Pharmaceutical composition for inhibiting the binding of von Willebrand factor to platelets

Abstract

To provide a pharmaceutical composition for inhibiting bonding between a VWF and a platelet, which comprises a low molecular weight compound as an active ingredient, is inexpensive and allows for oral administration.SOLUTION: It has been discovered that a compound having a predetermined skeleton and a compound to give a predetermined platelet mobility inhibit bonding between a VWF and GPIbα, a membrane glycoprotein receptor of a platelet, and also discovered that a pharmaceutical composition comprising these compounds can be widely used as an agent for preventing thrombotic diseases such as myocardial infarction.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for inhibiting the binding of von Willebrand factor to platelets. [Background technology]

[0002] The interaction between von Willebrand factor (VWF) in the vascular wall and platelet membrane glycoprotein GPIbα is essential for the development of thrombotic diseases such as myocardial infarction (Non-Patent Literature 1). Epidemiological studies have shown that patients with VWF deficiency have a lower risk of developing myocardial infarction (Non-Patent Literature 2). It is hoped that drugs that inhibit the binding of GPIbα to VWF would have a preventive effect against thrombotic diseases such as myocardial infarction, but such drugs have not yet been realized.

[0003] Many factors other than the binding of GPIbα to VWF are involved in the development of myocardial infarction. Although rare, Thrombotic Thrombocytepenic Purpura (TTP) is a disease in which the onset of the disease can be directly prevented by inhibiting the binding of GPIbα to VWF. The main pathophysiology of TTP is the proliferation of microvessels and platelet consumption when the constitutive cleavage of VWF multimers by ADAMTS13 is inhibited. In the microvessels, a large number of platelets accumulate on VWF multimers via GPIbα. Caplacizumab, a nanobody of an anti-VWF antibody that inhibits the binding of VWF to GPIbα, has been approved in Europe and the United States as a treatment for TTP.

[0004] Although caplacizumab is a groundbreaking new drug for rare diseases, it has the following drawbacks: 1) As it is a variable region of immunoglobulin (antibody), its molecular size is large and it cannot be applied to oral therapy; 2) It is expensive compared to small molecule compounds; 3) Because anti-VWF antibodies constitutively inhibit the binding of VWF and GPIbα, there is a risk of causing bleeding complications such as VWF deficiency and GPIbα deficiency; 4) It is difficult to mass-produce and apply to the prevention and treatment of common diseases. In other words, while caplacizumab can be used for TTP cases, its drawbacks, such as the inability to administer it orally, make it unsuitable for use as a preventive agent for thrombotic diseases or myocardial infarction. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2004-251630 [Non-patent literature]

[0006] [Non-Patent Document 1] Nat Rev Dis Primers., 2019;5(1):39 [Non-Patent Document 2] J Thromb Haemost., 2015;13(11):1999-2003 [Non-Patent Document 3] J Clin Invest., 1998;101(2):479-86 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The present invention aims to provide an inexpensive, orally administered pharmaceutical composition containing a low molecular weight compound as an active ingredient for inhibiting the binding of VWF to platelets. [Means for solving the problem]

[0008] As a result of intensive studies to solve the above problems, the present inventors have found that a compound having a predetermined skeleton or a compound that causes a predetermined platelet mobility inhibits the binding between von Willebrand factor (VWF) present on the inner wall surface of blood vessels and GPIbα, which is a platelet membrane glycoprotein receptor. Further, the present inventors have found that a pharmaceutical composition containing these compounds can be widely used as a preventive agent for thrombotic diseases such as myocardial infarction, and thus completed the present invention.

