WO2017119417A1 - Clec-2 antagonist, platelet aggregation inhibitor, antithrombotic agent, antimetastatic agent, antiarthritic agent, compound having porphyrin skeleton, and method for producing same - Google Patents

Abstract

Disclosed is a CLEC-2 antagonist comprising a compound represented by either general formula (1) or general formula (2). (In general formula (1), M represents H₂ or one of Group 2 to 12 elements, R¹ represents a vinyl group or a 1-hydroxyethyl group, and X represents a Group 1 element.) (In general formula (2), M represents H₂ or one of Group 2 to 12 elements, and R² represents a phenyl group, a sulfophenyl group, a carboxyphenyl group, or a 1-methylpyridinium-4-yl group.)

Description

CLEC-2 antagonist, platelet aggregation inhibitor, antithrombotic agent, antimetastatic agent, antiarthritic agent, compound having porphyrin skeleton, and method for producing the same

The present invention has a CLEC-2 antagonist, a platelet aggregation inhibitor, an antithrombotic agent, an anti-metastatic agent, an anti-arthritic agent, and a porphyrin skeleton that can specifically inhibit the function of the platelet activating receptor CLEC-2 The present invention relates to a compound and a production method thereof.

Platelets play a central role in arterial thrombosis such as myocardial infarction and cerebral infarction. Antiplatelet drugs have a market size of about 2 trillion yen worldwide, but the side effects of bleeding are a problem. According to a large cohort study, the use of antiplatelet drugs for primary prevention is currently not recommended because the risk of bleeding outweighs the benefits. Therefore, an antiplatelet drug with few side effects of bleeding is desired.

About 90% of cancer deaths are caused by metastasis, followed by cancer-related thrombosis. However, no drug that suppresses metastasis has been reported so far. On the other hand, attempts have been made to prevent cancer-related thrombosis by administering heparin or warfarin to cancer patients, but there is a problem that side effects of bleeding occur, particularly after chemotherapy.

On the other hand, the platelet activating receptor CLEC-2 was discovered by the present inventors, and was identified on platelets as a receptor for rhodocytin, a snake venom that activates platelets (for example, Non-patent Document 1). reference). The present inventors have found that the in vivo ligand of CLEC-2 is a membrane protein podoplanin expressed in certain types of cancer cells (see, for example, Non-Patent Document 2).

As a mouse deficient in CLEC-2 function, thrombus formation is suppressed in CLEC-2 deficient bone marrow chimeric mice and mice deficient in CLEC-2 by injecting anti-podoplanin antibody. The present inventors have shown that there is no significant extension (see, for example, Non-Patent Documents 3 to 5). In addition, the present inventors have reported that anti-podoplanin antibodies suppress lung metastasis of cancer in a mouse lung metastasis model (see, for example, Non-Patent Document 6).

Therefore, it is expected that drugs targeting CLEC-2 can be antithrombotic, antiplatelet and antimetastatic drugs with few side effects of bleeding. However, no drugs targeting CLEC-2 have been identified, and research and development of platelet aggregation inhibitors with few bleeding side effects, antimetastatic agents that suppress cancer metastasis, and candidate compounds thereof are desired. It is rare.

This invention makes it a subject to solve the said various problems in the past and to achieve the following objectives. That is, the present invention relates to a CLEC-2 antagonist capable of specifically suppressing the function of the platelet activating receptor CLEC-2, a platelet aggregation inhibitor, an antithrombotic agent, an antimetastatic agent, an antiarthritic agent, and a metal ion An object of the present invention is to provide a compound in which a ligand having a porphyrin skeleton is coordinated, and a method for producing the same.

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> A CLEC-2 antagonist comprising a compound represented by any one of the following general formula (1) and the following general formula (2).
[General formula (1)]

(In the general formula (1), M represents any one of H ₂ and Group 2 to 12 elements, R ¹ represents either a vinyl group or a 1-hydroxyethyl group, and X represents Group 1) Represents an element.)
[General formula (2)]

(In the general formula (2), M represents any one of H ₂ and Group 2 to 12 elements, and R ² represents a phenyl group, a sulfophenyl group, a carboxyphenyl group, and 1-methylpyridinium-4-yl.) Represents any of the groups.)
<2> The compound according to <1>, wherein the compound represented by the general formula (1) is a compound represented by any one of the following general formula (1-A) and the following general formula (1-B): CLEC-2 antagonist.
[General Formula (1-A)]

(In the general formula (1-A), M represents any one of H ₂ , Co, Zn, Ni, and Pd, and X represents a Group 1 element.)
[General formula (1-B)]

(However, in the general formula (1-B), M represents any of H ₂ , Co, Zn and Cu.)
<3> The CLEC-2 antagonist according to <2>, wherein the compound represented by the general formula (1-B) is a compound represented by the following structural formula B4.
[Structural formula B4]

<4> The CLEC-2 antagonist according to any one of <1> to <3>, wherein the compound represented by the general formula (2) is a compound represented by the following structural formula C1.
[Structural formula C1]

<5> A platelet aggregation inhibitor comprising the CLEC-2 antagonist according to any one of <1> to <4>.
<6> An antithrombotic agent comprising the CLEC-2 antagonist according to any one of <1> to <4>.
<7> An antimetastatic agent comprising the CLEC-2 antagonist according to any one of <1> to <4>.
<8> An anti-arthritic drug comprising the CLEC-2 antagonist according to any one of <1> to <4>.
<9> A compound represented by any one of the following general formula (1-A) and the following general formula (1-B).
[General Formula (1-A)]

(In the general formula (1-A), M represents any one of Co, Zn, Ni and Pd, and X represents a Group 1 element.)
[General formula (1-B)]

(In the general formula (1-B), M represents any one of Co, Zn and Cu.)
<10> A salt of protoporphyrin and a metal acetate are reacted with either dimethyl sulfoxide or acetic acid at 70 ° C. to 95 ° C. for 1 hour to 30 hours in the presence of water, and the following general formula (1-A) It is a manufacturing method of the compound characterized by including the process of obtaining the compound represented.
[General Formula (1-A)]

(In the general formula (1-A), M represents any one of Co, Zn, Ni and Pd, and X represents a Group 1 element.)
<11> A compound represented by the following general formula (1-B) is obtained by reacting hematoporphyrin and metal acetate in the presence of either methanol or acetic acid at 15 ° C. to 30 ° C. for 5 hours to 30 hours. It is a manufacturing method of the compound characterized by including a process.
[General formula (1-B)]

(In the general formula (1-B), M represents any one of Co, Zn and Cu.)

According to the present invention, a CLEC-2 antagonist capable of solving the above-described problems and achieving the above-mentioned object and capable of specifically suppressing the function of the platelet-activating receptor CLEC-2, platelet aggregation inhibition Agents, antithrombotic agents, antimetastatic agents, antiarthritic agents, compounds in which a ligand having a porphyrin skeleton is coordinated to a metal ion, and a method for producing the same can be provided.

