Novel Olsalazine compound and composition for preventing or treating thrombocytopenia comprising the same

Abstract

One aspect relates to olsalkene, a method for manufacturing the same, a composition for preventing or treating thrombocytopenia containing the same, and the use thereof. According to one aspect, it was confirmed that olsalkene is not cleaved by azoreductase (AzoR) in the body and binds to biliverdin reductase B (BLVRB), thereby inhibiting the reduction of biliverdin (BV) to bilirubin (BR). In addition, it was confirmed that it promotes platelet production through the inhibition of the activity of biliverdin reductase B. Therefore, the olsalkene can be usefully utilized as a treatment for thrombocytopenia, a health functional food, etc., by playing a role in promoting platelet production by inhibiting the activity of biliverdin reductase B.

Description

Technology Field

[0001] One example of the present invention relates to Olsalkene (OSK), stereoisomers thereof or pharmaceutically acceptable salts thereof, a method for preparing the same, a composition for the prevention or treatment of thrombocytopenia comprising the same as an active ingredient, and the use of said composition. Background Technology

[0003] Platelets originate from megakaryocytes (MKs) that differentiate from pluripotent hematopoietic stem cells. Platelets play a pivotal role in various biological processes, including blood clotting and thrombosis, as well as inflammation, neovascularization, innate immunity, adaptive immune responses, and tumor metastasis. Therefore, regulating platelet counts and controlling specific platelet responses are key objectives for new drugs treating platelet-related diseases. Platelet dysregulation can lead to various bleeding disorders, including thrombocytopenia. The standard treatment for thrombocytopenia is to increase platelet counts through platelet induction or platelet transfusion. Conversely, thrombocytosis refers to a condition in which there is an excessive amount of platelets in the blood, in contrast to thrombocytopenia. Analysis of populations of primary (essential) and secondary (reactive) thrombocytopenia through large-scale platelet transcriptome sequencing revealed that biliverdin reductase B (BLVRB) regulates platelet production through MK differentiation by regulating reactive oxygen species (ROS).

[0004] In the heme degradation pathway, BLVRB utilizes NAD(P)H downstream of heme oxygenase(s)-1 (inducible HMOX1) and -2 (constitutive HMOX2) to reduce biliverdin (BV)-IXβ to bilirubin (BR)-IXβ. BR, the product of this process, acts as a potent antioxidant and exhibits cytoprotective effects. However, high concentrations can cause toxicity. Therefore, the BV / BR redox cycle controlled by BLVRB plays a crucial role in regulating reactive oxygen species (ROS). A study using induced pluripotent stem cells (iPSCs) expressing a loss-of-function BLVRB mutant (BLVRBS111L) showed a significant increase in ROS accumulation and proliferation in modified CD34+ / BLVRBS111L hematopoietic stem cells (HSCs), as measured by MK colony formation (CFU-MK). On the other hand, wild-type BLVRB did not exhibit this effect. The loss-of-function mutant (BLVRBS111L) induces MK differentiation and, consequently, promotes platelet production through ROS accumulation. Therefore, effectively removing the antioxidant BR by inhibiting BLVRB activity can lead to a new strategy to increase platelet production by utilizing BLVRB's unique redox regulation within the heme degradation pathway.

[0005] Xanthen dyes and acridine-containing compounds have been developed to inhibit BLVRB activity. These compounds are structurally similar to flavin mononucleotide (FMN), a natural BLVRB substrate. Among the xanthen dyes, erythrosin B and phloxine B have emerged as the most potent inhibitors. Molecular modeling and X-ray crystallography studies revealed that erythrosin B and phloxine B bind to the active site of BLVRB. Furthermore, the hydrogen bonding network within the BLVRB active site was elucidated, and the crucial role of the S111 residue in catalytic activity was highlighted. Interestingly, linear Weaver-Burk plot analysis indicated that these inhibitors do not bind as deeply as the natural substrate FMN, but rather bind similarly to the binding pocket of BLVRB while acting in a non-competitive manner. Both erythrosin B and phloxine B have been utilized as food coloring agents, and phloxine B has also exhibited antimicrobial activity against various Gram-positive bacteria. However, chronic administration of erythrosine B was found to be associated with the promotion of thyroid tumors in mice. Furthermore, NMR and dynamic light scattering (DLS) experiments confirmed that both erythrosine B and floxin B induce multiplexing of BLVRB, thereby reducing therapeutic efficacy. Therefore, novel drug candidates targeting BLVRB are needed to treat platelet disorders.

[0006] To pursue this goal, a drug repurposing approach was used to screen for new candidate substances, resulting in the identification of 20 potential inhibitors. Among the inhibitors derived from the repurposing of FDA-approved drugs, olsalazine (OSA) exhibits the strongest inhibitory affinity. Recent studies report that olsalazine is degraded in various human gastrointestinal environments. Olsalazine contains diazenyl bonds that are easily cleaved by abundant azoreductase (AzoR).

[0007] Therefore, the inventors focused on developing a new compound that is not cleaved by AzoR while maintaining the inhibitory, biochemical, and biophysical properties of olsalazine. To this end, the inventors devised a new chemical system in which the diazenyl bond of olsalazine is replaced with an alkene bond, thereby completing olsalkene (OSK, (E)-5,5'-(ethene-1,2-diyl)bis(2-hydroxybenzoic acid)). Prior art literature

[0009] Korean Registered Patent 10-1996880 The problem to be solved

[0010] One aspect is to provide an olsalkene represented by the following chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0011] [Chemical Formula 1]

[0012]

[0013] Another aspect is to provide a method for manufacturing the above-mentioned Olsalkene.

[0014] Another aspect is to provide a pharmaceutical composition for the prevention or treatment of thrombocytopenia comprising the above-mentioned Olsalkene and a pharmaceutically acceptable salt thereof as an active ingredient.

