JP5224333B2 - Triprenylphenol compound and thrombolysis promoter - Google Patents

Description

  The present invention relates to a triprenyl phenol compound and a thrombolysis promoter.

Some microbial metabolites having a triprenylphenol skeleton have important physiological activities. For example, it is known that a specific triprenyl phenol compound obtained from a filamentous fungus has a physiologically active action against phenomena important to the living body such as a thrombolysis promoting action and an angiogenesis inhibiting action (Patent Documents 1 to 3). ).
Moreover, as another triprenyl phenol compound having a three-dimensional structure different from that of the above triprenyl phenol compound, Patent Document 4 discloses a triprenyl phenol compound having hair growth activity. Non-Patent Document 1 discloses a triprenyl phenol compound having antibacterial activity and antifungal activity.

Triprenylphenol compounds obtained by cultivation with filamentous fungi lead to conformational changes of plasminogen (Plg), resulting in sensitivity to activation by plasminogen activator (PA) and fibrin of plasminogen It has been suggested to increase the binding ability and consequently promote thrombolysis (Non-Patent Document 2). It is known that a compound having two triprenyl phenol skeletons obtained when ornithine is added as an amino acid (hereinafter referred to as orniprabin) has a particularly strong effect of promoting such thrombolysis (Patent Document 2). .
Thus, a compound having a triprenylphenol skeleton exhibits high activity value because it exhibits various activities depending on its steric structure and substituents.

These various active triprenylphenol compounds are required to be obtained more efficiently because of their complex structures. As such a production method, a method of producing a microorganism by culturing it has been developed (Patent Documents 1 to 3).
On the other hand, Non-Patent Document 3 and Patent Document 5 disclose triprenyl phenol compound analogs having no side chain bonded to the lactam nitrogen atom in chromanlactam. This analog has no antithrombolytic activity and has antibacterial activity, and has been shown to be usable as an intermediate in the synthesis of a compound having a side chain on the chromanlactam nitrogen atom.

JP 2002-65288 A JP 2004-224737 A Japanese Patent Laid-Open No. 2004-224738 International Publication No. 98/56940 Pamphlet International Publication No. 2007/111203 Pamphlet J. Org. Chem., (1992), Vol.57, pp.6700-6703 FEBS Letter, (1997) Vol.418, pp.58-62 J. Antibiot., (2007) 60 (7), pp.463-468

In general, a side effect of thrombolytic agents and coagulation inhibitors is that they tend to bleed. With respect to thrombolytic agents such as tPA, this solution has been achieved by increasing fibrin affinity. On the other hand, the above-mentioned triprenyl phenol compound has both an action of promoting the activation of plasminogen and an activity of promoting both the binding of plasminogen to fibrin. However, the conventional triprenyl phenol compound is superior in the activity of promoting the activation of plasminogen to the activity of promoting the binding of plasminogen to fibrin in comparison of relative activities. In order to properly exert thrombolysis, it was necessary to increase the activity of promoting the binding of plasminogen to fibrin to cause thrombolysis locally in this relative activity comparison.
Accordingly, an object of the present invention is to provide a novel triprenyl phenol compound having an excellent activity of promoting the binding of plasminogen to fibrin relative to the activity of promoting the activation of plasminogen in comparison of relative activities. And providing a thrombolysis-promoting agent containing the same.

The triprenyl phenol compound of the present invention has the following formula (I) (wherein X is —CHY—C (CH ₃ ) ₂ Z and Y and Z are —H or —OH, respectively, To form a single bond, L ¹ represents a linking group which is a C 1-4 alkylene group having a carboxyl group, L ² represents a linking group represented by —NH—C (═O) —, R represents a 9-fluorenylalkyloxy group having an alkyloxy group having 1 to 3 carbon atoms.)

In the above formula, L ¹ preferably has 4 carbon atoms. In the above formula, R is preferably a 9-fluorenylmethoxy group.
The triprenyl phenol compound of the present invention is preferably one represented by the following formula (IA) or (IB).

  The thrombolytic agent of the present invention contains the above triprenyl phenol compound as an active ingredient.