FIG. 1 is a diagram for explaining the flow and results of primary screening in Example 1. FIG. 2 is a diagram for explaining the results of secondary screening in Example 1. FIG. 3 is a graph showing the evaluation results of protoporphyrin IX (H ₂ -PP) in Example 1 using a flow cytometer. FIG. 4 is a graph showing the measurement results of the protoporphyrin zinc complex (Zn-PP) of Example 2 by UV-vis spectrum and fluorescence spectrum. FIG. 5 is a diagram showing the results of ¹ H-NMR measurement of the protoporphyrin zinc complex (Zn—PP) of Example 2. FIG. 6 is a view showing the evaluation results of a protoporphyrin zinc complex (Zn—PP) in Example 2 using a flow cytometer. FIG. 7 is a graph showing the measurement results of the protoporphyrin nickel complex (Ni—PP) of Example 3 by UV-vis spectrum and fluorescence spectrum. FIG. 8 is a view showing the evaluation results of the protoporphyrin nickel complex (Ni—PP) in Example 3 using a flow cytometer. FIG. 9 is a graph showing the measurement results of the protoporphyrin cobalt complex (Co-PP) of Example 4 by UV-vis spectrum and fluorescence spectrum. FIG. 10 is a view showing the evaluation results of the protoporphyrin cobalt complex (Co-PP) in Example 4 using a flow cytometer. FIG. 11 is a graph showing the measurement results of the protoporphyrin palladium complex (Pd-PP) of Example 5 by UV-vis spectrum and fluorescence spectrum. FIG. 12 is a view showing the evaluation results of a protoporphyrin palladium complex (Pd-PP) in Example 5 using a flow cytometer. FIG. 13 is a diagram showing the evaluation results of hematoporphyrin (H ₂ -HP) in Example 6 using a flow cytometer. FIG. 14 shows the measurement results of UV-vis spectrum and fluorescence spectrum of the hematoporphyrin zinc complex (Zn—HP) of Example 7. FIG. 15 shows the results of ¹ H-NMR measurement of the hematoporphyrin zinc complex (Zn—HP) of Example 7. FIG. 16 is a view showing the evaluation results of a hematoporphyrin zinc complex (Zn—HP) in Example 7 using a flow cytometer. FIG. 17 shows the measurement results of the hematoporphyrin copper complex (Cu—HP) of Example 8 using the UV-vis spectrum and fluorescence spectrum. FIG. 18 is a diagram showing the evaluation results of a hematoporphyrin copper complex (Cu—HP) in Example 8 using a flow cytometer. FIG. 19 shows the measurement results of the hematoporphyrin cobalt complex (Co—HP) of Example 9 using UV-vis spectrum and fluorescence spectrum. FIG. 20 is a diagram showing the evaluation results of a hematoporphyrin cobalt complex (Co—HP) in Example 9 using a flow cytometer. FIG. 21 is a diagram showing the results of evaluation by flow cytometer of 5,10,15,20-tetrakis (p-sulfophenyl) -21H, 23H-porphyrin (TPPS) in Example 10. FIG. 22 is a diagram showing the results of evaluation by flow cytometer of p-toluenesulfonate (TMPyP4OTs) of α, β, γ, δ-tetrakis (1-methylpyridinium-4-yl) porphyrin in Example 11. FIG. 23 is a diagram showing the evaluation results of the platelet aggregation inhibitory effect on human platelet aggregation. FIG. 24 is a diagram showing the evaluation results of the platelet aggregation inhibitory effect on mouse platelet aggregation. FIG. 25 is a photograph showing the state of the lung on the 14th day after the start of compound administration (DMSO or Co-HP) in a mouse lung metastasis model. FIG. 26 is a graph showing the weight of the lung on the 14th day from the start of compound administration (DMSO or Co-HP) in a mouse lung metastasis model. FIG. 27 is a diagram showing the effect of prolonging the vascular occlusion time using a mouse in vivo iron chloride thrombus formation model. FIG. 28 is a diagram showing the effect on bleeding time in mice in vivo. FIG. 29 is a graph showing the degree of inflammation of rheumatoid arthritis in a K / BxN mouse serum transfer arthritis model using wild-type mice and CLEC-2 deficient bone marrow chimeric mice as recipient mice. FIG. 30 shows the effect of Co-HP administration on rheumatoid arthritis in a K / BxN mouse serum transfer arthritis model using wild-type mice as recipient mice. FIG. 31 is a photograph visualizing platelet adhesion of human lymphatic epithelial cells (LEC). FIG. 32 is a graph showing a quantified platelet adhesion area of human lymphatic epithelial cells (LEC).

(CLEC-2 antagonist)
The CLEC-2 antagonist of the present invention comprises a compound having a porphyrin skeleton represented by any of the following general formula (1) and the following general formula (2).
The CLEC-2 antagonist is a drug having an action of suppressing the function of CLEC-2 which is a platelet activation receptor. The CLEC-2 antagonist preferably has an action of suppressing the binding between CLEC-2 and podoplanin, which is a ligand in vivo of CLEC-2.

-CLEC-2-
The CLEC-2 has an official name: C-type lectin-like receptor-2 and was identified as a receptor for rhodocytin, a snake venom that activates platelets (Blood. 2006; 107 (2): 542-549. ). In humans, it is known to be specifically expressed only in platelets.
On the other hand, podoplanin, an in vivo ligand for CLEC-2, is a membrane glycoprotein rich in sialic acid, and is expressed in squamous cell carcinoma, malignant mesothelioma, etc. It was known. Further, it has been known that it has platelet aggregation activity and cancer metastasis promoting activity, and it has been predicted that a receptor exists in platelets. However, until the platelet receptor is identified as CLEC-2, It took 20 years.
When the CLEC-2 is activated by podoplanin, platelet aggregation is promoted and thrombus formation is promoted.
In contrast, the CLEC-2 antagonist of the present invention functions as an antagonist that suppresses the function of CLEC-2 by specifically binding to CLEC-2 or competing with podoplanin.

<< Compound Represented by Formula (1) >>
[General formula (1)]

(In the general formula (1), M represents any one of H ₂ and Group 2 to 12 elements, R ¹ represents either a vinyl group or a 1-hydroxyethyl group, and X represents Group 1) Represents an element.)

In the general formula (1), M is any one of H ₂ and Group 2 to 12 elements, and H ₂ , Group 8 elements, Group 9 elements, Group 10 elements, Group 11 elements, Group 12 elements are preferred, and H ₂ , Fe, Co, Ni, Pd, Cu, and Zn are more preferred.
The Group 2 elements and the like are based on a new notation of the International Pure Applied Chemical Association (IUPAC), and the Group 2 to 12 elements are Group IIA to Group VIIIA and IB of the former IUPAC notation. Corresponding to the Group and Group IIB respectively.

In the general formula (1), X is a Group 1 element, preferably H, Li, Na, or K, and more preferably H or Na.

The compound represented by the general formula (1) is preferably any one of a compound represented by the following general formula (1-A) and a compound represented by the following general formula (1-B).

<<< Compound represented by formula (1-A) >>>
The compound represented by the following general formula (1-A) is a compound having a protoporphyrin skeleton in which R ¹ is a vinyl group in the general formula (1).
[General Formula (1-A)]

(In the general formula (1-A), M represents any one of H ₂ , Co, Zn, Ni, and Pd, and X represents a Group 1 element.)

Examples of the compound represented by the general formula (1-A) include a protoporphyrin IX represented by the following structural formula A1, a protoporphyrin zinc complex represented by the following structural formula A2, and a structural formula represented by the following structural formula A3. Examples thereof include a protoporphyrin nickel complex, a protoporphyrin cobalt complex represented by the following structural formula A4, and a protoporphyrin palladium complex represented by the following structural formula A5. Among these, a protoporphyrin IX represented by the following structural formula A1, a protoporphyrin zinc complex represented by the following structural formula A2, and a protoporphyrin cobalt complex represented by the following structural formula A4 are preferable, and represented by the following structural formula A1. Protoporphyrin IX is more preferred.

<<< Compound represented by formula (1-B) >>>
The compound represented by the following general formula (1-B) is a compound having a hematoporphyrin skeleton in which R ¹ is a 1-hydroxyethyl group and X is H (hydrogen) in the general formula (1). is there.
[General formula (1-B)]

(However, in the general formula (1-B), M represents any of H ₂ , Co, Zn and Cu.)

The compound represented by the general formula (1-B) is specifically represented by hematoporphyrin represented by the following structural formula B1, hematoporphyrin zinc complex represented by the following structural formula B2, and represented by the following structural formula B3. A hematoporphyrin copper complex and a hematoporphyrin cobalt complex represented by the following structural formula B4. Among these, hematoporphyrin represented by the following structural formula B1, hematoporphyrin copper complex represented by the following structural formula B3, and hematoporphyrin cobalt complex represented by the following structural formula B4 are preferable, and CLEC-2, podoplanin, A hematoporphyrin cobalt complex represented by the following structural formula B4 is more preferable in that the activity of suppressing the binding of is particularly high.

<< Compound Represented by Formula (2) >>
[General formula (2)]

(In the general formula (2), M represents any one of H ₂ and Group 2 to 12 elements, and R ² represents a phenyl group, a sulfophenyl group, a carboxyphenyl group, and 1-methylpyridinium-4-yl.) Represents any of the groups.)

In the general formula (2), M is any of H ₂ and Group 2 to 12 elements, and H ₂ , Group 8 elements, Group 9 elements, Group 10 elements, Group 11 elements, Group 12 elements are preferred, and H ₂ , Fe, Co, Ni, Pd, Cu, and Zn are more preferred.

