Thrombolytic or hemolytic agents based on DO3A containing gadolinium complexes

Abstract

The present invention has discovered that DO3A-based substances containing gadolinium complexes, which are conventionally used as anti-inflammatory agents, have a novel effect of inhibiting thrombin-induced thrombosis or dissolving blood, without being involved in the platelet action function of stopping bleeding at an injury or wound site, and discloses the applicability of DO3A-based substances containing gadolinium complexes to novel uses as thrombosis inhibitors or blood coagulants.

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition based on DO3A containing a gadolinium complex, and more specifically, to an invention that provides that the pharmaceutical composition, which has been conventionally known as an anti-inflammatory agent, has a novel effect of inhibiting thrombosis or dissolving blood. [Background technology]

[0002] Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the COX-1 and COX-2 enzymes involved in the synthesis of prostaglandins, and through this action produce analgesic, anti-inflammatory and antipyretic effects. COX-1 activates prostaglandins and thromboxane A2, and is responsible for defensive actions such as protecting the gastric mucosa, maintaining kidney function and platelet hemostasis, while COX-2 activates inflammatory mediators and cytokines in various tissues such as vascular endothelial cells, causing pain and inflammation.

[0003] NSAIDs are classified into non-selective NSAIDs and COX-2 selective NSAIDs based on the selectivity of the COX substances. Non-selective NSAIDs have the fatal weakness of inhibiting the protective effect of the gastric mucosa and causing antiplatelet effects, which frequently cause gastrointestinal side effects such as peptic ulcers, because they inhibit both COX-1 and COX-1. COX-2 selective NSAIDs were then introduced with the hope that they could selectively inhibit only COX-2, thereby resolving the various side effects caused by blocking COX-1 while maintaining anti-inflammatory and analgesic effects.

[0004] COX-2 selective NSAIDs are known to reduce the incidence of gastrointestinal complications, but there are still theories regarding cardiovascular and neotoxic side effects. When NSAIDs inhibit COX-2, the production of prostacyclin is suppressed, resulting in reduced antiplatelet effects, sodium and water retention, and the platelet coagulation effect of COX-1 is further promoted, increasing the risk of thrombosis and embolism, which increases the occurrence of cardiovascular events.

[0005] Meanwhile, diflunisal is known as a non-selective COX inhibitor or a weak COX-1 selective inhibitor. Korean Patent Nos. 10-2018-0033426 and 10-2020-0119574 disclose that a compound in which diflunisal is bound to DO3A containing a Gd complex and a compound in which diflunisal is bound to DO3A can be used as a selective COX-2 inhibitor and an anti-inflammatory agent, respectively. Based on the contents of these two patents, it is well known that a compound in which diflunisal is bound to DO3A acts as a selective COX-2 inhibitor and an anti-inflammatory agent, particularly an anti-inflammatory agent for brain inflammatory diseases. However, as can be seen from the contents of Korean Patent No. 10-2020-0119574, its effect as an anti-inflammatory agent has been proven, but its effect on thrombosis inhibition has not been disclosed. Summary of the Invention [Problem to be solved by the invention]

[0006] One object of the present invention is to provide that a pharmaceutical composition conventionally developed as an anti-inflammatory agent has a novel medical effect as an antithrombotic agent. [Means for solving the problem]

[0007] A thrombus inhibitor or hemolytic agent for one object of the present invention includes a compound having a structure represented by the following Chemical Formula 1. The compound having the structure of the following Chemical Formula 1 is a substance that has been conventionally known for use as an anti-inflammatory agent. In the prior art, the cardiovascular side effects were resolved by co-administering the compound with another thrombus inhibitor. However, the inventors of the present invention have found that the compound having the structure of the following Chemical Formula 1 provides a new effect in inhibiting thrombus or dissolving blood in addition to the anti-inflammatory effect, and therefore provide that the compound having the structure of the following Chemical Formula 1 can be used for a new medical use as a thrombus inhibitor or hemolytic agent.