  According to the present invention, in a relative activity comparison, a novel triprenyl phenol compound having an enhanced effect of promoting the binding of plasminogen to fibrin relative to the activity of promoting plasminogen activation, and Can be provided.

The triprenyl phenol compound of the present invention has the above formula (I) (wherein X is —CHY—C (CH ₃ ) ₂ Z and Y and Z are —H or —OH, respectively, To form a single bond, L ¹ represents a linking group which is a C 1-4 alkyl group having a carboxyl group, L ² represents a linking group represented by —NH—C (═O) —, R represents a 9-fluorenylalkyloxy group having an alkyloxy group having 1 to 3 carbon atoms.

  The triprenylphenol compound of the present invention, unlike the conventional triprenylphenol compound, has an action of promoting the binding of plasminogen to fibrin with respect to the activity of promoting the activation of plasminogen in comparison of relative activities. As a result, plasminogen is concentrated on the fibrin deposited in the tissue, and the effect can be exerted locally.

  In addition, since the triprenyl phenol compound of the present invention has a fluorene group represented by R, fluorescence emission based on the fluorene skeleton is possible. It can be used to study drug absorption, kinetics, and metabolism.

In general formula (I), it is preferable that Y and Z together represent a single bond from the viewpoint of the activity of the obtained triprenyl phenol compound.
In general formula (I), the number of carbon atoms of the alkylene group in the linking group represented by L ¹ is preferably 2 or more and more preferably 4 from the viewpoint of the activity of the triprenyl phenol compound. Further, at least one carboxyl group in L ¹ may be present, and two or more carboxyl groups may be present, and may be present in any part of the alkylene group. From the viewpoint of activity, it is preferable to have one carboxyl group, and it is more preferable to be at a site far from the chromanlactam nitrogen atom of the triprenylphenol skeleton.
In the general formula (I), the linking group represented by L ² is —NH—C (═O) — from the viewpoint of the activity of the triprenyl phenol compound.
In the general formula (I), the 9-fluorenylalkyloxy group having an alkyloxy group having 1 to 3 carbon atoms represented by R has an alkyloxy group having 2 or less carbon atoms from the viewpoint of the activity of the compound. And is most preferably a 9-fluorenylmethoxy (Fmoc) group.

  Specific examples of preferred side chains linked to the chromanlactam nitrogen atom in the triprenyl phenol compound of the present invention include the following.

  The triprenyl phenol compound of the present invention is particularly preferably a compound represented by the formula (IA) or (IB) shown below from the viewpoint of activity. In the present invention, the compound of the following formula (IA) may be referred to as SMTP-46, and the compound of the formula (IB) may be referred to as SMTP-47.

The triprenyl phenol compound of the present invention can be obtained by either a chemical production method by chemical synthesis or a biological production method using living organisms.
When the triprenyl phenol compound of the present invention is produced by chemical synthesis, it is preferable to use an intermediate having no side chain on the chromanlactam nitrogen atom in the triprenyl phenol skeleton from the viewpoint of production efficiency. Examples of such intermediates include those disclosed in WO 2007/111203.

  In order to produce the triprenylphenol compound of the present invention by a biological production method, a filamentous fungus can be used as an organism. From the viewpoint of production efficiency, a filamentous fungus belonging to the genus Stachybotris is preferable. Stachybotrys microspora) is more preferable, and S. microspora IFO30018 strain is more preferable.

In the biological production method, it can be more easily obtained from the viewpoint of production efficiency that the method comprises culturing in a medium in which a specific nitrogen compound is added to the filamentous fungus. Examples of such a production method include those disclosed in the aforementioned WO 2007/111203.
Briefly describing this production method, a culture step of culturing filamentous fungi in a culture solution containing a specific added amine compound described later, and a separation step of separating the triprenyl phenol compound from the culture after the culture step Is included.
The added amine compound only needs to be present during the culturing process of the filamentous fungus and may be present from the initial stage of the culture, but is preferably added at the middle stage of the culture from the viewpoint of production efficiency.