[0009] That is, the present invention is as follows. [1] A pharmaceutical composition for inhibiting the binding between von Willebrand factor (VWF) present on the inner wall surface of blood vessels and platelet membrane glycoprotein receptor GPIbα, comprising as an active ingredient a compound (1) represented by the following general formula (1) or a compound (2) represented by the following general formula (2), or a pharmacologically acceptable salt thereof. General formula (1): [Chemical formula] (In general formula (1), R A and R B are each a phenyl group, an acyclic saturated aliphatic hydrocarbon group having 1 to 6 carbon atoms, an acyclic unsaturated aliphatic hydrocarbon group having 2 to 6 carbon atoms, an alicyclic saturated hydrocarbon group having 3 to 6 members, an alicyclic unsaturated hydrocarbon group having 4 to 6 members, a saturated heterocyclic group having 5 or 6 members, or an unsaturated heterocyclic group having 5 or 6 members, and these groups may have one or more substituents selected from a saturated hydrocarbon group having 3 or fewer carbon atoms, an unsaturated hydrocarbon group having 3 or fewer carbon atoms, a saturated hydrocarbon oxy group having 2 or fewer carbon atoms, and an unsaturated hydrocarbon oxy group having 2 carbon atoms.) General formula (2): [Chemical formula] (In general formula (2), R C and R DX and Y are, respectively, a cyclopentenyl group, a cyclopentadienyl group, a phenyl group, a saturated heterocyclic group of 5 or 6 members, or an unsaturated heterocyclic group of 5 or 6 members, and these groups may have one or more substituents selected from saturated hydrocarbon groups having 4 or fewer carbon atoms and unsaturated hydrocarbon groups having 4 or fewer carbon atoms; X and Y are, respectively, -O- or -NH-. [2] Compound (1) represented by the general formula (1) or compound (2) represented by the general formula (2) [ka] [ka] The pharmaceutical composition described in [1]. [3] A pharmaceutical composition comprising the following compound (3) or a pharmacopositically acceptable salt thereof as an active ingredient, for inhibiting the binding of von Willebrand factor (VWF), which is present on the inner wall surface of blood vessels, to the platelet membrane glycoprotein receptor GPIbα. Compound (3): A compound in which, when blood to which the test compound has been added is injected at a certain rate into a flow chamber in which a flow channel is formed with a layer of VWF provided on the flow channel wall, and platelets attached to the layer of VWF provided on the flow channel wall of the flow chamber move along the flow channel wall of the flow chamber due to shear stress from the blood passing through the flow chamber, the platelet mobility is measured to be greater than 1.010 and less than 1.966. [4] The pharmaceutical composition according to [3], wherein the platelet mobility is 1.314 or higher and 1.416 or lower. [5] A pharmaceutical composition for preventing thrombosis or myocardial infarction, comprising as an active ingredient a compound (1) represented by the following general formula (1), a compound (2) represented by the following general formula (2), or a compound (3) below, or a pharmacoposly acceptable salt thereof. General formula (1): [ka] (In general formula (1), R A and R B Each of these groups is a phenyl group, an acyclic saturated aliphatic hydrocarbon group having 1 to 6 carbon atoms, an acyclic unsaturated aliphatic hydrocarbon group having 2 to 6 carbon atoms, an alicyclic saturated hydrocarbon group having 3 to 6 membered rings, an alicyclic unsaturated hydrocarbon group having 4 to 6 membered rings, a saturated heterocyclic group having 5 or 6 membered rings, or an unsaturated heterocyclic group having 5 or 6 membered rings. These groups may have one or more substituents selected from saturated hydrocarbon groups having 3 or fewer carbon atoms, unsaturated hydrocarbon groups having 3 or fewer carbon atoms, saturated hydrocarbon oxy groups having 2 or fewer carbon atoms, and unsaturated hydrocarbon oxy groups having 2 carbon atoms. General formula (2): [ka] (In general formula (2), R C and R D X and Y are, respectively, a cyclopentenyl group, a cyclopentadienyl group, a phenyl group, a saturated heterocyclic group of 5 or 6 members, or an unsaturated heterocyclic group of 5 or 6 members, and these groups may have one or more substituents selected from saturated hydrocarbon groups having 4 or fewer carbon atoms and unsaturated hydrocarbon groups having 4 or fewer carbon atoms; X and Y are, respectively, -O- or -NH-. Compound (3): A compound in which, when blood to which the test compound has been added is injected at a certain rate into a flow chamber in which a flow channel is formed with a layer of VWF provided on the flow channel wall, and platelets attached to the layer of VWF provided on the flow channel wall of the flow chamber move along the flow channel wall of the flow chamber due to shear stress from the blood passing through the flow chamber, the platelet mobility is measured to be greater than 1.010 and less than 1.966. [6] A method for screening compounds useful for inhibiting the binding of von Willebrand factor (VWF), which is present on the inner wall surface of blood vessels, to the platelet membrane glycoprotein receptor GPIbα, A process in which blood to which the test compound has been added is injected at a certain rate into a flow chamber in which a flow channel is formed with a layer of VWF provided on the flow channel wall surface. Platelets attached to the layer of VWF provided on the flow channel wall of the flow chamber, A step of measuring the mobility of platelets as they move along the flow path wall of the flow chamber due to receiving shear stress from the blood passing through the flow chamber. A method comprising the step of selecting a compound whose platelet mobility is greater than 1.010 and less than 1.966. [7] The method according to [6], wherein the platelet mobility is 1.314 or higher and 1.416 or lower. [Effects of the Invention]

[0010] The present invention provides an inexpensive, orally administered pharmaceutical composition containing a low-molecular-weight compound as an active ingredient for inhibiting the binding of vegetative whole fat (VWF) to platelets. Furthermore, it provides a pharmaceutical composition that promotes the detachment of platelets from VWF without causing bleeding complications, and can be used for the prevention or treatment of recurrence of thrombotic diseases such as myocardial infarction. [Brief explanation of the drawing]

[0011] [Figure 1] This photograph illustrates the method of platelet analysis using ImageJ. A photograph of a platelet at the moment it adheres to a glass wall (start of movement) and a photograph of a platelet after it has rolled (end of movement) are superimposed at 50% transmittance. A circle approximating the platelet and a straight line connecting the center points of the platelets at the start and end of movement are shown. [Figure 2] This figure shows the results of measuring platelet mobility using the compound of the present invention. [Modes for carrying out the invention]

[0012] The embodiments will be described in detail below with reference to the attached drawings, but this does not limit the scope of the invention as claimed.

[0013] [Pharmaceutical composition containing compound (1) or compound (2)] One embodiment of the present invention is a pharmaceutical composition for inhibiting the binding of von Willebrand factor (VWF) present on the inner wall surface of blood vessels to the platelet membrane glycoprotein receptor GPIbα, which contains, as an active ingredient, a compound (1) represented by the general formula (1), a compound (2) represented by the general formula (2), or a pharmacologically acceptable salt thereof.

[0014] Compound (1) is a compound represented by the following general formula (1). General formula (1):

Chemical formula

[0015] In general formula (1), R A and R B are each a phenyl group, an acyclic saturated aliphatic hydrocarbon group having 1 to 6 carbon atoms, an acyclic unsaturated aliphatic hydrocarbon group having 2 to 6 carbon atoms, an alicyclic saturated hydrocarbon group having 3 to 6 members, an alicyclic unsaturated hydrocarbon group having 4 to 6 members, a saturated heterocyclic group having 5 or 6 members, or an unsaturated heterocyclic group having 5 or 6 members. These groups may have one or more substituents selected from a saturated hydrocarbon group having 3 or fewer carbon atoms, an unsaturated hydrocarbon group having 3 or fewer carbon atoms, a saturated hydrocarbon oxy group having 2 or fewer carbon atoms, and an unsaturated hydrocarbon oxy group having 2 carbon atoms at any position.

[0016] In the present specification, the aliphatic hydrocarbon group may be either linear or branched, and the position and number of unsaturated bonds in various unsaturated groups are not particularly limited. Also, in the present specification, the heteroatom contained in the ring structure of the saturated heterocyclic group having 5 or 6 members is not particularly limited, and examples include a nitrogen atom, an oxygen atom, a sulfur atom, etc. The position and number of heteroatoms contained in one heterocyclic ring are also not particularly limited. Unless otherwise specified, the compounds in the present specification may be any stereoisomers and any geometric isomers, and may also be mixtures in any ratio thereof.

[0017] ​​Examples of acyclic saturated aliphatic hydrocarbon groups having 1 to 6 carbon atoms include alkyl groups such as methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, 1-methylbutyl group, 2-methylbutyl group, 3-methylbutyl group, 1-ethylpropyl group, neopentyl group, n-hexyl group, 1-methylpentyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 1,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2,3-dimethylbutyl group, 3,3-dimethylbutyl group, 1-ethylbutyl group, and 2-ethylbutyl group.

