JP2012188396A - COMPOUND HAVING INHIBITORY ACTIVITY TO TAFIa PROTEIN - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a compound that bonds to a TAFIa protein, and can inhibit the cleavage of a substrate by the protein.SOLUTION: The compound that has inhibitory activity to the TAFIa protein may be an organoselenium compound that has a basic functional group, an acid functional group, and a selenium atom that can coordinate to a zinc of the TAFIa protein. A dimer in which the organoselenium compounds bond to each other through a diselenide binding, and pharmaceutically acceptable salts or hydrate thereof are also provided.

Description

  The present invention relates to a compound having an inhibitory activity against TAFIa protein.

  The first cause of death in developed countries including Japan is malignant neoplasm (cancer), second is heart disease, and third is cerebrovascular disease. Many of heart diseases and cerebrovascular diseases are thrombotic diseases such as myocardial infarction and cerebral infarction caused by arteriosclerosis, and are caused by fibrin thrombus formed in the artery. Thrombus is composed of fibrin fibers entangled with aggregated platelets, so treatment of such thrombotic diseases (thrombosis) requires activation of a fibrinolytic system that cuts fibrin fibers and separates the aggregated platelets. is there. Therefore, in the current treatment of thrombosis, tissue plasminogen activator (tPA) that activates plasmin, which plays a major role in fibrinolysis, is used.

  TPA and plasminogen have low but affinity for the amino acid, lysine (Lys, side chain Lys), which is incorporated in the molecules of many fibrin fibers. The fibrinolytic system begins with the binding of these molecules. Similarly, tPA bound to the Lys residue activates the plasminogen bound to the Lys residue in the nearby fibrin fiber molecule by limited degradation to produce plasmin, which is a proteolytic enzyme. Plasmin cleaves between Lys and its carboxy-terminal (C-terminal) amino acid, so that the Lys residue is exposed at the C-terminal of the fibrin fiber at the initial stage of the fibrinolytic system. And tPA and plasminogen show higher affinity for the C-terminal Lys residue than the side chain Lys, efficiently produce plasmin via the C-terminal Lys, and accelerate fibrin fiber cleavage. That is, the C-terminal Lys of the fibrin fiber functions as a tPA and plasminogen receptor that accelerates fibrinolysis, and fibrinolysis is completed (see FIG. 1). However, since tPA has a high risk of bleeding after administration and may exhibit particularly lethal bleeding side effects, development of a new thrombolytic drug is desired.

  On the other hand, in recent years, thrombin activated fibrinolysis inhibitor (TAFI) has been discovered as an enzyme precursor in human plasma, and this precursor is activated by thrombin, and activated TAFI (TAFIa) is a line. It has been revealed that the activity of the lytic system is inhibited (Non-patent Document 1). Furthermore, TAFI is a single-chain glycoprotein with a molecular weight of about 58 kDa that is mainly synthesized in the liver and released into plasma (Non-Patent Document 2), procarboxypeptidase B (procarboxypeptidase B), procarboxypeptidase R (procarboxypeptidase). It is also known to be the same enzyme precursor as R) and procarboxypeptidase U.

  Moreover, the following is known about the activation mechanism of TAFI to TAFIa and its influence on the fibrinolytic system. That is, TAFI having a molecular weight of 57 kDa is hydrolyzed by thrombin or plasmin at the position of arginine (Arg) 92, and the activated peptide of 23 kDa is cleaved to become active TAFI (TAFIa) having a molecular weight of 35 kDa (Non-Patent Document). 2, 3). TAFIa has a carboxypeptidase B-like activity, that is, an activity of specifically cleaving Lys or Arg residues at the C-terminal of the peptide. Therefore, as described above, the C-terminal Lys residue that accelerates fibrinolysis is exposed in fibrin fibers that have undergone partial degradation, but since TAFIa can be cleaved using it as a substrate, TAFIa activity is associated with fibrin fibers. The C-terminal Lys residue is cleaved to inhibit the fibrinolytic system (see FIG. 1).

  In clinical studies, in patients with venous thrombosis, a high TAFI antigen level is a risk factor for venous thrombosis (Non-patent Document 4) and in acute ischemic cerebral infarction, plasma TAFI antigen level is high. (Non-patent Document 5) suggests that TAFI is an exacerbation factor of thrombosis.

  Furthermore, in animal experiments conducted by the present inventors, it has been proved that inhibiting TAFIa activity has a very low risk of bleeding compared to tPA, and a sufficient effect of thrombolysis can be obtained. It has been strongly suggested that the inhibitor may be a novel thrombolytic drug with extremely few bleeding side effects (Non-patent Document 6).

  However, at present, a compound having an inhibitory activity against TAFIa protein that can withstand clinical application in terms of specificity of inhibitory activity and pharmacokinetics has not yet been developed.

Eaton DL. Et al. Biol. Chem. 1994, 266, 21833-2121838 Bajzer L. Et al. Biol. Chem. , 1996, 271, 16603-16608 Bajzer L. Et al. Biol. Chem. 1995, 270, 14477-14484. Eichinger S. Blood, 2004, 103, 3773-3776. Leebeeek FW. Et al. Thromb Haemost. 2005, 3 volumes, 2211-2218 Muto Y. Et al., Crit Care Med. May 2009, Vol. 37, No. 5, pp. 1744-1749

  This invention is made | formed in view of the subject which the said prior art has, and it aims at providing the compound which bind | bond | couples to TAFIa protein and can inhibit the cutting | disconnection of the substrate by this protein.

  In order to achieve the above-mentioned object, the present inventors have a structure required when a substrate such as fibrin fiber is cleaved (hydrolyzed) by a TAFIa protein, that is, a zinc atom and a TAFIa protein necessary for the hydrolysis reaction of the substrate. Arginine residue at position 125, aspartic acid residue at position 256 of TAFIa protein for determining substrate specificity, asparagine residue at position 142 and arginine residue at position 143 of TAFIa protein necessary for binding to the substrate (See Buma BN., Thromb Res, 2001, 101, 329-354 for the correlation between the three-dimensional structure of the TAFIa protein and the cleavage of the substrate.)

  As a result, the basic functional group capable of coordinating to the 256th aspartic acid residue of TAFIa protein and the 125th arginine residue, 142th asparagine residue and 143rd arginine residue of TAFIa protein A compound containing an acidic functional group to be obtained, and further selecting a functional group containing selenium as a functional group capable of coordinating with zinc has a very high inhibitory activity against TAFIa protein. The headline and the present invention were completed.

More specifically, the present invention provides the following.
<1> An organic selenium compound having an inhibitory activity against a TAFIa protein, comprising a basic functional group, an acidic functional group, and a selenium atom capable of coordinating with zinc of the TAFIa protein , Dimers in which the organic selenium compounds are bonded to each other via a diselenide bond, and pharmacologically acceptable salts or solvates thereof.
<2> A compound having inhibitory activity against TAFIa protein, which is an organic selenium compound represented by the following general formula (1), a dimer in which the organic selenium compounds are bonded to each other via a diselenide bond, and These pharmacologically acceptable salts or solvates.

(In formula (1), R ¹ represents —R ⁴ —NH ₂ , —R ⁴ —C (═NH) NH ₂ , —R ⁴ —NH—C (═NH) NH ₂ , or the following formula (2) R ² represents a group represented by —COOR ⁷ , and R ³ represents a hydrogen atom, —C (═O) R ⁵ , —C (═O) R ⁶ —Ar, or — A group represented by C (═O) Ar, R ⁴ represents a linear, branched or cyclic alkylene group having 1 to 6 carbon atoms, and R ⁵ is a straight chain having 1 to 6 carbon atoms. R ⁶ represents a linear, branched or cyclic alkylene group having 1 to 6 carbon atoms, R ⁷ represents a hydrogen atom and 1 to 6 carbon atoms. A linear, branched or cyclic alkyl group, or an aryl group, and Ar represents an aryl group.