When an added amine compound is added in the middle of the culture, the filamentous fungus culturing step includes the first culturing step using a restricted medium having an amine compound content of 0.5% by mass or less and the added amine compound. And a second culturing step with a production medium.
When a limited medium in which the content of amine compound is limited to 0.5% by mass is used as the medium used in the first culturing step, after the middle stage of culturing to move to the second culturing step, a larger amount than before is used. Intermediate compounds can be obtained. In addition, by producing a large amount of the intermediate compound in this way and then performing the second culturing step with the production medium containing aminobenzoic acid or a derivative thereof, the target triprenylphenol is efficiently and selectively selected. A compound can be obtained. In the present specification, the “amine compound” includes an added amine compound unless otherwise specified.

The amine compound that can be contained in the restriction medium acts as a nitrogen source for growth of filamentous fungi in the restriction medium, a growth promoting factor, or a production promoting factor for the triprenyl phenol compound precursor. As a form of addition, it can be used as a natural mixture such as yeast extract, bouillon, peptone, tryptone, soy bean meal, pharma media, corn steep liquor, fish meat extract, etc., or as a purified compound. Since natural mixtures contain a wide variety of amine compounds, it is preferable to limit the amount in a limiting medium.
In this case, the amine compound is preferably 0.5% by mass or less with respect to the total volume of the restricted medium, preferably from 0.01 to 0.5% by mass from the viewpoint of fungal growth, production amount and production selectivity. Preferably it can be 0.1 mass%-0.3 mass%. When it exceeds 0.5 mass%, things other than the target compound are produced at the same time, resulting in inferior selectivity and reduced production efficiency. On the other hand, if less than 0.01% by mass, the activity of the filamentous fungus may be inferior, which is not preferable. Moreover, when adding a refinement | purification compound as an amine compound, the quantity and kind of the range in which the growth of the filamentous fungus used for production and the production of a triprenyl phenol compound precursor occur favorably are used.

  In the second culture step after the middle stage of culture, a production medium containing an added amine compound is used. Here, the “intermediate culture period” can be a predetermined period from the start of the culture for reliably continuing the first culture step, preferably the second day after the start of the culture, more preferably the fourth day or later. . If this period is too short, for example, if the culture in the production medium is started immediately after the start of the culture, the amount of intermediate compound necessary to obtain the target triprenyl phenol compound may be insufficient, and the The prenylphenol compound may not be produced.

  The production medium used in the second culture step can be composed of the same composition as that of the restriction medium except that it contains an added amine compound for obtaining the target triprenylphenol compound. For this reason, the culturing in the second culturing step may be performed by adding an added amine compound to the restriction medium used in the first culturing step, or the newly prepared added amine compound-containing medium is added as it is. May be.

Examples of the added amine compound for obtaining the target triprenyl phenol compound include N _δ -Fmoc-L-orithine and N _α -Fmoc-L-ornithine. By adding N _δ -Fmoc-L-ornithine or the like to the production medium, the filamentous fungus is incorporated as a side chain of the chromanlactam nitrogen atom, and the triprenyl phenol compound of the present invention is produced.
The added amine compound that can be contained in the production medium in the second culture step may be present in the medium in an amount necessary to obtain the target triprenylphenol compound. From the viewpoint of production amount, it is preferably 0.01% by mass to 1% by mass, and more preferably 0.1% by mass to 0.5% by mass.

  In addition to the above components, the restriction medium and the production medium contain, in addition to the above components, an additive component of a synthetic medium usually used for culturing the above microorganisms for the purpose of promoting the production of compounds by the microorganisms. Examples of additional components that can be added to the restriction medium include nutrient sources such as glucose, sucrose, dextrin, animal oil, vegetable oil, vitamins such as chlorine, nitric acid, sulfuric acid, phosphoric acid, sodium, potassium, calcium, magnesium, cobalt , And other inorganic salts capable of generating ions.