[0008] [ka]

[0009] In the above Chemical Formula 1, A is *-(CH2) n -A 1 - indicates *, n represents an integer of 0 to 5, A 1 are *-NH-*, *-COO-*, *-CO-*, *-NR 1 -*, *-CH2-*, *-CONH-*, or *-O-*, Linker: *-L 1 -NHCO-L 2 -*, *-L 1 -OR 2 -OL 2 -*, *-L 1 -CH2-L 2 -*, *-L 1 -NR 3 -L 2 -*, or *-L 1 -COO-L 2 - indicates *, L 1 represents a linear or branched (C1-C30) alkyl group; L 2 represents a single bond or a linear or branched (C1-C30) alkyl group; R 1and R 3 each independently represents hydrogen or linear or branched (C1-C10) alkyl; R 2 represents a linear or branched (C1-C10) alkyl group; B is

[0010] [ka]

[0011] where * denotes a binding site.

[0012] Preferably, the thrombus inhibitor or hemolytic agent of the present invention has a structure represented by the following chemical formula 2 in which Diflunisal is bound to DO3A containing a gadolinium complex.

[0013] [ka]

[0014] When blood vessels in the body are damaged and bleeding occurs, hemostasis is achieved through the primary and secondary hemostasis processes. Primary hemostasis is achieved by platelets adhering and aggregating to the site of the damaged blood vessel to stop bleeding, while secondary hemostasis is achieved by clotting factors, which activate the clotting factors to form hard fibrin from the aggregated platelets to completely stop bleeding. In primary hemostasis, platelets change shape from a disk to a sphere and adhere to and aggregate with collagen in the blood vessel. The adhered platelets are activated and release calcium (Ca 2+The secreted ADP binds to surrounding platelets to activate them, resulting in platelet aggregation. Secondary hemostasis involves the formation of hard fibrin through the activation of blood coagulation factors. Activation of blood coagulation factors produces thrombin through the intrinsic and extrinsic pathways, which then acts on fibrinogen to form fibrin polymers. Fibrin or fibrin polymers are bound to the primary hemostatic plug, which is made up of platelet adhesion and aggregation in primary hemostasis, to form a stronger and harder secondary hemostatic plug, achieving complete hemostasis.

[0015] The thrombus inhibitor or hemolytic agent of the present invention is characterized by inhibiting platelet aggregation induced by thrombin, which is a secondary hemostasis. The thrombus inhibitor or hemolytic agent of the present invention has the effect of inhibiting platelet aggregation induced by thrombin, which is a secondary hemostasis, rather than inhibiting platelet adhesion at the damaged site, which is a primary hemostasis.

[0016] In one embodiment, the thrombus inhibitor or hemolytic agent can control integrin αIIBβ3 activation. Integrin αIIBβ3 is a protein involved in adhesion and is a substance that mediates platelet aggregation and interaction with other adjacent tissues. The thrombus inhibitor or hemolytic agent of the present invention can inhibit integrin αIIBβ3 activation in a concentration-dependent manner. In the present invention, "concentration-dependent" means that the degree of effect increases as the concentration increases, or the degree of effect decreases as the concentration decreases. That is, the higher the concentration of the thrombus inhibitor or hemolytic agent of the present invention, the more effectively it can inhibit integrin αIIBβ3 activity involved in platelet aggregation.

[0017] In one embodiment, the thrombus inhibitor or hemolytic agent can inhibit the secretion of adenosine triphosphate (ATP). Adenosine triphosphate is a substance released by platelets when they are activated, and can bind to P2Y2 receptors on the surface of vascular endothelial cells. The present invention can affect platelet activity by inhibiting the secretion of ATP.

[0018] Another object of the present invention is to provide an anti-inflammatory agent having a structure represented by the following formula 1, characterized in that it has antithrombotic or hemolytic properties. The anti-inflammatory agent has the advantage that it does not require co-administration with other drugs for antithrombotic purposes, since it has antithrombotic or hemolytic properties.

[0019] [ka]

[0020] In the above Chemical Formula 1, A is *-(CH2) n -A 1 - indicates *, n represents an integer of 0 to 5, A 1 are *-NH-*, *-COO-*, *-CO-*, *-NR 1 -*, *-CH2-*, *-CONH-*, or *-O-*, Linker: *-L 1 -NHCO-L 2 -*, *-L 1 -OR 2 -OL 2 -*, *-L 1 -CH2-L 2 -*, *-L 1 -NR 3 -L 2 -*, or *-L 1 -COO-L 2 - indicates *, L 1 represents a linear or branched (C1-C30) alkyl group; L2 represents a single bond or a linear or branched (C1-C30) alkyl group; R 1 and R 3 each independently represents hydrogen or linear or branched (C1-C10) alkyl; R 2 represents a linear or branched (C1-C10) alkyl group; B is

[0021] [ka]

[0022] where * denotes a binding site.