(In the formula (2), R ⁸ represents a hydrogen atom or a halogen atom, and n represents an integer of 0 to 3.)

  According to the present invention, it is possible to provide a compound that binds to the TAFIa protein and has an inhibitory activity against the cleavage of the substrate by the protein.

It is the schematic which shows the dissolution mechanism (fibrinolysis, fibrinolysis) of the fibrin thrombus by tPA, plasminogen, and plasmin, and the inhibition mechanism with respect to this fibrinolysis by TAFIa protein.

  First, compounds having inhibitory activity against the TAFIa protein of the present invention will be described. The compound having inhibitory activity against the TAFIa protein of the present invention contains a basic functional group, an acidic functional group, and a selenium atom that can coordinate to zinc of the TAFIa protein, the organic selenium compound, It is a dimer in which organic selenium compounds are bonded via a diselenide bond, and a pharmacologically acceptable salt or solvate thereof.

  The TAFIa protein according to the present invention is also referred to as an activated thrombin activated fibrinolytic inhibitor, and the TAFI protein (molecular weight 57 kDa) is hydrolyzed at the arginine residue at position 92 by the hydrolysis of the TAFI protein (molecular weight 57 kDa). It is a protein with a molecular weight of 35 kDa that is produced by cleaving the activated peptide. The TAFIa protein according to the present invention has carboxypeptidase B-like activity (activity that specifically cleaves Lys or Arg residues at the peptide C-terminus).

  As such TAFIa protein, a typical human-derived protein includes a protein consisting of 309 amino acids from alanine at position 115 to valine at position 423 among the proteins specified by GenBank accession number NP_001863.2. Also, the amino acid sequence of a protein can be mutated in nature (ie, non-artificially). Therefore, the TAFIa protein in the present invention includes such natural mutants.

  The basic functional group in the compound having inhibitory activity against the TAFIa protein of the present invention is a group capable of coordinating to the 256th aspartic acid residue of the TAFIa protein, and the acidic functional group is the TAFIa protein. Of the arginine residue at position 125, the asparagine residue at position 142, and the arginine residue at position 143 (the amino acid residue is positioned at the N-terminal amino acid residue of the TAFIa protein as position 1). Is the position of the case).

  As the aspartic acid residue at position 256, the arginine residue at position 125, the asparagine residue at position 142, and the arginine residue at position 143 of the TAFIa protein according to the present invention, typically, GenBank accession number NP_001863.2 is used. When the methionine residue located on the most N-terminal side of the protein specified in (1) is defined as the first, the 370th aspartic acid residue, the 239th arginine residue, the 256th asparagine residue, respectively. Group and the arginine residue located at position 257.

  Zinc of the TAFIa protein according to the present invention is contained in the active center of the protein. A TAFIa protein catalytic group is formed by coordinating the water molecule with a 67-position histidine residue, a 70-position glutamic acid residue and a 196-position histidine residue of the TAFIa protein. The compound of the present invention can inhibit cleavage of the substrate by TAFIa protein by coordinating a selenium atom to the zinc.

  In addition, the compound having inhibitory activity against the TAFIa protein of the present invention includes an organic selenium compound represented by the following general formula (1), a dimer in which the organic selenium compounds are bonded via a diselenide bond, and These are pharmacologically acceptable salts or solvates.

[In the formula (1), R ¹ represents —R ⁴ —NH ₂ , —R ⁴ —C (═NH) NH ₂ , —R ⁴ —NH—C (═NH) NH ₂ , or the following formula (2) R ² represents a group represented by —COOR ⁷ , and R ³ represents a hydrogen atom, —C (═O) R ⁵ , —C (═O) R ⁶ —Ar, or — A group represented by C (═O) Ar, R ⁴ represents a linear, branched or cyclic alkylene group having 1 to 6 carbon atoms, and R ⁵ is a straight chain having 1 to 6 carbon atoms. R ⁶ represents a linear, branched or cyclic alkylene group having 1 to 6 carbon atoms, R ⁷ represents a hydrogen atom and 1 to 6 carbon atoms. Represents a linear, branched or cyclic alkyl group, or an aryl group, and Ar represents an aryl group. ]

[In Formula (2), R ⁸ represents a hydrogen atom or a halogen atom, and n represents an integer of 0 to 3. ].

Such a compound of the present invention is represented by R ^2, which can be coordinated to the arginine residue at position 125, the asparagine residue at position 142, and the arginine residue at position 143 of the TAFIa protein as shown in the Examples below. TAFIa contains an acidic functional group, a basic functional group represented by R ¹ that can coordinate to the aspartic acid residue at position 256 of the TAFIa protein, and a selenium atom that can coordinate to zinc of the TAFIa protein. It can inhibit the cleavage of the substrate by the protein.

R ^{1 in the} formula (1) is —R ⁴ —NH ₂ , —R ⁴ —C (═NH) NH ₂ , —R ⁴ —NH—C (═NH) NH ₂ , or the formula (2) The group represented. R ⁴ in the formula (1) is a linear, branched or cyclic alkylene group having 1 to 6 carbon atoms, R ⁸ in the formula (2) is a hydrogen atom or a halogen atom, n is an integer of 0-3.

Examples of the “linear, branched or cyclic alkylene group having 1 to 6 carbon atoms” in R ⁴ include a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, a pentamethylene group, a hexamethylene group, Examples thereof include an ethylethylene group, a dimethylethylene group, a butylethylene group, a cyclohexylene group, and a cyclopentylene group. From the viewpoint of easy coordination to the aspartic acid residue at position 256 of the TAFIa protein, R ⁴ is preferably a linear alkylene group having 4 carbon atoms.

Examples of the “halogen atom” in R ⁸ of the formula (2) include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. From the viewpoint of easy coordination with the aspartic acid residue at position 256 of the TAFIa protein, in the formula (2), R ⁸ is preferably a hydrogen atom or a chlorine atom, and n is preferably 0. .

R ^{2 in the} formula (1) is a group represented by —COOR ⁷ , and R ⁷ is a hydrogen atom, a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms, or an aryl group. is there.

Examples of the “linear, branched or cyclic alkyl group having 1 to 6 carbon atoms” in R ⁷ include a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, Examples include i-butyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, hexyl group, cyclopropyl group, cyclobutyl group, cyclopentyl group, and cyclohexyl group. Furthermore, examples of the “aryl group” in R ⁷ include a phenyl group and an alkoxyphenyl group (for example, methoxyphenyl group, ethoxyphenyl group, propyloxyphenyl group, i-propyloxyphenyl group, butoxyphenyl group, i-butoxy group). Phenyl group, t-butoxyphenyl group), alkylphenyl group (for example, methylphenyl group, ethylphenyl group, dimethylphenyl group, propylphenyl group, mesityl group, methylethylphenyl group, i-propylphenyl group, butylphenyl group, i-butylphenyl group, t-butylphenyl group), 1-naphthyl group, 2-naphthyl group, 1-anthracenyl group, 2-anthracenyl group, 9-anthracenyl group, and pentafluorophenyl group.

From the viewpoint of easy coordination to the arginine residue at position 125, the asparagine residue at position 142, and the arginine residue at position 143 of the TAFIa protein, R ² is preferably —COOH or —COOC ₂ H ₅ . .

R ^{3 in the} formula (1) is a group represented by a hydrogen atom, —C (═O) R ⁵ , —C (═O) R ⁶ —Ar, or —C (═O) Ar, and R ⁵ Is a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms, R ⁶ is a linear, branched or cyclic alkylene group having 1 to 6 carbon atoms, Ar is An aryl group.