Among inorganic salts, in particular, inorganic salts capable of generating metal ions, and from the viewpoint of increasing the production amount of the product and production efficiency, it can be preferably added to a restricted medium. Examples of such metal ions include magnesium ions, cobalt ions, iron ions, calcium ions, potassium ions, and sodium ions.
The addition amount of these metal ions is 0.001% by mass to 0% as magnesium sulfate heptahydrate in the case of magnesium ions with respect to the total volume of the medium from the viewpoint of product production and fungal growth. 0.5% by mass (more preferably 0.01% by mass to 0.1% by mass), in the case of cobalt ions, 0.00001% by mass to 0.01% by mass (more preferably 0%) as cobalt chloride hexahydrate. 0.0001% by mass to 0.005% by mass), in the case of iron ions, 0.0001% by mass to 0.1% by mass (more preferably 0.0005% by mass to 0%) as iron (II) sulfate heptahydrate. 0.05 mass%), in the case of calcium ions, 0.00001 mass% to 0.1 mass% (more preferably 0.0001 mass% to 0.05 mass%) as calcium chloride dihydrate, In case 0.002% by mass to 2% by mass (more preferably 0.05% by mass to 0.5% by mass) as dipotassium phosphate or potassium nitrate; Mass% to 2 mass% (more preferably 0.05 mass% to 0.5 mass%).
These inorganic salts and metal ions may be used alone or in combination of two or more.

The first culture step using the restriction medium is continued until the middle stage of culture in which a sufficient amount of the intermediate compound is obtained to efficiently obtain the target triprenylphenol compound. From the viewpoint of efficient production of the triprenylphenol compound, the second culturing step using the production medium is preferably performed after 2 days from the start of cultivation of the filamentous fungus, more preferably from 4 days after the start of the culture.
The second culture step is terminated by stopping the culture when the amount of triprenylphenol compound produced is maximum. The period of the second culture step varies depending on the state of the microorganism and the size of the culture system, but is generally 1 to 5 days, and preferably 1 to 3 days from the viewpoint of production.

The first and second culturing steps in this production method are usually performed by static culture or shaking culture using the above-mentioned medium. When shaking culture is applied, it may be performed at a speed usually applied in fungal culture. For example, a rotary shaker of TB-25S (amplitude 70 mm) manufactured by Takasaki Kagaku Co., Ltd. has a capacity of 100 ml in a 500 ml flask. When it is set as the amount of culture media, it is 30 rpm-240 rpm, Preferably it can be set as 160 rpm-200 rpm.
The culture temperature in the first and second culture steps can be appropriately set according to the growth conditions of fungi at various temperatures, but is generally 4 to 50 ° C, preferably 15 to 37 ° C, more preferably 20 -30 ° C, most preferably room temperature (25 ° C). Outside this range, a triprenyl phenol compound cannot be produced efficiently. Moreover, generally the pH of the culture medium used can be 3-9, Preferably it can be set to 5-6.

  In addition, you may provide a preculture process in order to stabilize the production ability by microorganisms before the 1st and 2nd culture process. The medium used in the preculture step may be a normal growth medium used for maintaining microorganisms.

The obtained triprenyl phenol compound can be obtained by collecting and purifying from the culture. The recovery / purification method may be any means as long as it can recover and purify the triprenylphenol compound released into the medium, and examples thereof include liquid chromatography, solvent extraction, and crystallization. The product is preferably recovered and purified in two or more stages from the viewpoint of recovery efficiency.
In these recovery / purification methods, it is preferable to select a solvent or the like by utilizing the fact that the triprenyl phenol compound is lipophilic.
When collecting and purifying the triprenylphenol compound from the culture, it is preferable to remove the cells from the culture in advance. In that case, a solvent such as methanol is added to the culture to extract the triprenylphenol compound in the cells, and filtration or the like may be used for the subsequent removal of the cells.

