WO2022145617A1 - Method for synthesizing novel compounds potassium all-trans retinoate and potassium 9-cis retinoate, and pharmaceutical composition for cardiovascular treatment comprising same - Google Patents

Abstract

Provided is a pharmaceutical composition for cardiovascular treatment comprising a potassium all-trans retinoate compound and a potassium 9-cis retinoate compound. It has confirmed that the pharmaceutical composition is metabolically stable without being dissolved or destroyed in water and artificial gastric juice. The pharmaceutical composition has the effects of inhibiting disorders, observed in the process of pathogenesis of atherosclerosis, which cause an inflammatory response due to decreased immunity, accumulate cholesterol in the blood by the ingestion of cholesterol, and are caused in the process of decomposing cholesterol in macrophages, inhibiting stenosis due to cholesterol which is deposited in blood vessels by blood coagulation of cholesterol, fibrinogel, and P-selection, inhibiting the process of a plaque phenomenon caused by calcium ions, alleviating a developed disease, or removing the plaque.

Description

Method for synthesizing novel compounds POTASSIUM ALL-TRANS RETINOATE and POTASSIUM 9-CIS RETINOATE, and pharmaceutical composition for cardiovascular treatment comprising the same

The present invention provides a method for absorption of 9-cis β-carotene into the intestines without decomposition in the stomach in consideration of the human body absorption and biologic synthesis route so that 9-cis β-carotene is not excreted during oral administration. It relates to a method for synthesizing Potassium all-trans retinoate and Potassium 9-cis retinoate, which can be easily delivered and can increase absorption, and a pharmaceutical composition for cardiovascular treatment comprising the same.

Research and development of 9-cis β-carotene as a cardiovascular treatment, that is, atherosclerosis treatment and angina pectoris treatment has progressed a lot. 9-cis β-carotene is one of the carotenoid components and is a general term for a group of yellow, orange, and red pigments widely seen in the biological world. It is an unstable substance that is easy to do. In addition, it is not soluble in water, and unlike flavonoid pigments or betalain pigments with the same color, it dissolves well in solvents that dissolve fat, such as benzene and ether.

9-cis β-carotene is a powerful antioxidant known to lower the risk of cancer and cardiovascular disease. In addition, it has a protective effect against skin damage caused by sunlight and prevents the generation of wrinkles or age spots, and even exhibits an effect of delaying aging. And, it is a carotenoid-based substance that prevents complications from diabetes, improves lung function, and has antibacterial action.

9-cis β-carotene is a different isomer from the well-known all-trans β-carotene. When administered orally, 9-cis β-carotene is absorbed into the body through the stomach and intestines and is separated into 9-cis retinoid and all-trans retinoid. Thereafter, 9-cis retinoic acid and all-trans retinoic acid are biosynthesized in the human body by liver metabolism. 9-cis retinoic acid in the form of biosynthesized retinoic acid acts on the retinoid X receptor of macrophages.

It is a kind of nuclear receptor that regulates gene expression. It forms several heterodimers with subfamily 1 nuclear receptors CAR, FXR, LXR, PPAR, PXR, RAR, TR and VDR. LXL/RXR and PPAR/RXR heterodimers are involved in the pathogenesis of atherosclerosis. That is, in macrophage metabolism, LDL (low-density lipoprotein) is broken down into HDL (high-density lipoprotein) and released into the blood.

All-trans retinoic acid is a vitamin A derivative that acts by binding to two nuclear receptor families (retinoic acid receptors (RAR) and retinoid X receptors (RXR (RXR)) within the cell. Normalization of follicular keratinization and cohesion of keratinocytes. As a result, follicle occlusion and microcomedon formation are reduced, and all-trans retinoic acid inhibits inflammation and platelet activation, and has the effect of reducing the expression of P-selection and fibrinogen.

However, 9-cis β-carotene has a problem in that most of it is not absorbed and is excreted with a LogP value of >9, which measures the absorption rate in the intestine when administered orally. Accordingly, the present invention develops a chemical formulation that is not decomposed from the stomach in an isolated form, is easily delivered to the intestine, and increases the absorption rate in consideration of the human body absorption and biologic synthesis route of 9-cis β-carotene, and the solubility of the substance in water is improved. An object of the present invention is to provide a pharmaceutical composition for cardiovascular treatment, including Potassium all-trans retinoate and Potassium 9-cis retinoate, which improves absorbability by increasing and is not easily destroyed by gastric juice and can be safely delivered to the intestine.

The present invention is a powerful antioxidant and 9-cis β-carotene, which lowers the risk of cancer and cardiovascular disease, has a LogP value of >9 that measures absorption in the intestine when orally administered. To provide a method for synthesizing Potassium all-trans retinoate and Potassium 9-cis retinoate in chemical formulations that are not degraded in the stomach and easily delivered to the intestine and increase absorption, and a pharmaceutical composition for cardiovascular treatment or a pharmaceutical composition for inhibiting blood clotting comprising the same want to

The present invention is an all-trans retinal synthesis step for synthesizing all-trans retinal by inducing a Knoevenagel condensation reaction of β-C15 aldehyde and 3-Methyl-2-butenal with the method for synthesizing Potassium all-trans retinoate and Potassium 9-cis retinoate. (go); preparing a solution of all-trans retinal in step (a) in methanol, and reacting it with a solution of Na2PO4 and KMnO4 in sterile distilled water (DW) to synthesize all-trans retinoic acid (B); Methyl b-formylcrotonate is added to EtOH and maintained by adding a 50% aqueous KOH solution, and after adding a 38.8% ethanol solution of 9-cis phosphonium chloride, a 50% aqueous KOH solution is added to maintain the reaction. Synthesis step of 9-cis retinoic acid (C ); To each of the all-trans retinoic acid of step (b) and 9-cis retinoic acid of step (c), after adding hexane, KOH was added dropwise to synthesize Potassium all-trans retinoate and Potassium 9-cis retinoate (d) may have been made

The present invention provides a pharmaceutical composition in the form of Potassium all-trans retinoate and Potassium 9-cis retinoate synthesized using all-trans retinoic acid and 9-cis retinoic acid as precursors as a pharmaceutical composition for cardiovascular treatment.

The present invention is a powerful antioxidant and 9-cis β-carotene, which has an effect of lowering the risk of cancer and cardiovascular disease, has a LogP value of >9, which measures the absorption rate in the intestine when administered orally, and prevents most of it from being absorbed and excreted. There is an effect that can be applied as a cardiovascular treatment that can be administered, that is, a treatment for atherosclerosis and treatment for angina.