Examples of the “linear, branched or cyclic alkyl group having 1 to 6 carbon atoms” in R ⁵ include, for example, methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, Examples include i-butyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, hexyl group, cyclopropyl group, cyclobutyl group, cyclopentyl group, and cyclohexyl group.

Examples of the “linear, branched or cyclic alkylene group having 1 to 6 carbon atoms” in R ⁶ include, for example, a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, a pentamethylene group, a hexamethylene group, Examples thereof include an ethylethylene group, a dimethylethylene group, a butylethylene group, a cyclohexylene group, and a cyclopentylene group.

Examples of the “aryl group” in R ³ include a phenyl group and an alkoxyphenyl group (for example, methoxyphenyl group, ethoxyphenyl group, propyloxyphenyl group, i-propyloxyphenyl group, butoxyphenyl group, i-butoxyphenyl group). , T-butoxyphenyl group), alkylphenyl group (for example, methylphenyl group, ethylphenyl group, dimethylphenyl group, propylphenyl group, mesityl group, methylethylphenyl group, i-propylphenyl group, butylphenyl group, i- Butylphenyl group, t-butylphenyl group), 1-naphthyl group, 2-naphthyl group, 1-anthracenyl group, 2-anthracenyl group, 9-anthracenyl group, and pentafluorophenyl group.

From the viewpoint of easy coordination to zinc of the TAFIa protein, R ³ is preferably a hydrogen atom, —C (═O) C ₂ H ₅ , —C (═O) C ₂ H ₄ -phenyl group or — _{C (= O) C 3 H} 6 - is a phenyl group.

  The compounds of the present invention also include dimers in which the organic selenium compounds of the present invention are bonded via a diselenide bond, and pharmacologically acceptable salts or solvates thereof.

  Such a pharmacologically acceptable salt is not particularly limited and may be appropriately selected depending on the intended purpose. For example, hydrochloride, sulfate, hydrobromide, nitrate, hydrosulfate, phosphate , Acetate, lactate, succinate, citrate, maleate, hydroxy maleate, tartrate, fumarate, methanesulfonate, p-toluenesulfonate, camphorsulfonate, sulfamic acid Salt, mandelate, propionate, glycolate, stearate, malate, ascorbate, pamonate, phenylacetate, glutamate, benzoate, salicylate, sulfanilate, 2- Acetoxybenzoate, ethanedisulfonate, oxalate, isethionate, formate, trifluoroacetate, ethyl succinate, lactobionate, glucone Salt, glucoheptonate, 2-hydroxyethanesulfonate, benzenesulfonate, paratoluenesulfonate, lauryl sulfate, aspartate, adipate, hydroiodide, nicotinate, oxalate , Picrate, thiocyanate, and undecanoate. Moreover, there is no restriction | limiting in particular as a solvate, According to the objective, it can select suitably, A hydrate shall also be included.

  As the compound of the present invention, from the viewpoint of easy coordination to the TAFIa protein, the aspartic acid residue at position 256, the arginine residue at position 125, the asparagine residue at position 142, the arginine residue at position 143, and the zinc atom. The following compounds are more preferable.

  Furthermore, the compound of the present invention is particularly preferably the compound DD22 and the compound DD28 in the above formula from the viewpoint of higher inhibitory activity against the TAFIa protein, and from the viewpoint of higher specificity for the TAFIa protein. Particularly preferred is the compound DD28 in the above formula.

  The compounds of the present invention include all isomers and isomer mixtures such as geometric isomers, optical isomers based on asymmetric carbon, stereoisomers, and tautomers. In addition, the compounds of the present invention also include compounds that exhibit the desired activity after undergoing metabolism such as oxidation, reduction, hydrolysis, and conjugation in vivo, and the present invention further includes oxidation, reduction, hydrolysis and the like in vivo. Also included are compounds that undergo metabolism to produce the compounds of the present invention.

Moreover, there is no restriction | limiting in particular as a synthesis method of the compound of this invention, For example, the synthesis method as shown in a following formula is mentioned. In the following formula, R ^{1 to} R ³ are as described above.

As shown in the above formula, first, a compound represented by R ³ OH was added to an anhydrous toluene suspension of Woolins reagent (Woolins' reagent, Ph ₂ P ₂ Se ₄ , where “Ph” represents a phenyl group). In addition, by heating and refluxing, a selenol body represented by R ³ SeH is obtained. Next, an alkene compound having a group represented by R ¹ and a group represented by R ² is added to the resulting selenol body to cause a Michael addition reaction, whereby a group represented by R ¹ , R ² And an organic selenium compound having a group represented by R ³ can be obtained.

In such a case, in order to protect the amino group in R ¹ , the alkene compound may be a compound into which a protecting group is introduced. Such protective groups are not particularly limited, and examples thereof include tert-butoxycarbonyl group (Boc), benzyloxycarbonyl group, 9-fluorenylmethyloxycarbonyl group, 2,2,2-trichloroethoxycarbonyl group, allyloxycarbonyl Groups. Further, in the case where a compound into which such a protecting group is introduced is used, an organic selenium compound having a group represented by R ¹ , a group represented by R ² and a group represented by R ³ as required. You may use for the deprotection reaction according to the protecting group which introduce | transduced. As this deprotection reaction, for example, when a tert-butoxycarbonyl group is introduced, a deprotection reaction under strongly acidic conditions such as trifluoroacetic acid and 4N hydrochloric acid can be mentioned.

  As mentioned above, although the suitable synthesis method of the compound of this invention was demonstrated, the synthesis method of the compound of this invention is not limited to the said method. Moreover, since the specific manufacturing method of the compound of this invention is shown by the below-mentioned Example, if it is those skilled in the art, referring the said general description and the specific description of an Example, reaction The compound of the present invention is produced by appropriately modifying or modifying these methods as necessary while appropriately selecting the raw materials, reaction reagents, reaction conditions (for example, solvent, reaction temperature, reaction time) and the like. It is possible.

  In addition, as described above and as shown in the Examples below, the compound of the present invention has a high inhibitory activity against TAFIa protein and can dissolve fibrin thrombus formed in the body. Cerebral infarction, angina pectoris, pulmonary embolism, chronic arterial occlusion, acute arterial thrombosis / embolism, vasculitis syndrome, diabetic gangrene, thrombophlebitis / deep venous thrombosis, thrombotic thrombocytopenic purpura / hemolysis Various thrombotic diseases such as uremic toxin, disseminated intravascular coagulation syndrome, antiphospholipid antibody syndrome, etc., and diabetes, hyperlipidemia, inflammation, etc., in which thrombotic tendency associated with decreased in vivo fibrinolysis is pointed out In order to prevent thrombus formation as a pharmaceutical composition to be administered for the prevention of thrombosis in a device (eg, catheter, stent, heart valve, artificial blood vessel, dialysis device) placed in the body, It can be suitably used as a coating the pharmaceutical composition.

  Such a pharmaceutical composition can be formulated by a known pharmaceutical method. For example, capsule, tablet, pill, liquid, powder, granule, fine granule, film coating, pellet, troche, sublingual, chewing agent, buccal, paste, syrup, suspension, To be used orally or parenterally as elixirs, emulsions, coatings, ointments, plasters, cataplasms, transdermal preparations, lotions, inhalants, aerosols, injections, suppositories, etc. Can do.

  In these preparations, a pharmacologically acceptable carrier, specifically, sterile water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, fragrance Agent, excipient, vehicle, preservative, binder, diluent, tonicity agent, soothing agent, extender, disintegrant, buffer, coating agent, lubricant, colorant, sweetener, viscous It can be appropriately combined with agents, flavoring agents, solubilizing agents or other additives.