<Thrombolysis promoter>
The thrombus dissolution promoter of the present invention is characterized by containing the above triprenyl phenol compound as an active ingredient. Here, as the triprenyl phenol compound, those described above may be used alone or in combination.
In the thrombolytic agent, the triprenylphenol compound can be contained in the thrombolytic agent in a form that is usually applicable as a pharmaceutical, such as a free form, a pharmaceutically acceptable salt, or an ester.
Further, the dosage form of the present thrombolytic agent can be appropriately changed according to various administration forms. Examples of oral dosage forms include tablets, capsules, powders, fine granules, granules, solutions or syrups, and parenteral dosage forms include injections, drops, suppositories, inhalants, A patch etc. can be mentioned.
In order to maintain these forms, additives such as well-known solvents and excipients that can be used in these applications can be included.

  The thrombolytic agent of the present invention can be administered at an appropriate dose depending on age, weight, and symptoms. For example, in the case of intravenous administration, the amount of active ingredient per day for an adult is 1 to 25 mg / kg. In the case of administration or oral administration, administration of 2 to 200 mg / kg as an active ingredient amount per day for an adult is preferable, and the administration period can be arbitrarily determined according to age and symptoms.

In addition, the triprenyl phenol compound of the present invention has a 9-fluorenylalkyloxy group as a side chain, and therefore can emit fluorescence based on the fluorin ring. Thereby, this triprenyl phenol compound can be used as a labeling compound for plasminogen, and the localization and behavior of plasminogen can be detected optically. It can also be used as a reagent for studying drug absorption, kinetics, and metabolism.
When used as such a labeling compound, those skilled in the art can appropriately select the concentration and form of use. Generally, the triprenylphenol compound of the present invention is used at a concentration of 0.01 μM to 1 mM. can do.

  Examples of the present invention will be described below, but the present invention is not limited thereto. Further,% in the examples is based on mass / volume unless otherwise specified.

[Example 1]
Synthesis of Compound SMTP-46 The amine to be added was synthesized as follows. N _α -Fmoc-L-ornithine is obtained by adding 5 ml of TFA (trifluoroacetic acid) to 5 ml of 150 mg / ml THF (tetrahydrofuran) solution of N _δ -Boc (tert-butoxycarbonyl) -N _α -Fmoc-L-ornithine. It was prepared by reacting for hours and deprotecting the Boc group. Concentrated to dryness, dissolved in 5 ml of chloroform, applied to 4.5 g silica gel (1.0 cm × 13 cm), eluted with a mixed solvent of chloroform: methanol = 75: 25 and purified. This was concentrated to dryness to obtain 136.4 mg of N _α -Fmoc-L-ornithine.

  The spore of Stachybotrys microspora IFO30018 strain (Fermentation Laboratory) was inoculated into a 500 ml Erlenmeyer flask containing 100 ml of the seed culture medium, and seed culture was performed at 180 rpm and 25 ° C. for 4 days using a rotary shaker. As the seed culture medium, glucose (4%), soybean meal (0.5%), dry bouillon (0.3%), and powdered yeast extract (0.3%) are dissolved in water, and the pH is adjusted to 5. The antifoaming agent CB442 (0.01%) (0.1 g / ml acetone solution added at 1 ml / L) (Nippon Yushi Chemical Co., Ltd.) was added, and 100 ml each was dispensed to the incubator, and then the autoclave ( 121 ° C., 15 min) was used.

5 ml of this culture solution was inoculated into a 500 ml 1 Erlenmeyer flask containing 100 ml of the main culture medium, and main culture was carried out at 180 rpm and 25 ° C. for 5 days using a rotary shaker.
The main culture medium (restriction medium) is sucrose (5%), powdered yeast extract (0.1%), NaNO ₃ (0.3%), K ₂ HPO ₄ (0.1%), MgSO ₄ · 7H ₂ O (0.05%), KC1 (0.05%), CoCl ₂ .6H ₂ O (0.00025%), FeSO ₄ .7H ₂ O (0.0015%), CaCl ₂ .2H ₂ O ( 0.00065%) is dissolved in water, adjusted to pH 5.8 using HCl, and defoamer CB442 (0.01%) (0.1 g / ml acetone solution is added at 1 ml / L) (Nippon Oils Chemicals, Japan) was added, and 100 ml each was dispensed to the incubator, and then autoclaved (121 ° C., 15 min).