[0015] Another aspect is to provide a pharmaceutical preparation for the prevention or treatment of thrombocytopenia comprising the above-mentioned pharmaceutical composition.

[0016] Another aspect is to provide a health functional food composition for the prevention or improvement of thrombocytopenia, comprising the above-mentioned Olsalkene and a pharmaceutically acceptable salt thereof as an active ingredient. means of solving the problem

[0018] To achieve the above objective, an olsalkene represented by the following chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is provided.

[0019] [Chemical Formula 1]

[0020]

[0021] In addition, the present invention provides a method for manufacturing the olsalkene, comprising the following steps:

[0022] A step of preparing an intermediate by reacting 5-formyl-2-hydroxybenzoic acid with methyl iodide;

[0023] A step of preparing a compound represented by the following chemical formula 2 by reacting the above intermediate with TiCl4; and

[0024] A step of reacting BBr3 with a compound represented by chemical formula 2.

[0025] [Chemical Formula 2]

[0026] Effects of the invention

[0028] The inventors developed olsakene, a derivative that retains the biochemical and biophysical properties of olsalazine. The inventors confirmed that, unlike conventional olsalazine, olsakene is not cleaved by azoreductase (AzoR) in the body but binds to biliverdin reductase B (BLVRB), thereby inhibiting the reduction of biliverdin (BV) to bilirubin (BR). Furthermore, they confirmed that it promotes platelet production through the inhibition of biliverdin reductase B activity.

[0029] Through this, Olsalken can be usefully utilized as a treatment for thrombocytopenia and a health functional food by inhibiting the activity of biliverdin reductase B and promoting platelet production. Brief explanation of the drawing

[0031] Figure 1 shows the partial purification and enzyme kinetics of the AzoR enzyme. Figure 1A shows the SDS-PAGE results displaying the protein profile after purification via anion exchange chromatography. Figure 1B shows the enzyme activity analysis of AzoR, indicated by the decrease in absorbance at 340 nm over time, measured by the reduction of menadione in the eluted fraction. Figure 2 shows the cleavage of the diazenyl bond of olsalazine (OSA) to produce 5-aminosalicylic acid (5-ASA) by azoreductase (AzoR) containing cell extract. Figure 2A is the olsalazine of Figure 2B and the NAD of Figure 2C. + and shows the 1D 1H NMR spectra of OSA treated with AzoR activity and NADH factions, along with the reference spectrum of 5-ASA in Fig. 2D. Fig. 2B shows all chemical structures of OSA, and Fig. 2C shows NAD + And in Fig. 2D, all chemical structures using the AzoR catalyst of 5-ASA, NADH and OSA are shown above the corresponding spectra. Fig. 3 shows Olsalazine (green), NAD + This is the DOSY spectrum of olsalazine (blue) treated with cell extracts containing AzoR and NADH (red), 5-ASA (magenta), and AzoR and NADH. Figure 4 shows the 5-ASA binding of half-holo BLVRB. Figure 4A shows 0.1 mM half-holo BLVRB in the absence (black) and presence (red) of 1 mM 5-ASA. 1 H- 15Figure 4B shows the chemical shift difference between half-holo BLVRB and BLVRB in complexes with OSK (black) and 5-ASA (red). Figure 4C shows NMR titration data for binding to half-holo BLVRB. Figure 4D shows the surface representation of 5-ASA-bound half-holo BLVRB generated using the crystal symmetry of the previously reported PDB structure (7ERA). Figure 5 shows the 1D 1H NMR spectra, where Figure 5A shows the fraction of AzoR activity and OSK treated with NADH, Figure 5B shows OSK and Figure 5C shows NADH. Figure 6 is a thermal image of the isothermal titration calorimetry of BLVRB through the titration of OSK (Figure 6A) and OSA (Figure 6B). Figure 7 shows the binding topology of OSK within the substrate pocket as revealed by NMR spectroscopy. Figure 7A shows half-holo BLVRB titrated with OSK. 1 H- 15 Figure 7B shows the N HSQC spectrum. Figure 7B shows the chemical shift difference between a half-holo BLVRB and a BLVRB complexed with OSK. Figure 7C shows the surface of a half-holo BLVRB containing FMN generated using the crystal symmetry of a previously reported PDB structure (1HE4). Figure 7D shows the surface of a half-holo BLVRB using OSK derived from the crystal structure. Figure 8 compares the structures of BLVRB, olasalazine (OSA, gray), and olsalken (OSK, red) within the complex. Figure 8A represents the backbone of BLVRB as a ribbon. Figure 5B shows the interaction locations of BLVRB and OSA or OSK superimposed. Figure 8C is a schematic diagram of the interaction network of the BLVRB:OSK complex. Figure 9 shows the linear Weaver-Burk plots of BLVRB using the inhibition mechanism (Figure 9A), OSK (Figure 9B), and OSA (Figure 9C). Figure 10 shows apo-BLVRB's 1 H- 15 As N HSQC spectra, Fig. 10A shows OSK, Fig. 10B shows OSA, and Fig. 10C shows the data overlaid with the addition of PhlB (absence (black) and presence (red)). Fig. 10D shows NMR titration data for the apo-BLVRB binding of OSK, and Fig. 10E shows NMR titration data for OSA. Specific details for implementing the invention

[0032] The present invention will be described in detail below.

[0033] To achieve the above objective, an olsalkene represented by the following chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is provided.

[0034] [Chemical Formula 1]

[0035]

[0036] The term "olsalkene" above refers to (E)-5,5'-(ethene-1,2-diyl)bis(2-hydroxybenzoic acid), an olsalazine derivative in which the diazenyl bond of olsalazine is replaced with an alkene bond. The inventors confirmed that it inhibits BLVRB activity without being cleaved by azoreductase (AzoR) in the body.