  When the compound of the present invention is applied as a pharmaceutical composition, known pharmaceutical compositions (for example, antithrombotic agents (anticoagulants and platelet aggregation inhibitors), thrombolysis) used for the prevention of diseases associated with thrombus formation. You may use together with an agent (for example, tPA), the other substance which has a fibrinolysis acceleration | stimulation activity, an antihypertensive agent, a blood glucose regulator, a lipid lowering agent, and an antiarrhythmic agent).

  Such pharmaceutical composition can be used for animals including humans, but there is no particular limitation for animals other than humans, and it should be used for various livestock, poultry, pets, laboratory animals, etc. Can do. Specific examples include, but are not limited to, pigs, cows, horses, sheep, goats, chickens, ducks, ostriches, ducks, dogs, cats, rabbits, hamsters, mice, rats, and monkeys.

  When such a pharmaceutical composition is administered, the dosage is appropriately selected according to the age, weight, symptom, health condition, type of composition, etc. of the subject. For example, the dose of the composition of the present invention per dose is 1 mg / kg body weight to 10 mg / kg body weight.

  A product (eg, pharmaceutical, reagent) containing the compound of the present invention or instructions thereof may be labeled with an indication that it is used to dissolve thrombus. Here, “labeled product or instructions” means that the product body, container, packaging, etc. are marked, or instructions, package inserts, promotional materials, or other printed materials that disclose product information. It means that the display is attached to. The indication that it is used to dissolve the thrombus may include information about the mechanism by which the thrombus is dissolved by administering the compound of the present invention. Examples of the mechanism include information on promoting the fibrinolytic system by cleaving the C-terminal Lys residue of the fibrin fiber to inhibit the function of the TAFIa protein that inhibits the fibrinolytic system. In addition, the display indicating that it is used for dissolving a thrombus may include information on the use for the prevention or treatment of a thrombotic disease.

EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not limited to a following example. In addition, the compounds obtained in the following Examples and Preparation Examples are nuclear magnetic resonance spectroscopy (NMR: ¹ H NMR, ¹³ C NMR), electrospray ionization mass spectrometry (ESI-MS), high resolution mass spectrometry. (HRMS). The multiplicity in ¹ H-NMR is as follows: s = singlet (single line), d = doublelet (double line), t = triplet (triple line), q = quintet (quadruple line), m = multiplet (multiplexing) Line), dd = double doublet, and bs = broad singlet. J represents a coupling constant. Furthermore, the parentheses in NMR () indicate the solvent used for the analysis.

  (Preparation Example 1)

Selenol ester 1 was synthesized as described in the scheme shown in the above formula. Specifically, first, propionic acid (190 μL, 2.55 mmol, 3 eq) was added to an anhydrous toluene (toluene, 1 mL) suspension of Woolins' reagent (225.8 mg, 0.42 mmol, 2 eq), and the mixture was heated to reflux for 2 hours. Selenopropionic acid was prepared. Subsequently, the reaction solution was cooled to 0 ° C., a solution of ethyl ester 1 (240.6 mg, 0.84 mmol) in toluene (500 + 250 + 250 μL) was added, and the mixture was stirred at 70 ° C. for 41 hours to cause a Michael addition reaction. Subsequently, the reaction liquid was returned to room temperature and the solvent was distilled off. The obtained residue was purified by silica gel column chromatography, and selenol ester 1 (114.5 mg, 32.1%) was obtained from the eluate of 15% ethyl acetate (AcOEt) / hexane (Hexane). ¹ H NMR, ¹³ C NMR, ESI-MS, and HRMS of selenol ester 1 obtained are shown below.

_{^{1 H NMR (CDCl 3) δ1.17}} (3H, t, J = 7.5Hz, SeCOCH 2 C H 3), 1.26 (3H, t, J = 7.1Hz, COOCH 2 C H 3), 1 _{.3~1.8 (8H, m, C H} 2 x4), 1.44 (9H, s, Boc), 2.63 (3H, m, C H, SeCOC H 2 CH 3), 3.08 ( _{4H, m, C H 2 NHBoc} , C H 2 Se), 4.14 (2H, q, J = 7.1Hz, COOC H 2 CH 3), 4.53 (1H, m, N H).
¹³ C NMR (CDCl ₃ ) δ9.7, 14.5, 26.3, 26.8, 26.9, 28.6 ( ₃ carbons), 30.1, 33.0, 40.7, 41.7, 46.5, 60.8, 79.3, 156.2, 174.9, 202.6.
ESI-MS m / z (%): 448 (M + Na, 20), 447 (M + Na, 20), 446 (M + Na, 100), 445 (M + Na, 10), 444 (M + Na, 50), 443 (M + Na, 18) ), 442 (M + Na, 18), 424 (25), 423 (20), 422 (75), 421 (13), 420 (38), 419 (10), 418 (8), 392 (8), 391 (6), 390 (40), 389 (3), 388 (6), 387 (6), 386 (6), 326 (13), 325 (9), 324 (63), 323 (5), 322 (31), 321 (11), 320 (11).
_{HRMS (ESI) calcd for C 18} H 33 NNaO 5 80 Se 446.14216, found 446.14188.

Example 1
Next, as described in the scheme shown in the above formula, the selenol ester 1 obtained in Preparation Example 1 was deprotected to obtain Compound A. That is, concentrated hydrochloric acid (HCl, 1 mL) was added to selenol ester 1 (25.9 mg, 0.061 mmol), and the mixture was stirred at 100 ° C. for 2 hours. Subsequently, the solvent of the reaction liquid was distilled off to obtain Compound A (16.5 mg, 98.3%). ¹ H NMR, ESI-MS, and HRMS of Compound A (Compound DD9) obtained below are shown.

¹ H NMR (D ₂ O) δ 1.40 (8H, m, C H ₂ x4), 1.67 (8H, m, C H ₂ x4), 2.84 (2H, m, C H x2), 2 .99 (4H, t, J = 7.5 Hz, C H ₂ NH ₂ x2), 3.13 (4H, dd, J = 7.1, 2.4 Hz, C H ₂ Sex2).
ESI-MS m / z (%): 481 (M + H, 25), 480 (M + H, 8), 479 (M + H, 35), 478 (22), 477 (M + H, 100), 476 (M + H, 22), 475 (M + H, 92), 474 (M + H, 35), 473 (M + H, 55), 472 (M + H, 15), 471 (M + H, 22), 470 (M + H, 12), 469 (M + H, 3).
HRMS (ESI) calcd for C ₁₆ H ₃₃ N ₂ O ₄ ⁸⁰ Se ₂ 477.077780, found 477.07708.

(Example 2)
Next, as described in the scheme shown in the above formula, the selenol ester 1 obtained in Preparation Example 1 was deprotected to obtain amino form A. Specifically, dist H ₂ O (50 μL) and trifluoroacetic acid (TFA, 950 μL) were added to selenol ester 1 (19.2 mg, 0.045 mmol) cooled to 0 ° C. and stirred for 1 hour. Subsequently, the solvent of the reaction solution was distilled off to obtain amino form A (19.9 mg) quantitatively. ¹ H NMR, ESI-MS, and HRMS of the amino compound A (compound DD10) obtained are shown below.