The day of inoculation was defined as day 0 of culture, and on day 4 of culture (96 hours later), 100 mg of N _α -Fmoc-L-ornithine was dissolved in 1 ml of DMSO (dimethylsulfoxide) and added to the medium to obtain a production medium. The culture was continued. About 24 hours later, 200 ml of methanol was added to terminate the culture. Then, it extracted by shaking for about 3 hours at 180 rpm and 25 degreeC using the rotary shaker.

  From 300 ml of the obtained culture extract, cells were removed using a Buchner funnel to obtain a culture supernatant. Concentration was performed using a rotary evaporator under reduced pressure by a water pump. MeOH was added thereto, and the dissolved fraction was pretreated with Lichrolut (registered trademark) RP-18 (100 mg) (MERCK KGaA, Darmstadt, Germany). Reversed phase HPLC is a column; Inertsil PREP-ODS (diameter 30 × 250 mm) (GL Science Co., Ltd., Tokyo, Japan), temperature: 40 ° C., flow rate: 25 ml / min, detection wavelength: 260 nm, developing solvent: 0.1% (Vol / vol) It was performed with 85% methanol containing formic acid, and a peak having a retention time of 13.5 minutes was collected. Methanol was distilled off using a rotary evaporator and then freeze-dried. N-Hexane was added to the dried product and the mixture was stirred and then centrifuged to collect insoluble matter. This operation was performed 3 times, and the insoluble matter was dissolved in methanol and then filtered. The filtrate was concentrated and dried to obtain 64.6 mg of a purified product of compound SMTP-46.

The properties of the compound SMTP-46 were confirmed as follows.
MALDI-TOF-MS was measured using Voyager-DE STR (Applied Biosystem) and α-cyano-4-hydroxycinnamic acid as a matrix in positive ion mode.
UV was measured in methanol using a 320 spectrophotometer (Hitachi).
As the FT-IR, JIR-WINSPEC50 (JEOL) was used. A sample dissolved in acetone was applied to rock salt and measured. NMR was measured using Alpha 600 (JEOL) at ¹ H 600 MHz, ¹³ C 150 MHz at room temperature. The sample was an approximately 10 mg / ml acetone-d ₆ solution.

The physicochemical properties of the compound SMTP-46 are shown below.
Molecular formula C ₄₃ H ₅₀ N ₂ O ₈
MALDI-TOF-MS (m / z)
Found (M + Na) ⁺ : 745.3524
Calculated: 745.3465 for C ₄₃ H ₅₀ N ₂ NaO ₈
UV λ _max nm (ε) 208 (95,063), 263 (30,628), 289 (7,946), 300 (9,824)
IR ν _max (NaCl) cm ^-1 3317, 3061, 2970, 2924, 2862, 1707, 1662, 1616, 1531, 1462, 1344, 1223, 1074, 850, 746, 540

[Example 2]
Synthesis of Compound SMTP-47 The amine to be added was synthesized as follows. N _δ -Fmoc-L-ornithine was prepared by adding 5 ml of TFA to 5 ml of 60 ml / ml THF solution of N _α -Boc-N _δ -Fmoc-L-ornithine and deprotecting the Boc group. did.

Pre-culture was performed in the same manner as in Example 1.
Main culture was carried out in the same manner as in Example 1 except that the organic amine compound added on the fourth day of main culture was N _δ -Fmoc-ornithine.
From 300 ml of the obtained culture extract, cells were removed using a Buchner funnel to obtain a culture supernatant. Concentration was performed using a rotary evaporator under reduced pressure by a water pump. From 300 ml of the obtained culture extract, cells were removed using a Buchner funnel to obtain a culture supernatant. Concentration was performed using a rotary evaporator under reduced pressure by a water pump. MeOH was added thereto, and the dissolved fraction was pretreated with Lichrolut RP-18 (100 mg). Reversed phase HPLC is a column; Inertsil PREP-ODS (diameter 30 × 250 mm), temperature 40 ° C., flow rate: 25 ml / min, detection wavelength: 260 nm, developing solvent: 85% methanol containing 0.1% (vol / vol) formic acid And a peak with a retention time of 9.2 minutes was collected. Methanol was distilled off using a rotary evaporator and then freeze-dried. N-Hexane was added to the dried product and the mixture was stirred and then centrifuged to collect insoluble matter. This operation was performed three times, the insoluble material was dissolved in methanol and then filtered, and this was concentrated and dried to obtain 35.0 mg of a purified product of compound SMTP-47.