[0037] The term "olsalazine" is a 5-aminosalicylic acid (5-ASA) derivative and is an anti-inflammatory drug originally used to treat ulcerative colitis. Olsalazine is known to inhibit BLVRB activity. However, there are studies showing that olsalazine is degraded in the human gastrointestinal environment because it contains diazenin bonds that are easily cleaved by abundant azoreductases.

[0038] The term "stereoisomer" above refers to compounds that have the same molecular formula and bonding structure but differ in the arrangement of atoms in three-dimensional space.

[0039] "Pharmaceuticalally acceptable salt" may mean a pharmaceutically acceptable salt, hydrate, solvate, or prodrug of a compound that retains the desired biological activity of the compound and exhibits minimal or undesirable toxicological effects.

[0040] The term "salt" above refers to an acidic and / or basic salt form formed from an inorganic and / or organic acid and a base. According to one embodiment, a salt of the compound may be formed by reacting an equal amount of the compound of the present invention with an equal amount of an acid or base in a medium such as a medium in which the salt precipitates or in an aqueous medium.

[0041] In addition, the present invention provides a method for manufacturing an olsalkene of claim 1, comprising the following steps:

[0042] A step of preparing an intermediate by reacting 5-formyl-2-hydroxybenzoic acid with methyl iodide;

[0043] A step of preparing a compound represented by the following chemical formula 2 by reacting the above intermediate with TiCl4; and

[0044] A step of reacting BBr3 with a compound represented by chemical formula 2.

[0045] [Chemical Formula 2]

[0046]

[0047] Meanwhile, the method for manufacturing olsalken is not limited to the specific synthesis conditions of the examples, and may be carried out by a person skilled in the art of organic chemistry by appropriately modifying it using known organic chemistry knowledge.

[0048] Unless otherwise specified, all numbers, values, and / or expressions used herein to represent amounts of ingredients, reaction conditions, compositions, and formulations should be understood to be modified by the term “approximately” in all cases, as these numbers are essentially approximations reflecting the various uncertainties of measurement that occur in obtaining these values ​​among other things. Furthermore, where numerical ranges are disclosed herein, such ranges are continuous and, unless otherwise indicated, include all values ​​from the minimum value of such range to the maximum value including said maximum value. Moreover, where such ranges refer to integers, they include all integers from the minimum value to the maximum value including said maximum value, unless otherwise indicated.

[0049] In this specification, where a range is described for a variable, it will be understood that the variable includes all values ​​within the described range, including the described endpoints of the range. For example, the range “5 to 10” will be understood to include not only the values ​​5, 6, 7, 8, 9, and 10, but also any sub-ranges such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc., and any values ​​between integers valid for the category of the described range, such as 5.5, 6.5, 7.5, 5.5 to 8.5, and 6.5 to 9, etc. In addition, for example, the range “10% to 30%” will be understood to include all integers including values ​​such as 10%, 11%, 12%, 13%, etc. and up to 30%, as well as any sub-range such as 10% to 15%, 12% to 18%, 20% to 30%, etc., and any value between valid integers within the stated range category such as 10.5%, 15.5%, 25.5%, etc.

[0050] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of thrombocytopenia comprising olsalkene and a pharmaceutically acceptable salt thereof as an active ingredient.

[0051] The term "thrombocytopenia" refers to a condition in which the number of platelets in the blood falls below normal levels. Platelets play a crucial role in promoting hemostasis through coagulation during bleeding. When thrombocytopenia occurs, symptoms such as easy bruising or persistent bleeding may appear. The causes of thrombocytopenia are diverse and include autoimmune diseases, infections, drug side effects, and bone marrow abnormalities.

[0052] In one embodiment of the present invention, the thrombocytopenia may be one or more selected from the group consisting of non-immunogenic thrombocytopenia, immunogenic thrombocytopenia, and drug-induced thrombocytopenia.

[0053] In another embodiment of the present invention, the composition may inhibit the activity of biliverdin (BV)-Ixβ-reductase B (BLVRB).

[0054] In another embodiment of the present invention, the composition may inhibit the reduction of biliverdin (BV)-IXβ to bilirubin (BR)-IXβ.

[0055] The term "biliverdin (BV)-Ixβ-reductase B (BLVRB)" refers to an enzyme that plays an important role in heme metabolism. Specifically, biliverdin reductase refers to an enzyme that catalyzes the reaction of reducing biliverdin to bilirubin. Here, "biliverdin (BV)" is a green pigment produced during the breakdown of heme, and "bilirubin (BR)" is a yellow pigment that is ultimately excreted from the body through bile. Biliverdin reductase carries out the above reaction using NADPH as a coenzyme. This enzyme is found in liver cells, the spleen, and other tissues, and is known to possess antioxidant and cytoprotective functions.

[0056] The term "prevention" above refers to any act of suppressing or delaying the onset of thrombocytopenia, and the term "treatment" above refers to any act of improving or beneficially altering thrombocytopenia by administering a pharmaceutical composition according to one aspect.

[0057] In one aspect, the "pharmaceutical composition" may be provided as a pharmaceutical composition comprising an active ingredient alone or comprising one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0058] Specifically, the carrier may be, for example, a colloidal suspension, powder, saline solution, lipid, liposome, microsphere, or nano-spherical particle. These may form a complex with or be associated with a transport means and may be transported in vivo using a transport system known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption-enhancing substances, or fatty acids.