[0023] In one embodiment, the anti-inflammatory agent may have a structure represented by Formula 2:

[0024] [ka] Effect of the Invention

[0025] According to the present invention, a substance based on DO3A containing a gadolinium complex, which is known as an anti-inflammatory agent in the prior art, has a new effect of inhibiting thrombus or dissolving blood. In particular, the substance based on DO3A containing a gadolinium complex can effectively inhibit platelet aggregation induced by thrombin. Therefore, the thrombus inhibitor or blood dissolving agent of the present invention has the advantage that it is not necessary to administer it in combination with other thrombus inhibitors. [Brief description of the drawings]

[0026] [Figure 1] 1 is a diagram for evaluating the platelet aggregation inhibitory effect of MBP-11902 of the present invention according to Experimental Example 1. In the drawing, MBP-11902 is represented as MBP-02. [Diagram 2]1 is a diagram for comparatively evaluating the platelet aggregation inhibitory effects of MBP-11902 of the present invention and NSAID and MBP-11902L as comparative examples according to Experimental Example 2 of the present invention. In the drawing, MBP-11902 and MBP-119002L are indicated as MBP-02 and MBP-02L, respectively. [Diagram 3] FIG. 1 is a diagram for evaluating the platelet activation efficacy of MBP-11902 of the present invention according to Example 3 of the present invention. In the figure, MBP-11902 is designated as MBP-02A. [Figure 4] 1 is a diagram for investigating the effect of MBP-11902 of the present invention on ATP and TXB2 secretion from platelets according to Example 4 of the present invention, in which MBP-11902 is indicated as MBP-02. [Diagram 5] FIG. 1 is a diagram for evaluating the effect of MBP-11902 of the present invention on platelet Ca 2+ mobilization according to Experimental Example 3. In the drawing, MBP-11902 is represented as MBP-02. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Hereinafter, the embodiments of the present invention will be described in detail with reference to the accompanying drawings. The present invention can be modified in various ways and can have various forms, and specific embodiments are illustrated in the drawings and described in detail herein. However, it is not intended to limit the present invention to the specific disclosed forms, but it should be understood that the present invention includes all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. Similar reference numerals are used for similar components when describing the various drawings.

[0028] The terms used in this application are merely used to describe specific embodiments and are not intended to limit the present invention. A singular expression includes a plural expression unless the context clearly differs. In this application, the terms "include" or "have" are intended to specify the presence of features, steps, operations, components, parts, or combinations thereof described in the specification, and should be understood not to preclude the presence or additional possibility of one or more other features, steps, operations, components, parts, or combinations thereof.

[0029] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the present invention belongs. Terms as defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning they have in the context of the relevant art, and should not be interpreted in an idealized or overly formal sense unless expressly defined in this application.

[0030] Hereinafter, the thrombus inhibitor or hemolytic agent based on DO3A containing the gadolinium complex of the present invention will be described in more detail through specific examples and comparative examples. However, the examples of the present invention are merely some embodiments of the present invention, and the scope of the present invention is not limited to the following examples.

[0031] Example 1 <mbp-11902> The compound used in Example 1 of the present invention (hereinafter, MBP-11902) was provided by Etnova Theraputics, Inc. MBP-11902 in Example 1 of the present invention is represented as MBP-02 in the drawings.

[0032] Comparative Example 1 <nsaid> The compound (hereinafter, NSAID) used in Comparative Example 1 of the present invention was Diflunisal, provided by Etnova Theraputics.

[0033] Comparative Example 2 <mbp-11902l> The compound used in Comparative Example 2 of the present invention (hereinafter, MBP-11902L) is a ligand compound in which gadolinium has been removed from the compound in Example 1 of the present invention, and was provided by Etnova Theraputics, Inc. MBP-11902L in Comparative Example 2 of the present invention is represented as MBP-02L in the drawings.

