CLEC-2 and / or GPVI antagonists

Abstract

To provide a compound that inhibits platelet aggregation caused by platelet activation due to bonding between PDPN and CLEC-2, and also has high selectivity to an activation inducer.SOLUTION: A CLEC-2 and / or GPVI antagonist includes a compound represented by the general formula (1) (where n is an integer of 0-5; R1 represents a substituent on a ring of a phenyl group, R1 independently being a C1-C6 alkyl group or a C1-C6 alkoxy group; and Ar is a substituted or unsubstituted phenyl group or a substituted or unsubstituted naphthyl group).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to antagonists of CLEC-2 and / or GPVI. The present invention further relates to platelet aggregation inhibitors or antithrombotic agents comprising antagonists of CLEC-2 and / or GPVI. [Background technology]

[0002] Thrombosis occurs frequently in many diseases, both arterial and venous, and is essentially one of the leading causes of death among Japanese people. Arterial thrombosis, triggered by the rupture of arteriosclerotic plaques, can lead to cerebral infarction and myocardial infarction. Bedridden hospitalized patients frequently develop deep vein thrombosis in the lower limbs, which can lead to pulmonary infarction, a condition with a high mortality rate. Terminal cancer and infections such as sepsis frequently cause disseminated intravascular coagulation (DIC), in which blood clots form in small blood vessels throughout the body, leading to multiple organ failure.

[0003] Platelets play an important role in the development of these thrombosis events. Once activated, platelets release adenosine diphosphate (ADP) and thromboxane A2 (TxA2), which act on surrounding platelets, causing a chain reaction of platelet activation. Antiplatelet drugs currently in clinical use are ADP receptor inhibitors (e.g., clopidogrel) and cyclooxygenase inhibitors (e.g., aspirin) that suppress TxA2 production. While these drugs have a wide range of applications because they suppress chain reaction activation, they have the disadvantage of increasing the risk of bleeding, and the development of drugs with high selectivity for activation-inducing substances is desired (Non-Patent Document 1).

[0004] Podoplanin (PDPN) is a membrane protein containing a platelet activation domain. PDPN binds to C-type lectin-like receptor 2 (CLEC-2) on the platelet membrane, clustering CLEC-2 and activating platelets. PDPN is normally expressed in tissues and sites not in contact with platelets, such as lymphatic endothelial cells. However, PDPN is also expressed on certain cancer cells, vascular endothelial cells and macrophages activated during infection and inflammation, and macrophages accumulated in atherosclerotic lesions, where it contributes to thrombus formation via platelet activation. Therefore, compounds that can inhibit the binding of PDPN to CLEC-2 have attracted attention as they may exhibit selective antiplatelet effects against thrombosis associated with such inflammatory diseases.

[0005] As a compound that inhibits platelet aggregation, such as thrombus formation, resulting from platelet activation caused by the binding of PDPN to CLEC-2, Non-Patent Document 2 discloses a benzoylnitrostyrene derivative (2CP), and Non-Patent Document 3 and Patent Document 1 disclose cobalt-coordinated hematoporphyrin. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2017 / 119417 (Patent 6805470) [Non-patent literature]

[0007] [Non-Patent Document 1] Nat Rev Drug Discov., 19, 333, 2020. [Non-patent document 2] Oncotarget, 2015, 6, 42733. [Non-patent document 3] Blood Adv. 2018, 2, 2214. Summary of the Invention [Problem to be solved by the invention]

[0008] However, these two compounds have not yet been put to practical use. Therefore, there is a need for the development of compounds that can inhibit the interaction between PDPN and CLEC-2. Furthermore, to reduce the risk of bleeding, there is a need for the development of compounds that are highly selective for platelet activation inducers.

[0009] Therefore, an objective of the present invention is to provide a compound that inhibits platelet aggregation resulting from platelet activation caused by the binding of PDPN to CLEC-2 and that is highly selective for activation-inducing substances. [Means for solving the problem]

[0010] As a result of extensive research to achieve the above object, the present inventors have discovered a compound that inhibits PDPN-induced platelet aggregation and has high selectivity for substances that induce platelet activation.

[0011] According to the present invention, the following inventions are provided. [1] A CLEC-2 and / or GPVI antagonist comprising a compound represented by the following general formula (1): [ka] In the formula, n is an integer of 0 to 5, and R 1 are substituents on the ring of the phenyl group, each independently a C1-C6 alkyl group or a C1-C6 alkoxy group; Ar is a substituted or unsubstituted phenyl group or a substituted or unsubstituted naphthyl group; Ar, [ka] If m is an integer from 0 to 5, and R 2 are substituents on the ring of the phenyl group, each independently a C1-C6 alkyl group, a C1-C6 alkoxy group, or a C1-C6 halogenated alkyl group; Ar, [ka] or [ka] If m' is an integer from 0 to 5, and R 3 are substituents on the naphthyl ring, each independently a C1-C6 alkyl group, a C1-C6 alkoxy group, or a C1-C6 halogenated alkyl group. [2] In the general formula (1), R 1 are each independently a methyl group or a methoxy group, R 2 and 3 and each independently represent a methyl group, an ethyl group, an isopropyl group, or a trifluoromethyl group. [3] The antagonist according to [1] or [2], wherein n, m, and m' are each independently an integer of 0 to 3. [4] A platelet aggregation inhibitor comprising the antagonist according to any one of [1] to [3]. [5] An antithrombotic drug comprising the antagonist according to any one of [1] to [3]. [6] A method for antagonizing CLEC-2 and / or GPVI in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound having the general formula (1) defined in any one of [1] to [3]. [7] A method for inhibiting platelet aggregation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound having the general formula (1) defined in any one of [1] to [3]. [8] A method for treating or preventing thrombosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound having the general formula (1) defined in any one of [1] to [3]. [9] A compound having the general formula (1) as defined in any one of [1] to [3] for use in antagonizing CLEC-2 and / or GPVI.