Examples of the compound represented by the general formula (2) include 5,10,15,20-tetrakis (p-sulfophenyl) -21H, 23H-porphyrin (“TPPS”) represented by the following structural formula C1. P) of α, β, γ, δ-tetrakis (1-methylpyridinium-4-yl) porphyrin (sometimes referred to as “TMPyP”) represented by the following structural formula C2. Toluenesulfonate (sometimes referred to as “TMPyP4OTs”), tetraphenylporphyrin (sometimes referred to as “TPP”), 5,10,15,20-tetrakis (p-carboxyphenyl) -21H, 23H -Porphyrin (sometimes referred to as "TPPC"). Among these, TPPS represented by the following structural formula C1 is preferable.

[Structural formula C1]

[Structural formula C2]

(Platelet aggregation inhibitor and antithrombotic agent)
The platelet aggregation inhibitor of the present invention comprises the CLEC-2 antagonist of the present invention. The platelet aggregation inhibitor is a drug having an action of suppressing platelet aggregation.
The antithrombotic agent of the present invention comprises the CLEC-2 antagonist of the present invention. The antithrombotic drug is a drug having an action of suppressing the formation of a thrombus and preventing vascular occlusion.
It is preferable that the platelet aggregation inhibitor and the antithrombotic agent act via function suppression of CLEC-2 which is a platelet activating receptor, and podoplanin which is an in vivo ligand of CLEC-2 and CLEC-2 It is more preferable to have an action of suppressing the binding with.

(Anti-metastatic drug)
The antimetastatic agent of the present invention comprises the CLEC-2 antagonist of the present invention. The antimetastatic drug is a drug having an action of suppressing cancer cell metastasis.
The anti-metastatic agent preferably acts through suppression of the function of CLEC-2, which is a platelet activation receptor, and suppresses the binding of CLEC-2, which is a ligand in vivo of CLEC-2, to CLEC-2 More preferably, it has an action.

(Anti-arthritis drug)
The anti-arthritic agent of the present invention comprises the CLEC-2 antagonist of the present invention. The anti-arthritic drug is a drug having an action of suppressing joint inflammation, and examples thereof include an anti-rheumatic drug.
The anti-arthritic agent preferably acts through suppression of the function of CLEC-2, which is a platelet activating receptor, and suppresses the binding of CLEC-2 in vivo to podoplanin, which is a ligand of CLEC-2. More preferably, it has an action.

(Formulation)
The preparation includes at least one of the CLEC-2 antagonist of the present invention, a platelet aggregation inhibitor, an antithrombotic drug, an antimetastatic drug, and an antiarthritic drug, and further pharmacologically acceptable as necessary. Contains other ingredients such as a carrier.
The CLEC-2 antagonist, the platelet aggregation inhibitor, the antithrombotic agent, the antimetastatic agent, and the antiarthritic agent are represented by any one of the general formula (1) and the general formula (2). Or a pharmacologically acceptable salt, solvate or prodrug of the compound.

The preparation is prepared by formulating into any dosage form such as liquid, powder, granule, tablet, etc. according to a conventional method using a pharmacologically acceptable carrier such as dextrin and cyclodextrin, and an auxiliary agent. In addition to being used in other compositions (for example, eye drops, oral pharmaceuticals, etc.), it can also be used as an ointment, a solution for external use, a patch, and the like.
As said adjuvant, an excipient | filler, binder, a disintegrating agent, a lubricant agent, a stabilizer, a corrigent, a corrigent etc. can be used, for example.

Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid. Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, polyvinylpyrrolidone and the like. It is done. Examples of the disintegrant include dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, and lactose. Examples of the lubricant include purified talc, stearate, borax, and polyethylene glycol. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid, and the like. Examples of the flavoring agent or flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like.

The method for administering the preparation is not particularly limited, and administration conditions such as an administration route, administration timing, dosage, and administration schedule suitable for each drug can be appropriately selected according to the purpose.
There is no restriction | limiting in particular as said administration route, Although it can select suitably according to the objective, Intravenous administration, transdermal administration, and oral administration are preferable.

The dosage of the preparation is not particularly limited and may be appropriately selected depending on factors such as the disease state of the subject requiring treatment, body weight, etc., but preferably 1 mg to 1,000 mg per day. ˜200 mg is more preferred.

(Compound)
The compound of the present invention is a compound having a porphyrin skeleton represented by any one of the following general formula (1-A) and the following general formula (1-B).
The compound having a protoporphyrin skeleton represented by the general formula (1-A) can be preferably produced by the method for producing a compound represented by the general formula (1-A) of the present invention.
The compound having a hematoporphyrin skeleton represented by the general formula (1-B) can be preferably produced by the method for producing a compound represented by the general formula (1-B) of the present invention.

[General Formula (1-A)]

(In the general formula (1-A), M represents any one of Co, Zn, Ni and Pd, and X represents a Group 1 element.)

[General formula (1-B)]

(In the general formula (1-B), M represents any one of Co, Zn and Cu.)

(Method for producing compound)
<Method for Producing Compound Represented by General Formula (1-A)>
A method for producing a compound of the present invention is a method for producing a compound represented by the above general formula (1-A), comprising a salt of protoporphyrin and a metal acetate, either dimethyl sulfoxide or acetic acid, and water. Including a step of obtaining a compound represented by the following general formula (1-A) by reacting at 70 ° C. to 95 ° C. for 1 to 30 hours in the presence, and further including other steps such as a purification step, if necessary. .
The salt of protoporphyrin is not particularly limited as long as it is a salt of a compound in which M of the compound represented by the general formula (1-A) is H ₂ , and can be appropriately selected depending on the purpose. Examples thereof include protoporphyrin IX represented by the following structural formula A1.
Here, the metal in the metal acetate is any one of Co, Zn, Ni and Pd represented by M in the general formula (1-A).

When the compound represented by the general formula (1-A) is a protoporphyrin zinc complex represented by the following structural formula A2, the salt of protoporphyrin and zinc acetate are reacted in the presence of dimethyl sulfoxide and water. The reaction temperature is preferably 70 ° C. to 90 ° C. (eg, 80 ° C.), and the reaction time is preferably 1 hour to 10 hours (eg, 2 hours).

When the compound represented by the general formula (1-A) is a protoporphyrin nickel complex represented by the following structural formula A3, the salt of protoporphyrin and nickel acetate are reacted in the presence of dimethyl sulfoxide and water. The reaction temperature is preferably 70 ° C. to 90 ° C. (eg, 80 ° C.), and the reaction time is preferably 5 hours to 20 hours (eg, 8 hours).

When the compound represented by the general formula (1-A) is a protoporphyrin cobalt complex represented by the following structural formula A4, a salt of protoporphyrin and cobalt (II) acetate are added in the presence of acetic acid and water. The reaction temperature is preferably 80 ° C. to 95 ° C. (eg, 90 ° C.), and the reaction time is preferably 12 hours to 30 hours (eg, 24 hours).

When the compound represented by the general formula (1-A) is a protoporphyrin palladium lead complex represented by the following structural formula A5, a salt of protoporphyrin and palladium (II) acetate are present in the presence of acetic acid and water. The reaction temperature is preferably 80 ° C. to 95 ° C. (eg, 90 ° C.), and the reaction time is preferably 12 hours to 30 hours (eg, 24 hours).

<Method for Producing Compound Represented by General Formula (1-B)>
The method for producing a compound of the present invention is a method for producing a compound represented by the general formula (1-B), wherein hematoporphyrin and metal acetate are added at 15 ° C. to 15 ° C. in the presence of either methanol or acetic acid. It includes a step of obtaining a compound represented by the following general formula (1-B) by reacting at 30 ° C. for 5 to 30 hours, and further includes other steps such as a purification step as necessary.
The hematoporphyrin is a compound represented by the following structural formula B1.
Here, the metal in the metal acetate is any one of Co, Zn and Cu represented by M in the general formula (1-B).

When the compound represented by the general formula (1-B) is a hematoporphyrin zinc complex represented by the following structural formula B2, it is preferable to react hematoporphyrin and zinc acetate in the presence of methanol. The temperature is preferably 15 ° C. to 30 ° C. (eg, 25 ° C.), and the reaction time is preferably 5 hours to 20 hours (eg, 8 hours).