[0034] Experimental Example 1 (1) Evaluation of the efficacy of MBP-11902 in inhibiting platelet aggregation In order to evaluate the platelet aggregation activity of MBP-11902 according to Example 1 of the present invention, platelet aggregation was activated in rat plasma using platelet aggregation factors and agonists, thrombin, collagen, ADP (adenosine diphosphate), U46619 (thromboxane A2 agonist), and PMA (phorbol-12-myristate-13-acetate), and pretreated with a vehicle (distilled water) and various doses (100, 300, and 500 μM) of MBP-11902. Platelet aggregation levels were then measured using a 4-channel platelet lumi-aggregometer (Chronolog Corp, Havertown, PA), and the results are shown in FIG. 1.

[0035] FIG. 1 is a diagram for evaluating the platelet aggregation inhibitory effect of MBP-11902 of the present invention according to Experimental Example 1 of the present invention.

[0036] Referring to Figure 1, it was observed that MBP-11902 of the present invention effectively inhibited platelet aggregation induced by thrombin (0.05U / mL) compared to the control group (vehicle). Meanwhile, when observing the platelet aggregation effect depending on the concentration of MBP-11902 (25, 50, 100, 300 and 500μM), it was observed that there was no significant difference in the inhibitory effect depending on the concentration. Meanwhile, MBP-11902 of the present invention was not observed to have an effect on the inhibition of platelet aggregation induced by collagen (1μg / mL), ADP (10μM), U46619 (1μM) and PMA (1μM).

[0037] In summary, it was confirmed that MBP-11902 of the present invention inhibits platelet aggregation induced only by thrombin, but does not inhibit platelet aggregation in a concentration-dependent manner. This is because MBP-11902 shows an inhibitory effect on platelet aggregation through the secondary signaling pathway in platelet aggregation mediated by thrombin, but is limited in platelet aggregation through morphological deformation of platelets and the primary signaling pathway.

[0038] (2) Comparative evaluation of the platelet aggregation inhibitory efficacy of MBP-11902, NSAIDs and MBP-11902L In order to re-evaluate the platelet aggregation inhibitory effect of MBP-11902 using thrombin as the only intermediate in the platelet aggregation inhibitory effect of Experimental Example 1, the platelet aggregation was evaluated for the original NSAID, the intermediate MBP-11902L, and the main product MBP-11902 (100 μM each). The evaluation was performed using thrombin and collagen, and the platelet aggregation level was measured using a 4-channel platelet lumi-aggregometer (Chronolog Corp, Havertown, PA), and the results are shown in Figure 2.

[0039] FIG. 2 is a diagram for comparatively evaluating the platelet aggregation inhibitory efficacy of MBP-11902 of the present invention and NSAID and MBP-1192L as comparative examples according to Experimental Example 2 of the present invention.

[0040] Referring to Figure 2, it was observed that NSAID (100μ) significantly inhibited platelet aggregation induced by thrombin (0.05U / ml) and collagen (1μg / mL) compared to the control group (vehicle). Meanwhile, it was observed that the intermediate MBP-11902L (100μM) had no effect on the inhibition of platelet aggregation by both thrombin (0.05U / ml) and collagen (1μg / mL) compared to the control group (vehicle). Meanwhile, it was confirmed that MBP-11902 of the present invention inhibited platelet aggregation induced only by thrombin.

[0041] Considering the results, it is found that the original NSAID can significantly inhibit platelet activity, the intermediate MBP-11902L has no effect on platelet aggregation inhibition, and the MBP-11902 of the present invention inhibits platelet aggregation induced only by thrombin only through the secondary signaling pathway. Therefore, the original NSAID is excellent in inhibiting platelet aggregation, but it is considered to be accompanied by side effects on bleeding. On the other hand, the MBP-11902 of the present invention shows platelet aggregation inhibition effect only through the secondary signaling pathway with thrombin as the only mediator during platelet aggregation, and is therefore considered to inhibit platelet aggregation without side effects on bleeding through thrombin-mediated platelet aggregation, compared to the original NSAID and the intermediate MBP-11902L.