[10] A compound having the general formula (1) as defined in any one of [1] to [3] for use in a method for inhibiting platelet aggregation in a subject.

[11] A compound having the general formula (1) as defined in any one of [1] to [3] for use in a method for treating or preventing thrombosis in a subject.

[12] Use of a compound having the general formula (1) defined in any one of [1] to [3] in the manufacture of an antagonist of CLEC-2 and / or GPVI.

[13] Use of a compound having the general formula (1) defined in any one of [1] to [3] in the manufacture of a medicament for inhibiting platelet aggregation.

[14] Use of a compound having the general formula (1) defined in any one of [1] to [3] in the manufacture of a medicament for treating or preventing thrombosis.

[15] Use of a compound having the general formula (1) as defined in any one of [1] to [3] in a method for antagonizing CLEC-2 and / or GPVI in a subject.

[16] Use of a compound having general formula (1) as defined in any one of [1] to [3] in a method for inhibiting platelet aggregation in a subject.

[17] Use of a compound having the general formula (1) as defined in any one of [1] to [3] in a method for treating or preventing thrombosis in a subject. [Effects of the Invention]

[0012] According to the present invention, it is possible to provide compounds that inhibit platelet aggregation resulting from platelet activation through the binding of PDPN to CLEC-2 and that are highly selective for activation-inducing substances. According to the present invention, it is possible to provide antagonists of CLEC-2 and / or GPVI, platelet aggregation inhibitors, and antithrombotic agents. [Brief explanation of the drawings]

[0013] [Figure 1A]Figure 1 shows a schematic diagram of CLEC-2-mediated PDPN pull-down and slot blot assay of PDPN levels. In this assay, the amount of PDPN pulled down decreases when a test compound inhibits the binding of CLEC-2 to PDPN. [Figure 1B] FIG. 1 shows some of the results of measuring the amount of PDPN by slot blotting. [Figure 1C-1] 1B is a graph quantifying the inhibitory effects of Compound 1, Compound 2, Compound 4, and Compound 5. [Figure 1C-2] 1B is a graph quantifying the inhibitory effects of Compounds 7, 8, 10, and 11. [Figure 1C-3] 1B is a graph quantifying the inhibitory effects of Compound 3, Compound 9, Compound 6, and Compound 12.

[0014] [Figure 2-1] These results show that 12 CLEC-2 inhibitors completely inhibited the aggregation of washed human platelets (1 × 109 / mL) induced by PDPN-CHO cells (2 × 106 / mL) at 50 μM. Platelet aggregation increases the transparency (%) of the platelet suspension. (A) Compounds 1, 2, and 11; (B) Compounds 4, 5, and 6; (C) Compounds 3, 7, and 10; (D) Compounds 8, 9, and 12. [Figure 2-2] Continuation of Figure 2-1. (E) Cangrelor (3.12 μM). (F) To demonstrate the degree of aggregation inhibition, graphs show the relative area under the curve (AUC) values ​​for each compound added to the control (DMSO) aggregation curve at each measurement. Both compounds were shown to have inhibitory effects on PDPN-induced platelet aggregation. Cangrelor is abbreviated as "Cang" in the graph.

[0015] [Figure 3]The results show the concentration dependence of the inhibitory effect of Compound 1, Compound 5, and Compound 11 on aggregation of washed human platelets (1 x 109 / mL) induced by PDPN-CHO cells (2 x 106 / mL). (A) Compound 1, (B) Compound 5, (C) Compound 11. These compounds completely inhibit aggregation at concentrations of <6.25 μM.

[0016] [Figure 4-1] Figure 1 shows the effect of 12 CLEC-2 inhibitor compounds at 50 μM on CLEC-2 clustering-induced aggregation of human washed platelets (5 × 10 / mL) induced by anti-CLEC-2 polyclonal antibody (4 μg / mL): (A) Compound 1, Compound 5, Compound 11; (B) Compound 3, Compound 8, Compound 12; (C) Compound 2, Compound 6, Compound 9; (D) Compound 4, Compound 7, Compound 10. [Figure 4-2] Continued from Figure 4-1. Graph showing the relative area under the curve (AUC) values ​​for each compound added compared to the aggregation curves for (E) cangrelor (3.12 μM) and (F) the control (DMSO) at each measurement time. Unlike cangrelor, none of the compounds had much inhibitory effect on anti-CLEC-2 antibody-induced platelet aggregation. Cangrelor is abbreviated as "Cang" in the graph.

[0017] [Figure 5-1] Figure 1 shows the effect of 12 CLEC-2 inhibitors at 50 μM on thrombin (0.5 U / mL)-induced aggregation of human washed platelets (3 × 10 / mL): (A) Compound 1, Compound 5, Compound 11; (B) Compound 4, Compound 2, Compound 6; (C) Compound 3, Compound 7, Compound 10; (D) Compound 8, Compound 9, Compound 12. [Figure 5-2]Continuation of Figure 5-1. Graph showing the relative area under the curve (AUC) values ​​for each compound added compared to the aggregation curves for (E) cangrelor (6.25 μM) and (F) the control (DMSO) at each measurement. None of the compounds demonstrated inhibitory activity against thrombin-induced platelet aggregation. In the graph, cangrelor is abbreviated as Cang.

[0018] [Figure 6-1] Figure 1 shows the effect of 12 CLEC-2 inhibitors at 50 μM on ADP (20 μM) + fibrinogen (0.6 mg / mL)-induced aggregation of human washed platelets (3 × 10 / mL): (A) Compounds 1, 5, and 11; (B) Compounds 2, 6, and 7; (C) Compounds 8, 9, and 12; and (D) Compounds 3, 4, and 8. [Figure 6-2] Continued from Figure 6-1. Graphs showing the relative area under the curve (AUC) values ​​for (E) Compound 10, (F) Compound 12, (G) control (DMSO) at each measurement point, compared to the aggregation curves for (E) Compound 10, (F) cangrelor (3.1 μM), and (F) compound 12. Unlike cangrelor, none of the compounds had any inhibitory effect on ADP-induced platelet aggregation. Cangrelor is abbreviated as "Cang" in the graph.