[0059] When the above pharmaceutical composition is formulated, it may be prepared using diluents or excipients such as commonly used lubricants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, fillers, volume expanders, binders, wetting agents, disintegrants, and surfactants. Solid dosage forms for oral administration may include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above pharmaceutical composition. In addition, lubricants such as magnesium stearate and talc may also be used in addition to simple excipients. Liquid formulations for oral administration include suspensions, oral liquids, emulsions, syrups, etc., and may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents like water and liquid paraffin. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. Witepsol, macrogol, Tween 61, cacao gelatin, laurin gelatin, glycerogelatin, etc. may be used as bases for suppositories, and known diluents or excipients may be used when manufactured in the form of ophthalmic preparations.

[0060] The above pharmaceutical composition may be administered parenterally or orally, such as external application to the skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intra-arterial injection, intramedullary injection, intracardiac injection, intrathecal injection, transdermal injection, nasal injection, enteral injection, local injection, sublingual injection, or intrathoracic injection.

[0061] The above pharmaceutical composition is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0062] The above administration may be given once a day or divided into several doses. For example, it may be given every other day or once a week.

[0063] The above pharmaceutical composition may be provided by mixing it with a conventionally known pharmaceutical composition for the prevention or treatment of thrombocytopenia or a newly developed pharmaceutical composition for the prevention or treatment of thrombocytopenia. If the above pharmaceutical composition further comprises a pharmaceutical composition for the prevention or treatment of thrombocytopenia, it is important that an amount is mixed such that the maximum effect can be obtained with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

[0064] In addition, in one aspect, the pharmaceutical composition may be administered alone or in combination with other treatments for thrombocytopenia. That is, the pharmaceutical composition may be administered in conjunction with a known composition or other treatments for thrombocytopenia that have a preventive or therapeutic effect on thrombocytopenia, and may be administered simultaneously, separately, or sequentially, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art.

[0065] In addition, the present invention provides a pharmaceutical preparation for the prevention or treatment of thrombocytopenia comprising the above pharmaceutical composition.

[0066] The term "injectable" above refers to a solution, suspension, emulsion, or solid sterile preparation that is administered directly into internal tissues or organs, such as subcutaneously, intramuscularly, or into blood vessels, or is dissolved in a solvent or suspended therein for use.

[0067] The term "injectable" above refers to a drug that is inserted into the body through the urethra, anus, vagina, etc., and dissolves due to body heat or secretions to produce its medicinal effect.

[0068] The term "spray" above refers to a medicine that is sprayed out like a mist using a device.

[0069] The term "liquid formulation" above refers to a pharmaceutical form that provides a drug in liquid form, and is a dosage form containing an active ingredient in liquid form.

[0070] The term "patch" above refers to a preparation designed to provide a continuous therapeutic effect when applied to the skin. Patches have the advantage of a lower risk of side effects, such as gastrointestinal or liver damage associated with oral medications, and can be administered to patients for whom oral administration is difficult. Furthermore, since they are absorbed directly into the bloodstream through the skin without undergoing hepatic metabolism, they can produce the same therapeutic effect without side effects at a lower dose compared to oral medications.

[0071] In one embodiment of the present invention, the formulation may be an injectable formulation, an infusion formulation, a spray formulation, a liquid formulation, or a patch formulation.

[0072] In addition, the present invention provides a health functional food composition for the prevention or improvement of thrombocytopenia, comprising olsalkene and a pharmaceutically acceptable salt thereof as an active ingredient.

[0073] In one embodiment of the present invention, the thrombocytopenia may be one or more selected from the group consisting of non-immunogenic thrombocytopenia, immunogenic thrombocytopenia, and drug-induced thrombocytopenia.

[0074] In another embodiment of the present invention, the composition may inhibit the activity of biliverdin (BV)-Ixβ-reductase B (BLVRB).

[0075] In another embodiment of the present invention, the composition may inhibit the reduction of biliverdin (BV)-IXβ to bilirubin (BR)-IXβ.

[0076] The term "improvement" above may refer to any action that at least reduces parameters related to the condition being treated, for example, the severity of symptoms.

[0077] In one aspect, the above-mentioned health functional food may be used to prevent or improve thrombocytopenia, either simultaneously with or separately from a drug for treatment, before or after the onset of the disease.

[0078] In the above-mentioned health functional food, the active ingredient may be added directly to the food or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The amount of the active ingredient may be appropriately determined according to its purpose of use (for prevention or improvement). Generally, when manufacturing food or beverages, the above-mentioned health functional food may be added in an amount of about 15% by weight or less, specifically about 10% by weight or less, with respect to the raw materials. However, in the case of long-term consumption for the purpose of health and hygiene or health control, the above amount may be less than the above range.

[0079] The above-mentioned health functional food may be formulated into one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, by further including one or more of a carrier, a diluent, an excipient, and an additive. Foods to which a compound according to one aspect may be added include various types of food, powders, granules, tablets, capsules, syrups, beverages, gum, tea, vitamin complexes, health functional foods, etc.

[0080] Specific examples of the above carrier, excipient, diluent, and additive may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0081] In addition to containing the active ingredient mentioned above, the above-mentioned health functional food may contain other ingredients as essential components without special limitations. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients, such as in ordinary beverages. Examples of the above-mentioned natural carbohydrates may be monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; polysaccharides, e.g., dextrin, cyclodextrin, etc., and conventional sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used. The proportion of the above-mentioned natural carbohydrates may be appropriately determined by the choice of a person skilled in the art.

[0082] In addition to the above, a health functional food according to one aspect may contain various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients may be used independently or in combination, and the proportion of these additives may also be appropriately selected by a person skilled in the art.