In addition, according to the experimental example of the present invention, it was confirmed that it is not dissolved or destroyed in water and artificial gastric juice, and is metabolically stable. Disorders in the process of decomposing cholesterol in macrophages and cholesterol stenosis, which are deposited in blood vessels by blood coagulation by cholesterol, fibrinogel, and P-selection, and plaque caused by calcium ions It was confirmed that there is an effect of inhibiting the progression of the disease or of alleviating the disease and removing plaque.

Figures 1 to 19 of the present invention are the same as Figures 1 to 19 of the previously applied Korean Patent Application Nos. 10-2020-0185144 and 10-2021-90290.

1 shows the synthesis step of all-trans retinal and 9-cis phosphonium chloride (salt) of the present invention.

Figure 2 shows the all-trans retinoic acid synthesis step.

Figure 3 shows the synthesis step of 9-cis retinoic acid.

Figure 4 shows the Potassium all-trans retinoate synthesized according to the present invention.

5 shows Potassium 9-cis retinoate synthesized according to the present invention.

6 shows an IR analysis result according to Experimental Example 1 of the present invention.

7 shows ¹ H-NMR results according to Experimental Example 1 of the present invention.

8 is a UV-VIS spectrophotometer analysis result according to Experimental Example 1 of the present invention.

9 shows an IR analysis result according to Experimental Example 1 of the present invention.

10 shows ¹ H-NMR results according to Experimental Example 1 of the present invention.

11 is a UV-VIS spectrophotometer analysis result according to Experimental Example 1 of the present invention.

12 is a graph for confirming the concentration of the ISMC applied MTS Assay of P9CRA according to Experimental Example 2.

13 shows a Scatter Plot showing the distribution of changes in all genes according to Experimental Example 2.

14 shows an Intergrative Genomics Viewer (IGV) Mapping grip according to Experimental Example 2 of the present invention.

15 shows the number of genes corresponding to gene orthologs through DAVID analysis.

16 is a graph showing the concentration confirmation graph of the THP-1 cell applied MTS Assay of PATRA according to Experimental Example 3 of the present invention.

17 shows a scatter plot showing the distribution of changes in all genes according to Experimental Example 3 of the present invention.

18 shows an Intergrative Genomics Viewer (IGV) Mapping grip according to Experimental Example 3 of the present invention.

19 shows the number of genes corresponding to gene orthologs through DAVID analysis according to Experimental Example 3 of the present invention.

The present invention can provide a method for synthesizing all-trans retinoic acid and 9-cis retinoic acid from synthesized all-trans retinal and 9-cis phosphonium chloride (salt). In addition, after adding hexane to each of the synthesized all-trans retinoic acid and 9-cis retinoic acid, a pharmaceutical composition for cardiovascular treatment or blood clotting inhibition comprising the steps of obtaining the synthesized Potassium all-trans retinoate compound and Potassium 9-cis retinoate A pharmaceutical composition is provided.

The compounds all-trans retinal and 9-cis phosphonium chloride (salt) used in the synthesis of all-trans retinoic acid and 9-cis retinoic acid of the present invention are Korean Patent Application No. 10-2019-0137160 registered as an earlier application by the applicant of the present invention All-trans retinal and 9-cis phosphonium chloride (salt) produced during the synthesis of 9-cis β-carotene can be used (see Fig. 1)

1A shows the synthesis reaction of 2.2.6 trimethyl cyclohexanone and (Z)-3-Methylpent-2-en-4-yn-1-ol. 1) First, a solution of 10 g of 2.2.6 trimethyl cyclohexanone in 5 ml THF was stirred and mixed. 2) Stir 11.2 ml (1.5 fold) (Z)-3-Methylpent-2-en-4-yn-1-ol in 100 ml THF at 0-5° C. preferably at 0° C. and then into solution. Ethylmagnesiumbromide (3 folds) was added to a solution of 7 ml of etherial solution and stirred for 30 to 40 minutes until the temperature reached room temperature. 3) The mixture of 1) and 2) was saturated for 12 hours and then exchanged with Ammonium chloride (NH ₄ Cl). (The reaction was stopped by adding a saturated aqueous solution of ammonia chloride.) 4) This was purified on a silica gel column with hexane and ethylacetate solvent to obtain 13.5 g (yield 80%) of the product.

1B shows a process of mixing a Rochelle salt such as LiAlH4 as a reducing agent for reducing acetylene to E-ethylene. 1) Add 2g of lithium aluminum hydride (LiAlH4) and THF (100ml) slowly for safety and mix until emulsion of the mixed solution disappears. Then, 13.5 g of 2-Methyl-5-phenylpentan-2-ol formed above was mixed and stirred for 12 hours. 2) After cooling to 0°C, sodium sulfate (Na2SO4) was mixed and stirred for 30 minutes. 3) Filtered with Celite ^ㄾ filter to remove fine particles. 4) This was purified on a silica gel column with hexane and ethylacetate solvent to obtain 10.5 g (yield 70%) of the product.

1C shows a method for producing an aldehyde by the selective oxidation reaction of an alcohol. 1) A mixture of 5.75 g of manganese dioxide (MnO 2 ) was added to 10.5 g of the compound obtained in Example 2 above in 5 ml of DCM. 2) After completion of the reaction, the product was filtered through a Celite® filter and washed with DCM to obtain 7.40 g (yield 79%).

1D shows a method of synthesizing C ₁₈ hydroxy ester with Phosphonium ylides in the synthesized C ₁₅ Aldaehyde. 1) 7.0 g of the product obtained in Step 3 was dissolved in 10 ml of benzene, 24 g of Phosphonium ylides was added, and the reaction was allowed to proceed for 1 hour. 2) The stirred solvent was removed in vacuo to obtain 6.50 g of hydroxy ester (75%) by HPLC.

1E shows a reaction in which alcohol is dehydrated with HCOOH acid to form ethylene. Add 0.4 ml of 80% formic acid to 6.50 g of the above hydroxy ester solution dissolved in hexane. After stirring at room temperature for 12 hours, 3.9 g (yield 69%) was obtained.

1F shows a method of synthesizing C ₁₇ alcohol by mixing Rochelle salt and using THF as a reducing agent for reduction. 1) Slowly add 0.48 g of lithium aluminum hydride (LiAlH4) to THF (25ml) for safety and mix until emulsion of the mixed solution disappears. After completion, it was mixed with 3.5 g of the ester formed above and stirred for 12 hours. 2) After cooling to 0°C, sodium sulfate (Na2SO4) was mixed and stirred for 30 minutes. 3) Filtered with Celite filter to remove fine particles. 4) This was purified on a silica gel column with hexane and ethylacetate solvent to obtain a product of 2.19g (yield 70%).