[0019] [Figure 7-1] Figure 1 shows the effect of 12 CLEC-2 inhibitors at 50 μM on collagen (2 μg / mL)-induced aggregation of human washed platelets (1 × 10 / mL): (A) Compounds 5, 6, and 11; (B) Compounds 8, 9, and 12; (C) Compounds 4 and 7; and (D) Compounds 3 and 10. [Figure 7-2]Continuation of Figure 7-1. Graph showing the relative area under the curve (AUC) values ​​for (E) Compound 1, (F) Compound 2, and (G) Compound 11, (F) Cangrelor (3.12 μM), and (G) the control (DMSO) at each measurement. It was shown that all compounds have inhibitory effects on collagen-induced platelet aggregation, although to varying degrees. Cangrelor is abbreviated as "Cang" in the graph.

[0020] [Figure 8] 1 is a graph showing the concentration dependence of the inhibitory effect of selected compounds (A) Compound 1, (B) Compound 5, and (C) Compound 11 on collagen (2 μg / mL)-induced aggregation of human washed platelets (1 × 109 / mL).

[0021] [Figure 9-1] Figure 1 shows the inhibitory effects of 12 CLEC-2 inhibitors at 100 μM on aggregation of washed mouse platelets (1 × 10 / mL) induced by PDPN-CHO cells (2 × 10 / mL): (A) Compounds 1, 9, and 11; (B) Compounds 2, 3, and 6; (C) Compounds 5, 7, and 11; and (D) Compounds 4, 8, and 10. [Figure 9-2] Continuation of Figure 9-1. Graphs showing the relative area under the curve (AUC) values ​​for each compound added compared to the control (DMSO) aggregation curve at each measurement point for (E) cangrelor (3.1 μM) and (F) 100 μM. Cangrelor is abbreviated as Cang in the graph. [Figure 10] The results show the concentration dependence of the inhibitory effect of selected compounds, Compound 1, Compound 5, and Compound 11, on aggregation of washed mouse platelets (1 × 10 / mL) induced by PDPN-CHO cells (2 × 10 / mL). (A) Compound 1, (B) Compound 5, (C) Compound 11.

[0022] [Figure 11-1]The results show the inhibitory effects of 12 CLEC-2 inhibitors at 50 μM on thrombin (0.5 U / mL)-induced aggregation of washed mouse platelets (3 × 10 / mL): (A) Compounds 1, 5, and 11; (B) Compounds 3, 4, and 8; (C) Compounds 2, 10, and 12; and (D) Compounds 3, 4, and 8. [Figure 11-2] Continuation of Figure 11-1. (E) Graph showing the relative area under the curve (AUC) values ​​for the aggregation curves of cangrelor (3.1 μM) and (F) control (DMSO) at each measurement time. Cangrelor is abbreviated as Cang in the graph.

[0023] [Figure 12-1] Figure 1 shows the inhibitory effects of 12 CLEC-2 inhibitors at 50 μM on ADP (50 μM) + fibrinogen (0.6 mg / mL) aggregation of washed mouse platelets (5 × 10 / mL): (A) Compounds 1, 2, and 3; (B) Compounds 4, 5, and 6; (C) Compounds 7, 8, and 9; (D) Compounds 10, 11, and 12. [Figure 12-2] Continuation of Figure 12-1. (E) Graph showing the relative area under the curve (AUC) values ​​for the aggregation curves of cangrelor (3.1 μM) and (F) control (DMSO) at each measurement time. In the graph, cangrelor is abbreviated as Cang.

[0024] [Figure 13-1] Figure 1 shows the inhibitory effects of 12 CLEC-2 inhibitors at 50 μM on collagen (2 μg / mL)-induced aggregation of washed mouse platelets (1 × 10 / mL): (A) Compounds 2, 4, and 6; (B) Compounds 5, 8, and 12; (C) Compounds 3, 10, and 11; and (D) Compounds 1, 7, and 9. [Figure 13-2]Continuation of Figure 13-1. (E) Graph showing the relative area under the curve (AUC) values ​​for the aggregation curves of cangrelor (3.1 μM) and (F) control (DMSO) at each measurement time. In the graph, cangrelor is abbreviated as Cang.

[0025] [Figure 14] The results show the concentration dependence of the inhibitory effect of Compound 1, Compound 5, and Compound 11 on collagen (2 μg / mL)-induced aggregation of washed mouse platelets (1 × 10 / mL). (A) Compound 1, (B) Compound 5, (C) Compound 11.

[0026] [Figure 15] This figure shows an in silico compound docking simulation of the interaction between the collagen receptor GPVI and compound 5 (A) or compound 11 (B). CRP (collagen-related peptide) is shown as a reference. GPVI: ribbon model (cartoon model), compound 5 (A) and compound 11 (B): space-filling model, CRP: surface model.

[0027] [Figure 16] Electron micrographs of platelets are shown. untreated: image of untreated platelets, PDPN + DMSO: image of PDPN-treated platelets in the presence of DMSO (control), PDPN + Compound 5: image of PDPN-treated platelets in the presence of 50 μM Compound 5, collagen + DMSO: image of collagen-treated platelets in the presence of DMSO (control), collagen + Compound 5: image of collagen-treated platelets in the presence of 50 μM Compound 5. DETAILED DESCRIPTION OF THE INVENTION

[0028] The following description of the present invention may be based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0029] In this specification, unless otherwise specified, each term has the following meaning. Prevention means inhibiting the onset, reducing the risk of onset, or delaying the onset. Treatment means amelioration of a disease or symptom, suppression of progression, or the like. A therapeutically effective amount includes an amount that, when administered (as a single dose or over the course of multiple doses), promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of disability or impairment due to disease affliction. A therapeutically effective amount also includes a "prophylactically effective amount," which is an amount that, when administered to a subject at risk of developing a disease or at risk of suffering a recurrence of a disease, inhibits the onset or recurrence of the disease. By subject is meant a mammal, including a human.