When the compound represented by the general formula (1-B) is a hematoporphyrin copper complex represented by the following structural formula B3, hematoporphyrin and copper (II) acetate can be reacted in the presence of acetic acid. The reaction temperature is preferably 15 ° C. to 30 ° C. (eg, 25 ° C.), and the reaction time is preferably 12 hours to 30 hours (eg, 24 hours).

When the compound represented by the general formula (1-B) is a hematoporphyrin cobalt complex represented by the following structural formula B4, the hematoporphyrin and cobalt (II) acetate can be reacted in the presence of acetic acid. The reaction temperature is preferably 15 ° C. to 30 ° C. (eg, 25 ° C.), and the reaction time is preferably 12 hours to 30 hours (eg, 24 hours).

Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples.

(Example 1: Protoporphyrin IX)
<Search for compounds that suppress the binding of CLEC-2 to podoplanin (primary screening)>
Using a library of 15,000 kinds of low molecular weight compounds centering on natural products at the RIKEN Chemical Biology Research Base Facility, primary screening of compounds that inhibit the binding of CLEC-2 to podoplanin is performed by the following method. went.
An ELISA detection system capable of detecting the binding between the recombinant CLEC-2 and the recombinant podopranin was constructed, and primary screening was performed by measurement using the ELISA detection system.

-Preparation of recombinant CLEC-2-
Human CLEC-2-rFc2 was prepared as recombinant CLEC-2 according to the method described in the document “JBC, 2007; 282 (36): 25993-2601”.

-Preparation of recombinant podopranin-
As a recombinant podopranin, human podoplanin-hFc2-Biotin was prepared according to the method described in the literature “JBC, 2007; 282 (36): 25993-2601”.

-Measurement using ELISA detection system-
100 μL of a solid phase protein solution (PBS containing 1 μg / 100 mL of human CLEC-2-rFc2, pH 7.4) was added dropwise to a 96-well plate (Immulon (registered trademark) 2HB, manufactured by Thermo Scientific), and the plate was sealed. The reaction was carried out at 4 ° C. for 8 to 24 hours. After removing the solid phase protein solution, it was washed once with 400 μL of a washing solution (PBS containing 0.05 vol% Tween 20, pH 7.2). Next, 150 μL of a blocking agent (Super Block, manufactured by Scy Laboratories) was added, and the mixture was allowed to stand accurately for 5 minutes. After removing the blocking agent, washing was performed twice with 400 μL of the washing solution.

To each well of the obtained ELISA plate coated with the recombinant CLEC-2, 100 μL of each compound solution (10 μg / mL) of the low molecular compound library was dropped, and after removing the solution, washed with 400 μL of the washing solution three times. did. Subsequently, 100 μL of a detection protein solution (PBS containing 1 μg / 100 mL of human podoplanin-hFc2-Biotin, pH 7.4) was added dropwise and reacted at room temperature (25 ° C.) for 1 hour. After removing the detection protein solution, it was washed with 400 μL of the washing solution three times. 100 μL of a detection reagent (streptAvidin-HRP, Vector Laboratories, CA) was added dropwise and reacted for 1 hour. After removing the detection reagent, it was washed 3 times with 400 μL of washing solution. Next, 100 μL of TBM solution (T0440, manufactured by SIGMA-ALDRICH) was dropped and reacted in a dark room for 3 to 5 minutes. Then, 50 μL of a reaction stop solution (0.5 M HCl) was added, and a microplate reader (apparatus Name: Absorbance at 450 nm was measured with a multi-label counter, ARV0mx-U1, 1420-050J, manufactured by Perkin Elmer).

As a negative control, PBS was used instead of each compound solution and detection protein solution. As a positive control, PBS was used instead of the solution of each compound.
From the absorbance of each compound, the binding inhibition rate was calculated by the following formula 1. A compound having a binding inhibition rate of 40% or more was judged as a compound having binding activity to CLEC-2.
[Formula 1]
Binding inhibition rate (%) = 100− (absorbance of each compound−absorbance of negative control) / (absorbance of positive control−absorbance of negative control)

FIG. 1 is a diagram for explaining the flow and results of the primary screening in Example 1. In the positive control, a signal having a wavelength of 450 nm derived from the binding of CLEC-2 and podoplanin is observed. In contrast, as shown in FIG. 1, when Compound A that binds to CLEC-2 is administered, the binding between CLEC-2 and podoplanin is suppressed, and the signal observed in the positive control is reduced. On the other hand, when Compound B that does not bind to CLEC-2 (or does not suppress binding to podoplanin) is administered, the signal observed in the positive control does not change because binding between CLEC-2 and podoplanin is not suppressed. .

As described above, a small molecule that binds to CLEC-2 with respect to the binding between recombinant CLEC-2 and recombinant podopranin, using as an index whether or not each compound in the low molecular compound library suppresses the binding. Compound ligands were searched. As a result, 64 types of compounds including protoporphyrin IX (sometimes referred to as “H ₂ -PP”), which is a compound having a porphyrin skeleton, were obtained.

<Evaluation of compounds that suppress the binding of CLEC-2 to podoplanin (secondary screening)>
The 64 compounds obtained in the primary screening were then subjected to secondary screening by a flow cytometer using CLEC-2 expressing cultured cells.

-Preparation of CLEC-2 expressing cultured cells-
As CLEC-2 expressing cultured cells, human CLEC-2 expressing 293TRex cells were prepared according to the method described in the literature “J Virol. 2003; 77 (7): 4070-4080”.
This cell is a cell that expresses CLEC-2 by the addition of doxycycline by the Tet on system, and CLEC-2 is added by adding doxycycline at a final concentration of 1 μg / mL 24 hours before measurement with a flow cytometer. Expression was induced.

-Human podoplanin -Preparation of human Fc2-
As a recombinant podopranin, human podoplanin-human Fc2 was prepared according to the method described in the document “JBC, 2007; 282 (36): 25993-2601”.

-Measurement with a flow cytometer using CLEC-2 expressing cultured cells-
50 μL of PBS containing human CLEC-2 expressing 293TRex cells 2.5 × 10 ⁵ cells was added to a 1.5 mL microtube. 20 μg / mL of each compound was added to the microtube to a final concentration of 5 μg / mL and allowed to stand at 4 ° C. for 30 minutes. Next, human podoplanin-human Fc2 or human Fc2 is added so as to be 5 μg / mL (however, for compounds that emit autofluorescence in detection mode FL1, biotin-labeled human podoplanin-human Fc2 or biotin-labeled human Fc2 is added. And left at 4 ° C. for 30 minutes. Further, 1 mL of PBS was added for washing, excess compounds and recombinant proteins were removed, and 0.1 mL of PBS was added for resuspension. Thereafter, anti-human IgG antibody-FITC was added (however, with respect to a compound that exhibits autofluorescence in detection mode FL1, an APC label avidin was added) and left at 4 ° C. for 20 minutes. 300 μL of PBS was added to the obtained sample and analyzed in detection mode FL1 of a flow cytometer (device name: BD Accuri (registered trademark) C6, manufactured by BD Bioscience) (provided that autofluorescence was detected in detection mode FL1) Were analyzed in detection mode FL4).
As a negative control, a sample to which 1 μL of DMSO was added instead of the solution of each compound was used. As another negative control, a sample to which human Fc2 was added instead of human podoplanin-human Fc2 was used. As a positive control, a sample to which rhodocytin having a final concentration of 50 nM was added instead of the solution of each compound was used.

FIG. 2 shows a diagram for explaining the results of the secondary screening in Example 1. In the absence of the compound (see the left panel of FIG. 2), a peak shift derived from the binding of CLEC-2 and podoplanin is observed by substituting human podoplanin-human Fc2 as a negative control. On the other hand, when a compound that binds to CLEC-2 is administered (see the right panel of FIG. 2), when the binding between CLEC-2 and podoplanin is completely suppressed, the peak derived from the binding between CLEC-2 and podoplanin No shift is observed and a peak that completely overlaps the peak from the negative control is observed. In addition, when the binding between CLEC-2 and podoplanin is partially suppressed (not shown), depending on the degree of suppression, it is derived from the peak derived from the negative control and the binding between CLEC-2 and podoplanin. A peak in between is observed.