[0042] (3) Evaluation of the platelet activation efficacy of MBP-11902 In the platelet aggregation inhibition experiment in Experimental Example 1, MBP-11902 was confirmed to have a selective platelet aggregation inhibitory effect on the thrombin-mediated signal transduction pathway, so in this platelet activation experiment, an activation efficacy inhibition experiment was carried out using only thrombin. The efficacy of MBP-11902 on the activation regulation mechanism of α-granules, P-selectin, and integrin αIIBβ3, which are used as platelet activation biomarkers in the main process of inducing a positive feedback cycle in platelet activation, was evaluated using flow cytometry analysis (FCM), and the results are shown in Figure 3.

[0043] FIG. 3 is a diagram for evaluating the platelet activation efficacy of MBP-11902 of the present invention according to Example 3 of the present invention.

[0044] Referring to FIG. 3, it was observed that, compared with the control group (vehicle), MBP-11902 of the present invention significantly inhibited rat platelet integrin αIIBβ3 activation in a concentration (100, 300 and 500 μM) dependent manner, but had no inhibitory effect on the activity of α-granules and P-selectin.

[0045] These results suggest that MBP-11902 of the present invention does not affect platelet granule secretion, but selectively inhibits platelet-platelet aggregation activity by activating integrin αIIBβ3 present on the platelet cell membrane.

[0046] (4) Evaluation of MBP-11902's inhibition of platelet activity by measuring ATP and TXB2 secretion During platelet aggregation, ATP (Adenosine triphosphate) and TXB2 (Thromboxane B2) are released and activate specific receptors on the outer wall of platelets through autocrine and paracrine mechanisms, promoting platelet aggregation. Therefore, the secretion and activation of ATP and TXB2 in platelet aggregation reactions are known to be the main mechanisms of thrombus aggregation reactions.

[0047] In the present invention, to confirm the ATP and TXB2 secretion activity of MBP-11902, platelet aggregation activity was increased with thrombin, and then MBP-11902 was treated at different concentrations (100, 300, and 500 μM) and the ATP and TXB2 generation concentrations were measured using a luciferin / luciferase reagent (Chronolog) and a TXB2 generation analysis kit. The results are shown in Figure 4.

[0048] FIG. 4 is a diagram for investigating the effect of MBP-11902 of the present invention on ATP and TXB2 secretion from platelets according to Example 4 of the present invention.

[0049] 4, it was confirmed that, compared to the control group (vehicle), only platelets treated with MBP-11902 significantly inhibited rat platelet ATP secretion in a concentration-dependent manner. Meanwhile, no significant difference was observed in TXB2 generation. These results indicate that MBP-11902 of the present invention affects platelet activity through regulating ATP secretion in platelet activity, but does not affect TXB2 generation.

[0050] (5) Platelet Ca in MBP-11902 2+ Evaluation of the impact on mobilization Platelet agonists stimulate other receptors on platelets to release intracellular stored Ca 2+ secrete Ca from outside the platelets 2+ By accepting Ca into the platelet cytoplasm, 2+ Behavior (Ca 2+ Such Ca mobilization occurs. 2+ The basic mechanism of action is important for platelet activation, including platelet α-granule, P-selectin and integrin αIIBβ3 activation regulation mechanism. Therefore, it is believed that MBP-11902 of the present invention can regulate Ca during platelet activation. 2+ We investigated whether it plays an important role in behavior.

[0051] Rat platelets (1 × 10 8 Platelets were treated with vehicle or various doses (100, 300, and 500 μM) of MBP-11902 for 10 min at 37°C, and platelets were activated with thrombin after incubation with the FLIPR Calcium 5 Assay kit for 30 min at 37°C in the dark. Cytoplasmic Ca 2+ The concentrations were measured using a spectrofluorometer (Spectramax I3, Molecular Devices) with an input wavelength of 485 nm and an emission wavelength of 525 nm, and the results are shown in FIG.

[0052] FIG. 5 shows the platelet Ca of MBP-11902 according to Experimental Example 3 of the present invention. 2+ FIG. 1 is a diagram for evaluating the effect on mobilization.

[0053] Referring to Figure 5, platelet Ca 2+ These results suggest that thrombin-induced Ca2+ expression is not regulated by MBP-11902. 2+ These results suggest that concentration-dependent platelet activation is not regulated by MBP-11902.

[0054] The results of Experimental Examples 1 to 5 of the present invention are summarized as follows.