[0030] (CLEC-2 and / or GPVI antagonists) The CLEC-2 and / or GPVI antagonist of the present invention includes an N,3-diaryl-2-tetrazole-propanamide compound, and more specifically, includes a compound represented by the following general formula (1):

[0031] The CLEC-2 and / or GPVI antagonist has the effect of suppressing the function of CLEC-2 and / or GPVI, which are receptors on the platelet membrane for substances that induce platelet activation.

[0032] CLEC-2 (C-type lectin-like receptor 2) is a membrane protein expressed on platelets. The in vivo ligand for CLEC-2 is PDPN (podoplanin). PDPN is a membrane protein with a platelet activation domain and is constitutively expressed in lymphatic epithelium, kidney podocytes, and other tissues. CLEC-2 activation by PDPN promotes platelet aggregation and thrombus formation. The antagonist of the present invention functions as an antagonist that inhibits the function of CLEC-2 by specifically binding to CLEC-2 or competing with PDPN.

[0033] Glycoprotein VI (GPVI) is a type 1 transmembrane receptor expressed on megakaryocytes and platelets. It has two immunoglobulin domains, including a collagen-binding domain, and is involved in collagen-mediated platelet activation. Known agonists of GPVI include collagen (type I and type III) and cross-linked collagen-related peptide (CRP) (Journal of Thrombosis and Hemostasis 18(1):61-65, 2007). At the site of vascular injury, platelet GPVI binds to collagen at the injury site and is activated as part of a multistep platelet aggregation reaction, resulting in the formation of a platelet thrombus. The antagonist of the present invention functions as an antagonist that inhibits the function of GPVI by specifically binding to GPVI or by competing with collagen, CRP, etc.

[0034] The CLEC-2 and / or GPVI antagonist preferably has the effect of inhibiting the binding between CLEC-2 and PDPN and / or the effect of inhibiting the binding between GPVI and a GPVI agonist (e.g., collagen or CRP), and more preferably has the effect of inhibiting the binding between CLEC-2 and PDPN and the effect of inhibiting the binding between GPVI and a GPVI agonist (e.g., collagen or CRP).

[0035] (Compound represented by general formula (1)) [ka] In the formula, n is an integer of 0 to 5, and R 1 are substituents on the ring of the phenyl group, each independently a C1-C6 alkyl group or a C1-C6 alkoxy group; Ar is a substituted or unsubstituted phenyl group or a substituted or unsubstituted naphthyl group; Ar, [ka] If m is an integer from 0 to 5, and R 2 are substituents on the ring of the phenyl group, each independently a C1-C6 alkyl group, a C1-C6 alkoxy group, or a C1-C6 halogenated alkyl group, preferably a C1-C3 alkyl group, a C1-C3 alkoxy group, or a C1-C3 halogenated alkyl group; Ar, [ka] or [ka] If m' is an integer from 0 to 5, and R 3 are substituents on the naphthyl ring, each independently a C1-C6 alkyl group, a C1-C6 alkoxy group, or a C1-C6 halogenated alkyl group, preferably a C1-C3 alkyl group, a C1-C3 alkoxy group, or a C1-C3 halogenated alkyl group.

[0036] In general formula (1), n ​​is preferably an integer of 0 to 3, more preferably an integer of 1 to 3, and even more preferably 1 or 2. 1 are preferably each independently a methyl group or a methoxy group.

[0037] Ar, [ka] In this case, m is preferably an integer of 0 to 3, more preferably an integer of 1 to 3, and even more preferably 1 or 2. 2 are preferably each independently a methyl group, an ethyl group, an isopropyl group, or a trifluoromethyl group.

[0038] Ar, [ka] or [ka] In this case, m' is preferably an integer of 0 to 3. 3 is optional, m' may be 0, and Ar in general formula (1) may be unsubstituted 1-naphthyl or 2-naphthyl. When m' is an integer of 1 or greater, R 3 may each independently be a methyl group, an ethyl group, an isopropyl group, or a trifluoromethyl group.

[0039] The compound used in the present invention is more preferably a compound represented by the general formula (1): n is 1 or 2, and R 1 are each independently a methyl group or a methoxy group, and Ar, [ka] where m is 1 or 2, and R 2 are each independently a methyl group, an ethyl group, an isopropyl group, or a trifluoromethyl group; Ar is an unsubstituted naphthyl group.

[0040] R 1 , R 2 , and R 3 The substitution positions of R are each independently arbitrary. 2 When is an isopropyl group, it is preferably in the para position relative to the nitrogen atom.

[0041] A "C1-C6 alkyl group" means a straight or branched chain alkyl group having from 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1-ethylpropyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, hexyl, 1-methylpentyl, 2-methylpentyl, 1,1-dimethylbutyl, and 1,3-dimethylbutyl.

[0042] "C1-C6 alkoxy group" means an alkyl group as defined above that is bonded to a carbon atom of the main chain via an oxygen atom, and non-limiting examples include methoxy and ethoxy groups.

[0043] The term "C1-C6 halogenated alkyl group" refers to an alkyl group substituted with one or more halogen atoms, and examples thereof include a fluoromethyl group, a difluoromethyl group, and a trifluoromethyl group.

[0044] The compounds of general formula (1) may be in all stereoisomers (e.g., cis-trans isomers) and all optical isomers (e.g., R and S enantiomers), and in such cases, may be used in any of these configurations or in mixtures of multiple isomers, i.e., racemates and other mixtures of such isomers. While all stereoisomers are encompassed within the scope of the present invention, those skilled in the art will recognize that certain stereoisomers may be preferred.

[0045] The compound represented by the general formula (1) may be used as its pharmaceutically acceptable salt, or as a hydrate or solvate thereof. Examples of the salt include mineral acid salts such as hydrochloride and sulfate, and salts with organic acids such as acetic acid, propionic acid, tartaric acid, fumaric acid, maleic acid, malic acid, oxalic acid, succinic acid, citric acid, and benzoic acid. In addition to salts and free compounds, any hydrates or solvates thereof may also be used as active ingredients. Examples of solvents that can form the above solvates include ethanol, isopropanol, acetone, ethyl acetate, and methylene chloride.