[0083] The above-mentioned health functional food may be provided in combination with a conventionally known health functional food for the prevention or improvement of thrombocytopenia or a newly developed health functional food for the prevention or improvement of thrombocytopenia. If the above-mentioned health functional food further includes a health functional food for the prevention or improvement of thrombocytopenia, it is important that an amount is mixed such that the maximum effect can be obtained with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

[0084] In addition, the above-mentioned health functional food may be consumed alone or in combination with the above-mentioned health functional food for the prevention or improvement of thrombocytopenia. The above-mentioned health functional food may be consumed in conjunction with a known composition having an effect of preventing or improving thrombocytopenia or another health functional food for the prevention or improvement of thrombocytopenia, and may be consumed simultaneously, separately, or sequentially, and may be consumed as a single or multiple times. It is important to consume an amount that obtains maximum effect with a minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art.

[0085] The present invention will be explained in more detail below through examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0088] Examples

[0089] Experimental Materials and Methods

[0090] 1. Chemical substances

[0091] Olsalazine, Floxin-B, FMN, NADH, NAD + , NADPH, NADP + All chemicals, including methylhydroquinone (2-MHQ), were purchased from Sigma Aldrich (USA) unless otherwise specified. 15 N-ammonium chloride, 13 C-glucose and 2 H-dimethyl sulfoxide (d6-DMSO) was purchased from Cambridge Isotope Laboratories.

[0093] 2. Chemical Synthesis of Olsalken (OSK)

[0094] All reagents, starting materials, and anhydrous organic solvents with a purity of over 99.9% were sourced from Sigma Aldrich, TCI. Thin-layer chromatography (TLC) was performed using silica gel 60 F. 254 The study was performed on Merck aluminum sheets using [method] and visualized by staining with UV light, phosphomolyvic acid, and KMnO4. For the purification of the compounds, column chromatography was performed on Merck silica gel 60 (230-400 mesh). 1 The 1H NMR spectrum was recorded on a Bruker DRX-400 spectrometer. Chemical shift ( δ) It is reported as parts per million (ppm) measured relative to an internal standard and the coupling constant ( J) It was expressed in Hertz (Hz). The product mass was measured using a Shimadzu (MALDI-TOF) mass spectrometer.

[0095] [All Salken's Synthesis]

[0096]

[0098] 5-formyl-2-hydroxybenzoic acid (1 g, 0.0060 mol), methyl iodide (1.49 ml, 0.0240 mol), and dried K2CO3 (2.91 g, 0.0210 mol) were added to dry DMF solvent contained in a 100 ml two-necked round-bottom flask stored overnight in a nitrogen gas environment with a magnetic stirrer, and the reaction progress was monitored via TLC analysis. After completion, the crude mixture was quenched with water (3 × 25 ml), extracted with dichloromethane, and purified by column chromatography to obtain methyl 5-formyl-2-methoxybenzoate (950 mg). Separately, Zn powder (1.01 g, 0.0224 mol) was taken in THF at 0°C, TiCl4 (2.12 g, 0.0112 mol) was added for 30 minutes, the solution was heated continuously from room temperature to 60°C for 2 hours, and then the reaction mixture, methyl 5-formyl-2-methoxybenzoate (500 mg, 0.0028 mol) was added dropwise to THF and refluxed for 4 hours, then further quenched with K2CO3 and extracted with DCM-provided (E)-dimethyl 5,5'-(ethene-1,2-diyl)bis(2-methoxybenzoate) (150 mg).

[0099] At -78℃ DCM ( EBBr3 (240 μL, 0.0014) was added to a 10 ml round-bottom flask containing a )-dimethyl 5,5'-(ethene-1,2-diyl)bis(2-methoxybenzoate) (100 mg, 0.0003 mol) solution and stirred at room temperature for 5 hours. Then, 1 ml of 1 M HCl was added for quenching and stirred well for 15 minutes, after which the solvent was removed under reduced pressure, the crude mass was dissolved in ethyl acetate, and washed twice with water and salt water. The resulting mixture was dissolved in 95% ethanol, and then potassium hydroxide solution was added and stirred under reflux conditions for 3 hours. After precipitation occurred, the reaction mixture was extracted by acidifying it again with 1 M HCl and brine solution and recrystallized in ethyl acetate solvent. Finally, a colorless crystalline solid of (E)-5,5'-(ethene-1,2-diyl)bis(2-hydroxybenzoic acid) (60 mg, 72%) was obtained. 1 It was further confirmed through H NMR and mass spectrum analysis. 1 ¹H NMR (CDCl₃, 400 MHz) δ 6.96 (d, 2H, J = 8.64 Hz), 7.12 (s, 2H), 7.79 (d, 2H, J = 8.56 Hz), 7.96 (s, 2H); C 16 H 12 MALDI-TOF m / z calcd for O6: 300.27, 322.5044 (M+Na) found respectively.

[0101] 3. Expression and Partial Purification of AzoR

[0102] E. coli DH5α cells were cultured in LB medium to obtain AzoR protein at 37°C. Various hydrophilic quinones encoding AzoR protein acpD Because it improves gene mRNA levels, 600nm (OD) 600When the optical density of the cells reached 0.8, 0.5 mM methylhydroquinone (2-MHQ) was added to the culture medium. Cells were harvested by centrifugation after 3–4 hours, and anion exchange column chromatography using a Hitrap-Q HP column (Cytiva) was performed to concentrate AzoR protein. The column was pre-calibrated with buffer (pH 8.0, 25 mM Tris-HCl), and the protein was eluted by applying a 1.0 M NaCl gradient. The AzoR activity of the eluted fraction was measured by an enzyme activity assay using 0.1 mM menadione and 0.1 mM NADH, and the decrease in absorption at 340 nm was measured as in previously reported methods. A 0.1 mM stock solution of menadione was prepared in 100% DMSO. The eluted fraction was diluted 100-fold prior to analysis. Finally, the elution fraction with the highest activity was diluted 1,000-fold in phosphate-based saline (PBS) buffer and used for the cleavage analysis of olsalazine, to which 1% DMSO was additionally added to dissolve 0.1 mM menadione.