1G shows a method for producing Aldehyde by selective oxidation of C ₁₇ alcohol. 1) To 1.50 g of the obtained product, 0.58 g of MnO 2 was added to 30 ml of DCM, and the mixture was stirred at room temperature for 30 minutes. 2) After completion of the reaction, the product was filtered through a Celite filter, and the Celite filter was washed with DCM. The solvent was removed under reduced pressure, and the compound 1.10 g (yield 74%) was confirmed by HPLC.

1H shows the reaction of alkylating ketone with methyl group through Grignard reaction. 1) To a stirred solution of 1.10 g of the above aldehyde in 20 ml THF at 0 °C was added 0.5 ml of Methylmagnesium bromide (MeMgBr). The solution was stirred at 0 °C for 30 min. 2) After completion of the reaction, quenched with a saturated solution of NHCl and extracted with ether. The extracted ether layer was washed with water and brine solution and dried over anhydrous sodium sulfate (NaSO4). The solvent was removed in vacuo, and the chromatographically purified compound was obtained in 55% yield.

I of FIG. 1 shows the process of inducing the Hydro Oxidation reaction. 1) Add 0.50 g of the above extract in 20 ml of DCM, add 0.18 g of MnO 2 , and stir the mixture at room temperature. After completion of the reaction, the product was filtered through a Celite filter. 2) Washed with DCM. The solvent was removed in vacuo and 0.39 g (79%) of the pure compound was obtained after chromatographic purification.

1 J shows the process of C = C coupling reaction of Aldehyde or ketone as an RO group. 1) To a suspension of a 50% dispersion of 4 mg sodium hydride (NaH) in 20 ml of THF, 0.14 ml of methyl diethylphosphonoacetate was added, and the reaction mixture was stirred until cooled and clear. 2) 1 g of ketone in THF was added dropwise, and the reaction was stirred until the reaction was completed at room temperature. The excess NaH was quenched with water and the reaction mixture was extracted with ether. The ether layer was washed with water, brine solution. 3) dried over anhydrous Na2SO4. The solvent was distilled off under reduced pressure, and methyl retinoate (0.88 g, 70% yield) was obtained by chromatographic purification.

1K shows a method for obtaining an alcohol group by reducing the ester group with a LiAlH4 reducing agent. 1) 0.106 g of lithium aluminum hydride (LiAlH4) was mixed with THF (45 ml), 0.88 g of the ester formed above was mixed, and the mixture was stirred for 12 hours. 2) After cooling to 0°C, sodium sulfate (Na2SO4) was mixed and stirred for 30 minutes. 3) Filtered with Celite ^ㄾ filter to remove fine particles. 4) This was purified on a silica gel column with hexane and ethylacetate solvent to obtain a product of 0.56 g 9-cis-retinol (yield 70%).

L in FIG. 1 represents a reaction for replacing an alcohol group with a phosphonium salt. 1) Drop a solution of 0.812 g PPh3HBr in methanol to a solution of 0.56 g 9-cis-retinol dissolved in 2.5 mL methanol. 2) Stir the mixed solution for 1 hour at room temperature under Argon filling. 3) Remove the solvent with a rotary vacuum. 4) Wash the by-products 5-6 times with 25mL hexane. 0.86 g of phosphonium salt (yield 70%) was obtained.

M in FIG. 1 shows the step of synthesizing all-trans retinal by inducing a reaction of Knoevenagel condensation between β-C15 aldehyde and 3-Methyl-2-butenal. 1) To a solution of 0.31 g of 3-Methyl-2-butenal in 8 mL of water, quickly add 0.12 g of β-C15 aldehyde and stir at room temperature. 2) After 5 minutes, filter the resulting solid and dry it. 0.39 g (yield 95%) of all trans retinal was obtained.

I. Synthesis and analysis of Potassium all-trans retinoate and Potassium 9-cis retinoate

1. All-trans retinoic acid synthesis step

Figure 2 shows the all-trans retinoic acid synthesis step. For the synthesis of all-trans retinoic acid of the present invention, a 0.25 M solution of all-trans retinal synthesized as shown in FIG. 1 was prepared in methanol, and 1 equivalent of Na2PO4 and KMnO4 were dissolved in sterile distilled water (DW) to prepare a 0.25M solution. Mix and stir at room temperature for 30 minutes. For crystallization of all-trans retinoic acid, it is dissolved in IPA (isopropyl alcohol, isopropanol) at 60~70℃, cooled slowly to 0~5℃ to crystallize, and the crystals are filtered and dried before storage.

2. Synthesis of 9-cis retinoic acid

Figure 3 shows the synthesis step of 9-cis retinoic acid. For the synthesis of 9-cis retinoic acid, methyl b-formylcrotonate is added to EtOH (80 mL), and 50% KOH aqueous solution is added for 20 minutes, and maintained at 0-5°C. And after adding 9-cis phosphonium chloride 38.8% ethanol solution for 20 minutes and maintaining it at 0-5 ℃, KOH 50% aqueous solution is added for 20 minutes, and the reaction is further maintained at 0-5 ℃.

Thereafter, sterile distilled water (DW) is added and the orange suspension is stirred for about 10 minutes to form a transparent orange, and then DW and MC are mixed 2:1 again and injected, and stirred for 10 minutes. After that, it is extracted about 3 times with MC. The MC layer solution is filtered, dried under vacuum, added with MeOH, stirred at 0-5° C. for 2 hours, and filtered. Crystallization (Crystallization) is dissolved in IPA (isopropyl alcohol, isopropanol) at 60 ~ 70 ℃ as described above, then slowly cooled to 0 ~ 5 ℃ to crystallize, filter, and store after drying.

3. Potassium all-trans retinoate and Potassium 9-cis retinoate synthesis step

4 shows Potassium all-trans retinoate obtained according to the synthesis of the present invention, and FIG. 5 shows Potassium 9-cis retinoate. After adding hexane to each of all-trans retinoic acid and 9-cis retinoic acid obtained in the all-trans retinoic acid synthesis step and the 9-cis retinoic acid synthesis step, the internal temperature was raised from room temperature to 40°C. After that, KOH was dissolved in a small amount of D.W and slowly added dropwise, stirred for 5 minutes while maintaining the temperature, and after the reaction was completed, the liquid phase was removed and the precipitate was dried, as shown in FIGS. 4 to 5, two types of Potassium retinoate was obtained.