The binding inhibition rate was calculated by the following formula 2. A compound having a binding inhibition rate of 40% or more was judged as a compound having binding activity to CLEC-2.
[Formula 2]
Binding inhibition rate (%) = 100-100 × (TN) / (PN)
In the above formula, P, N, and T are as follows.
P: mean fluorescent intensity (MFI) derived from compound positive control (human podoplanin-human Fc2, DMSO)
N: MFI derived from negative binding control (human Fc2, DMSO)
T: MFI derived from administration of compound

As described above, with respect to the binding between CLEC-2 expressing cells and recombinant podopranin, whether or not each compound identified in the primary screening suppresses the binding (binding suppression rate of 40% or more) is used as an index. A compound that suppresses the binding between CLEC-2 and podoplanin was searched.
As a result, protoporphyrin IX (H ₂ -PP), which is a compound having a porphyrin skeleton, was obtained. FIG. 3 shows the evaluation results of protoporphyrin IX (H ₂ -PP) in Example 1 using a flow cytometer. The binding inhibition rate of protoporphyrin IX was 86.2%.

In order to optimize the protoporphyrin IX obtained in the secondary screening of Example 1 as shown in Examples 2 to 5 and 7 to 9 below, protoporphyrin metal complex and hematoporphyrin are used as analogs of protoporphyrin IX. A metal complex was prepared.
Further, in Examples 2 to 11 below, CLEC-2 and CLEC-2 were obtained in the same manner as in the secondary screening of Example 1, except that the compounds to be evaluated were similar to the protoporphyrin IX of Examples 2 to 11. Evaluation and optimization of compounds that inhibit the binding to podoplanin were performed.

(Example 2: Protoporphyrin zinc complex)
<Synthesis and Identification of Protoporphyrin Zinc Complex (Zn-PP)>
Zinc acetate (390 mg, 1.78 mmol) was added to a solution obtained by adding 20 mL of dimethyl sulfoxide (DMSO) and 5 mL of distilled water to protoporphyrin disodium salt (manufactured by Tokyo Chemical Industry Co., Ltd., 100 mg, 0.165 mmol). And stirred for 2 hours. Then, it cooled to room temperature (25 degreeC), ethanol was added and crystallized, and the obtained precipitation was wash | cleaned with distilled water, and the purple precipitate (109 mg) was obtained with yield 98%.
The obtained compound was identified by ¹ H-NMR, ultraviolet-visible light absorption spectrum (UV-vis spectrum), and fluorescence spectrum, and it was found that a protoporphyrin zinc complex was obtained. The reaction formula is shown in the following reaction formula (1). FIG. 4 shows the measurement results of the UV-vis spectrum and fluorescence spectrum of Example 2. The measurement result by ¹ H-NMR of Example 2 is shown in FIG.
UV-vis spectrum measurement was performed using UV-1800 (manufactured by Shimadzu Corporation), and fluorescence spectrum measurement was performed using FP-6300 (manufactured by JASCO Corporation). In the figure, the UV-vis spectrum is indicated by a solid line. The fluorescence spectrum is shown by a broken line. ^{For 1} H-NMR measurement, AVANCE 400 (manufactured by Bruker) was used, and measurement was performed in heavy DMSO.

[Reaction Formula (1)]

(In the reaction formula (1), Zn (OAc) ₂ represents zinc acetate, and DMSO represents dimethyl sulfoxide.)

<Evaluation of binding inhibition between CLEC-2 and podoplanin>
In the secondary screening of Example 1, binding of CLEC-2 and podoplanin was performed in the same manner as in the secondary screening of Example 1, except that a protoporphyrin zinc complex having a final concentration of 5 μg / mL was used as the compound to be evaluated. Evaluation of compounds that inhibit the above was performed. The evaluation result by the flow cytometer of a protoporphyrin zinc complex is shown in FIG. The binding inhibition rate of the protoporphyrin zinc complex was 71.2%.

(Example 3: Protoporphyrin nickel complex)
<Synthesis and Identification of Protoporphyrin Nickel Complex (Ni-PP)>
To a protoporphyrin disodium salt (Tokyo Chemical Industry Co., Ltd., 100 mg, 0.165 mmol) was added DMSO (20 mL) and distilled water (5 mL), nickel acetate (610 mg, 2.47 mmol) was added to the solution, and the mixture was heated and stirred at 80 ° C. for 8 hours. did. Then, it cooled to room temperature (25 degreeC), methanol was added and crystallized, and the obtained precipitation was wash | cleaned with distilled water. This precipitate was recrystallized with acetic acid-distilled water to obtain a deep purple precipitate (71 mg) with a yield of 65%.
The obtained compound was identified by UV-vis spectrum and fluorescence spectrum, and it was found that a protoporphyrin nickel complex was obtained. The reaction formula is shown in the following reaction formula (2). FIG. 7 shows the measurement results of the UV-vis spectrum and fluorescence spectrum of Example 3.

[Reaction Formula (2)]

(However, in the reaction formula (2), Ni (OAc) ₂ represents nickel acetate and DMSO represents dimethyl sulfoxide.)

<Evaluation of binding inhibition between CLEC-2 and podoplanin>
In the secondary screening of Example 1, binding of CLEC-2 and podoplanin was performed in the same manner as in the secondary screening of Example 1, except that a protoporphyrin nickel complex having a final concentration of 5 μg / mL was used as the evaluation target compound. Evaluation of compounds that inhibit the above was performed. FIG. 8 shows the evaluation results of the protoporphyrin nickel complex using a flow cytometer. The binding inhibition rate of the protoporphyrin nickel complex was 50.4%.

(Example 4: Protoporphyrin cobalt complex)
<Synthesis and Identification of Protoporphyrin Cobalt Complex (Co-PP)>
When 20 mL of acetic acid was added to and dissolved in protoporphyrin disodium salt (Tokyo Chemical Industry Co., Ltd., 100 mg, 0.165 mmol), cobalt acetate (II) tetrahydrate (410 mg, 1.65 mmol) was added to the solution. Stir for hours. Thereafter, after cooling to room temperature (25 ° C.), the reaction mixture was added to 150 mL of distilled water and cooled to about 4 ° C. to obtain a deep purple precipitate (102 mg) at a yield of 93%.
As a result of identification of the obtained compound by UV-vis spectrum and fluorescence spectrum, it was found that a protoporphyrin cobalt complex was obtained. The reaction formula is shown in the following reaction formula (3). FIG. 9 shows the measurement results of the UV-vis spectrum and fluorescence spectrum of Example 4.

[Reaction Formula (3)]

(In the reaction formula (3), Co (OAc) ₂ represents cobalt acetate (II).)

<Evaluation of binding inhibition between CLEC-2 and podoplanin>
In the secondary screening of Example 1, binding of CLEC-2 and podoplanin was performed in the same manner as in the secondary screening of Example 1, except that a protoporphyrin cobalt complex having a final concentration of 5 μg / mL was used as the compound to be evaluated. Evaluation of compounds that inhibit the above was performed. The evaluation result by the flow cytometer of a protoporphyrin cobalt complex is shown in FIG. The binding inhibition rate of the protoporphyrin cobalt complex was 79.4%.

(Example 5: Protoporphyrin palladium complex)
<Synthesis and Identification of Protoporphyrin Palladium Complex (Pd-PP)>
To a protoporphyrin disodium salt (manufactured by Tokyo Chemical Industry Co., Ltd., 50 mg, 0.083 mmol) was added 10 mL of acetic acid and dissolved therein, palladium (II) acetate (92 mg, 0.415 mmol) was added, and the mixture was heated and stirred at 90 ° C. for 24 hours. . Then, after cooling to room temperature (25 ° C.), the reaction mixture is added to 50 mL of distilled water to crystallize, and the resulting precipitate is recrystallized with DMSO-methanol and DMSO-distilled water to obtain a quantitative yield. Deep purple precipitate (65 mg) was obtained.
The obtained compound was identified by UV-vis spectrum and fluorescence spectrum, and it was found that a protoporphyrin palladium complex was obtained. The reaction formula is shown in the following reaction formula (4). FIG. 11 shows the measurement results of the UV-vis spectrum and fluorescence spectrum of Example 5.