_{^{1 H NMR (CD 3 OD)}} δ1.13 (3H, t, J = 7.5Hz, SeCOCH 2 C H 3), 1.24 (3H, t, J = 7.1Hz, COOCH 2 C H 3), _{1.40 (4H, m, C H} 2 x2), 1.64 (4H, m, C H 2 x2), 2.61 (3H, m, C H, SeCOC H 2 CH 3), 2.91 ( 2H, t, J = 7.5 Hz, C H ₂ NH ₂ ), 3.06 (2H, m, C H ₂ Se), 4.13 (2H, q, J = 7.1 Hz, COOC H ₂ CH ₃ ).
ESI-MS m / z (%): 327 (M + H, 2), 326 (M + H, 21), 325 (M + H, 17), 324 (M + H, 100), 323 (M + H, 8), 322 (M + H, 53) ), 321 (M + H, 19), 320 (M + H, 19), 280 (3), 379 (2), 278 (17), 277 1), 276 (8), 275 (3), 274 (3), 270 (6), 269 (4), 268 (32), 267 (2), 266 (16), 265 (6), 264 (6), 224 (14), 223 (14), 222 (50), 221 (3), 220 (37), 219 (13), 218 (14), 140 (27).
_{HRMS (ESI) calcd for C 13} H 26 NO 3 80 Se 324.10779, found 324.10839.

  (Example 3)

Selenol ester 2 was synthesized as described in the scheme shown in the above formula. That is, first, propionic acid (310 μL, 4.16 mmol, 3 eq) was added to a suspension of Woolins' reagent (371.1 mg, 0.70 mmol, 2 eq) in anhydrous toluene (1 mL), and the mixture was heated to reflux for 2 hours, and selenopropion was added. The acid was prepared. Subsequently, the reaction solution was cooled to 0 ° C., a solution of exomethylene 1 (346.3 mg, 1.35 mmol) in toluene (500 + 500 + 250 μL) was added, and the mixture was stirred at 70 ° C. for 18 hours to cause a Michael addition reaction. Subsequently, the reaction liquid was returned to room temperature and the solvent was distilled off. The obtained residue was purified by silica gel column chromatography, and selenol ester 2 (235.3 mg, 44.3%) was obtained from the eluate of 30% AcOEt / Hexane. ¹ H NMR, ¹³ C NMR, ESI-MS, and HRMS of selenol ester 2 obtained below are shown.

_{^{1 H NMR (CDCl 3) δ1.18}} (3H, t, J = 7.5Hz, SeCOCH 2 C H 3), 1.3~1.8 (8H, m, C H 2 x4), 1.44 ( 9H, s, Boc), 2.64 (3H, m, C H , SeCOC H ₂ CH ₃ ), 3.09 (4H, m, C H ₂ NHBoc, C H ₂ Se), 4.60 (1H, m, NH ).
¹³ C NMR (CDCl ₃ ) δ9.7, 25.9, 26.7, 26.8, 28.6 (3 cabons), 30.0, 32.8, 40.6, 41.7, 46.3 79.4, 156.3, 179.6, 202.6.
ESI-MS m / z (%): 421 (M + Na, 1), 420 (M + Na, 20), 419 (M + Na, 18), 418 (M + Na, 100), 417 (M + Na, 9), 416 (M + Na, 50) ), 415 (M + Na, 18), 414 (M + Na, 17), 364 (7), 363 (5), 362 (40), 361 (3), 360 (19), 359 (6), 368 (7) , 298 (6), 297 (3), 296 (33), 295 (2), 294 (15), 293 (5), 292 (40), 224 (7), 223 (3), 222 (44) , 221 (3), 220 (30), 219 (7), 218 (11), 217 (2), 216 (3).
HRMS (ESI) calcd for C ₁₆ H ₂₉ NNaO ₅ ⁸⁰ Se 418.11086, found 418.11011.

Next, as described in the scheme shown in the above formula, the obtained selenol ester 2 was deprotected to obtain amino form B. Specifically, dist H ₂ O (50 μL) and trifluoroacetic acid (950 μL) were added to selenol ester 2 (24.3 mg, 0.062 mmol) cooled to 0 ° C. and stirred for 1 hour. The solvent of the reaction solution was distilled off to obtain amino form B (25.3 mg) quantitatively. ¹ H NMR, ¹³ C NMR, ESI-MS, and HRMS of the amino compound B (Compound DD12) obtained below are shown.

_{^{1 H NMR (CD 3 OD)}} δ1.14 (3H, t, J = 7.5Hz, SeCOCH 2 C H 3), 1.42 (4H, m, C H 2 x2), 1.66 (4H, m _{, C H 2 x2), 2.58} (1H, m, C H), 2.65 (2H, q, J = 7.5Hz, SeCOC H 2 CH 3), 2.92 (2H, t, J = 7.5 Hz, C H ₂ NH ₂ ), 3.06 (2H, m, C H ₂ Se).
¹³ C NMR (CD ₃ OD) δ 9.8, 26.6, 27.1, 27.6, 28.3, 33.4, 40.6, 42.2, 47.4, 178.2, 203. 7).
ESI-MS m / z (%): 299 (M + H, 1), 298 (M + H, 17), 297 (M + H, 12), 296 (M + H, 88), 295 (M + H, 6), 294 (M + H, 44) ), 293 (M + H, 16), 292 (M + H, 17), 243 (1), 242 (8), 241 (4), 240 (40), 239 (2), 238 (21), 237 (7) , 236 (8), 225 (4), 224 (34), 223 (18), 222 (100), 221 (9), 220 (95), 219 (32), 218 (36), 217 (1) , 216 (4).
_{HRMS (ESI) calcd for C 11} H 22 NO 3 80 Se 296.07649, found 296.07723.

  Example 4

Selenol ester 3 was synthesized as described in the scheme shown in the above formula. That is, first, 4-phenylbutyric acid (349.3 mg, 2.13 mmol, 3.5 eq) was added to an anhydrous toluene (1 mL) suspension of Woolins' reagent (240.6 mg, 0.45 mmol, 3 eq) for 2 hours. Heated to reflux. The reaction solution was returned to room temperature, a solution of exomethylene 1 (153.6 mg, 0.60 mmol) in anhydrous toluene (500 + 250 + 250 μL) was added, and the mixture was stirred at 70 ° C. for 18 hours to cause a Michael addition reaction. Subsequently, after returning a reaction liquid to room temperature, it refine | purified by silica gel column chromatography, and selenol ester 3 (135.7 mg, 46.9%) was obtained from the 20% AcOEt / Hexane elution part. ¹ H NMR of selenol ester 3 obtained is shown below.

¹ H NMR (CD ₃ OD) δ 1.2-1.8 (8H, m, C H ₂ x4), 1.42 (9H, s, Boc), 1.95 (2H, m, C H ₂ CH ₂ Ph), 2.56 (1H, m, C H ), 2.63 (4H, m, C H ₂ Ph, C H ₂ CO), 3.03 (4H, m, C H ₂ NH, C H ₂ Se), 7.16 (3H, m, Ph- H ), 7.26 (2H, m, Ph- H ).

Next, as described in the scheme shown in the above formula, the obtained selenol ester 3 was deprotected to obtain amino form C. Specifically, dist H ₂ O (50 μL) and trifluoroacetic acid (950 μL) were added to selenol ester 3 (17.2 mg, 0.036 mmol) cooled to 0 ° C. and stirred for 1 hour. The solvent of the reaction solution was distilled off to obtain amino form C (19.3 mg) quantitatively. ¹ H NMR and ¹³ C NMR for the amino form C (compound DD19) obtained are shown below.

¹ H NMR (CD ₃ OD) δ1.42 (4H, m, C H ₂ x2), 1.66 (4H, m, C H ₂ x2), 1.94 (2H, m, C H ₂ CH ₂ Ph ), 2.58 (1H, m, C H ), 2.64 (4H, m, C H ₂ Ph, C H ₂ CO), 2.91 (2H, m, C H ₂ NH ₂ ), 3. _{07 (2H, m, C H} 2 Se), 7.16 (3H, m, Ph- H), 7.26 (2H, m, Ph- H).
¹³ C NMR (CD ₃ OD) δ 26.8, 27.2, 28.3, 28.4, 33.4, 36.7, 40.6, 47.4, 48.0, 127.1, 129. 45 (2 carbons), 129.49 (2 carbons), 142.5, 178.1, 202.8.