In addition, the pharmaceutical compositions for cardiovascular treatment Potassium all-trans retinoate and Potassium 9-cis retinoate can be synthesized using all-trans retinoic acid and 9-cis retinoic acid known as Tretinoin and Alitretinoin as precursors.

Tretinoin (All-trans retinoic acid) is a treatment for acute promyeloid leukemia (undifferentiated myeloid leukemia, immature myeloblastic, mature myeloblastic, myelocytic monocytic, monocytic, megakaryotic and erythroleukemia). It is prescribed to be adjustable at 30 - 52 mg/m2 once a day, and is used as a treatment for suppressing bleeding tendencies such as intravascular coagulation disease (DIC).

Alitretinoin (9-cis retinoic acid) is known to treat severe/recalcitrant chronic hand eczema. For muscle efficacy and effectiveness, it is prescribed for adults with chronic severe hand eczema (PGA) who do not respond to strong topical steroid therapy for at least 4 weeks, to take 10 - 30 mg once a day with or immediately after a meal.

Hereinafter, the purity and yield of Potassium all-trans retinoate and Potassium 9-cis retinoate obtained from the above steps were confirmed by analysis through ① IR, ② ¹ H-NMR, and ③ UV-VIS spectrophotometer.

<Experimental Example 1> Potassium all-trans retinoate purity and yield confirmation

In order to confirm the purity and yield of the Potassium all-trans retinoate obtained according to the steps of the present invention, ① IR, ② ¹ H-NMR, ③ UV-VIS spectrophotometer analysis was performed.

6 shows an IR analysis result according to Experimental Example 1 of the present invention. As a result of analysis in the solid state with Thermo's iS50 ATR equipment, a clear symmetric stretch at 1400 cm ^-1 and a strong asymmetric stretch at 1600 cm ^-1 were found. This means that the carboxylic acid functional group of All trans retinoic acid was substituted with the caboxylate salt. Also, a significantly lower stretch was confirmed in the vicinity of 1700 cm ^-1 compared to the reagent All trans retinoic acid. This means that it has a property of a bond that is significantly different from that of the starting material.

7 shows ¹ H-NMR results according to Experimental Example 1 of the present invention. Bruker's 500MHz NMR equipment was used, and as the NMR solvent, DMSO was applied to the starting material (All trans retinoic acid) and the synthetic material (Potassium all trans retinoate). A~n above the peak on the NMR spectrum means hydrogen corresponding to a~n of each material structure drawn on the upper left of the analysis data. Comparing ¹ H-NMR spectrum of ATRA and PATRA, ATRA pattern is maintained while yellow It can be observed that the part marked with is shifted to the right. This means that electrons are concentrated when -OH of ATRA is substituted with O ^- K ⁺ .

8 shows the results of UV-VIS spectrophotometer analysis according to Experimental Example 1 of the present invention. Using Scinco's S-3100 equipment and PerkinElmer's Lambda 365 equipment (by concentration), the PATRA compound dissolved in water was placed in a disposable plastic cell and UV-VIS spectrophotometer was measured. As a result of the analysis, UV absorbed by all trans retinoic acid Spectrum results of similar patterns were shown in the wavelength range (300-400nm). Through these results, it can be confirmed that the basic retinoic acid structure did not change while having the property of dissolving in water due to the binding of potassium. (The noise peak at 200nm is generated by the disposable plastic cell and is ignored)

In addition, the concentration range of the PATRA compound was set in units of 15ppm - 30ppm, and UV-VIS Spectrophotometer was measured at 5ppm intervals, and a calibration curve with reliability (R ² ) 0.9925 value (closer to 1, higher reliability) was drawn through the result. . This is later used to measure the purity of the material to be synthesized. UV-VIS spectrophotometer; The absorbance curve was shown in the 250nm-450nm region, and the highest absorbance value was shown in the wavelength band 340nm.

<Experimental Example 2> Potassium 9-cis retinoate purity and yield confirmation

In order to confirm the purity and yield of the Potassium 9-cis retinoate obtained according to the steps of the present invention, ① IR, ② ¹ H-NMR, ③ UV-VIS spectrophotometer analysis was performed.

9 shows an IR analysis result according to Experimental Example 1 of the present invention. As a result of analysis in the solid state with Thermo's iS50 ATR equipment, a clear symmetric stretch at 1400 cm ^-1 and a strong asymmetric stretch at 1600 cm ^-1 are shown. This means that the carboxylic acid functional group of 9-cis retinoic acid was substituted with the caboxylate salt, and a significantly lowered stretch was confirmed in the vicinity of 1700 cm ^-1 compared to the reagent 9-cis retinoic acid. This means that it has a property of a bond that is significantly different from that of the starting material.

10 shows ¹ H-NMR results according to Experimental Example 1 of the present invention. Bruker's 500MHz NMR equipment was used, and as the NMR solvent, DMSO was applied to the starting material (9-cis retinoic acid) and the synthetic material (Potassium 9-cis retinoate) in the same way. A~n above the peak on the NMR spectrum means hydrogen corresponding to a~n of each material structure drawn on the upper left of the analysis data. Comparing the ¹ H-NMR spectrum of 9CRA and P9CRA, it can be observed that the pattern of 9CRA is maintained while the part marked in yellow is shifted to the right. This means that electrons are concentrated when -OH of ATRA is substituted with O ^- K ⁺ .

11 shows the results of UV-VIS spectrophotometer analysis according to Experimental Example 1 of the present invention. Using PerkinElmer's Lambda 365 equipment, the P9CRA compound dissolved in water was placed in a disposable plastic cell and UV-VIS spectrophotometer was measured. As a result of the analysis, a similar pattern was observed in the UV wavelength band (300-400 nm) absorbed by 9-cis retinoic acid. Spectrum results are shown. Through these results, it can be confirmed that the basic 9-cis retinoic acid structure did not change while having the property of dissolving in water due to the binding of Potassium.

In addition, the concentration range of the P9CRA compound was set in units of 20ppm to 35ppm, and UV-VIS Spectrophotometer was measured at 5ppm intervals, and a calibration curve of reliability (R ² ) 0.9941 value (closer to 1, higher reliability) was drawn through the result. . This is later used to measure the purity of the material to be synthesized. UV-VIS spectrophotometer: Absorbance curve was shown in the 290nm-400nm region, and the highest absorbance value was shown in the wavelength band 337nm.

I. Efficacy evaluation of Potassium all-trans retinoate and Potassium 9-cis retinoate

In order to establish a new mechanism of action and evaluate the efficacy of the Potassium all-trans retinoate and Potassium 9-cis retinoate type compounds completed by further synthesizing raw materials that can act on cardiovascular treatment using alitretinoin and Tretinoin as precursors, cardiovascular disease-related The pathway was confirmed through NGS test and analysis of the mechanism of action.