[0055] The MBP-11902 of the present invention does not affect thrombin-mediated platelet activity, but significantly inhibits platelet-platelet aggregation, and thus plays a more important role in secondary platelet-platelet aggregation inhibition than primary platelet-mediated thrombosis inhibition. It was confirmed that the MBP-11902 of the present invention has an effect on thrombolysis while replacing the side effects of NSAIDs, which are existing therapeutic agents, without affecting hemostatic activity. Therefore, the MBP-11902 of the present invention can be used as a functional raw material for improving blood circulation or a therapeutic agent due to its effect on thrombolysis.

[0056] Materials and Analytical Methods for Experimental Examples 1 to 5 (1)Materials Human thrombin, thromboxan A2 analogue (U46619), PMA, PGE1, dimethyl sulfoxide (DMSO), ADP, fibrinogen and all reagents were purchased from Sigma (St. Louis, MO). D-Phe-Pro-Arg-chloromethyl ketone (PPACK) was purchased from EMD Millipore (Billerica, MA). Phycoerythrin (PE)-conjugated isotype control IgGs, PE anti-mouse CD62P (P-selectin) antibody and PE anti-mouse αIIBβ3 (JON / A) antibody were purchased from Emfret Analytics (Eibelstadt, Germany). Ca 2+ The indicator FLIPR Calcium Assay Kit was purchased from Molecular Devices (Sunnyvale, Calif., USA) and used in this experiment.

[0057] (2) Platelet isolation method Platelets from male C57BL / 6 (6-8 weeks) were collected from the abdominal vein of rats using a syringe pretreated with citrate-dextrose solution (ACD, Sigma). To obtain platelet-rich plasma (PRP), after centrifugation at 300 × g for 20 min at room temperature, the plasma separated from erythrocytes was supplemented with 0.5 μM PGE1 and centrifuged again at 700 × g for 4 min. The precipitated platelets were washed with HEPES-Tyrode buffer (5 mM HEPES / NaOH, pH 7.3, 5 mM glucose, 136 mM NaCl, 12 mM NaHCO3, 2.7 mM KCl) containing 10% ACD, and then centrifuged again at 700 × g for 5 min. The precipitated platelets were diluted with HEPES-Tyrode buffer at 3 × 10 8 The cells were adjusted to a concentration of 1000 cells / mL and then used in this experiment.

[0058] (3) Platelet aggregation analysis method Rad platelets 3 x 10 8 Platelets were placed in HEPES-Tyrode's buffer at a concentration of 100 cells / mL and pretreated with vehicle (distilled water) and various doses (100, 300, and 500 μM) of MBP-11902 for 15 min at 37°C, and then activated with known agonists in the platelet aggregation system: collagen, thrombin, ADP, U46619 (endoperoxide / thromboxane analogs), and PMA (PKC activator). Platelet aggregation was measured by inducing platelet aggregation at 1,000 rpm in a platelet aggregation system (Chronolog Corp, Havertown, PA) at 37°C.

[0059] (4)ATP secretion measurement Rad platelets were pretreated with vehicle (distilled water) and various doses (100, 300, and 500 μM) of MBP-11902 for 10 min at 37°C, and then activated with thrombin, a platelet aggregation agonist, at 37°C. Platelet activation was terminated by centrifugation, and the supernatant was collected to measure adenosine triphosphate (ATP) secretion. ATP secretion was measured using an ATP assay kit (Biomedical Research Service Center, Buffalo, NY) on a GloMax® Explorer Multimode Microplate Reader (Promega).

[0060] (5) TXB2 generation measurement Rad platelets were pretreated with vehicle (distilled water) and various doses (100, 300, and 500 μM) of MBP-11902 for 10 min at 37°C, and then activated with thrombin, a platelet aggregation agonist, at 37°C. Platelet activity was terminated after 5 min by the addition of 2 mM EGTA containing 0.1 M KCl and 5 mM indomethacin. The mixture was centrifuged at 6,000 × g for 3 min, and the thromboxane (TXB2) generation level was measured using a TXB2 generation kit (Enzo Life Sciences, Farmingdale, NY).