The properties of the compound SMTP-47 were confirmed as follows.
MALDI-TOF-MS, UV, and FT-IR were measured in the same manner as in Example 1. NMR was measured using Alpha 600 (JEOL) at ¹ H 600 MHz, ¹³ C 150 MHz at room temperature. The sample was an approximately 10 mg / ml acetone-d ₆ solution.
The physicochemical properties of the compound SMTP-47 are shown below.
Molecular formula C ₄₃ H ₅₀ N ₂ O ₈
MALDI-TOF-MS (m / z)
Found (M + Na) ⁺ : 745.3575
Calculated: 745.3465 for C ₄₃ H ₅₀ N ₂ NaO ₈
UV λ _max nm (ε) 208 (86,394), 263 (29,328), 289 (7,368), 300 (9,102)
IR ν _max (NaCl) cm ^-1 3329, 3064, 2968, 2926, 2866, 1701, 1670, 1620, 1533, 1462, 1356, 1252, 1157, 1076, 847, 744, 542

[Example 3]
Regarding the triprenyl phenol compounds SMTP-46 and SMTP-46 obtained in Example 1 and Example 2, the binding activity of plasminogen to fibrin was performed as follows, and the performance was evaluated.
As a comparative example, the following SMTP-19 (see WO 2007/11203) was used.

Iodine-labeled plasminogen ( ¹²⁵ I-plasminogen) was prepared as follows. To a polypropylene tube coated with iodegen, add 10 μl of 50 mM sodium phosphate buffer (pH 7.4), 1.5 μl Na ¹²⁵ I (100 mCi / ml) and 50 μl-plasminogen (46.74 μM) for 10 minutes. Reacted on ice. Thereafter, 50 μl of 0.5 M sodium phosphate buffer (pH 7.4) was added, and the reaction was stopped by allowing to stand at room temperature for 15 minutes. Unreacted Na ¹²⁵ I was removed using Sephadex G-25. A 1 ml plastic syringe was filled with 0.9 ml Sephadex G-25 (equilibration buffer was PBS / 0.01% Triton X-100) and centrifuged at 1600 g for 4 minutes to collect ¹²⁵ I-plasminogen. The specific radioactivity was about 250 cpm / ng.

The binding of plasminogen to fibrin was evaluated as follows. Fibrinogen dissolved in PBS (20 mM sodium phosphate, 150 mM NaCl, pH 7.4) was added to a 96-well flat bottom microplate at 2 μg / well and dried at 37 ° C. for 3 days or more. 75 μl of 0.68 U / ml thrombin in PBS was added and reacted at 37 ° C. for 3-24 hours to obtain fibrin plates. The plate was washed twice with 100 μl PBS / T (PBS / 0.01% Tween 80) and once with 200 μl PBS, 200 μl of PBS containing 5 mg / ml gelatin was added, and the mixture was allowed to stand at 37 ° C. for 1 hour. 100 nM plasminogen (the ratio of ¹²⁵ I-plasminogen in plasminogen is 50%) and a sample were added to a 50 μl reaction system, and incubated at 37 ° C. for 1 hour. Washed twice with 200 μl PBS and once with 100 μl PBS. 50 μl of 0.2 M NaOH, 2% (w / v) sodium dodecyl sulfate solution was added and treated at 37 ° C. for 15 minutes, and the radioactivity of 40 μl of the supernatant was measured with a γ-counter and quantified. The specific binding between fibrin and plasminogen was calculated by subtracting the measured value in the presence of 200 mM 6-aminohexanoic acid.