In addition, based on the analysis data of the chemical and biological properties of physical properties, the amount of drug applied to blood and blood vessels related cells was determined through NTS Assay to determine the amount of drug applied as a cardiovascular treatment agent.

Next-Generation Sequencing (NGS) by applying Potassium 9-cis-retinoate (P9CRA) to fibroblast cells and Primary Human Intestinal Smooth Muscle Cells (ISMC) to confirm the gene response of drugs in vascular cells ) and analyzed the data.

To confirm the gene response of the drug in blood cells, Potassium All trans retinoate (PATRA) was applied to mononuclear cells (THP-1 cells), Next-Generation Sequencing (NGS) was performed, and data were analyzed.

<Experimental Example 1> Chemical and biological stability of Potassium all trans retinoate (PATRA) & Potassium 9-cis-retinoate (P9CRA)

In Example 1 of the present invention, the chemical and biological stability of Potassium all trans retinoate (PATRA) & Potassium 9-cis-retinoate (P9CRA) was performed in vitro and in vivo. The purpose of this study is to test and evaluate Potassium all trans retinoate (PATRA) and Potassium 9-cis-retinoate (P9CRA) in vitro/in vivo ADME. This may include solubility tests, plasma metabolic stability tests, and liver micrometabolism tests.

For solubility test, an excess of the test drug was added to a test tube containing 1 mL of DDW or artificial gastric juice (SGF), and the mixture was shaken incubated at room temperature for 48 hours. After reaching equilibrium solubility, the test tube was centrifuged at 3000 rpm for 10 min. After taking the supernatant, the undissolved test drug was removed using a syringe filter. Filtrate was placed in a test tube and analyzed.

For the metabolic stability test in plasma, mouse plasma, rat plasma and human plasma stored in a -20C freezer were thawed. The final DMSO concentration of the test drug was less than 1%. (The final test drug concentration was 20 μM) (Procaine drug was used as a positive control) The metabolic reaction of the test drug was started immediately after the test drug was added, and samples were sampled for 0, 15, 30, 60, 120, 180, 240 minutes. (However, procaine is sampled for 5, 10, 30, 60, and 120 minutes) For sample treatment, ice cold acetonitrile containing IS was added and voltexing was performed for 30 seconds to terminate the metabolic reaction. The metabolic test sample after completion of the reaction was centrifuged at 14000 rpm for 15 minutes. The supernatant was separated, placed in a test tube, and analyzed by HPLC system.

For liver microsome metabolism test, MLM, RLM, and HLM fractions stored in a -80℃ freezer were thawed. Preincubate the MLM, RLM, and HLM fractions dispersed in NADPH and metabolic test buffer for 5 minutes (1 mg/mL microsomal protein concentration). The final concentration of DMSO of the test drug was less than 1% (final concentration was 20 μM). (If necessary, use a drug such as buspirone as a positive control) The metabolic reaction of the test drug was started immediately after the test drug was added, and samples were sampled for 0, 15, 30, 60, and 120 minutes. For sample treatment, ice cold acetonitrile containing IS was added, and rapid voltexing was performed for 30 seconds to terminate the metabolic reaction. The metabolic test sample after completion of the reaction was centrifuged at 14000 rpm for 15 minutes. The supernatant was separated, placed in a test tube, and analyzed by HPLC system.

The analysis method is as follows. As the analysis equipment, UPLC-DAD equipment, Agilent HPLC 1290 infinite II system was used. HPLC analysis conditions were HECTOR-A C18 column (250 mm x 4.6 mm ID., 5 μm) for the column, FDDW (0.1% FA+5% acetic acid):ACN = 30:70 for the mobile phase, Flow rate: 1 mL/ min, Injection vol: 10 μL, Total run time: 30 min, Wavelength: 349 nm.

For the calibration curve, a standard solution of at least 5 concentrations was prepared for the test substance, treated by the pretreatment method described above, and analyzed by UHPL-DAD.

The evaluation method is as follows. As a solubility test, an excess of the test drug was quantitatively analyzed after incubation for 48 hours. The metabolic stability test evaluated the metabolic stability of the test drug in plasma (mouse, rat, human) and liver microsomes (mouse, rat, human).

The test results are as follows. Table 1 below shows the solubility test results. (n=3) The equilibrium solubility of P9CRA and PATRA in water was measured to be 3.76 μg/mL and 1.18 μg/mL, respectively, whereas their solubility in artificial gastric juice was relatively decreased to 0.18 μg/mL and 0.69 μg/mL, respectively. was measured. That is, through the meaning of the test, neither P9CRA nor PATRA was dissolved or destroyed in water and artificial gastric juice.

Tables 5 to 7 below show the results of liver microsome metabolic stability. In liver microsomal stability, both P9CRA and PATRA showed similar stability in mice (% remaining 83.1% vs 83.4% at 30 min). In rats, PATRA was more stable than P9CRA, and both compounds were stable (% remaining 78.3% vs 84.3% at 30 min). In humans, the liver microsomal metabolic stability of P9CRA was not as good as that of PATRA, and P9CRA was relatively more stable (% remaining 64.5% vs 86.7% at 30 min).

Solubility test result

compound	Solubility in distilled water (DDW) (μg/mL)	Solubility in Artificial Gastric Fluid (SGF) (μg/mL)
P9CRA	3.76±1.08	0.18±0.11
PATRA	1.18±0.52	0.69±0.17

Tables 2 to 4 below show the results of the metabolic stability test in plasma. In terms of plasma stability, both P9CRA and PATRA were metabolically stable without any differences between species (mouse, rat, human).