[0061] (6) Flow cytometry analysis (FCM) P-selectin and integrin αIIBβ3 activation, known as biomarkers of platelet activity, were measured using a flow cytometer (Gonalos, Beckman Coulter). Rad platelets were pretreated with vehicle (distilled water) and various doses (100, 300, and 500 μM) of MBP-11902 for 15 minutes at 37°C. For platelet activation, Rad platelets were treated with thrombin for 5 minutes at 37°C, and then the activity was measured using P-selectin and integrin αIIBβ3.

[0062] (7) Ca 2+ Behavioral Analysis Rad platelets (1×10 8 Rad platelets (100, 300, and 500 μM) were suspended in CaCl2-free HEPES-Tyrode's buffer and pretreated with vehicle (distilled water) and various doses (100, 300, and 500 μM) of MBP-11902 for 10 min at 37 °C. Rad platelets were incubated with CaCl for 30 min in the dark. 2+ Staining (FLIPR Ca 2+ Platelet Ca analysis kit) and activated with 0.05 U / mL thrombin. 2+ Levels were measured using a spectrofluorometer (Spectramax I3, Molecular Devices) using a wavelength of 505 nm.

[0063] (8) Statistical processing Statistical analysis of the data was performed using GraphPad Prism 5. Statistical significance was analyzed by multiple group ANOVA, Dunnett's test for comparisons of multiple groups, and Student's t-test for comparisons of two groups. P values ​​lower than 0.05 were considered significant.

[0064] Although the present invention has been described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and variations of the present invention can be made without departing from the spirit and scope of the present invention as set forth in the appended claims. < / nsaid>

Claims

1. A thrombus inhibitor or hemolytic agent having a structure represented by the following chemical formula 1: 【Chemistry 1】 In the above Chemical Formula 1, A is *-(CH ２ ) ｎ -A １ - indicates *, n represents an integer of 0 to 5, A １ *-NH-*, *-COO-*, *-CO-*, *-NR １ -*, *-CH ２ -*, *-CONH-*, or *-O-*; The linker is *-L １ -NHCO-L ２ -*, *-L １ -O-R ２ -O-L ２ -*, *-L １ -CH ２ -L ２ -*, *-L １ -NR ３ -L ２ -* or *-L １ -COO-L ２ - indicates *, L １ represents a linear or branched (C1-C30) alkyl group; L ２ represents a single bond or a linear or branched (C1-C30) alkyl group; R １ and R ３ each independently represents hydrogen or a linear or branched (C1-C10) alkyl; R ２ represents a linear or branched (C1-C10) alkyl group; B is 【Chemistry 2】 where * denotes a binding site.

2. The thrombus inhibitor or hemolytic agent according to claim 1 , wherein the thrombus inhibitor or hemolytic agent has a structure represented by the following chemical formula 2: 【Chemistry 3】

3. The thrombus inhibitor or hemolytic agent according to claim 1, which inhibits platelet aggregation induced by thrombin.

4. The thrombus inhibitor or hemolytic agent according to claim 3 , which inhibits integrin αIIBβ3 activation in a concentration-dependent manner.

5. The thrombus inhibitor or hemolytic agent according to claim 3, which inhibits secretion of ATP (Adenosine triphosphate).

6. It has a structure represented by the following chemical formula 1: Anti-inflammatory agents characterized by having antithrombotic or hemolytic properties: 【Chemistry 4】 In the above Chemical Formula 1, A is *-(CH ２ ) ｎ -A １ - indicates *, n represents an integer of 0 to 5, A １ *-NH-*, *-COO-*, *-CO-*, *-NR １ -*, *-CH ２ -*, *-CONH-*, or *-O-*; The linker is *-L １ -NHCO-L ２ -*, *-L １ -O-R ２ -O-L ２ -*, *-L １ -CH ２ -L ２ -*, *-L １ -NR ３ -L ２ -* or *-L １ -COO-L ２ - indicates *, L １ represents a linear or branched (C1-C30) alkyl group; L ２ represents a single bond or a linear or branched (C1-C30) alkyl group; R １ and R ３ each independently represents hydrogen or a linear or branched (C1-C10) alkyl; R ２ represents a linear or branched (C1-C10) alkyl group; B is 【Chemistry 5】 where * denotes a binding site.

7. The anti-inflammatory agent according to claim 6, wherein the anti-inflammatory agent has a structure represented by the following formula 2: 【Chemistry 6】

Images