[0104] 4. Expression and Purification of BLVRB

[0105] N-terminal His6-tagged BLVRB was expressed in E. coli BL21 DE3 strains using the pET-21b protein expression vector. Transformed E. coli Cells were cultured in LB and M9 media at 37°C to produce unlabeled and isotope-labeled ( 15 N or 13 C / 15 N) BLVRB protein was expressed. To induce protein expression, the culture medium OD 600Upon reaching 0.8, 0.5 mM IPTG was added, and cells were collected after 3–4 hours and lysed by sonication in buffer (pH 8.0, 25 mM Tris-HCl, 500 mM NaCl, 10 mM β). The BLVRB protein was first purified by His-tag affinity column chromatography using a HisTrap HP column (Cytiva). To remove the His-tag, thrombin (~5 units per mg protein) was added to the protein eluent, and the mixture was dialyzed overnight at 4°C in buffer (pH 8.0, 50 mM Tris-HCl, 1 mM dithiothreitol). Since thrombin bound to the HiTrap-Q HP column at pH 8.0, ion exchange column chromatography was performed with buffer (pH 8.0, 25 mM Tris-HCl, and 1 mM dithiothreitol), and then Elution was performed using a 1.0 M NaCl gradient to elute BLVRB and thrombin at ~150 and ~300 mM NaCl, respectively. To separate His6-tagged BLVRB from completely cleaved BLVRB, a HisTrap HP column was attached alongside a HiTrap-Q column. BLVRB proteins were further purified by size exclusion chromatography (SEC) using a HiLoad 16 / 600 Superdex 75 (Cytiva) with unbuffered solution (50 mM NaCl and 1 mM dithiothreitol). Protein fractions were concentrated using an ultracentrifugation filter on a 10 kDa MWCO (Millipore) and stored at -70°C prior to use.

[0106] Purified BLVRB was denatured in a buffer (pH 7.5, 25 mM Tris-HCl, 10 mM β-mercaptoethanol, and 6.0 M urea) and then applied to a HisTrap HP column. After washing with a buffer (pH 5.0, 25 mM Na-acetate, 10 mM β-mercaptoethanol, and 6.0 M urea), it was eluted with an additional 1.0 M NaCl gradient. After 2-fold dilution with a buffer (pH 5.0, 20 mM Na-acetate, 10 mM β-mercaptoethanol, and 6.0 M urea), apo-BLVRB was further purified by cation exchange column chromatography using a HiTrap-SP column (Cytiva). Protein elution was performed with a 1.0 M NaCl gradient in the presence of 6.0 M urea. The protein eluent was reconjugated by dialyzing in a buffer (pH 6.5, 50 mM Bis-tris, 150 mM NaCl, and 1 mM DTT). After removing aggregates (~20%) by centrifugation, the solution was purified by SEC using a HiLoad 16 / 600 Superdex 75 with a buffer (pH 6.5, 10 mM Bis-tris, 50 mM NaCl, and 1 mM DTT). The apo-BLVRB solution was concentrated and stored at -70°C. Upon thawing the frozen sample, approximately 10% of apo-BLVRB precipitates, but whether the structure is maintained is 1 H- 15 It was confirmed through the N HSQC spectrum.

[0108] 5. Crystallization and Structural Determination of the BLVRB:OSK Complex

[0109] 1mM NADPH + The protein was added to the BLVRB protein solution and further purified by size exclusion chromatography in buffer (pH 8.0, 20 mM Tris-HCl, 150 mM NaCl, and 1 mM DTT) using a HiLoad 16 / 600 Superdex 75. The protein fraction was concentrated using a 10 kDa MWCO ultracentrifuge filter. The protein supernatant (14.8 mg / ml) was integrated and stored at -70°C before use.

[0110] BLVRB crystals were grown in a buffer solution (pH 6.5, 0.1 M bis-tris, 1.9–2.0 M ammonium sulfate) using the hanging drop vapor diffusion method at 18°C. To immerse OSK molecules, the crystals were immersed in 1 mM OSK and 0.2 mM NADP. + Incubated at 18°C ​​for 1 day in the same crystallization buffer containing [the substance]. Since the OSK stock solution (100 mM) was prepared with 100% DMSO, 1% DMSO was additionally present in the immersion solution. Crystals of the BLVRB:OSK complex were loop-frozen and rapid-frozen in liquid nitrogen using 20–25% glycerol as a cryoprotectant.

[0111] X-ray diffraction data were collected at beamline 5C of the Pohang Accelerator Laboratory. After processing the diffraction data using the HKL-2000 program, the structure was solved via molecular substitution (MR) using previously reported BLVRB coordinates (PDB code, 7ERA). The structural model was refined by alternating model building using the Coot program and refinement using the PHENIX software package. Structural visualization was performed using the Chimera program. Schematic diagrams of the interaction networks for OSK and OSA combined with BLVRB were generated using LIGPLOT.

[0113] 6. Enzyme activity analysis.

[0114] Enzyme kinetics were monitored using the absorbance of NADPH at 340 nm with a SpectraMax Gemini EM Microplate Reader (Molecular Devices). Enzyme reactions were performed by adding 0.5 μM BLVRB, 100 μM FMN, and 100 μM NADPH to PBS buffer containing 0.5% DMSO in the presence of various concentrations of inhibitors (OSK and OSA). The initial reaction rate was estimated from data collected over 5 minutes and fitted to the Michaelis-Menten equation. The inhibition model and inhibition constants were determined using an in-house Python script for nonlinear least-squares regression fitting.