[0018] Examples of acyclic unsaturated aliphatic hydrocarbon groups having 2 to 6 carbon atoms include vinyl group, allyl group, isopropenyl group, 1-butenyl group, 2-butenyl group, 3-butenyl group, 1-methyl-1-propenyl group, 1-methyl-2-propenyl group, 2-methyl-1-propenyl group, 1,3-butadienyl group, 1-pentenyl group, 2-pentenyl group, 3-pentenyl group, 4-pentenyl group, 1,3-pentadienyl group, 2,4-pentadienyl group, 1-hexenyl group, 2-hexenyl group, 3-hexenyl group, 4-hexenyl group, and 5-hexenyl group. Examples include the 2,4-hexadienyl group, 3,5-hexadienyl group, ethynyl group, 1-propynyl group, 2-propynyl group, 1-butynyl group, 2-butynyl group, 3-butynyl group, 1,3-butadiinyl group, 1-pentynyl group, 2-pentynyl group, 3-pentynyl group, 4-pentynyl group, 1,3-pentadynyl group, 1-hexynyl group, 2-hexynyl group, 3-hexynyl group, 4-hexynyl group, 5-hexynyl group, 2,4-hexadienyl group, 3,5-hexadienyl group, and 1,3,5-hexatriinyl group.

[0019] Examples of 3- to 6-membered alicyclic saturated hydrocarbon groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups.

[0020] Examples of alicyclic unsaturated hydrocarbon groups with 4 to 6 member rings include 1-cyclobutenyl group, 2-cyclobutenyl group, 1,3-cyclobutadienyl group, 1-cyclopentenyl group, 2-cyclopentenyl group, 3-cyclopentenyl group, 1,3-cyclopentadienyl group, 2,4-cyclopentadienyl group, l-cyclohexenyl group, 2-cyclohexenyl group, 3-cyclohexenyl group, 1,3-cyclohexadienyl group, 1,4-cyclohexadienyl group, 2,4-cyclohexadienyl group, and 2,5-cyclohexadienyl group.

[0021] Specific examples of saturated heterocyclic groups with five members include pyrrolidine-1-yl, pyrrolidine-2-yl, pyrrolidine-3-yl, pyrazolidine-1-yl, pyrazolidine-4-yl, imidazolidined-1-yl, imidazolidined-2-yl, 1,2,3-triazolidine-1-yl, 1,2,4-triazolidine-1-yl, and 1,2,4-triazol Examples include lysine-3-yl group, tetrazolidine-1-yl group, tetrazolidine-5-yl group, tetrahydrofuran-2-yl group, tetrahydrofuran-3-yl group, 1,2-dioxolan-3-yl group, 1,2-dioxolan-4-yl group, 1,3-dioxolan-2-yl group, 1,3-dioxolan-4-yl group, tetrahydrothiophene-2-yl group, tetrahydrothiophene-3-yl group, 1,2-dithiolan-3-yl group, 1,2-dithiolan-4-yl group, 1,3-dithiolan-2-yl group, and 1,3-dithiolan-4-yl group.

[0022] Specific examples of saturated heterocyclic groups with six members include piperidine-1-yl, piperidine-2-yl, piperidine-3-yl, piperidine-4-yl, piperazine-1-yl, piperazine-2-yl, 1,3,5-triazinan-1-yl, 1,3,5-triazinan-2-yl, 1,2,4,5-tetradinan-1-yl, 1,2,4,5-tetradinan-3-yl, tetrahydropyran-2-yl, tetrahydropyran-3-yl, tetrahydropyran-4-yl, 1,2-dioxan-3-yl, 1,2-dioxan- Examples include 4-yl group, 1,3-dioxan-2-yl group, 1,3-dioxan-4-yl group, 1,3-dioxan-5-yl group, 1,4-dioxan-2-yl group, thian-2-yl group, thian-3-yl group, thian-4-yl group, 1,3-dithian-2-yl group, 1,3-dithian-4-yl group, 1,3-dithian-5-yl group, 1,4-dithian-2-yl group, morpholine-2-yl group, morpholine-3-yl group, morpholine-4-yl group, thiomorpholine-2-yl group, thiomorpholine-3-yl group, and thiomorpholine-4-yl group.

[0023] Examples of five-membered unsaturated heterocyclic groups include imidazole-1-yl group, imidazole-2-yl group, 1,2,4-triazolin-1-yl group, pyrrole-1-yl group, pyrrole-2-yl group, pyrrole-3-yl group, pyrazole-1-yl group, pyrazole-3-yl group, pyrazole-4-yl group, pyrazole-5-yl group, 1,2,3-triazole-1-yl group, 1,2,3-triazole-4-yl group, Examples include 1,2,4-triazole-1-yl group, 1,2,4-triazole-4-yl group, tetrazole-1-yl group, tetrazole-4-yl group, furan-2-yl group, furan-3-yl group, thiophene-2-yl group, thiophene-3-yl group, 1,3-oxazole-2-yl group, 1,3-oxazole-4-yl group, 1,3-thiazole-2-yl group, and 1,3-thiazole-4-yl group.

[0024] Examples of six-membered unsaturated heterocyclic groups include pyridine-2-yl, pyridine-3-yl, pyridine-4-yl, pyrimidine-2-yl, pyrimidine-4-yl, pyrimidine-5-yl, pyrazine-2-yl, pyridazine-3-yl, pyridazine-4-yl, 1,3,5-triazine-2-yl, 1,2,4,5-tetrazin-3-yl, 3,4-dihydro-2H-pyran-5-yl, 3,4-dihydro-2H-pyran-6-yl, 3,6-dihydro-2H-pyran-4-yl, and 3,6-dihydro-2H-thiopyran-4-yl.

[0025] The substituents that the above group may have are described below. Examples of saturated hydrocarbon groups with 3 or fewer carbon atoms include methyl, ethyl, n-propyl, isopropyl, and cyclopropyl groups. Examples of unsaturated hydrocarbon groups having 3 or fewer carbon atoms include vinyl groups, allyl groups, isopropenyl groups, ethynyl groups, 1-propynyl groups, and 2-propynyl groups. Examples of saturated hydrocarbon oxy groups with two or fewer carbon atoms include methoxy groups and ethoxy groups. Examples of carbon-2 unsaturated hydrocarbon oxy groups include vinyloxy groups and ethynyloxy groups.