Results of plasma metabolic stability test of mouse (n=3)

time (minutes)	% remaining of P9CRA	% remaining of PATRA
0	100.0±0.0	100.0±0.0
15	101.5±4.2	96.1±2.6
30	98.9±1.8	94.4±3.1
60	99.0±6.4	94.3±1.5
120	101.2±8.3	93.6±4.2
180	92.2±3.6	93.6±1.3
240	86.1±0.6	93.5±3.3

Results of plasma metabolic stability test in rats (n=3)

hours (minutes)	% remaining of P9CRA	% remaining of PATRA
0	100.0±0.0	100.0±0.0
15	100.7±1.1	100.4±2.0
30	102.3±2.6	101.4±2.5
60	101.3±1.3	101.0±2.2
120	102.6±2.4	97.6±3.2
180	99.8±2.8	99.3±3.2
240	99.5±0.7	102.0±3.7

Results of human (n=3) plasma metabolic stability test

hours (minutes)	% remaining of P9CRA	% remaining of PATRA
0	100.0±0.0	100.0±0.0
15	101.4±1.5	106.4±4.8
30	101.9±1.5	106.2±4.2
60	103.4±1.7	106.2±4.3
120	105.4±2.5	107.1±2.8
180	103.4±1.5	104.7±2.7
240	103.8±1.2	99.0±4.7

Results of liver microsomal metabolic stability in mouse (n=3)

hours (minutes)	% remaining of P9CRA	% remaining of PATRA
0	100.0±0.0	100.0±0.0
15	92.5±3.0	95.1±1.9
30	83.1±5.5	83.4±2.8
60	78.5±4.0	75.9±2.6
120	70.8±5.2	67.7±2.3

Results of liver microsomal metabolic stability in rats (n=3)

time (minutes)	% remaining of P9CRA	% remaining of PATRA
0	100.0±0.0	100.0±0.0
15	84.0±3.4	88.4±5.3
30	78.3±2.7	84.3±7.8
60	67.6±4.0	73.5±3.7
120	58.5±2.8	67.3±4.0

Results of liver microsomal metabolic stability in human (n=3)

hours (minutes)	% remaining of P9CRA	% remaining of PATRA
0	100.0±0.0	100.0±0.0
15	80.0±7.5	90.7±1.8
30	64.4±5.0	86.7±1.5
60	48.7±0.9	79.3±3.3
120	39.6±1.2	73.9±7.2

<Experimental Example 2> Fibroblast cell differentiation ISMC and Potassium 9-cis-retinoate (P9CRA) applied NGS

First, count 5 x 10 ⁵ fibroblast cells, mix them with fibroblast media containing 1% FBS and 10ng/ml TGF-beta 1, and spray them in a T-75 flask coated with type IV collagen, and then in a 5% CO ₂ incubator. Differentiate for about 3 days. For differentiation, 1 x 10 ⁶ Primary Human Intestinal Smooth Muscle Cells (ISMC) were cultured in a T-75 flask, and incubated overnight in a 5% CO ₂ incubator.

In order to confirm the concentration of 9-cis-retinoate (P9CRA) without cytotoxicity, the MTS Assay shown in FIG. 12 was used, and the cells were sprayed so that the optimal concentration was 1.25uM. After culturing for 24 hours in a 5% CO ₂ incubator, 1 ml of trizol reagent was sprayed on 75T-flask for RNA isolation. After waiting at room temperature for 5 minutes, scrape the cells well with a scraper, transfer the destroyed cells to a 1.5ml e-tube, store them in a -80 freezer, and transfer the samples to an NGS analysis institution (ebiogen).

13 shows a Scatter Plot showing the distribution of changes in all genes according to Experimental Example 2. When the increased (increased) / decreased (decreed) genes were identified and graphed by the total number of mRNAs expressed by 24,426 treatment with Potassium 9-cis-retinoate (P9CRA) in ISMC, between control cells and drug (P9CRA) applied cells It shows the distribution of log2 (Normalized data) for each gene.

The horizontal axis represents log2 (Normalized data) of the control group, and the vertical axis represents log2 (Normalized data) of the experimental group (P9CRA). The genes located in the black oblique line in the middle are genes having the same expression value in both samples, and the red and green lines indicate the double standard increase/decrease, respectively.

Genes located above the red slanted line are genes that are more than doubled in the experimental group compared to the control group, and genes located below the green slanted are genes that are down 2 times or more in the experimental group compared to the control group. The increased number of red genes was 592 and the decreased number of green genes was 452.

14 shows an Intergrative Genomics Viewer (IGV) Mapping grip according to Experimental Example 2 of the present invention. The Intergrative Genomics Viewer (IGV) observed the gene pattern by confirming the result of mapping the reads to the reference genome on the image. The BAM file was reconstructed using the organized database for the locations where gene insertions and deletions frequently occur according to P9CRA drug treatment.

A red mark of the control shown in FIG. 14 indicates a heterogenotype. The colored part of the coverage of the Bam file is the heterogenotype, that is, the base part where Single Nucleotide polymorphism (SNP) has occurred. The nucleotide is gray, the red is T (Thymine), and the green is A (Adenine). , blue is C (Cytosine), yellow is G (Guanine), and the color change means the part where the base mutation has occurred, and the purple part is when the base is inserted when comparing the sequence of the reference genome with the sample. indicates the part

As a result, although it cannot be said that there is no heterogeneous change according to P9CRA drug treatment, it can be confirmed that it has significantly disappeared compared to the control. In other words, it can be confirmed that the P9CRA drug prevents the mutation of cells and protects the mutation that can occur in primary Human Intestinal Smooth Muscle Cells (ISMC).

Table 8 shows the KEGG Mapper Search Results according to Experimental Example 2 of the present invention. In order to understand the mechanism of action of the P9CRA drug, the pathway analysis was carried out using the KEGG mapping method to classify the gene group having a total of 27 pathways and the field of action of each group as follows, find detailed gene items in the relevant category, and classify their roles did As shown in Table 8, it was confirmed that it was related to inflammation, blood, cholesterol, calcium membrane receptor-related mRNA and coding protein.