  (Example 5)

Selenol ester 4 was synthesized as described in the scheme shown in the above formula. That is, first, 3-phenylpropionic acid (327.3 mg, 2.18 mmol, 3.5 eq) was added to an anhydrous toluene (1 mL) suspension of Woolins' reagent (247.4 mg, 0.47 mmol, 3 eq). Heated to reflux for hours. The reaction solution was returned to room temperature, a solution of exomethylene 1 (158.1 mg, 0.61 mmol) in anhydrous toluene (500 + 250 + 250 μL) was added, and the mixture was stirred at 70 ° C. for 16 hours to cause Michael addition reaction. Subsequently, after returning a reaction liquid to room temperature, it refine | purified with the silica gel column chromatography, and selenol ester 4 (108.4 mg, 37.5%) was obtained from the 25% AcOEt / Hexane elution part. ¹ H NMR of selenol ester 4 obtained below is shown.

¹ H NMR (CD ₃ OD) δ 1.2 to 1.8 (8H, m, C H ₂ x4), 1.43 (9 H, s, Boc), 2.56 (1 H, m, C H ), 2 _{.94 (4H, m, C H} 2 Ph, C H 2 CO), 3.03 (4H, m, C H 2 NH, C H 2 Se), 7.17 (3H, m, Ph- H), 7.25 (2H, m, Ph- H ).

Next, as described in the scheme shown in the above formula, the obtained selenol ester 4 was deprotected to obtain amino form D. Specifically, dist H ₂ O (50 μL) and trifluoroacetic acid (950 μL) were added to selenol ester 4 (27.1 mg, 0.058 mmol) cooled to 0 ° C. and stirred for 1 hour. The solvent of the reaction solution was distilled off to obtain amino form D (29.4 mg) quantitatively. ¹ H NMR and ¹³ C NMR for the amino form D (compound DD20) obtained are shown below.

¹ H NMR (CD ₃ OD) δ 1.39 (4H, m, C H ₂ x2), 1.60 (4H, m, C H ₂ x2), 2.59 (1H, m, C H ), 2. 92 (2H, m, C H ₂ NH ₂ ), 2.95 (4H, m, C H ₂ Ph, C H ₂ CO), 3.06 (2H, m, C H ₂ Se), 7.17 ( 3H, m, Ph-H), 7.26 (2H, m, Ph-H).
¹³ C NMR (CD ₃ OD) δ 26.8, 27.1, 27.6, 28.3, 32.3, 33.4, 40.6, 47.3, 50.0, 50.3, 127. 4, 129.4 (2 carbons), 129.5 (2 carbons), 141.2, 178.1, 202.1.

  (Preparation Example 2)

Selenol ester 5 was synthesized as described in the scheme shown in the above formula. Specifically, first, propionic acid (130 μL, 1.74 mmol, 3.5 eq) was added to an anhydrous toluene (1 mL) suspension of Woolins' reagent (198.0 mg, 0.37 mmol, 3 eq), and the mixture was heated to reflux for 2 hours. The reaction solution was returned to room temperature, a solution of exomethylene 2 (150.7 mg, 0.49 mmol) in anhydrous toluene (1000 + 250 + 250 μL) was added, and the mixture was stirred at 70 ° C. for 16 hours to cause a Michael addition reaction. Next, after returning the reaction solution to room temperature, it was purified by silica gel column chromatography (25 g), and eluted from 2.5% ammonia-methanol (NH ₃ : MeOH = 1: 9) / dichloromethane (CH ₂ Cl ₂ ). Selenol ester 5 (192.6 mg, 88.3%) was obtained. ¹ H NMR, ¹³ C NMR, ESI-MS, and HRMS of selenol ester 5 obtained are shown below.

¹ H NMR (CDCl ₃ ) δ 1.16 (3H, t, J = 7.1 Hz, COOC H ₂ CH ₃ ), 1.17 (3 H, t, J = 7.5 Hz, COC H ₂ CH ₃ ), 1 .53 (9H, s, Boc) , 2.63 (2H, q, J = 7.5Hz, COCH 2 C H 3), 2.80 (1H, m, C H), 2.91 (2H, m _{, C H 2 CH), 3.07} (2H, d, J = 6.2Hz, C H 2 Se), 4.07 (2H, q, J = 7.1Hz, COOCH 2 C H 3), 7. 48 (1H, dd, J = 8.6, 2.3 Hz, Py-4), 7.88 (1H, d, J = 8.6 Hz, Py-5), 8.02 (1H, bs, NH) ), 8.08 (1H, d, J = 2.3 Hz, Py-2).
¹³ C NMR (CDCl ₃ ) δ 9.7, 14.4, 26.0, 28.6 ( ₃ carbons), 35.5, 41.7, 48.0, 61.1, 81.1, 112.2, 128.4, 138.9, 148.2, 151.1, 152.7, 173.7, 202.0.
ESI-MS m / z (%): 448 (M + H, 8), 447 (M + H, 22), 446 (M + H, 23), 445 (M + H, 100), 444 (M + H, 13), 443 (M + H, 53) ), 442 (M + H, 20), 441 (M + H, 19), 392 (3), 391 (15), 390 (13), 389 (73), 388 (7), 387 (35), 386 (15) , 385 (15).
_{HRMS (ESI) calcd for C 19} H 29 N 2 O 5 80 Se 1 445.12417, found 445.12508.

  (Example 6)

Next, as described in the scheme shown in the above formula, the selenol ester 5 obtained in Preparation Example 2 was deprotected to obtain Compound B. That is, concentrated hydrochloric acid (1 mL) was added to selenol ester 5 (24.3 mg, 0.055 mmol), and the mixture was stirred at 100 ° C. for 1 hour. After returning the reaction solution to room temperature, the solvent was distilled off, and dist H ₂ O and ether were added. Then, after removing the ether layer, the solvent was distilled off to obtain Compound B (14.9 mg, 92.0%). Compound B was purified with a cation exchange column OASIS MCX cartridge, and the solvent at the elution portion of 1-2% NH ₃ / MeOH was distilled off. Subsequently, the product was dissolved in 4M HCl, and the solvent was distilled off to obtain Compound B (10.8 mg, 66.7%, approximately 1: 1 mixture). ¹ H NMR, ESI-MS, and HRMS of Compound B (Compound DD22) obtained below are shown.

¹ H NMR (CD ₃ OD) δ 2.90 (4H, d, J = 6.7 Hz, C H ₂ CHx2), 3.13 (6H, m, C H x2, C H ₂ Sex2), 7.01 ( 2H, d, J = 9.1 Hz, Py-4x2), 7.71 (2H, s, Py-5x2), 7.87 (2H, d, J = 9.1 Hz, Py-2x2).
¹³ C NMR (CD ₃ OD) δ 31.5 and 31.6 (1: 1, 2 carbons), 34.5 and 34.6 (1: 1, 2 carbons), 48.8 and 48.9 (1: 1, 2 carbons), 114 .9 (2 carbons), 125.0 (2 carbons), 135.1 (2 carbons), 147.0 (2 carbons), 154.7 (2 carbons), 176.2 (2 carbons).
ESI-MS m / z (%): 523 (M + H, 3), 522 (M + H, 5), 521 (M + H, 19), 520 (M + H, 13), 519 (M + H, 57), 518 (M + H, 15 ), 517 (M + H, 52), 516 (M + H, 20), 515 (M + H, 30), 514 (M + H, 8), 513 (M + H, 10), 512 (M + H, 3), 511 (M + H, 2) , 276 (9), 275 (30), 274 (80), 273 (78), 272 (43), 271 (18), 270 (5), 269 (5), 268 (5), 267 (8) , 266 (6), 265 (5), 262 (8), 261 (5), 260 (100), 259 (75), 258 (54), 257 (20), 256 (8), 208 (13) , 207 (35), 180 (30), 1 79 (75).
_{HRMS (ESI) calcd for C 18} H 23 N 4 O 4 80 Se 2 519.00497, found 519.00600.