“Double accelerating activity” refers to fibrin and plasminogen produced by an SMTP compound when the value of specific binding between fibrin and plasminogen is 1 when a reaction solution (control) containing no SMTP compound is used. The concentration at which the specific binding of is doubled. Further, “10-fold promoting activity” represents a concentration at which specific binding between fibrin and plasminogen by the SMTP compound is 10-fold.
The results are shown in Table 3.

  As shown in Table 3, both SMTP-46 and SMTP-47 have a stronger effect of promoting the binding of plasminogen to fibrin compared to SMTP-19, and plasminogen to fibrin in vivo. It was suggested that by promoting the binding of gen, a local reaction is induced.

[Example 4]
The triprenyl phenol compounds SMTP-46 and SMTP-47 obtained in Example 1 and Example 2 were subjected to thrombolytic activity as follows as the activity to promote plasminogen activation by the urokinase catalyst. evaluated.
As a comparative example, SMTP-19 (see WO2007 / 11203) was used.

Utilizing that plasmin cleaves the peptide bond of the synthetic chromogenic substrate VLK-pNA (Val-Leu-Lys-p-nitroanilide) to produce p-nitroarinin (pNA), the yellow color absorbed at 405 nm of pNA By measuring the color development, the plasminogen activation promoting activity of the sample is measured. An MTP-500 type microplate reader (Corona Electric Co., Ltd.) was used as a measuring device, and measurement was performed with a 96-well round bottom microplate.
The measurement conditions were a kinetic measurement at 37 ° C. and a dual wavelength of 405 nm (activity) -630 nm (background), and measurement was performed 60 times per minute.

The purified sample was an aqueous sodium salt solution. It was diluted with TBS / T (50 mM Tris-HCl, 100 mM NaCl and 0.01% Tween 80, pH 7.4) to obtain a measurement sample. To 15 μl of sample, 35 μl of each reaction solution (prepared by TBS / T to have final concentrations of 0.1 mM VLK-pNA, 50 nM Glu-plg, 50 U / ml u-PA) was added, and 50 μl / well. The measurement was performed in triplicate for each concentration.
Moreover, it reacted using the reaction liquid which does not contain u-PA as a blank, and the value was subtracted from the value obtained by said reaction. Absorbance against the square of time was plotted, the slope was taken as the initial reaction rate, and comparison was made with no triprenylphenol compound added as a control, and the degree of activity of each compound was determined.

“10-fold accelerating activity concentration” represents a concentration at which 10-fold accelerating activity is obtained when the value when a reaction solution (control) containing no SMTP compound is used is 1. “Maximum promoting activity” represents the magnification and concentration at which the promotion of plasminogen activation by the SMTP compound is maximized.
The results are shown in Table 4.

  As shown in Table 4, plasminogen activation promoting activity was observed in SMTP-46 and SMTP-47. This activity is equivalent to or stronger than SMTP-19, suggesting that it can be used as a thrombolytic agent.

  As described above, the compound of this example is superior in the activity of promoting the binding of plasminogen to fibrin in comparison with the activity of promoting the activation of plasminogen in comparison of relative activities. It is clear that it can be used as an effective thrombolytic agent to cause local thrombolysis by increasing the binding of plasminogen to fibrin and promoting the activation of plasminogen at that site .

Claims (5)

The following formula (I) (wherein X is —CHY—C (CH ₃ ) ₂ Z, Y and Z are each —H or —OH, or together form a single bond, L ¹ represents a linking group which is a C 1-4 alkylene group having a carboxyl group, L ² represents a linking group represented by —NH—C (═O) —, and R represents an alkyl having 1 to 3 carbon atoms. Represents a 9-fluorenylalkyloxy group having an oxy group.).
The triprenyl phenol compound according to claim ¹ , wherein L ¹ has 4 carbon atoms.   The triprenyl phenol compound according to claim 1 or 2, wherein R is a 9-fluorenylmethoxy group. The triprenyl phenol compound according to claim 1, which is a triprenyl phenol compound represented by the following formula (IA) or (IB).
  The thrombolysis promoter which contains the triprenyl phenol compound of any one of Claims 1-4 as an active ingredient.