KEGG Mapper Search Result

hsa04080 Neuroactive ligand-receptor interaction - Homo sapiens (human) (2) hsa:9170 LPAR2; lysophosphatidic acid receptor 2: inflammatory response hsa:6915 TBXA2R; thromboxane A2 receptor: blood clotting hsa04979 Cholesterol metabolism - Homo sapiens (human) (2) hsa:19 ABCA1; ATP binding cassette subfamily A member 1: cholesterol metabolism hsa:348 APOE; apolipoprotein E: cholesterol metabolism hsa05200 Pathways in cancer - Homo sapiens (human) (2) [Cancer network viewer] hsa:1050 CEBPA; CCAAT enhancer binding protein alpha: cholesterol metabolism hsa:9170 LPAR2; lysophosphatidic acid receptor 2: inflammatory response hsa04020 Calcium signaling pathway - Homo sapiens (human) (2) hsa:490 ATP2B1; ATPase plasma membrane Ca 2+ transporting 1 hsa:6915 TBXA2R; thromboxane A2 receptor; blood clot hsa04261 Adrenergic signaling in cardiomyocytes - Homo sapiens (human) (1) hsa:490 ATP2B1; ATPase plasma membrane Ca 2+ transporting 1: Calcium transmembrane transporter activity hsa04015 Rap1 signaling pathway - Homo sapiens (human) (1) hsa:9170 LPAR2; lysophosphatidic acid receptor 2: inflammatory response hsa04978 Mineral absorption - Homo sapiens (human) (1) hsa:490 ATP2B1; ATPase plasma membrane Ca 2+ transporting 1: Calcium transmembrane transporter activity hsa05130 Pathogenic Escherichia coli infection - Homo sapiens (human) (1) hsa:9170 LPAR2; lysophosphatidic acid receptor 2: inflammatory response hsa04972 Pancreatic secretion - Homo sapiens (human) (1) hsa:490 ATP2B1; ATPase plasma membrane Ca 2+ transporting 1: Calcium transmembrane transporter activity hsa04022 cGMP-PKG signaling pathway - Homo sapiens (human) (1) hsa:490 ATP2B1; ATPase plasma membrane Ca 2+ transporting 1: Calcium transmembrane transporter activity hsa05202 Transcriptional misregulation in cancer - Homo sapiens (human) (1) hsa:1050 CEBPA; CCAAT enhancer binding protein alpha: cholesterol metabolism hsa04024 cAMP signaling pathway - Homo sapiens (human) (1) hsa:490 ATP2B1; ATPase plasma membrane Ca 2+ transporting 1: Calcium transmembrane transporter activity hsa04975 Fat digestion and absorption - Homo sapiens (human) (1) hsa:19 ABCA1; ATP binding cassette subfamily A member 1: cholesterol metabolism hsa04970 Salivary secretion - Homo sapiens (human) (1) hsa:490 ATP2B1; ATPase plasma membrane Ca 2+ transporting 1: Calcium transmembrane transporter activity hsa04932 Non-alcoholic fatty liver disease - Homo sapiens (human) (1) hsa:1050 CEBPA; CCAAT enhancer binding protein alpha: cholesterol metabolism hsa00100 Steroid biosynthesis - Homo sapiens (human) (1) hsa:6713 SQLE; squalene epoxidase: cholesterol metabolism hsa01100 Metabolic pathways - Homo sapiens (human) (1) hsa:6713 SQLE; squalene epoxidase: cholesterol metabolism hsa04925 Aldosterone synthesis and secretion - Homo sapiens (human) (1) hsa:490 ATP2B1; ATPase plasma membrane Ca2+ transporting 1 hsa04810 Regulation of actin cytoskeleton - Homo sapiens (human) (1) hsa:9170 LPAR2; lysophosphatidic acid receptor 2: inflammatory response hsa04611 Platelet activation - Homo sapiens (human) (1) hsa:6915 TBXA2R; thromboxane A2 receptor; blood clot hsa04928 Parathyroid hormone synthesis, secretion and action - Homo sapiens (human) (1) hsa:9935 MAFB; MAF bZIP transcription factor B: cholesterol metabolism hsa04072 Phospholipase D signaling pathway - Homo sapiens (human) (1) hsa:9170 LPAR2; lysophosphatidic acid receptor 2: inflammatory response hsa02010 ABC transporters - Homo sapiens (human) (1) hsa:19 ABCA1; ATP binding cassette subfamily A member 1: cholesterol metabolism hsa05010 Alzheimer disease - Homo sapiens (human) (1) hsa:348 APOE; apolipoprotein E: cholesterol metabolism hsa04961 Endocrine and other factor-regulated calcium reabsorption-Homo sapiens (human) (1) hsa:490 ATP2B1; ATPase plasma membrane Ca 2+ transporting 1 ; Calcium transmembrane transporter activity hsa04151 PI3K-Akt signaling pathway - Homo sapiens (human) (1) hsa:9170 LPAR2; lysophosphatidic acid receptor 2: inflammatory response hsa05221 Acute myeloid leukemia - Homo sapiens (human) (1) hsa:1050 CEBPA; CCAAT enhancer binding protein alpha: cholesterol metabolism

GenomeNet Database Resources were analyzed based on the gene category items and search result data of Kyoto University Bioinformatics Center. 15 shows the number of genes corresponding to the gene ortholog through DAVID analysis.

The number of genes corresponding to the gene ortholog through DAVID analysis is as follows. DAVID analysis was carried out to identify the category of mRNA that acts the most among the mRNAs related to inflammation, blood, cholesterol, and calcium membrane receptors above, and the target mRNA for cholesterol metabolism and inflammatory reaction mechanisms (secondary alcohol action mechanism, steroid action mechanism) As shown in FIG. 15 , it was confirmed that is significantly reduced (Down Count).

In conclusion, as shown in Table 9, genes that differed more than twice when calculated as control/9CRA out of a total of 10 genes by P9CRA applied to ISMC through NGS evaluation were identified as a total of 10 types as shown in Table 9. It can be confirmed that the protein is involved in atherosclerosis in the cardiovascular system, and the fold change value from the green solid line indicating a 2-fold or more change in the NGS Fold Change value as shown in FIG. 12 can be summarized as the evaluation result in Table 9.

Atherosclerosis is a process that starts from inflammation and develops into 'atheromatous plaque' by combining with Ca ²⁺ ions in the process of being wrapped in blood, encapsulated in the blood vessel wall, and coagulated. Therefore, P9CRA causes an inflammatory response due to a decrease in human immunity, which can be seen in the atherosclerosis pathogenesis process, accumulates in the blood through cholesterol intake, and a disorder in the process of decomposing cholesterol in macrophages and cholesterol and fibrils. It has been shown that it has a role in inhibiting the progress of cholesterol stenosis, which is deposited in blood vessels by blood coagulation by Nogel, P-selection, and plaque phenomenon caused by calcium ions, or alleviating the disease and removing plaque. It was confirmed through gene pathway analysis.

In more detail, LPAR2 mRNA that causes an inflammatory response, that is, Lysophosphatidic acid receptor 2, is a lipid signaling molecule that is a G-protein-coupled receptor, and converts the factor X protein encoded by Gene 2 that contributes to Ca ²⁺ movement and intravascular cellular response. + 1.026 = 3.026-fold inhibition. Due to this, it inhibits TBXA2R mRNA 2 + 0.939 = 2.939 fold, which causes blood to clot, interacts with G-protein, that is, thromboxane A2 to inhibit platelet aggregation, and is related to all cholesterol metabolism. , APOE, INSIGI, MafB, or ATP-binding cassette (ABC) transporter, is an ABC protein that transports a variety of molecules across the extracellular/intrinsic membrane, enhances the cholesterol efflux pump role in the cellular lipid clearance pathway, and is absorbed from the small intestine. Adipose major apolipoprotein (APOE) present in the post-blood or lymphatic system, encodes an endoplasmic reticulum membrane protein that activates increases in plasma cholesterol and triglycerides and clearance of VLDL residues, and regulates adipogenesis and glucose homeostasis and is a sterol regulatory element binding protein (SREBP), cleavage activation protein (SCAP), and sterol-sensing domains such as 3-hydroxy-3-methyl glutaryl-coenzyme A (HMG-CoA) reductase activate binding and negotiation actions.