[0116] 7. Isothermal Titration Calorific Value (ITC)

[0117] Isothermal Titration Calorimetry (ITC) experiments were performed using a Microcal Auto-iTC200 (Malvern Instruments) in a buffer solution (pH 6.5, 50 mM Bis-tris, 50 mM NaCl, 0.1 mM TCEP) at 25°C. 0.09 mM BLVRB and 1.1 mM drugs (OSK or OSA) were loaded into a calorimetry cell (200 μl) and a syringe (40 μl), respectively. ITC data were analyzed after subtracting the heat from blank injections measured under the same conditions but without BLVRB. Data processing was performed using Origin software provided by the manufacturer, and fitting was performed using a single-site coupled model.

[0119] 8. NMR Experiment

[0120] All NMR experiments were performed at 25°C using a Bruker 800 MHz spectrometer. 0.2 mM 15 N-labeled BLVRB or apo-BLVRB is prepared in a buffer (pH 6.5, 50 mM Bis-tris, 50 mM NaCl, 5% D2O) and depending on the presence or absence of drug molecules 1 H- 15N Heteronuclear Single Quantum Coherence (HSQC) spectra were recorded. One-dimensional (1D) for monitoring enzyme reactions 1 H spectra were performed in PBS buffer. Chemical shift perturbation (CSP) data (Δ 2 + [(Δ 2 Using the square root formula 1 H and 15 The relative effect of N from CS was normalized and presented. Diffusion-order spectroscopy (DOSY) spectra were measured using ledbpgppr2s with a diffusion delay of 80 milliseconds, a relaxation delay of 2 seconds, and 32 gradient intensity increments.

[0122] Experimental results

[0123] 1. Confirmation of prevention of azoreductase cleavage by substituting diazenyl with an alkene bond to enhance stability

[0124] The inventors of the present invention have partially purified E. coli ( E. coli Using the AzoR protein, we tested whether OSA was cleaved into 5-aminosalicylic acid (5-ASA) (Fig. 1). We enhanced the expression of AzoR by adding methylhydroquinone (2-MHQ) to the cell culture.

[0125] As a result, when OSA is cultured with AzoR and NADPH, the diazenyl bond of OSA is cleaved and 5-ASA, an active compound responsible for the therapeutic effect on intestinal diseases, which is OSA's original use, is formed (Figs. 2 and 3). In addition, the binding of 5-ASA, a product of the AzoR catalyst, to BLVRB is significantly reduced (Fig. 4).

[0126] To this end, the inventors developed a new synthesis method that replaces diazenyl bonds with alkene bonds while maintaining a trans configuration.

[0127] As a result, an olsalken (OSK) was generated, and it was confirmed that this olsalken remained intact and was not cut by AzoR (Fig. 5).

[0129] 2. Thermodynamic Analysis of the BLVRB and OSK Combination

[0130] Thermodynamic quantities such as ΔH and ΔS were characterized using an isothermal titration calorimeter (ITC) that directly measures the heat generated during the formation of the BLVRB-OSK composite at a constant temperature.

[0131] As a result, the dissociation constant of OSK (K D The value was found to be 140 nm (Table 1, Fig. 6), indicating that OSK acts as a potent and efficient binder for BLVRB. Comparative analysis with the previously identified xanthen-based inhibitor Floxin B revealed that OSK exhibits a tighter binding affinity. Furthermore, as indicated by the enthalpy contribution observed in the ITC data, OSK demonstrated more specific interactions with BLVRB compared to xanthen-based inhibitors (Table 1). The binding of OSK is primarily driven by enthalpy changes, reflecting specific interactions between BLVRB and the compound. The difference in thermodynamic values ​​between OSA and OSK can be explained by the substitution of diazenyl bonds with alkene bonds. In summary, the findings of the ITC study support the existence of specific interactions between BLVRB and OSK.

[0132] [Table 1] Representative ITC data for the combination of each drug and BLVRB

[0133]

[0135] 3. Combined analysis of BLVRB and OSK

[0136] Protein-based NMR spectroscopy is gaining prominence as a highly effective method for identifying intermolecular interactions, including those involving small molecules and proteins. Furthermore, protein-based NMR methods provide valuable insights into binding sites crucial for optimizing the positioning of small molecules within complexes. Generally, this includes 1 H- 15It involves monitoring changes in the chemical shift of the N HSQC spectrum. Assigning amide resonances is essential for interpreting the HSQC spectrum, and this is performed by the HNCA and HN(CO)CA spectra, which provide 95% of the backbone resonance assignments.

[0137] Accordingly, the inventors titrated OSK in gradually increasing amounts to monitor changes in chemical movement upon binding. 15 Using N-labeled BLVRB 1 H- 15 N HSQC experiments were performed. It was noted that previous studies have shown that the FMN binding site of BLVRB is utilized for OSA binding, and that Ser111 is a major residue in the catalytic mechanism of BLVRB.

[0138] Accordingly, by monitoring changes in the amide chemical shift in the HSQC spectrum, it was observed that when OSK binds to BLVRB, it induces changes in the amide resonance of the FMN binding site (Fig. 7A). In addition, chemical shift perturbation (CSP) was analyzed (Fig. 7B), and OSK was mapped to the BLVRB surface structure (Fig. 7C) to successfully verify the effect of OSK binding to the BLVRB surface (Fig. 7D). Through this analysis, it was clearly confirmed that the FMN binding site, specifically the xanthine ring of FMN, is the binding site of OSK.

[0140] 4. X-ray crystal structure analysis of the BLVRB-OSK complex

[0141] The inventors analyzed the structure of the complex through the determination of the BLVRB-OSK complex to analyze in detail the binding of Olsalken with BLVRB.

[0142] As a result, NADP + The crystal structure of BLVRB coupled to OSK was determined with a resolution of 1.7 Å. The crystal belongs to space group P21212, and each crystal asymmetric unit contains two BLVRB molecules.