[0026] The following describes preferred embodiments of the compound represented by general formula (1). R A and R B If the group is a phenyl group, a saturated heterocyclic group of 5 or 6 members, or an unsaturated heterocyclic group of 5 or 6 members, it is preferable that these groups have the above substituents. Also, R A and R B It is preferable that it includes rigid structures such as unsaturated bonds and cyclic structures. A and R BThis is because the inclusion of a rigid structure stabilizes the hydrophobic interaction between the compound represented by general formula (1) and the VWF of the blood vessel wall, making it possible to stably obtain the effect of inhibiting the interaction between the VWF of the blood vessel wall and the platelet membrane glycoprotein GPIbα. Examples of groups containing a rigid structure include phenyl groups, acyclic unsaturated aliphatic hydrocarbon groups having 2 to 6 carbon atoms, alicyclic saturated hydrocarbon groups having 3 to 6 membered rings, alicyclic unsaturated hydrocarbon groups having 4 to 6 membered rings, saturated heterocyclic groups having 5 or 6 membered rings, and unsaturated heterocyclic groups having 5 or 6 membered rings. Among these, phenyl groups having a cyclic structure, alicyclic saturated hydrocarbon groups having 3 to 6 membered rings, alicyclic unsaturated hydrocarbon groups having 4 to 6 membered rings, saturated heterocyclic groups having 5 or 6 membered rings, and unsaturated heterocyclic groups having 5 or 6 membered rings are preferred. Also, R A From the viewpoint of being advantageous in forming hydrophobic interactions between the compound represented by general formula (1) and the VWF of the blood vessel wall, for example, R in the compound dealt with in the examples described later A Examples of equivalent groups include the cyclohexyl group, cyclobutyl group, and 4-methoxyphenyl group. R B For the same reason, the R in the compound dealt with in the examples described later is B Examples of equivalent groups include the 1-methylimidazole-2-yl group, the 4-methoxyphenyl group, and the 1-methylpyrazole-4-yl group.

[0027] Compounds represented by general formula (1) are either known or can be produced by known methods.

[0028] Compound (1) is not particularly limited as long as it does not hinder the effect of inhibiting the binding of VWF to platelets in the pharmaceutical composition of the present invention, and examples include the following compounds. [ka] [ka]

[0029] Compound (2) is a compound represented by the following general formula (2). General formula (2): [ka]

[0030] In general formula (2), R C and R D These groups are, respectively, a cyclopentenyl group, a cyclopentadienyl group, a phenyl group, a saturated heterocyclic group of 5 or 6 members, or an unsaturated heterocyclic group of 5 or 6 members. These groups may have one or more substituents selected from saturated hydrocarbon groups having 4 or fewer carbon atoms and unsaturated hydrocarbon groups having 4 or fewer carbon atoms at any position. Furthermore, in general formula (2), X and Y are -O- or -NH-, respectively.

[0031] The cyclopentenyl group may be any of 1-cyclopentenyl, 2-cyclopentenyl, or 3-cyclopentenyl groups, and the cyclopentadienyl group may be any of 1,3-cyclopentadienyl or 2,4-cyclopentadienyl groups.

[0032] R C and R D The saturated heterocyclic group of a 5 or 6-membered ring and the unsaturated heterocyclic group of a 5 or 6-membered ring, represented by R, are as follows: A and R B Examples include saturated heterocyclic groups of 5 or 6 members and unsaturated heterocyclic groups of 5 or 6 members, as represented by the formula.

[0033] The substituents that the above group may have are described below. Examples of saturated hydrocarbon groups with 4 or fewer carbon atoms include methyl group, ethyl group, n-propyl group, and iso Examples include acyclic saturated aliphatic hydrocarbon groups such as propyl groups, n-butyl groups, isobutyl groups, sec-butyl groups, and tert-butyl groups; and alicyclic saturated hydrocarbon groups such as cyclopropyl groups and cyclobutyl groups. Examples of unsaturated hydrocarbon groups having 4 or fewer carbon atoms include vinyl groups, allyl groups, isopropenyl groups, 1-butenyl groups, 2-butenyl groups, 3-butenyl groups, 1-methyl-1-propenyl groups, 1-methyl-2-propenyl groups, 2-methyl-1-propenyl groups, 1,3-butadienyl groups, ethynyl groups, 1-propynyl groups, 2-propynyl groups, 1-butynyl groups, 2-butynyl groups, 3-butynyl groups, and 1,3-butadiinyl groups; and alicyclic unsaturated hydrocarbon groups such as 1-cyclobutenyl groups, 2-cyclobutenyl groups, and 1,3-cyclobutadienyl groups.

[0034] The following describes preferred embodiments of the compound represented by general formula (2). R C and R D The cyclopentenyl group, cyclopentadienyl group, phenyl group, saturated heterocyclic group of 5 or 6 members, and unsaturated heterocyclic group of 5 or 6 members represented by the formula may be unsubstituted, but it is preferable that they have the above substituents. The number of carbon atoms in the saturated hydrocarbon group and the unsaturated hydrocarbon group that are substituents is preferably 3 or less, and more preferably 2 or less. Also, R C and R D These may be different from each other, but it is preferable that they be the same group. From a drug delivery standpoint, X and Y are preferably -NH-. Furthermore, X and Y may be different from each other, but it is preferable that they be the same group, and it is particularly preferable that both be -NH-.

[0035] Compounds represented by general formula (2) are either known or can be produced by known methods.

[0036] Compound (2) is not particularly limited as long as it does not hinder the effect of inhibiting the binding of VWF to platelets in the pharmaceutical composition of the present invention, and examples include the following compounds. [ka]

[0037] In this specification, "pharmacologically acceptable" means suitable for pharmacological use. Examples of pharmacologically acceptable salts include: salts of alkali metals such as sodium and potassium; magnesium salts; salts of alkaline earth metals such as calcium; salts of hydrohalic acids such as hydrofluoric acid, hydrochloric acid, hydrobromic acid, and hydroiodic acid; salts of inorganic acids such as sulfuric acid, nitric acid, phosphoric acid, perchloric acid, and carbonic acid; salts of organic carboxylic acids such as formic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, hydroxyacetic acid, propionic acid, lactic acid, citric acid, tartaric acid, oxalic acid, benzoic acid, mandelic acid, butyric acid, fumaric acid, succinic acid, maleic acid, and malic acid; salts of acidic amino acids such as aspartic acid and glutamic acid; salts of basic amino acids such as arginine and lysine; ammonium salts; salts of aliphatic amines such as trimethylamine, triethylamine, and ethanolamine; and salts of sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and toluenesulfonic acid.