  (Example 7)

Next, as described in the scheme shown in the above formula, the selenol ester 5 obtained in Preparation Example 2 was deprotected to obtain Compound C. That is, dist H ₂ O (50 μL) and trifluoroacetic acid (950 μL) were added to selenol ester 5 (21.6 mg, 0.049 mmol) cooled to 0 ° C. and stirred for 1 hour. Subsequently, the solvent of the reaction solution was distilled off, dist H ₂ O and hexane were added, the hexane layer was removed, and then the solvent was distilled off to obtain Compound C (12.5 mg, 56.1%). ¹ H NMR, ¹³ C NMR, ESI-MS, and HRMS of Compound C (Compound DD23) obtained below are shown.

¹ H NMR (CD ₃ OD) δ 1.14 (3H, t, J = 7.4 Hz, COC H ₂ CH ₃ ), 1.19 (3 H, t, J = 7.1 Hz, COOC H ₂ CH ₃ ), _{2.66 (2H, q, J =} 7.4Hz, COCH 2 C H 3), 2.79 (1H, m, C H), 2.85 (1H, m, C H 2 CH), 2.97 (1H, m, C H ₂ CH), 3.10 (2H, m, C H ₂ Se), 4.09 (2H, q, J = 7.1 Hz, COOCH ₂ C H ₃ ), 6.98 ( 1H, d, J = 9.1 Hz, Py-5), 7.67 (1H, d, J = 2.1 Hz, Py-2), 7.84 (1H, dd, J = 9.1, 2. 1 Hz, Py-4).
¹³ C NMR (CD ₃ OD) δ9.7, 14.5, 26.1, 34.7, 42.2, 48.2, 62.1, 114.8, 124.8, 135.3, 147. 0, 154.9, 175.6, 203.1.
ESI-MS m / z (%): 349 (M + H, 3), 348 (M + H, 10), 347 (M + H, 60), 346 (M + H, 53), 345 (M + H, 100), 344 (M + H, 28 ), 343 (M + H, 95), 342 (M + H, 57), 341 (M + H, 59), 340 (M + H, 1), 339 (M + H, 6).
_{HRMS (ESI) calcd for C 14} H 21 N 2 O 3 80 Se 345.07174, found 345.08189.

  (Example 8)

Amino E was synthesized as described in the scheme shown in the above formula. Specifically, first, propionic acid (130 μL, 1.74 mmol, 6.5 eq) was added to an anhydrous toluene (1 mL) suspension of Woolins' reagent (208.2 mg, 0.39 mmol, 6 eq), and the mixture was heated to reflux for 2 hours. The reaction solution was returned to room temperature, a solution of exomethylene 3 (88.4 mg, 0.26 mmol) in anhydrous toluene (1000 + 250 + 250 μL) was added, and the mixture was stirred at 70 ° C. for 20 hours to cause Michael addition reaction. Next, after returning the reaction solution to room temperature, it was purified by silica gel column chromatography (15 g, 40 g, 30 g), and amino form E (84.9 mg, 86.5%) was obtained from the eluate of 1% AcOEt / Hexane. . ¹ H NMR, ¹³ C NMR, ESI-MS, and HRMS of the amino compound E (Compound DD29) obtained below are shown.

_{^{1 H NMR (CDCl 3) δ1.18}} (3H, t, J = 7.5Hz, COC H 2 CH 3), 1.19 (3H, t, J = 7.1Hz, COOC H 2 CH 3), 2 _{.65 (2H, q, J =} 7.5Hz, COCH 2 C H 3), 2.78 (1H, m, C H), 2.86 (2H, m, C H 2), 3.07 (2H , D, J = 6.1 Hz, C H ₂ Se), 4.09 (2H, q, J = 7.1 Hz, COOCH ₂ C H ₃ ), 4.78 (2H, bs, N H ), 7. 35 (1H, d, J = 2.0 Hz, Py-4), 7.79 (1H, d, J = 2.0 Hz, Py-2).
¹³ C NMR (CDCl ₃ ) δ9.7, 14.4, 25.9, 34.9, 41.7, 48.1, 61.1, 115.0, 125.1, 137.7, 146.6 , 153.7, 173.7, 202.0.
ESI-MS m / z (%): 383 (M + H, 5), 382 (M + H, 6), 381 (M + H, 45), 380 (M + H, 18), 379 (M + H, 100), 378 (M + H, 13 ), 377 (M + H, 50), 376 (M + H, 15), 375 (M + H, 17).
_{HRMS (ESI) calcd for C 14} H 20 ClN 2 O 3 80 Se 379.03277, found 379.03314.

  Example 9

Compound D was synthesized as described in the scheme shown in the above formula. That is, concentrated hydrochloric acid (1 mL) was added to amino compound E (32.6 mg, 0.086 mmol), and the mixture was stirred at 100 ° C. for 1 hour. After returning the reaction solution to room temperature, the solvent was distilled off. The obtained residue was purified with a cation exchange column SHISEIDO PCX cartridge, the solvent at the elution part of 5% NH ₃ / MeOH was distilled off and then dissolved in 4M HCl. After the solvent was distilled off, compound D (30.0 mg , 1: 1 mixture) was obtained quantitatively. ¹ H NMR, ¹³ C NMR, ESI-MS, and HRMS of Compound D (Compound DD28) obtained below are shown.

¹ H NMR (CD ₃ OD) δ 2.94 (4H, d, J = 7.0 Hz, C H ₂ CHx2), 3.07 (2H, m, C H x2), 3.20 (4H, m, C _{H 2 Sex2), 7.81 (2H} , s, Py-4x2), 8.14 (2H, d, J = 1.7Hz, Py-2x2).
¹³ C NMR (CD ₃ OD) δ 31.6, 31.7 (1: 1, 2 carbons), 34.37, 34.41 (1: 1, 2 carbons), 48.71, 48.74 (1: 1, 2 carbons), 119.8 (2 carbons), 125.8 (2 carbons), 134.6 (2 carbons), 145.9 (2 carbons), 152.0 (2 carbons), 176.06, 176.08 (1: 1, 2 carbons).
ESI-MS m / z (%): 591 (M + H, 10), 590 (M + H, 5), 589 (M + H, 30), 588 (M + H, 12), 587 (M + H, 48), 586 (M + H, 13) ), 585 (M + H, 37), 584 (M + H, 13), 583 (M + H, 20), 582 (M + H, 5), 581 (M + H, 8), 580 (M + H, 1), 579 (M + H, 1) .
_{HRMS (ESI) calcd for C 18} H 20 Cl 2 N 4 O 4 78 Se 80 Se 583.91999, found 583.92394.

<Evaluation of inhibitory activity against TAFIa>
About each compound obtained in Examples 1-9, the measurement of TAFIa inhibitory effect was measured by Suzuki K. et al. Et al, J Pharmacol Exp Ther. , 2004, 309, 607-615.

(A) Purification of TAFI from fresh human plasma First, 80 mM barium chloride was added to citric acid-collected human plasma and treated at 4 ° C. for 30 minutes. The supernatant after centrifugation at 10,000 rpm for 20 minutes was dialyzed against 20 mM HEPES buffer (pH 7.4) containing 0.15 M sodium chloride. Furthermore, the protein of 35-70% ammonium sulfate fraction was purified by chromatography using various columns of Q-Sepharose, Heparin-Sepharose, Sephacryl S-300 and Plasminogen-Sepharose.