[Reaction Formula (4)]

(In the reaction formula (4), Pd (OAc) ₂ represents palladium acetate (II).)

<Evaluation of binding inhibition between CLEC-2 and podoplanin>
In the secondary screening of Example 1, binding of CLEC-2 and podoplanin was performed in the same manner as in the secondary screening of Example 1, except that a protoporphyrin palladium complex having a final concentration of 5 μg / mL was used as the compound to be evaluated. Evaluation of compounds that inhibit the above was performed. FIG. 12 shows the evaluation results of the protoporphyrin palladium complex using a flow cytometer. The binding inhibition rate of the protoporphyrin palladium complex was 66.7%.

(Example 6: Hematoporphyrin)
<Evaluation of binding inhibition between CLEC-2 and podoplanin>
In the secondary screening of Example 1, CLEC was performed in the same manner as the secondary screening of Example 1, except that hematoporphyrin (manufactured by Wako Pure Chemical Industries, Ltd.) having a final concentration of 5 μg / mL was used as the evaluation target compound. -2 was evaluated for compounds that inhibit the binding of podoplanin. The evaluation results of hematoporphyrin using a flow cytometer are shown in FIG. The binding inhibition rate of hematoporphyrin was 90.2%.

(Example 7: Hematoporphyrin zinc complex)
<Synthesis and Identification of Hematoporphyrin Zinc Complex (Zn-HP)>
20 ml of methanol was added to hematoporphyrin (manufactured by Wako Pure Chemical Industries, Ltd., 100 mg, 0.167 mmol) and dissolved, 2 ml of a saturated zinc acetate / methanol solution was added, and the mixture was stirred overnight (8 hours) at room temperature (25 ° C.). Thereafter, the reaction mixture was added to 50 mL of distilled water and cooled to about 4 ° C. to obtain a purple precipitate (116 mg) in a quantitative yield.
The obtained compound was identified by ¹ H-NMR, UV-vis spectrum, and fluorescence spectrum, and it was found that a hematoporphyrin zinc complex was obtained. The reaction formula is shown in the following reaction formula (5). FIG. 14 shows the measurement results of the UV-vis spectrum and the fluorescence spectrum of Example 7. Further, FIG. 15 shows the measurement result of Synthesis Example 1 by ¹ H-NMR.

[Reaction Formula (5)]

(In the reaction formula (5), Zn (OAc) ₂ represents zinc acetate, and MeOH represents methanol.)

<Evaluation of binding inhibition between CLEC-2 and podoplanin>
In the secondary screening of Example 1, binding of CLEC-2 and podoplanin was performed in the same manner as in the secondary screening of Example 1, except that a hematoporphyrin zinc complex having a final concentration of 5 μg / mL was used as the compound to be evaluated. Evaluation of compounds that inhibit the above was performed. FIG. 16 shows the results of evaluation of the hematoporphyrin zinc complex using a flow cytometer. The binding inhibition rate of the hematoporphyrin zinc complex was 81.3%.

(Example 8: Hematoporphyrin copper complex)
<Synthesis and Identification of Hematoporphyrin Copper Complex (Cu-HP)>
To hematoporphyrin (Wako Pure Chemical Industries, Ltd., 100 mg, 0.167 mmol), 20 mL of acetic acid was added and dissolved, copper (II) acetate (300 mg, 1.65 mmol) was added, and the mixture was stirred at room temperature (25 ° C.) for 24 hours. . Thereafter, the reaction mixture was added to 150 mL of distilled water and cooled to about 4 ° C. to obtain a purple precipitate (162 mg) in a quantitative yield.
As a result of identification of the obtained compound by UV-vis spectrum and fluorescence spectrum, it was found that a hematoporphyrin copper complex was obtained. The reaction formula is shown in the following reaction formula (6). FIG. 17 shows the measurement results of the UV-vis spectrum and fluorescence spectrum of Example 8.

[Reaction Formula (6)]

(In the reaction formula (6), Cu (OAc) ₂ represents copper acetate (II).)

<Evaluation of binding inhibition between CLEC-2 and podoplanin>
In the secondary screening of Example 1, binding of CLEC-2 and podoplanin was performed in the same manner as in the secondary screening of Example 1, except that a hematoporphyrin copper complex having a final concentration of 5 μg / mL was used as the compound to be evaluated. Evaluation of compounds that inhibit the above was performed. FIG. 18 shows the results of evaluation of the hematoporphyrin copper complex using a flow cytometer. The binding inhibition rate of the hematoporphyrin copper complex was 84.0%.

(Example 9: Hematoporphyrin cobalt complex)
<Synthesis and Identification of Hematoporphyrin Cobalt Complex (Co-HP)>
When hematoporphyrin (Wako Pure Chemical Industries, Ltd., 100 mg, 0.167 mmol) was added with 20 mL of acetic acid, cobalt (II) acetate tetrahydrate (410 mg, 1.65 mmol) was added to the solution, and room temperature (25 ° C.) was added. Stir for 24 hours. Then, when extraction operation was performed by adding ethyl acetate and distilled water, a precipitate was formed. The precipitate was collected by filtration, the organic phase was dried over sodium sulfate, and the solvent was distilled off under reduced pressure. Since it was confirmed by TLC that the residue component and the precipitate obtained by the extraction operation were the same component, a deep purple precipitate (55 mg) was obtained in a yield of 50% by recrystallization from methanol-toluene.
As a result of identification of the obtained compound by UV-vis spectrum and fluorescence spectrum, it was found that a hematoporphyrin cobalt complex was obtained. The reaction formula is shown in the following reaction formula (7). FIG. 19 shows the measurement results of the UV-vis spectrum and fluorescence spectrum of Example 9.

[Reaction Formula (7)]

(However, Co (OAc) _{2 in the} reaction formula (7) represents cobalt acetate (II).)

<Evaluation of binding inhibition between CLEC-2 and podoplanin>
In the secondary screening of Example 1, binding of CLEC-2 and podoplanin was performed in the same manner as in the secondary screening of Example 1, except that the hematoporphyrin cobalt complex having a final concentration of 5 μg / mL was used as the evaluation target compound. Evaluation of compounds that inhibit the above was performed. FIG. 20 shows the results of evaluation of the hematoporphyrin cobalt complex using a flow cytometer. The binding inhibition rate of the hematoporphyrin cobalt complex was 101.1%.

(Example 10: 5,10,15,20-tetrakis (p-sulfophenyl) -21H, 23H-porphyrin)
<Evaluation of binding inhibition between CLEC-2 and podoplanin>
In the secondary screening of Example 1, instead of protoporphyrin IX having a final concentration of 5 μg / mL, the compound to be evaluated was replaced with 5,10,15,20-tetrakis (p-sulfo) represented by the following structural formula C1. Phenyl) -21H, 23H-porphyrin (manufactured by Tokyo Chemical Industry Co., Ltd., sometimes referred to as “TPPS”) was used in the same manner as in the secondary screening of Example 1, except that CLEC-2 and podoplanin Evaluation of compounds that inhibit binding was performed. FIG. 21 shows the results of evaluation using a TPPS flow cytometer. The binding inhibition rate of TPPS was 89.8%.

[Structural formula C1]

(Example 11: p-toluenesulfonate of α, β, γ, δ-tetrakis (1-methylpyridinium-4-yl) porphyrin)
<Evaluation of binding inhibition between CLEC-2 and podoplanin>
In the secondary screening of Example 1, instead of protoporphyrin IX having a final concentration of 5 μg / mL, the α, β, γ, δ-tetrakis (1-methyl) represented by the following structural formula C2 was used as the evaluation target compound. Except that p-toluenesulfonate (manufactured by Tokyo Chemical Industry Co., Ltd., sometimes referred to as “TMPyP4OTs”) of pyridinium-4-yl) porphyrin (sometimes referred to as “TMPyP”) was used. In the same manner as in the second screening of 1, the compounds that inhibit the binding of CLEC-2 and podoplanin were evaluated. The evaluation result by the flow cytometer of TMPyP4OTs is shown in FIG. The binding inhibition rate of TMPyP4OTs was 54.4%.

