaC inhibitors is a CFTR function improving agent, wherein the CFTR function improving agent is Ivacaftor, QBW251, VX-561, PTI-808. GLPG1837, GLPG2451, GLPG3067, Lumacaftor, Tezacaftor, VX-445, VX-440, VX-152, VX-659, FDL169, GLPG2222, GLPG2851, GLPG2737, PTI-801, PTI-801, PTI-801, PTI-801, PTI-801, MTI 30. The method according to any one of claims 24 to 29, wherein the method is at least one independently selected.
33. At least one drug independently selected from the group consisting of a CFTR function improver and an ENaC inhibitor is a CFTR function improver, wherein the CFTR function improver is independently from the group consisting of Ivacaftor, Lumacaftor, and Tezacaftor. 30. The method according to any one of claims 24 to 29, wherein the method is at least one selected from the group consisting of:
34. At least one drug independently selected from the group consisting of CFTR function improving agents and ENaC inhibitors is an ENaC inhibitor, wherein the ENaC inhibitor is at least one selected from the group consisting of QBW276, SPX-101 and AZD5634. 30. The method of any one of claims 24 to 29, wherein the method is a species.
35. 35. The method according to claim 24, wherein the pharmaceutically acceptable salt is a magnesium salt, a calcium salt, a zinc salt, a sodium salt, a tert-butylamine salt or a diisopropylamine salt.
36. The compound represented by the formula (I) or a pharmaceutically acceptable salt thereof is bis [5- (2-chlorobenzyl) -4-methoxy-6-methylpyrimidine-2-carboxylic acid] magnesium salt. 35. The treatment method according to any one of 24 to 34.

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