[0038] Furthermore, the compound according to this embodiment may produce a specific platelet mobility, as described in the section [Pharmaceutical composition containing compound (3)] below.

[0039] The compound may be commercially available, or it may be chemically synthesized by a method known to those skilled in the art.

[0040] The mechanism of action of the pharmaceutical composition according to this embodiment is as follows: In the bloodstream, platelets probabilistically come into contact with vegetative whole follicular (VWF) present on the vessel wall, thereby binding to the vessel wall via GPIbα on the platelets. The bound VWF and GPIbα are subjected to shear stress from the bloodstream, causing them to be pulled together and their structure to change. The compound contained in the pharmaceutical composition according to this embodiment enters the gap formed by the VWF and GPIbα, and in particular enters the gap formed by the VWF and GPIbα whose structure has changed due to being pulled together as described above. As a result, it promotes the detachment of platelets from the vessel wall, creating a condition in which platelets are less likely to adhere to the vessel wall. This makes it less likely for platelets to aggregate on the vessel wall, which is the subsequent thrombus formation step, and can even prevent thrombus formation.

[0041] The target diseases of the pharmaceutical composition according to this embodiment are not particularly limited, as long as it is used to inhibit the binding of VWF to platelets. Examples of target diseases include thrombotic diseases, and examples of thrombotic diseases include arterial thrombosis such as myocardial infarction and cerebral infarction.

[0042] The pharmaceutical composition according to this embodiment can be used for the prevention and / or treatment of these thrombotic diseases, but it is preferable to use it for the prevention of thrombotic diseases.

[0043] The target animal for administration of the pharmaceutical composition according to this embodiment is a mammal, but it is preferably a human.

[0044] The pharmaceutical composition according to this embodiment is preferably administered orally, but can also be administered parenterally. Examples of parenteral administration include intravenous administration, intra-arterial administration, and local injection.

[0045] The dosage form of the pharmaceutical composition according to this embodiment is preferably an oral dosage form such as a tablet, powder, granule, or capsule, but it may also be an injection, suspension, emulsifier, or other dosage form. These various dosage forms may contain commonly used excipients, bulking agents, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, solubilizers, antiseptics, colorants, flavoring agents, stabilizers, etc., and can be manufactured by methods known to those skilled in the art.

[0046] The compound content in the pharmaceutical composition according to this embodiment is not particularly limited as long as it does not hinder the effects of the present invention.

[0047] The dosage, frequency of administration, duration of administration, etc., of the pharmaceutical composition according to this embodiment are not particularly limited as long as they do not hinder the effects of the present invention, and can be appropriately adjusted depending on the age, sex, weight, type of disease, route of administration, etc., of the recipient. For example, when administered orally to a human adult (60 kg body weight), the dosage per compound (1) or compound (2) may be 0.001 to 100 mg per day, or 0.01 to 10 mg per day. Also, for example, administration 1 to 3 times a day may be carried out for 1 day or more, 5 days or more, 10 days or more, 15 days or more, 20 days or more, 1 month or more, 2 months or more, 3 months or more, 6 months or more, or 1 year or less. The duration of administration may be expressed as a range combining either the lower limit or upper limit mentioned above. For example, it may be 1 day or more and 1 year or less, or 5 days or more and 2 months or less.

[0048] [Pharmaceutical composition containing compound (3)] Another embodiment of the present invention is a pharmaceutical composition for inhibiting the binding of von Willebrand factor (VWF), which is present on the inner wall surface of blood vessels, to the platelet membrane glycoprotein receptor GPIbα, comprising compound (3) or a pharmaceutically acceptable salt thereof as an active ingredient.

[0049] Compound (3) is a compound in which, when blood to which the test compound has been added is injected at a certain rate into a flow chamber in which a flow channel is formed with a layer of VWF provided on the flow channel wall, and platelets attached to the layer of VWF provided on the flow channel wall of the flow chamber move along the flow channel wall of the flow chamber due to shear stress from the blood passing through the flow chamber, the platelet mobility is measured to be greater than 1.010 and less than 1.966.

[0050] Platelet mobility can be measured by methods known to those skilled in the art, for example, using the apparatus disclosed in Patent Document 1. The measurement method may include, for example, at least the following steps: A process of injecting blood to which the test compound has been added at a certain rate into a flow chamber in which a flow channel is formed, with a layer of VWF (very wide wave flow) provided on the flow channel wall; A step of measuring the degree of platelet mobility when platelets attached to a layer of VWF provided on the flow channel wall of the flow chamber move along the flow channel wall of the flow chamber due to shear stress from the blood passing through the flow chamber.

[0051] The above measurement method may optionally include additional steps as needed.

[0052] A flow chamber in which a channel is formed with a layer of VWF on the channel wall can be prepared in advance. The method for preparing this flow chamber is not particularly limited as long as a layer of VWF can be formed on the channel wall, but for example, a VWF solution with a final concentration of 20-30 μg / mL can be dropped onto a glass slide, and then dried for 1-5 hours in an environment of 30-40% relative humidity to solidify it.

[0053] The blood sample used to measure platelet mobility is not particularly limited, but for example, blood from a healthy person can be used.

[0054] When a blood sample is injected into a flow chamber, the blood is injected at a constant rate. This constant rate is preferably similar to the blood flow velocity in a living body. The blood flow velocity can be within a physiological range, but for example, it can be expressed as the shear rate on the glass wall of the flow chamber into which the blood sample is injected. The shear rate is, for example, 1000-2000 s. -1 It may be, preferably 1400-1600s -1 And more preferably 1500s -1 That is the case.

[0055] Platelet mobility can be calculated by determining the diameter A of a perfect circle approximating the platelet, and the length of the line B connecting the center point of the platelet when it begins to move due to shear stress after adhering to the wall, and the center point of the platelet when it stops moving, and then calculating the formula "length of line B ÷ diameter A". The center point of the platelet is the center when the platelet is approximated as a perfect circle.