[Structural formula C2]

As a result of Examples 2 to 11, it was found that all of the evaluated protoporphyrin IX analogs showed a binding inhibitory activity of 40% or more and suppressed the binding between CLEC-2 and podoplanin. In particular, the binding inhibition activity of Co-HP was high, and it was found that the binding was almost completely suppressed in the secondary screening evaluation system of Example 1 (when the compound was used at a final concentration of 5 μg / mL).

<Evaluation of platelet aggregation inhibitory effect on human platelet aggregation>
Protoporphyrin IX (H ₂ -PP) and hematoporphyrin cobalt complex (Co-HP) when platelet aggregation is induced by rhodocytin, collagen, or thrombin using human-derived platelets (PLT: 20 × 10 ⁴ / μL). ) Was evaluated according to the method described in the document “Blood 2003: 102 (4): 1367-1373”.

The evaluation results of the platelet aggregation inhibitory effect on human platelet aggregation are shown in FIG.
As a result, administration of 1.0 μg / mL (1.5 μM) of H ₂ -PP or Co-HP specifically inhibits rhodocytin-induced platelet aggregation, and in particular, almost completely suppresses co-HP administration. I understood.

<Evaluation of platelet aggregation inhibitory effect on mouse platelet aggregation>
Protoporphyrin IX (H ₂ -PP) and hematoporphyrin cobalt complex (Co-HP) when platelet aggregation was induced by rhodocytin, collagen, or thrombin using platelets derived from mice (PLT: 20 × 10 ⁴ / μL). ) Was evaluated according to the method described in the document “Blood 2003: 102 (4): 1367-1373”.

The evaluation results of the platelet aggregation inhibitory effect on mouse platelet aggregation are shown in FIG.
As a result, administration of 1.0 μg / mL (1.5 μM) of H ₂ -PP or Co-HP suppresses rhodocytin-induced platelet aggregation, and in particular, administration of 1.0 μg / mL (1.5 μM) of Co-HP. Was found to most inhibit rhodocytin-induced platelet aggregation.

<Evaluation of metastasis suppression effect using mouse lung metastasis model>
Using the mouse lung metastasis model transplanted with the melanoma cell line B16F10 cells, which is highly metastatic and expresses podoplanin, the metastasis inhibitory effect of hematoporphyrin cobalt complex (Co-HP) was evaluated by the following method. .

B16F10-GFP cells (manufactured by Anti-Cancer Japan Co., Ltd.) 1 × 10 ⁶ on day 0 for C57BL / 6 strain mice (8 weeks old, male, purchased from Japan Charles River Co., Ltd., each group n = 5) Cells / individuals were administered intravenously via the tail vein. Simultaneously on day 0, the compound was administered by administering 200 μL of 1% by weight DMSO, 100 μg / mL Co-HP from the orbit (20 μg per animal, 10 μg / mL as 2 mL of circulating blood volume). Thereafter, the compound was administered on the 2nd day, the 4th day, the 6th day, the 8th day, the 10th day, and the 12th day every 2 days, and the lungs were removed from the mice on the 14th day and the lung weight was measured. did.

A photograph showing the state of the lung on the 14th day from the start of compound administration (DMSO or Co-HP) in a mouse lung metastasis model is shown in FIG. In addition, FIG. 26 shows a graph showing the weight of the lungs on the 14th day from the start of compound administration (DMSO or Co-HP) in the mouse lung metastasis model.
As a result, transplantation of the melanoma cell line B16F10 cells produced a tumor in the lung (see FIG. 25, left panel) and increased lung weight, whereas Co-HP suppressed tumor development (FIG. 25, (See right panel) and also increased lung weight (FIG. 26, p = 0.0144; Dunn multiple comparison test).

<Evaluation of vascular occlusion time extension effect using mouse in vivo iron chloride thrombus formation model>
Using a thrombus formation model mouse in which a thrombus was formed with iron chloride, the effect of prolonging the vascular occlusion time by hematoporphyrin cobalt complex (Co-HP) was evaluated by the following method.

C57BL / 6 strain mice (8 weeks old, male, purchased from Charles River Japan Co., Ltd., each group n = 3) were vaporized 3% sevoflurane (manufactured by Maruishi Pharmaceutical Co., Ltd.) in a plastic box at 0.5 L / min. The mouse was placed in a box to introduce anesthesia. Anesthesia was maintained under this anesthesia condition until the end of the experiment. The compound was administered by administering 200 μL of 1% by mass DMSO or 100 μg / mL Co-HP from the orbit (20 μg per animal, 10 μg / mL as 2 mL of circulating blood volume). Next, the limbs were fixed with vinyl tape to expose the left femoral artery, and then the left femoral artery was detached from other tissues using a tapered tweezers, and infiltration of tissue fluid was prevented with a parafilm interposed therebetween. Using a laser blood flow meter (device name: ALF21RD, manufactured by Advance Co., Ltd.), use the blood flow of 60 mL / min / 100 g or more as a guide to search for the position with the highest blood flow and fix the sensor, and blood flow for 2 to 3 minutes Was measured to measure each stable parameter before injury (stimulation of thrombus formation by iron chloride administration).
Thrombus formation was induced by placing a filter paper immersed in an aqueous solution of 10% by mass iron chloride on the exposed left femoral artery. As a reference for clot formation and blood vessel occlusion, measurement with a laser blood flow meter was continued until a blood flow of 5 mL / min / 100 g or less continued for 1 min or longer. The vascular occlusion time was the time required from the administration of iron chloride to the occlusion start time when blood flow of 5 mL / min / 100 g or less continued for 1 min or more.

FIG. 27 shows the effect of prolonging the vascular occlusion time using the mouse in vivo iron chloride thrombus formation model.
As a result, it was found that the vascular occlusion time was significantly prolonged by Co-HP administration as compared with the control to which DMSO was administered.

<Evaluation of effects on bleeding time in mice in vivo>
As a side effect of general antithrombotic drugs, it has been known that there is an action (bleeding tendency) that makes blood hard to clot, and there is a risk of bleeding. Therefore, the effect of administration of hematoporphyrin cobalt complex (Co-HP) on the time from bleeding to hemostasis (bleeding time) was evaluated by the following method.

C57BL / 6 strain mice (8 weeks old, male, Japan Charles River Co., Ltd., each group n = 8) were vaporized with 3 mass% sevoflurane (manufactured by Maruishi Pharmaceutical Co., Ltd.) in a plastic box at 0.5 L / min. Anesthesia was introduced by placing the mouse in a box. Anesthesia was maintained under this anesthesia condition until the end of the experiment. The compound was administered by administering 200 μL of 1% by mass DMSO or 100 μg / mL Co-HP from the orbit (20 μg per animal, 10 μg / mL as 2 mL of circulating blood volume). Ten minutes later, the mouse tail was cut from the tip with a 2 mm razor, the stump was immersed in 37 ° C. physiological saline, and the bleeding time was recorded.
The reference for hemostasis was that no rebleeding occurred for 30 seconds or more, and the bleeding time was the time required from the tail cut to the start time of hemostasis.

FIG. 28 shows the effect on bleeding time in mice in vivo.
As a result, the bleeding time (mean ± standard deviation: 212.0 ± 86.4 seconds) in the Co-HP administration group was compared to the bleeding time (152.0 ± 52.6 seconds) of the control administered with DMSO. No statistically significant difference was observed, and therefore, it was found useful as an antithrombotic agent with few side effects of bleeding.

<Evaluation of effects on arthritis using K / BxN mouse serum transfer arthritis model>
The present inventors have newly found that CLEC-2-deficient bone marrow chimeric mice have an arthritis inhibitory effect. Therefore, the effect of administration of hematoporphyrin cobalt complex (Co-HP) on rheumatoid arthritis was evaluated by the following method.