(B) Activation of TAFI Next, 20 μL of TAFI was added to a thrombomodulin solution (thrombomodulin (rabbit lung derived, American Diagnostica).
Inc., product number # 237) in buffer A (50% Tris-HCl buffer (pH 7.4) containing 0.1% Lubrol, 0.1% BSA, 0.15 M sodium chloride) at 300 ng / After adding 20 μL of a solution obtained by dissolving at a concentration of mL) and treating at 25 ° C. for 3 minutes, a thrombin solution (thrombin (derived from human plasma, manufactured by Sigma,
20 μL of buffer No. T8885) was added to buffer B (0.1% BSA, 0.15 M NaCl, 50 mM Tris-HCl buffer (pH 7.4) containing 10 mM calcium chloride) at a concentration of 3 u / mL) Further, by treating at 25 ° C. for 20 minutes, TAFI was activated to obtain a TAFIa solution.

(C) Method for measuring TAFIa inhibitory activity Next, various inhibitors (in Examples 1 to 9) serially diluted in 25 μL of the obtained TAFIa solution with 50 mM Tris-HCl buffer (pH 7.6) containing 27 μM dithiothreitol. 30 μL of each compound obtained in this manner was adjusted as a 10-fold dilution series so that the final concentration thereof was 1 pM to 1 mM, and mixed well, followed by treatment at 25 ° C. for 10 minutes. Further, 25 μL of a substrate solution (Hip-Arg (manufactured by Sigma, H2508) dissolved in 50 mM Tris-HCl buffer (pH 7.4) to a concentration of 3.2 mM) was added and mixed well at 25 ° C. The reaction was allowed for 30 minutes. Next, 100 μL of 0.2M PIPES buffer (pH 7.5) containing 12.5% Tween was added to stop the reaction. Then, after 5 minutes, 100 μL of a color developing solution (2-methoxyethanol containing 1% cyanuric chloride) was added and left at room temperature for 15 minutes, and the absorbance at 405 nm was measured with a microplate reader (BioRad, Model 3550). did. From these measurement results, the concentration at which the inhibition rate was 50% was determined as the IC ₅₀ value. The obtained results are shown in Table 1.

<Evaluation of inhibitory activity against carboxypeptidase B (CPB)>
About each compound obtained in Examples 1-9, the measurement of CPB inhibitory action was performed as follows.

(D) CPB inhibitory activity assay method Porcine pancreatic carboxypeptidase B (CPB) (manufactured by Worthington Biochemical Corporation) dissolved in 50 mM Tris-HCl buffer (pH 7.6) at 32 U / L in 25 μL, 27 μM dithiothrei Various inhibitors serially diluted with a 50 mM Tris-HCl buffer solution (pH 7.6) containing Toll (each compound obtained in Examples 1 to 9 was 10 times so that the final concentration thereof was 1 pM to 1 mM. 30 μL of the solution prepared as a dilution series) was mixed well and treated at 25 ° C. for 10 minutes. Furthermore, 25 μL of a substrate solution (Hip-Arg (manufactured by Sigma, H2508) dissolved in 50 mM Tris-HCl buffer (pH 7.4) to a concentration of 3.2 mM) was added and mixed well, and 25 ° C. , Reacted for 30 minutes. Next, 100 μL of 0.2 M PIPES buffer (pH 7.5) containing 12.5% Tween was added to stop the reaction. After 5 minutes, 100 μl of a color developing solution (2-methoxyethanol containing 1% cyanuric chloride) was added and left at room temperature for 15 minutes, and then the absorbance at 405 nm was measured with a microplate reader (BioRad, Model 3550). From these measurement results, the concentration at which the inhibition rate was 50% was determined as the IC ₅₀ value. The obtained results are shown in Table 1.

<Evaluation of inhibitory activity against carboxypeptidase N (CPN)>
About each compound obtained in Examples 1-9, the measurement of the CPN inhibitory effect was performed as follows.

(E) CPN inhibitory activity measurement method Human plasma-derived carboxypeptidase N (CPN) (ELASTIN PRODUCTS COMPANY, INC.) Dissolved in 50 mM Tris-HCl buffer (pH 7.6) at 2 μg / ml in 25 μL, 27 μM Inhibitors serially diluted with 50 mM Tris-HCl buffer (pH 7.6) containing dithiothreitol (each compound obtained in Examples 1 to 9 was adjusted so that the final concentration thereof was 1 pM to 1 mM. (Liquid prepared as a 10-fold dilution series) 30 μL was added, mixed well, and treated at 37 ° C. for 10 minutes. Further, 25 μL of a substrate solution (Hip-Lys (manufactured by Sigma, H6750) dissolved in 50 mM Tris-HCl buffer (pH 7.4) to a concentration of 10 mM) was added and mixed well. Reacted for 1 minute. Next, 100 μL of 0.2 M PIPES buffer (pH 7.5) containing 12.5% Tween is added to stop the reaction. After 5 minutes, 100 μL of a color developing solution (2-methoxyethanol containing 1% cyanuric chloride) was added and left at room temperature for 15 minutes, and then the absorbance at 405 nm was measured with a microplate reader (BioRad, Model 3550). From these measurement results, the concentration at which the inhibition rate was 50% was determined as the IC ₅₀ value. The obtained results are shown in Table 1.

As is clear from the results shown in Table 1, the compounds of the present invention (Examples 1 to 9) were all excellent in inhibitory activity against TAFIa. In particular, the compounds of the present invention (Examples 6 and 9, compounds DD22 and DD28) had an IC ₅₀ value of 10 ⁻¹² to 10 ⁻¹¹ M and extremely high inhibitory activity against TAFIa. In addition, the compound of the present invention (Example 9, Compound DD28) had extremely low inhibitory activity against CPN and was excellent in terms of specificity to TAFIa.

  As described above, it is possible to provide a compound that binds to the TAFIa protein and can inhibit the cleavage of the substrate by the protein.

  Therefore, since the compound of the present invention has high inhibitory activity against TAFIa protein, it is useful as a pharmaceutical composition for the prevention or treatment of thrombotic diseases such as myocardial infarction and cerebral infarction.

Claims (2)

  An organic selenium compound having inhibitory activity against TAFIa protein, comprising a basic functional group, an acidic functional group, and a selenium atom capable of coordinating with zinc of TAFIa protein, the organic A dimer in which selenium compounds are bonded to each other through a diselenide bond, and a pharmacologically acceptable salt or solvate thereof. A compound having inhibitory activity against TAFIa protein, which is an organic selenium compound represented by the following general formula (1), a dimer in which the organic selenium compounds are bonded to each other through a diselenide bond, and these drugs Physiologically acceptable salt or solvate.
(In formula (1), R ¹ represents —R ⁴ —NH ₂ , —R ⁴ —C (═NH) NH ₂ , —R ⁴ —NH—C (═NH) NH ₂ , or the following formula (2) R ² represents a group represented by —COOR ⁷ , and R ³ represents a hydrogen atom, —C (═O) R ⁵ , —C (═O) R ⁶ —Ar, or — A group represented by C (═O) Ar, R ⁴ represents a linear, branched or cyclic alkylene group having 1 to 6 carbon atoms, and R ⁵ is a straight chain having 1 to 6 carbon atoms. R ⁶ represents a linear, branched or cyclic alkylene group having 1 to 6 carbon atoms, R ⁷ represents a hydrogen atom and 1 to 6 carbon atoms. A linear, branched or cyclic alkyl group, or an aryl group, and Ar represents an aryl group.
(In the formula (2), R ⁸ represents a hydrogen atom or a halogen atom, and n represents an integer of 0 to 3.)