[0056] This study shows that, under a predetermined flow rate, the greater the platelet mobility of the blood to which the compound has been added, the more the interaction between VWF present on the blood vessel wall and GPIBα on platelets is inhibited. On the other hand, if the mobility is too high, blood coagulation is inhibited, raising concerns about bleeding. Therefore, the platelet mobility is preferably within the following values.

[0057] The platelet mobility when using the compound in the pharmaceutical composition according to this embodiment is The pharmaceutical composition relating to the state is not particularly limited as long as it inhibits the binding of VWF to the platelet membrane glycoprotein receptor GPIbα. The lower limit of platelet mobility may be, for example, 1.010 or higher, 1.045 or higher, 1.052 or higher, 1.057 or higher, 1.110 or higher, 1.143 or higher, 1.170 or higher, 1.196 or higher, 1.215 or higher, 1.314 or higher, 1.374 or higher, 1.413 or higher, or 1.416 or higher, or greater than 1.010, greater than 1.045, greater than 1.052, greater than 1.057, greater than 1.110, greater than 1.143, greater than 1.170, greater than 1.196, greater than 1.215, greater than 1.314, greater than 1.374, greater than 1.413, or greater than 1.416. Furthermore, the upper limit of platelet mobility may be, for example, 1.966 or less, 1.716 or less, 1.691 or less, 1.674 or less, 1.626 or less, 1.617 or less, 1.458 or less, 1.416 or less, 1.413 or less, 1.374 or less, 1.340 or less, 1.314 or less, 1.196 or less, or 1.045 or less, or less than 1.966, less than 1.716, less than 1.691 or less, less than 1.674 or less, less than 1.626, less than 1.617, less than 1.458, less than 1.416, less than 1.413, less than 1.374, less than 1.340, less than 1.314, less than 1.196, or less than 1.045. Furthermore, platelet mobility may be expressed as a range that combines either the lower limit or upper limit mentioned above. For example, it may be greater than 1.010 and less than 1.966, greater than 1.143 and less than 1.626, or 1.314 or greater and 1.416 or less.

[0058] Compound (3) may be a compound represented by general formula (1) or general formula (2) as described in the section above on [Pharmaceutical compositions containing compound (1) or compound (2)].

[0059] The target diseases, target animals, route of administration, dosage form, compound content, dosage, frequency of administration, duration of administration, etc., of the pharmaceutical composition according to this embodiment, as well as the pharmacochemically acceptable salts of the compounds contained in the pharmaceutical composition, can be referenced from the description in the section [Pharmaceutical composition containing compound (1) or compound (2)] above.

[0060] [A pharmaceutical composition comprising compound (1), compound (2), or compound (3) for the purpose of preventing thrombosis or myocardial infarction] Another embodiment of the present invention is a pharmaceutical composition for preventing thrombosis or myocardial infarction, comprising as an active ingredient a compound (1) represented by general formula (1), a compound (2) represented by general formula (2), or a compound (3), or a pharmaceutically acceptable salt thereof.

[0061] General formula (1): [ka]

[0062] In general formula (1), R A and R B These are, respectively, a phenyl group and an acyclic saturated carbon atom with 1 to 6 carbon atoms. These are aliphatic hydrocarbon groups, acyclic unsaturated aliphatic hydrocarbon groups having 2 to 6 carbon atoms, alicyclic saturated hydrocarbon groups having 3 to 6 membered rings, alicyclic unsaturated hydrocarbon groups having 4 to 6 membered rings, saturated heterocyclic groups having 5 or 6 membered rings, or unsaturated heterocyclic groups having 5 or 6 membered rings. These groups may have one or more substituents at any position selected from saturated hydrocarbon groups having 3 or less carbon atoms, unsaturated hydrocarbon groups having 3 or less carbon atoms, saturated hydrocarbon oxy groups having 2 or less carbon atoms, and unsaturated hydrocarbon oxy groups having 2 carbon atoms. For details, refer to the description in the section above on [Pharmaceutical Compositions Containing Compound (1) or Compound (2)].

[0063] General formula (2): [ka]

[0064] In general formula (2), RC and R D These groups are, respectively, a cyclopentenyl group, a cyclopentadienyl group, a phenyl group, a saturated heterocyclic group of 5 or 6 members, or an unsaturated heterocyclic group of 5 or 6 members. These groups may have one or more substituents selected from saturated hydrocarbon groups having 4 or fewer carbon atoms and unsaturated hydrocarbon groups having 4 or fewer carbon atoms at any position. Furthermore, in general formula (2), X and Y are -O- or -NH-, respectively. For further details, refer to the description in the section above concerning [pharmaceutical compositions containing compound (1) or compound (2)].

[0065] Compound (3) is a compound in which, when blood to which the test compound has been added is injected at a certain rate into a flow chamber in which a flow channel is formed with a layer of VWF provided on the flow channel wall, and platelets attached to the layer of VWF provided on the flow channel wall of the flow chamber move along the flow channel wall of the flow chamber due to shear stress from the blood passing through the flow chamber, the platelet mobility is measured to be greater than 1.010 and less than 1.966. For further details, refer to the description in the section above titled [Pharmaceutical Compositions Containing Compound (3)].

[0066] The pharmaceutical composition according to this embodiment can prevent thrombosis by inhibiting the binding of von Willebrand factor (VWF), which is present on the inner wall surface of blood vessels, to the platelet membrane glycoprotein receptor GPIbα, through the mechanism of action of the pharmaceutical composition described above in [Pharmaceutical composition containing compound (1) or compound (2)]. Furthermore, as described in Non-Patent Literature 1, etc., the interaction between VWF on the blood vessel wall and platelet membrane glycoprotein GPIbα is essential for the development of thrombotic diseases such as myocardial infarction. Since the pharmaceutical composition according to this embodiment inhibits the binding of VWF to the platelet membrane glycoprotein receptor GPIbα, the VWF on the blood vessel wall and platelet membrane glycoprotein GPIbα cannot interact, thus preventing myocardial infarction.