[0046] Examples of the compound represented by the general formula (1) include, but are not limited to, the following. [ka] [ka]

[0047] The compound represented by the general formula (1) can be produced by chemical synthesis, or can be purchased from Namiki Shoji Co., Ltd. (Suppliers: ASINEX, Princeton Biomolecular, and Vitas-M Laboratory).

[0048] The ability of a test compound to inhibit the binding of CLEC-2 to its in vivo ligand, PDPN, can be evaluated using a pull-down slot blot system, an in vitro compound binding inhibitory activity evaluation system developed by the present inventors, as described in the Examples below (Watanabe N et al. (2019) A pull-down and slot blot-based screening system for inhibitor compounds of the podoplanin-CLEC-2 interaction. PLoS ONE 14(9): e0222331). Before using the in vitro compound binding inhibitory activity evaluation system, candidate compounds may be screened by performing an in silico compound docking simulation.

[0049] The anti-platelet aggregation activity of the compound represented by general formula (1) can be verified by adding various platelet activation inducers to a platelet suspension in the presence of a test compound and measuring the transmittance (%) of the platelet suspension using a platelet aggregation measuring device, as described in the Examples below. The transmittance (%) of the platelet suspension increases upon platelet aggregation. Examples of platelet activation inducers include, but are not limited to, PDPN (podoplanin), collagen, CRP (collagen-related peptide), thrombin, adenosine diphosphate (ADP), and thromboxane A2 (TxA2). Receptors for these include the podoplanin receptor (CELEC-2), collagen receptor (GPIb-IV-V complex, GPVI, GPIa / IIa), thrombin receptor (PAR1), ADP receptor (P2Y12), and thromboxane A2 receptor (TP).

[0050] The anti-platelet aggregation activity of a compound represented by general formula (1) can be verified by observing platelet morphology using an electron microscope or the like. Normally, platelets have a smooth, even surface. However, during bleeding, platelet activation inducers alter the intracellular cytoskeleton, producing numerous long protrusions. At the same time, cell adhesion factors are newly expressed on the cell membrane. In vitro, when a platelet activation inducer is added to a platelet suspension, numerous long protrusions usually appear on the platelets. When a specific platelet activation inducer is added to a platelet suspension in the presence of a test compound, if no protrusions are observed or the number of protrusions is reduced compared to the absence of the test compound, it can be said that the test compound inhibits platelet activation caused by that specific platelet activation inducer.

[0051] The CLEC-2 and / or GPVI antagonists of the present invention can potently inhibit PDPN-induced platelet aggregation (complete inhibition at <6.25 μM). They can also inhibit collagen-induced platelet aggregation (complete inhibition at <12.5 μM). On the other hand, the CLEC-2 and / or GPVI antagonists of the present invention do not inhibit platelet aggregation induced by anti-CLEC-2 antibodies, thrombin, or ADP, but can selectively inhibit platelet aggregation induced by PDPN and collagen. Compounds highly selective for platelet activation-inducing substances are thought to have a lower risk of bleeding than compounds that can be used with multiple activation-inducing substances.

[0052] (platelet aggregation inhibitor and antithrombotic agent) The CLEC-2 and / or GPVI antagonist preferably has the effect of inhibiting the binding between CLEC-2 and PDPN and / or the effect of inhibiting the binding between GPVI and a GPVI agonist (e.g., collagen or CRP), and more preferably has the effect of inhibiting the binding between CLEC-2 and PDPN and the effect of inhibiting the binding between GPVI and a GPVI agonist (e.g., collagen or CRP).

[0053] The platelet aggregation inhibitor of the present invention includes the CLEC-2 and / or GPVI antagonist of the present invention. The platelet aggregation inhibitor is a drug having an effect of inhibiting platelet aggregation.

[0054] The antithrombotic drug of the present invention includes the CLEC-2 and / or GPVI antagonist of the present invention. The antithrombotic drug is a drug that has the effect of suppressing thrombus formation and preventing vascular occlusion.

[0055] The platelet aggregation inhibitor and the antithrombotic agent preferably act through the inhibition of the function of CLEC-2 and / or GPVI, which are receptors for platelet activation-inducing substances on the platelet membrane, and more preferably have the effect of inhibiting the binding of CLEC-2 to its in vivo ligand PDPN and / or the effect of inhibiting the binding of GPVI to its agonist (e.g., collagen or CRP).

[0056] (Pharmaceutical composition) Pharmaceutical compositions containing at least one of the CLEC-2 and / or GPVI antagonist of the present invention, the compound represented by general formula (1), a platelet aggregation inhibitor, and an antithrombotic agent, and further containing other ingredients such as pharmaceutically acceptable additives, as necessary, are also within the scope of the present invention.

[0057] Examples of the pharmaceutically acceptable additives include isotonicity agents, pH adjusters, buffers, stabilizers, cryoprotectants, antibiotics, etc. Specific examples include water, ethanol, sodium chloride, glucose, albumin, etc. Other examples of pharmaceutically acceptable additives include excipients, binders, lubricants, etc. Examples of excipients include lactose, corn starch, sucrose, glucose, sorbitol, crystalline cellulose, silicon dioxide, etc. Examples of binders include polyvinyl alcohol, ethyl cellulose, methyl cellulose, gum arabic, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, etc. Examples of lubricants include magnesium stearate, talc, silica, etc.

[0058] The pharmaceutical composition is formulated in an optimal dosage form depending on the route of administration. The pharmaceutical composition may be a solid preparation such as a tablet, granule, fine granule, powder, or capsule, or an oral preparation such as a liquid, jelly, or syrup, or a parenteral preparation such as an injection, suppository, ointment, or patch.