100 μL / Body of arthritis-induced K / BxN mouse serum (bred at Yamanashi University and collected serum) was intraperitoneally administered to recipient mice on the day of treatment (Day 0) and the second day after treatment (Day 2). (In Fig. 29-30, black arrowhead). As the recipient mice, wild-type bone marrow chimeric mice (15-week-old bone marrow chimeric C57BL / 6 male mice transplanted into wild-type mice irradiated with wild-type mouse fetal liver, FIG. 29, n = 5; C57BL / 6 strain mice, 8 weeks old, male, purchased from Japan Charles River Co., Ltd.), or CLEC-2 deficient bone marrow chimeric mice (platelet-specific CLEC-2 deficient mice fetal liver transplanted 15 weeks old Bone marrow chimeric C57BL / 6 male mice, FIG. 29, n = 5; wild-type mice were C57BL / 6 strain mice, 8 weeks old, male, purchased from Charles River Japan Co., Ltd.).
Further, in wild-type mice, 200 μg / mL Co-HP was administered at 100 μL / Body via Day 0, 2, 4, and 6 from the orbital venous plexus (FIG. 30, Co-HP administration group n = 5). On the other hand, PBS was similarly administered as a control (FIG. 30, control group n = 6).
The forefoot thickness, the front ankle thickness, the heel thickness, and the rear foot thickness were measured with a digital caliper once a day until the 10th day after treatment (Day 10). In each individual, the total of these measured values was used as an index of the degree of arthritis. The results are shown as mean values ± standard deviation, and a significant difference test was performed by Student's t-test.

FIG. 29 shows the degree of inflammation of rheumatoid arthritis in a K / BxN mouse serum transfer arthritis model using wild type mice (WT) and CLEC-2 deficient bone marrow chimeric mice (CLEC-2 KO) as recipient mice. FIG. 30 shows the effect of Co-HP administration on rheumatoid arthritis in a K / BxN mouse serum transfer arthritis model using wild-type mice as recipient mice.
From the results in FIG. 29, the degree of rheumatoid arthritis inflammation was significantly suppressed in CLEC-2 deficient bone marrow chimeric mice compared to wild type mice. Therefore, it was suggested that CLEC-2 is involved in rheumatoid arthritis.
From the results of FIG. 30, the degree of rheumatoid arthritis inflammation was significantly suppressed by Co-HP administration. Presumably, the combination of podoplanin expressed on the intra-articular synoviocytes and platelet CLEC-2 may activate platelets and promote inflammation.
From these results, it was suggested that CLEC-2 antagonists including Co-HP could be used as anti-arthritic agents such as anti-rheumatic drugs.

<Evaluation of effects on thrombus formation on lymphatic endothelium>
It is known that thrombi are formed not only on the vascular endothelium but also on the lymphatic endothelium. Therefore, the effect of administration of hematoporphyrin cobalt complex (Co-HP) on the thrombus formed on the lymphatic endothelium was evaluated by the following method.

Human lymphatic epithelial cells (LEC) are cultured in a monolayer on a parallel plate flow chamber (ibidi μ-slide VI ^0.1 , ibiTreat, ibidi) and endothelial cells containing 1% by weight bovine serum albumin (BSA) The LEC was blocked for 2 hours using a growth medium (trade name: EGM-2, manufactured by Lonza).
Human whole blood from healthy volunteers using 100 ng / mL Argatroban (trade name: Novastan, manufactured by Mitsubishi Tanabe Pharma Corporation) and 5 U / mL heparin (trade name: heparin Na injection, manufactured by Mochida Pharmaceutical Co., Ltd.) Collected. The blood was pretreated with endothelial cell growth medium containing 5 μM 3-dihexyloxycarbocyanine iodide (DiOC ₆ , Thermo Fisher Scientific) and compound (Co-HP) or control solvent (DMSO) for 10 minutes. went. The obtained blood sample was perfused into the parallel plate flow chamber at a shear rate of 400 s ⁻¹ for 10 minutes. Platelet adhesion was visualized using a fluorescent video microscope, and still images were taken after 1, 3, 5, 7, and 10 minutes from the start of perfusion. The still image after 10 minutes passed, and the platelet adhesion area was quantified using image processing software Image J (URL: https://imagej.nih.gov/ij/).

A photograph visualizing platelet adhesion of human lymphatic epithelial cells (LEC) and a graph quantifying the platelet adhesion area are shown in FIGS. 31 and 32, respectively.
As a result, it was suggested that the administration of hematoporphyrin cobalt complex (Co-HP) significantly suppresses thrombus formation on the lymphatic endothelium.

Lymphedema may occur after lymph node dissection in malignant tumor surgery. There are 50,000 patients with upper limb lymphedema and 70,000 patients with lower limb lymphedema in Japan, and this number is increasing year by year. When lymphedema becomes chronic, it progresses to elephantization and fibrosis, and repeats infection, or QOL becomes extremely low. As one of the treatments for lymphedema, there is a treatment method in which lymphatic vein anastomosis is performed and the lymph fluid in the lymphatic vessels is perfused into the vein. This is an effective treatment, but the long-term patency rate is only 40%. One of the causes that the anastomosis is blocked is the formation of a thrombus at the anastomosis. Presumably, when blood flows back into the lymphatic vessels, platelets CLEC-2 and the podoplanin of the lymphatic endothelium are combined to form a thrombus. If the binding between CLEC-2 and podoplanin can be suppressed, it is considered that occlusion after lymphangiovascular anastomosis can be prevented to some extent.
From the results of this experiment, it became clear that thrombus formation on the lymphatic endothelium was remarkably suppressed by Co-HP. This suggests that a CLEC-2 antagonist such as Co-HP can be used as an antithrombotic drug at sites other than blood vessels such as lymph vessel vascular anastomoses.

Claims (11)

1. A CLEC-2 antagonist comprising a compound represented by any one of the following general formula (1) and the following general formula (2).
   [General formula (1)]
   

   

   (In the general formula (1), M represents any one of H ₂ and Group 2 to 12 elements, R ¹ represents either a vinyl group or a 1-hydroxyethyl group, and X represents Group 1) Represents an element.)
   [General formula (2)]
   

   

   (In the general formula (2), M represents any one of H ₂ and Group 2 to 12 elements, and R ² represents a phenyl group, a sulfophenyl group, a carboxyphenyl group, and 1-methylpyridinium-4-yl.) Represents any of the groups.)
2. 2. The CLEC-2 antagonist according to claim 1, wherein the compound represented by the general formula (1) is a compound represented by any one of the following general formula (1-A) and the following general formula (1-B). Agent.
   [General Formula (1-A)]
   

   

   (In the general formula (1-A), M represents any one of H ₂ , Co, Zn, Ni, and Pd, and X represents a Group 1 element.)
   [General formula (1-B)]
   

   

   (However, in the general formula (1-B), M represents any of H ₂ , Co, Zn and Cu.)
3. The CLEC-2 antagonist according to claim 2, wherein the compound represented by the general formula (1-B) is a compound represented by the following structural formula B4.
   [Structural formula B4]
   

   
4. The CLEC-2 antagonist according to any one of claims 1 to 3, wherein the compound represented by the general formula (2) is a compound represented by the following structural formula C1.
   [Structural formula C1]
   

   
5. A platelet aggregation inhibitor comprising the CLEC-2 antagonist according to any one of claims 1 to 4.
6. An antithrombotic drug comprising the CLEC-2 antagonist according to any one of claims 1 to 4.
7. An antimetastatic drug comprising the CLEC-2 antagonist according to any one of claims 1 to 4.
8. An anti-arthritic drug comprising the CLEC-2 antagonist according to any one of claims 1 to 4.
9. A compound represented by any one of the following general formula (1-A) and the following general formula (1-B).
   [General Formula (1-A)]
   

   

   (In the general formula (1-A), M represents any one of Co, Zn, Ni and Pd, and X represents a Group 1 element.)
   [General formula (1-B)]
   

   

   (In the general formula (1-B), M represents any one of Co, Zn and Cu.)
10. A salt of protoporphyrin and a metal acetate are reacted with either dimethyl sulfoxide or acetic acid at 70 ° C. to 95 ° C. for 1 hour to 30 hours in the presence of water and represented by the following general formula (1-A) The manufacturing method of the compound characterized by including the process of obtaining a compound.
    [General Formula (1-A)]
    

    

    (In the general formula (1-A), M represents any one of Co, Zn, Ni and Pd, and X represents a Group 1 element.)
11. A step of reacting hematoporphyrin and metal acetate in the presence of either methanol or acetic acid at 15 ° C. to 30 ° C. for 5 to 30 hours to obtain a compound represented by the following general formula (1-B) A method for producing a compound characterized by the above.
    [General formula (1-B)]
    

    

    (In the general formula (1-B), M represents any one of Co, Zn and Cu.)

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