[0067] Regarding the target diseases, target animals, route of administration, dosage form, compound content, dosage, frequency of administration, duration of administration, etc. of the pharmaceutical composition according to this embodiment, as well as each group and substituent of the compound contained in the pharmaceutical composition, the compounds specifically listed, pharmacologically acceptable salts, platelet mobility, etc., refer to the descriptions in the above sections [Pharmaceutical composition containing compound (1) or compound (2)] or [Pharmaceutical composition containing compound (3)].

[0068] [Screening method for pharmaceutical compositions] Another embodiment of the present invention is a method for screening compounds useful for inhibiting the binding of von Willebrand factor (VWF), which is present on the inner wall surface of blood vessels, to the platelet membrane glycoprotein receptor GPIbα, A process in which blood to which the test compound has been added is injected at a certain rate into a flow chamber in which a flow channel is formed with a layer of VWF provided on the flow channel wall surface. A step of measuring the mobility of platelets when platelets attached to a layer of VWF provided on the flow channel wall of the flow chamber move along the flow channel wall of the flow chamber due to shear stress from the blood passing through the flow chamber. The method includes the step of selecting a compound whose platelet mobility is greater than 1.010 and less than 1.966.

[0069] To measure the platelet mobility in the screening method according to this embodiment, each step can be performed by referring to the description in the section [Pharmaceutical composition containing compound (3)] above.

[0070] In the screening method according to this embodiment, compounds are selected based on the fact that the lower limit of platelet mobility is, for example, 1.010 or higher, 1.045 or higher, 1.052 or higher, 1.057 or higher, 1.110 or higher, 1.143 or higher, 1.170 or higher, 1.196 or higher, 1.215 or higher, 1.314 or higher, 1.374 or higher, 1.413 or higher, or 1.416 or higher, or greater than 1.010, greater than 1.045, greater than 1.052, greater than 1.057, greater than 1.110, greater than 1.143, greater than 1.170, greater than 1.196, greater than 1.215, greater than 1.314, greater than 1.374, greater than 1.413, or greater than 1.416, and The upper limit of platelet mobility can be selected based on, for example, whether it is 1.966 or less, 1.716 or less, 1.691 or less, 1.674 or less, 1.626 or less, 1.617 or less, 1.458 or less, 1.416 or less, 1.413 or less, 1.374 or less, 1.340 or less, 1.314 or less, 1.196 or less, or 1.045 or less, or whether it is less than 1.966, less than 1.716, less than 1.691 or less, less than 1.674, less than 1.626, less than 1.617, less than 1.458, less than 1.416, less than 1.413, less than 1.374, less than 1.340, less than 1.314, less than 1.196 or less, or less than 1.045. The range of platelet mobility may be expressed as a combination of either the lower limit or upper limit mentioned above. For example, it may be greater than 1.010 and less than 1.966, greater than 1.143 and less than 1.626, or greater than or equal to 1.314 and less than or equal to 1.416.

[0071] By selecting compounds based on the platelet mobility described above, it is possible to select compounds that inhibit the interaction between VWF present on the blood vessel wall and GPIBα in platelets. On the other hand, the compounds selected in this way do not inhibit normal blood coagulation to the extent that it would cause concerns about bleeding in the recipient. [Examples]

[0072] The present invention will be described below using examples, but the present invention is not limited to these examples.

[0073] [in silico screening] The three-dimensional binding structure of VWF and GPIbα, which are responsible for platelet adhesion, was calculated using molecular dynamics simulations as an accumulation of femtosecond-level motions of the constituent atoms and surrounding water molecules. Shear stress, acting on platelets attached to the blood vessel wall, was applied to GPIbα, which supports platelet adhesion, and the structure of the intermolecular gap immediately before detachment from VWF was identified. Small molecules that enter the gap between the two molecules immediately before detachment were screened using in silico screening. The resulting small molecules are those that penetrate the gap between VWF and GPIbα immediately before detachment, promoting the detachment of both molecules. These in silico-screened compounds were purchased from Namiki Trading Co., Ltd. and used as test compounds.

[0074] [Test compound] Each test compound was dissolved in 10% DMSO (Sigma-Aldrich) and prepared as a Stock solution. (Daiichi Sankyo Co., Ltd.) Stored at -80°C at 1 mM. When actually using the test compound, it was prepared with 1.0% DMSO to a final concentration of 100 μM and dissolved in blood.

[0075] [Blood specimen] Ten milliliters of blood were collected from healthy adults who had not taken any medication for more than 10 days. First, to maintain blood fluidity, the antithrombin drug Argatroban (Tanabe Mitsubishi Pharma Corporation) was prepared in HEPES buffer to a final concentration of 100 μM, and 1 mL of this solution was prepared in a 50 mL centrifuge tube. A sterile 19G butterfly needle and a 20 mL syringe were taken out and connected. The upper arm was tourniqueted, and the 19G butterfly needle was inserted into the brachial vein through the alcohol-disinfected skin, and 10 mL of blood was collected at a rate of 1 mm / s. After blood collection, the tourniquet was released, and hemostasis was performed after needle removal. The collected blood was quickly transferred to the pre-prepared centrifuge tube containing Argatroban at a rate of 1 mm / s via the wall and mixed to perform anticoagulation treatment.

[0076] [Experimental equipment] A known experimental apparatus for measuring platelet mobility was used (Patent Document 1). The specific configuration is as follows: To reproduce a simulated blood flow in vitro, the apparatus configuration shown in Figure 1 of Patent Document 1 was used. The chamber section is as shown in Figure 3 of Patent Document 1, and specifically consists of a flow chamber, syringe pump, fluid delivery tube, waste fluid tube, and waste fluid tank. The detailed configuration of the flow chamber is as shown in Figure 4 of Patent Document 1. 10 μL of VWF (Very Wide Fluid) solution (dissolved in HEPES buffer) with a final concentration of 25 μg / mL was dropped onto a glass slide (24 mm wide x 50 mm long x 0.17 mm thick), and the solid phase layer was prepared by drying for 3 hours in an environment of relative humidity 30-40%.