[0059] The pharmaceutical composition of the present invention is preferably produced under conditions that comply with regulations for manufacturing and quality control of drugs and quasi-drugs (good manufacturing practice, GMP).

[0060] The method of administering the pharmaceutical composition is not particularly limited, and administration conditions such as the administration route, administration timing, dosage, administration schedule, etc. can be appropriately selected depending on the purpose. The administration route is not particularly limited, and can be appropriately selected depending on the purpose, but intravenous administration, transdermal administration, and oral administration are preferred.

[0061] The dosage of the pharmaceutical composition is not particularly limited and can be selected appropriately depending on factors such as the disease state and body weight of the subject requiring treatment, but is preferably 1 mg to 1,000 mg, and more preferably 10 mg to 200 mg per day. [Example]

[0062] The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.

[0063] Example 1 Measurement of inhibitory activity against PDPN / CLEC-2 interaction Compounds 1 to 12 were purchased from Namiki Shoji Co., Ltd. (suppliers: ASINEX, Princeton Biomolecular, and Vitas-M Laboratory). [Table 1] JPEG0007733916000016.jpg182154JPEG0007733916000017.jpg207154JPEG0007733916000018.jpg167154

[0064] Experimental Method 1) We constructed a pull-down assay system in which PDPN was captured by Fc-CLEC-2 fused to immunoglobulin Fc region (Fc-CLEC-2) by mixing it with lysate from PDPN-overexpressing HeLa cells (Fig. 1A). The Fc-CLEC-2 / PDPN complex or free Fc-CLEC was captured by Protein A-coupled beads (KANEKA KanCapA). TM The cells were collected using a centrifugal filter and washed with a UFC30DV00 ultra-free filter unit (Millipore). Proteins were eluted with GlyHCl (pH 2.8) and adsorbed onto a polyvinylidene difluoride (PVDF) membrane using a slot blot (Millipore). After blocking the PVDF membrane, CLEC-2 and PDPN were incubated with anti-CLEC-2 antibody (AF1718, Novus Biologicals) and anti-PDPN antibody (NZ-1, Angio Bio Co.), respectively, followed by a horseradish peroxidase (HRP)-conjugated secondary antibody. Chemiluminescence was detected, and the signal was recorded using an image analyzer (ATTO, Tokyo, Japan). The slot intensity was quantified using image analysis software (NIH, Image J). When a test compound was added and only PDPN precipitation was inhibited, the compound specifically inhibited the binding of CLEC-2 to PDPN.

[0065] 2) Fc-CLEC-2 was prepared as follows. RNA was prepared from human peripheral blood mononuclear cells and reverse-transcribed to obtain cDNA. The sequence corresponding to the extracellular domain of CLEC-2 (positions 351-890 of GenBank Accession No. NM_016509) was amplified by PCR using the following primers, which contain BamH1 or EcoRV restriction sites: CLEC-2-F: 5'-CGTGGATCCATGCTGGGGATTTGGTCTGTCAT (SEQ ID NO: 1) CLEC-2-R: 5'-CGTGATATCTTAAGGTAGTTGGTCCACCTTGG (SEQ ID NO: 2)

[0066] This was ligated into the Fc fusion protein expression vector pFUSEN-hG1Fc (InVIVOGen), transfected into HEK293 cells, and the medium was collected. Fc-CLEC-2 was purified from the medium using an AKTA chromatography system (GE Healthcare) and a HiTrap Protein A HP column (GE Healthcare).

[0067] 3) PDPN-expressing cells were established as follows. RNA was extracted from TE11 cells (RIKEN CELL BANK), a human esophageal carcinoma squamous cell line, and cDNA was obtained. The full-length PDPN protein coding region was amplified using the following primers, which contain the Age1 or BamH1 restriction site (GenBank: AF390106.1). PDPN-F: 5'-ACGACCGGTATGTGGAAGGTGTCAGCTCT (SEQ ID NO: 3) PDPN-R: 5'-CGTGGATCCTTAGGGCGAGTACCTTCCCG (SEQ ID NO: 4)

[0068] The PCR amplification product was ligated into the lentiviral expression vector CSII-MCS-Venus (RIKEN BRC DNA BANK) to generate a lentiviral vector. HeLa or CHO cells were infected with the lentivirus, and stably expressing cells were separated using a FACS (FACSAria, BD Biosciences).

[0069] result The results of measuring the inhibitory effect of 12 compounds (1 through 12) on the binding of CLEC-2 to PDPN are shown in Figures 1C-1 to 1C-3. The relative luminescence intensity upon addition of each compound is plotted, with the luminescence intensity of the PDPN slot in the presence of a control (DMSO) being set at 1. All 12 compounds exhibited binding inhibition rates of 85% or higher at 4 mM. Among the 12 test compounds, compounds 1, 5, 6, 7, and 11 exhibited particularly high binding inhibition and relatively high inhibitory effects. The binding inhibition rates at 2 mM were 84% for compound 1, 82% for compound 5, 97% for compound 6, 75% for compound 7, and 69% for compound 11.

[0070] Example 2 Platelet aggregation measurement Blood was obtained from healthy volunteers and mice using sodium citrate as an anticoagulant. The blood was centrifuged at 200 × G for 10 minutes to recover platelet-rich plasma, followed by the addition of apyrase (Sigma) and prostaglandin I2 (Cayman Chemical) and centrifugation at 1000 × G for 10 minutes. The platelets were then centrifuged again in Tyrode's solution in the presence of apyrase and prostaglandin I2, and then collected at 1 × 10 9 Washed platelets were suspended in Tyrode's solution at a density of 1 / mL. Platelet aggregation was measured using a 4-channel platelet aggregometer PA-200 (Kowa Co., Ltd.). The test compound was added 30 seconds before the addition of the inducer. Cangrelor (Cangrelor Tetrasodium, Selleckchem.com, Catalog No. S3737) was used as a control drug.