[0143] The overall structure of BLVRB complexed with OSK is very similar to the previously determined structure. For example, the backbone RMSD of the complex is BLVRB-NADP + -FMN(PDB ID: 1HE4) and BLVRB-NADP + Compared to the OSA (PDB ID: 7ERA) complex, the values ​​were 1.06 and 0.17 Å, respectively, indicating a high structural similarity between the OSK-combined BLVRB and the base substrate and the OSA-combined BLVRB (Fig. 8A).

[0144] Specifically, the binding site of OSK is NADP + It is closely associated with the nicotinamide moiety of the cofactor, and the xanthine ring of FMN adopts an orientation similar to xanthine-based inhibitors such as floxin B and erythrosine (Fig. 8B). The main driving force of OSK binding is NADP + These are ring stack interactions between the nicotinamide moiety and OSK, and hydrophobic interactions associated with residues S111, F113, W116, L125, V128, P152, and H153 (Fig. 8C). Additionally, favorable interactions are facilitated by positively charged residues R78, K120, R124, R170, and K178. In particular, OSK shifts the side chain of W116 (as observed in the FMN complex) toward the binding pocket by approximately 2.5 Å, and the side chain of R78 forms hydrogen bonds with OSK, potentially enhancing the binding of the drug compound. Furthermore, the orientation of R78 can strengthen hydrophobic interactions between the drug compound and BLVRB.

[0146] 5. Confirmation of BLVRB activity inhibition using Olsalken

[0147] The enzymatic kinetics of BLVRB in the presence of NADPH were investigated using the inhibitors OSK and OSA. The inhibition mechanism of BLVRB by OSK or OSA is shown in Fig. 9A. (Here, E represents BLVRB and I represents OSK or OSA.)

[0148] As a result, linear Weaver-Burk plots are competitive (K ic ) and non-competitive (K iu ) showed a mixed inhibition pattern characterized by an inhibition constant (Figs. 9B to 9C). The linear Weaver-Burk plot of OSK showed a maximum velocity (V) of 4.80 ± 0.16 μM / min. m ) and Michaelis constant (K) of 61.73 ± 5.28 μM m It showed ). A competitive inhibition constant (K) of 559.95 ± 100.98 nM. ic ) suggests that OSK competes with the substrate (FMN) to bind to the active site of BLVRB. Meanwhile, a non-competitive inhibition constant (K) of 606.14 ± 61.32 nM. iu ) is OSK is NADP + This indicates that the NADPH complex binds to both BLVRB, altering its conformation and inhibiting enzymatic activity (Fig. 9B). In the case of OSA, V of 4.71 ± 0.09 μM / min m and K of 61.57 ± 2.75 μM m Similar dynamics are observed. Competitive (K ic = 551.83 ± 38.65 nM) and non-competitive (K iu (= 787.53 ± 55.17 nM) The inhibition constant is that OSA also competes with the substrate for active site binding, and NADP + and suggests that the NADPH complex interacts with BLVRB (Fig. 9C).

[0149] Overall, the mixed inhibition pattern observed in OSK and OSA suggests that these inhibitors are NADP + This suggests that the NADPH complex can bind to both BLVRB and affect BLVRB activity. Additionally, both OSK and OSA can bind to apo-BLVRB, which is distinct from the xanthine-based inhibitor Floxin-B, implying better inhibitory activity for OSK (Fig. 10).

[0151] These experimental results indicate that the Olsalken of the present invention inhibits the activity of BLVRB and can be usefully utilized for the prevention and treatment of thrombocytopenia.

[0153] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

Claim 1 delete Claim 2 A method for preparing an olsalkene represented by the following chemical formula 1, comprising the following steps: reacting 5-formyl-2-hydroxybenzoic acid with methyl iodide to prepare an intermediate; reacting the intermediate with TiCl4 to prepare a compound represented by the following chemical formula 2; and reacting the compound represented by the following chemical formula 2 with BBr3. [Chemical Formula 1] [Chemical Formula 2] Claim 3 A pharmaceutical composition for the prevention or treatment of thrombocytopenia comprising, as an active ingredient, an olsalkene represented by the following chemical formula 1 and a pharmaceutically acceptable salt thereof. [Chemical Formula 1] Claim 4 A pharmaceutical composition according to claim 3, wherein the thrombocytopenia is one or more selected from the group consisting of non-immunogenic thrombocytopenia, immunogenic thrombocytopenia, and drug-induced thrombocytopenia. Claim 5 A pharmaceutical composition according to claim 3, wherein the composition inhibits the activity of biliverdin (BV)-Ixβ-reductase B (BLVRB). Claim 6 A pharmaceutical composition according to claim 3, characterized in that the composition inhibits the reduction of biliverdin (BV)-IXβ to bilirubin (BR)-IXβ. Claim 7 A pharmaceutical preparation for the prevention or treatment of thrombocytopenia comprising the pharmaceutical composition of claim 3. Claim 8 The pharmaceutical formulation of claim 7, wherein the formulation is an injectable formulation, an infusion formulation, a spray formulation, a liquid formulation, or a patch formulation. Claim 9 A health functional food composition for the prevention or improvement of thrombocytopenia, comprising as active ingredients an olsalkene represented by the following chemical formula 1 and a pharmaceutically acceptable salt thereof. [Chemical Formula 1] Claim 10 A health functional food composition according to claim 9, wherein the thrombocytopenia is one or more selected from the group consisting of non-immunogenic thrombocytopenia, immunogenic thrombocytopenia, and drug-induced thrombocytopenia. Claim 11 A health functional food composition according to claim 9, characterized in that the composition inhibits the activity of biliverdin (BV)-Ixβ-reductase B (BLVRB). Claim 12 A health functional food composition according to claim 9, characterized in that the composition inhibits the reduction of biliverdin (BV)-IXβ to bilirubin (BR)-IXβ.

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