[0077] [Measurement of platelet mobility] Platelet mobility was measured at room temperature (20-27°C). A glass slide glass with VWF (Very Whole Wood Foam) solidified was placed in a flow chamber. 1 mL of blood was taken into a 5 mL centrifuge tube, and FITC abciximab (Centcore) was added to a final concentration of 10 μg / mL to fluorescently label the platelets. Then, reagents were added to the following final concentrations: Caplacizumab (Creative Biolabs, model TAB-234) 400 nM, DMSO 1.0%, and test compound 100 μM (dissolved in DMSO 1.0%). The prepared blood was filled into a 2 mm inner diameter, 3 mm outer diameter, 500 mm long silicone delivery tube. A Parafilm-wrapped tube connector was attached to one end of the delivery tube, and a three-way stopcock was attached to the other end. 1 mL of the prepared blood was introduced through the tube connector using a micropipette, and the three-way stopcock was closed. A 20 mL syringe was attached to the syringe pump, and the three-way stopcock of the liquid delivery tube was attached to the syringe. A tube connector wrapped in Parafilm was connected to the inlet of the flow chamber. A silicone waste liquid tube with a tube connector attached was also connected to the outlet of the flow chamber, and a waste liquid tank was prepared at the end of it. The shear rate applied to the glass wall was 1500 s. -1 To achieve this, the syringe pump's extrusion speed was set to 0.37 mL / min, and blood was introduced into the flow chamber. Note that 1500 s was used on the wall. -1 It is known that the binding of GPIbα to VWF is essential for platelet adhesion under the shear rate conditions (Non-Patent Literature 3). The image display device (PC) was started and video was acquired. Video acquisition began immediately after blood flow, and the focus was manually adjusted. The first field of view in focus from the acquired video was output as an AVI file. The output video file was analyzed using the open-source image processing software ImageJ (Figure 1). In this analysis, platelets that were already attached at the time of video recording were excluded, and only newly attached platelets were analyzed. These were included in the study. In addition, platelets that came into contact with other platelets during adhesion or rolling, newly adhered but not completely immobile, still moving at the end of the video, or showing signs of movement were excluded from the analysis. To calculate the rolling distance of each platelet, images of the frame in which the platelet first appeared in the field of view and the frame in which it stopped moving were extracted from the video of each platelet and superimposed with a transparency of 50% to create an image. Images were processed using ImageJ. The superimposed images were loaded into ImageJ, and the circle tool was used to enclose the larger platelet with a perfect circle, obtaining the diameter A of the circle approximating the platelet. For platelets that were not perfectly round, such as ellipses, a perfect circle was created so that the area was the same from the center point of the platelet. The center points of the platelets at the start and end of their movement were connected by a straight line B. The center point was the center of the perfect circle approximating the platelet. The diameter A of the circle and the length of line B were calculated, and "length of line B ÷ diameter A" was calculated. This operation was performed for all the extracted platelet images. In this way, the platelet mobility was determined as a parameter that normalizes the movement distance for different platelet sizes. Based on the results, the average mobility was calculated. Statistical significance was determined using a 95% confidence interval.

[0078] [Table 1]

[0079] Table 1 shows the results obtained in this analysis. The platelet mobility when using DMSO was 1.010±0.133, and when using Caplacizumab it was 1.966±0.340. In contrast, the mobility when using the compounds of the present invention was 1.196±0.144, 1.314±0.144, 1.416±0.201, 10.10.374±0.317, 15.15.413±0.303, and 13.045±0.629 (mean ± 95% confidence interval). In other words, all of the compounds of the present invention showed a higher mean mobility than when using DMSO and a lower mean mobility than when using Caplacizumab. In particular, compounds 9, 4, 10, and 15 showed statistically significant differences compared to when using DMSO or Caplacizumab. T-tests were performed, and statistically significant differences were observed for samples 9, 4, 10, and 15, with P<0.05 (Figure 2). These results suggest that the compounds of the present invention have an inhibitory effect on the binding of VWF and GPIbα, and that they have a low risk of causing bleeding complications.

Claims

1. The active ingredient is a compound (1) represented by the general formula (1) below or a pharmacologically acceptable salt thereof, which interacts with von Willebrand factor (VWF) present on the inner wall surface of blood vessels and platelets. A pharmaceutical composition for inhibiting binding to the membrane glycoprotein receptor GPIbα. General formula (1): 【Chemistry 1】 (In general formula (1), R Ａ However, the group is a phenyl group or a 4-6 membered alicyclic saturated hydrocarbon group, and these groups may have one or more substituents selected from saturated hydrocarbon groups having 3 or less carbon atoms, unsaturated hydrocarbon groups having 3 or less carbon atoms, saturated hydrocarbon oxy groups having 2 or less carbon atoms, and unsaturated hydrocarbon oxy groups having 2 carbon atoms. R B is a phenyl group, an acyclic saturated aliphatic hydrocarbon group having 1 to 6 carbon atoms, or an unsaturated heterocyclic group with 5 or 6 members, and these groups may have one or more substituents selected from saturated hydrocarbon groups having 3 or less carbon atoms, unsaturated hydrocarbon groups having 3 or less carbon atoms, saturated hydrocarbon oxy groups having 2 or less carbon atoms, and unsaturated hydrocarbon oxy groups having 2 carbon atoms.

2. The compound (1) represented by the general formula (1) is 【Chemistry 2-1】 or 【Chemistry 2-2】 The pharmaceutical composition according to claim 1.

3. A pharmaceutical composition for preventing thrombosis or myocardial infarction, comprising a compound (1) represented by the following general formula (1) or a pharmacoposly acceptable salt thereof as an active ingredient. General formula (1): 【Transformation 3】 (In general formula (1), R Ａ However, the group is a phenyl group or a 4-6 membered alicyclic saturated hydrocarbon group, and these groups may have one or more substituents selected from saturated hydrocarbon groups having 3 or less carbon atoms, unsaturated hydrocarbon groups having 3 or less carbon atoms, saturated hydrocarbon oxy groups having 2 or less carbon atoms, and unsaturated hydrocarbon oxy groups having 2 carbon atoms. R B is a phenyl group, an acyclic saturated aliphatic hydrocarbon group having 1 to 6 carbon atoms, or an unsaturated heterocyclic group with 5 or 6 members, and these groups may have one or more substituents selected from saturated hydrocarbon groups having 3 or less carbon atoms, unsaturated hydrocarbon groups having 3 or less carbon atoms, saturated hydrocarbon oxy groups having 2 or less carbon atoms, and unsaturated hydrocarbon oxy groups having 2 carbon atoms.

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