[0071] Platelet aggregation inducers were used at the following final concentrations: i) PDPN-CHO cells (2 × 10 6 / mL), ii) Anti-CLEC-2 antibody, antigen affinity purified polyclonal goat IgG (AF1718, R&D Systems, 4 μg / mL) iii) Human thrombin (Sigma, 0.5 U / mL) iv) ADP (Nacalai, 20 μM) + human fibrinogen (Wako, 0.6 mg / mL) v) collagen (Revohem collagen Sysmex AW-993-82, 2 μg / mL), PDPN-expressing CHO cells were prepared as described in Example 1, 3).

[0072] result Inhibitory effect on human platelet aggregation i) Inhibitory effect on PDPN-induced platelet aggregation The inhibitory effects of 12 compounds (1 to 12) on platelet aggregation induced by PDPN-CHO cells were measured. Figures 2A–E show the transmittance (%) of platelet suspensions. Platelet aggregation increases the transmittance (%) of platelet suspensions. After the addition of PDPN-CHO cells, the transmittance (%) of the DMSO control increased, indicating the occurrence of platelet aggregation. In the presence of any of the 12 compounds (1 to 12) (50 μM), the transmittance (%) did not increase after the addition of PDPN-CHO cells (Figures 2A–D). These results indicate that 12 compounds (1 to 12) completely inhibit PDPN-induced platelet aggregation at 50 μM. Even in the presence of the control drug cangrelor (3.12 μM), the transmittance (%) did not increase after the addition of PDPN-CHO cells (Figure 2E). Figure 2-2F is a graph showing the relative area under the curve (AUC) of each compound added to the control (DMSO) aggregation curve at each measurement to indicate the degree of aggregation inhibition. All 12 compounds, from Compound 1 to Compound 12, were shown to have an inhibitory effect on PDPN-induced platelet aggregation.

[0073] Furthermore, we measured the permeability (%) of platelet suspensions in the presence of three compounds: Compound 1, Compound 5, and Compound 11, at concentrations of 6.25 μM, 12.5 μM, and 25 μM. The results in Figure 3 indicate that the inhibitory effects of at least Compound 1, Compound 5, and Compound 11 on PDPN-induced platelet aggregation are concentration-dependent.

[0074] ii) Inhibitory effect on anti-CLEC-2 antibody-induced platelet aggregation The inhibitory effects of 12 compounds (1 to 12) on platelet aggregation induced by anti-CLEC-2 antibody-induced CLEC-2 clustering were measured. Figures 4A–E show the transmittance (%) of platelet suspensions. After the addition of CLEC-2 antibody, the transmittance (%) of the DMSO control increased, indicating the occurrence of platelet aggregation. The transmittance (%) also increased after the addition of CLEC-2 antibody in the presence of any of the 12 compounds (1 to 12) (50 μM) (Figures 4A–D). These results indicate that the 12 compounds (1 to 12) at a concentration of 50 μM had little inhibitory effect on anti-CLEC-2 antibody-induced platelet aggregation. In the presence of the control drug cangrelor (3.12 μM), the transmittance (%) did not increase after the addition of anti-CLEC-2 antibody, indicating that anti-CLEC-2 antibody-induced platelet aggregation was inhibited (Figure 4E). Figure 4-2F is a graph showing the relative area under the curve (AUC) values ​​for each compound added to the control (DMSO) aggregation curve at each measurement to indicate the degree of aggregation inhibition. Compounds 1 to 12 were shown to have almost no inhibitory effect on anti-CLEC-2 antibody-induced platelet aggregation, unlike cangrelor.

[0075] iii) Thrombin-induced platelet aggregation The inhibitory effect of 12 compounds (1 to 12) on thrombin-induced platelet aggregation was measured. The transmittance (%) increased after the addition of thrombin in the presence of any of the 12 compounds (1 to 12) (50 μM) and the control drug cangrelor (6.25 μM) (Figures 5-1A-D and 5-2E). Figure 5-2F is a graph showing the relative area under the curve (AUC) of each compound added to the control (DMSO) aggregation curve at each measurement to indicate the degree of aggregation inhibition. Compounds 1 to 12 and cangrelor were shown to have little inhibitory effect on thrombin-induced platelet aggregation.

[0076] iv) ADP-induced platelet aggregation The inhibitory effect of 12 compounds, Compounds 1 through 12, on platelet aggregation induced by ADP was measured. ADP was added together with fibrinogen. ADP is one of the coagulation mediators in the platelet aggregation cascade that progresses continuously from platelets activated by injury, and is released from storage granules during activation. Fibrinogen and other platelets bind to the surface of activated platelets, causing them to aggregate. However, since ADP stimulation alone does not induce aggregation in washed platelets, as in this system, fibrinogen is usually added together.

[0077] The % transmittance of the DMSO control increased rapidly within a few minutes after the addition of ADP, indicating that ADP activated platelets and caused platelet aggregation (Figure 6-1, A-D). The results in Figure 6-1, A-D, show that the 12 compounds, Compounds 1 through 12, had little inhibitory effect on ADP-induced platelet aggregation at a concentration of 50 μM. In the presence of the control drug, Cangrelor (3.1 μM), the % transmittance did not increase after the addition of ADP, and ADP-induced platelet aggregation was inhibited (Figure 6-2, E). Figure 6-2, F, shows the relative area under the curve (AUC) of each compound added to the aggregation curve of the control (DMSO) at each measurement time point to indicate the degree of aggregation inhibition. Compounds 1 through 12, unlike Cangrelor, were shown to have no inhibitory effect on ADP-induced platelet aggregation.

[0078] v) Collagen-induced platelet aggregation After the addition of collagen cells, the % transmittance of the DMSO control increased, indicating the occurrence of collagen-induced platelet aggregation. In the presence of any of the 12 compounds 1 to 12 (50 μM), the increase in % transmittance after the addition of collagen cells was suppressed compared to the DMSO control, although the degree varied depending on the compound (Figures 7-1A-D and 7-2E). In particular, compounds 1, 2, 3, 4, 10, 11, and 12 showed little increase in % transmittance. These results indicate that the 12 compounds 1 to 12 inhibit collagen-induced platelet aggregation at 50 μM. Even in the presence of the control drug cangrelor (3.12 μM), the % transmittance did not increase after the addition of collagen (Figure 7-2F). Figure 7-2G shows the relative area under the curve (AUC) of each compound added to the aggregation curve of the control (DMSO) at each measurement time to indicate the degree of aggregation inhibition. All 12 compounds, Compounds 1 to 12, were shown to have inhibitory effects on collagen-induced platelet aggregation.

[0079] Furthermore, the transmittance (%) of the platelet suspension was measured in the presence of Compound 1, Compound 5, and Compound 11 at concentrations of 12.5 μM, 25 μM, and 50 μM. The results in Figure 8 show that the inhibitory effects of at least Compound 1, Compound 5, and Compound 11 on collagen-induced platelet aggregation are concentration-dependent. Of the three compounds, Compound 11 exhibits a stronger aggregation inhibitory effect.

[0080] About Cangrelor The control drug, cangrelor, is a competitive inhibitor of the ADP receptor. Cangrelor inhibited all platelet aggregation inducers except thrombin-induced aggregation, raising concerns about the risk of bleeding.

[0081] Inhibitory effect on mouse platelet aggregation Using mouse platelets, the inhibitory effects of 12 compounds (1 to 12) on PDPN-, thrombin-, ADP-, and collagen-induced platelet aggregation were measured. Similar results were obtained using human platelets (Figures 9–14). Compared to human platelets, the inhibitory effects on PDPN- and collagen-induced platelet aggregation in mouse platelets were weaker. This may be due to the slight differences between the amino acid sequences of CLEC-2 and GPVI in humans and mice, resulting in weaker binding of the compounds to mouse proteins.

[0082] Example 3 In silico compound docking simulation Docking simulations were performed in silico to dock Compounds 5 and 11 into the CRP (collagen-related peptide) binding site of GPVI, a collagen receptor.

[0083] This docking simulation allows us to determine the binding affinity and inhibition pattern of each of Compound 5 and Compound 11 to the collagen-binding site of GPVI. The three-dimensional structure of GPVI, the target of compound docking, was obtained from the Protein Data Bank (Protein Data Bank: http: / / pdbj.org / ) (GPVI, PDB ID: 5OU9). Docking simulations were performed using ASEDock (Goto, J.; Kataoka, R.; Muta, H.; Hirayama, N. “ASEDock-docking based on alpha spheres and excluded volumes” J Chem Inf Model., 2008, 48(3), 583-590.).

[0084] (result) Docking simulations demonstrated that compounds 5 and 11 each readily bind to the predicted CRP binding site of GPVI. The mode of inhibition was competitive. The strength of the interaction determined from the docking simulations was evaluated using the GBVI / WSA dG, which corresponds to the binding free energy (Corbeil, CR; Williams, CI; Labute, P. Variability in docking success rates due to dataset preparation. J. Comput.-Aided Mol. Des. 2012, 26, 775-786.). The GBVI / WSA dG values ​​for compounds 5 and 11 were -7.947 and -8.007 kcal / mol, respectively, indicating that both compounds readily bind to the predicted CRP binding site of GPVI.

[0085] Example 4 Platelet morphology Washed mouse platelets (1 × 10 9 PDPN-CHO cells (2 × 10 / mL) were cultured in the presence of compound 5 (50 μM) or DMSO (control). 6 Platelet morphology was observed under a scanning electron microscope (JEOL JSM-6510LV) after adding either 0.1 μg / mL or collagen (2 μg / mL).

[0086] Figure 16 shows electron micrographs of platelets. Normally, platelets have a smooth surface without any irregularities (untreated). Platelets treated with PDPN (PDPN + DMSO) exhibit numerous long projections. This indicates that the platelets are activated by PDPN. On the other hand, platelets treated with PDPN in the presence of Compound 5 (PDPN + Compound 5) are no different from untreated platelets (untreated) and do not exhibit projections. This indicates that the inhibition of platelet aggregation is due to inhibition of activation.

[0087] Platelets treated with collagen exhibited numerous long projections (collagen + DMSO), indicating that the platelets were activated by collagen. On the other hand, platelets treated with collagen in the presence of Compound 5 (collagen + Compound 5) exhibited no projections, similar to untreated platelets (untreated).

[0088] These results confirmed that the CLEC-2 / GPVI dual inhibitors of the present invention, such as Compound 5, inhibit platelet activation induced by PDPN and collagen, thereby inhibiting aggregation. [Sequence List Free Text]

[0089] SEQ ID NOs: 1 to 4: Primers

Claims

1. A CLEC-2 and / or GPVI antagonist comprising a compound represented by the following general formula (1): 【Chemical 1】 In the formula, n is an integer from 0 to 5, and R １ are substituents on the ring of the phenyl group, each independently a C1-C6 alkyl group or a C1-C6 alkoxy group, Ar is a substituted or unsubstituted phenyl group or a substituted or unsubstituted naphthyl group; Ar is, 【Chemistry 2】 If m is an integer from 0 to 5, and R ２ are substituents on the ring of the phenyl group, each independently a C1-C6 alkyl group, a C1-C6 alkoxy group, or a C1-C6 halogenated alkyl group; Ar is, 【Chemistry 3】 or 【Chemistry 4】 If m' is an integer from 0 to 5, and R ３ are substituents on the naphthyl ring, each independently a C1-C6 alkyl group, a C1-C6 alkoxy group, or a C1-C6 halogenated alkyl group.

2. In the general formula (1), R １ are each independently a methyl group or a methoxy group, R ２ and ３ and each independently represent a methyl group, an ethyl group, an isopropyl group, or a trifluoromethyl group.

3. 3. The antagonist according to claim 1, wherein n, m, and m' are each independently an integer of 0 to 3.

4. A platelet aggregation inhibitor comprising the antagonist according to any one of claims 1 to 3.

5. An antithrombotic drug comprising the antagonist according to any one of claims 1 to 3.

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