Evaluation results according to Experimental Example 2

No.	Gene Category	Target Effect / NGS Fold Change	evaluation target
			Protein Coding (Analysis after on-chip test)
efficacy
One	Blood coagulation	inhibitor / 0.939 decrease	G-protein	(TBXA2R) Thromboxane A2 receptor
2	Inflammatory response	inhibitor / 1.126 decrease	Tissue factor X (factor X)	lysophosphatidic acid receptor 2 (LPAR2)
3	calcium transmembrane transporter activity	inhibitor / 0.724 decrease	ATPase plasma membrane Ca2+ transporting 1	(ATP2B1) ATPase plasma membrane Ca2+ transporting 1
4	cholesterol metabolic process	Induce/ 6.674 increase	cholesterol efflux regulatory protein (CERP)_ABCA1	(ABCA1) ATP binding cassette subfamily A member 1
		inhibitor / 0.992 decrease	Apolipoprotein B	(APOBR) apolipoprotein B receptor
		Induce/ 1.099 increase	Apolipoprotein E	(APOE) apolipoprotein E
		inhibitor / 0.941 decrease	C/EBP	(CEBPA) CCAAT enhancer binding protein alpha
		Induce/ 1.003 increase	Insulin-induced gene 1 protein	(INSIGI) insulin induced gene 1
		Induce/ 1.307 increase	Transcription factor MafB	MafB
		inhibitor / 0.931 decrease	squalene epoxidase	(SQLE) Squalene monooxygenase

Each gene showed an improvement effect of 8.674, 3.099, 3.003, and 3.307. On the other hand, the effect of reducing SQLE (2.931 times), that is, apolipoprotein B48 receptor macrophage receptor binds triglyceride (TG) and increases plasma triglycerides, and through apolipoprotein B48 receptor foam cell formation, endothelial dysfunction and atherosclerosis It is a pathogenic mRNA and inhibits APOBR (2.992 fold).

This mRNA shows that the activity of C/EBP protein encoded by CEBPA can regulate the expression of genes involved in cell cycle regulation as well as body weight homeostasis, and that the mutation is suppressed by the gene mRNA, which is an acute myeloid leukemia pathogen. The value is 2.941 times.

Also, SQLE mRNA, Squalene monooxygenase, is a gene that coats a substance used as an inhibitor of a component called squalene epoxidase. This protein has an antifungal mechanism of action. The multidrug inhibitory mechanism of action is one method of treating hypercholesterolemia.

<Experimental Example 3> NGS applied with THP-1 cells and Potassium All trans retinoate (PATRA)

After mixing the number of THP-1 cells with the cell media as many as 5 x 10 ⁶ , spray them in a T-75 flask, and incubate in a 5% CO ₂ incubator overnight. PATRA is sprayed on the cells to an optimal concentration of 0.625uM at a non-cytotoxic concentration through the MTS Assay shown in FIG. 16, and incubated in a 5% CO ₂ incubator for 24 hours.

After that, the cell suspension in the flask is transferred to a 50ml tube and centrifuged at 1300rpm, 3min, 25°C for cell down. After removing the supernatant and spraying 1ml of trizol reagent for RNA isolation on the remaining cell pellet, pipetting so that the cells are well destroyed, transfer the trizol solution containing the cells to a 1.5ml e-tube and store the sample in -80 freezer It was delivered to an NGS institution (ebiogen).

17 shows a scatter plot showing the distribution of changes in all genes according to Experimental Example 3 of the present invention. In THP-1 cells, the total number of mRNAs expressed by the treatment with Potassium All-trans retinoate (P9CRA) was 24,426, and the increased (increased) / decreased (decreed) genes were identified and graphed. Control cells and cells applied with the drug (PATRA) It shows the distribution of log2 (Normalized data) for each gene in between. The horizontal axis represents log2 (Normalized data) of the control group, and the vertical axis represents log2 (Normalized data) of the experimental group (PATRA).

The genes located in the black oblique line in the middle are genes having the same expression value in both samples, and the red and green lines indicate the double standard increase/decrease, respectively. Genes located above the red slanted line are genes that are more than doubled in the experimental group compared to the control group, and genes located below the green slanted are genes that are down 2 times or more in the experimental group compared to the control group. The increased number of red genes was 47, and the decreased number of green genes was 195.

18 shows an Intergrative Genomics Viewer (IGV) Mapping grip according to Experimental Example 3 of the present invention. The Intergrative Genomics Viewer (IGV) observed the gene pattern by confirming the result of mapping the reads to the reference genome on the image. The BAM file was reconstructed using a database organized for the locations where gene insertions and deletions frequently occur according to PATRA drug treatment.

A control red mark in FIG. 18 indicates a heterogenotype. The colored part in the coverage of the Bam file in FIG. 18 is the base part where the heterogenotype, that is, single nucleotide polymorphism (SNP) occurred, and the nucleotide is gray, and the green is A (Adenine), as a result PATRA It is confirmed that the heterozygous type according to the drug treatment produces SNPs compared to the control. That is, it can be confirmed that the PATRA drug can show a mutation in the A (adenine) genotype in monocytes (THP-1 cells).

Table 10 shows the KEGG Mapper Search Results according to Experimental Example 3 of the present invention. In order to understand the mechanism of action of PATRA drugs, the pathway analysis was carried out using the KEGG mapping method, and the gene group having a total of 35 pathways and the field of action of each group were divided, and the detailed gene items of the related categories were found and their roles were divided. It was confirmed that it was related to the mRNA and coding proteins related to blood coagulation, anti-tumor, hematopoiesis and antioxidant in Table 10. GenomeNet Database Resources were analyzed based on the gene category items and search result data of Kyoto University Bioinformatics Center.

19 shows the number of genes corresponding to gene orthologs through DAVID analysis according to Experimental Example 3 of the present invention. The number of genes corresponding to the gene ortholog through DAVID analysis is as follows. DAVID analysis was carried out to confirm the category that acts the most among the blood coagulation, anti-tumor, hematopoietic, and antioxidant-related mRNAs, and the cellular component action mechanism and the blood clotting action mechanism target mRNA significantly increased (Up). Count) was confirmed. Table 11 shows the evaluation results according to Experimental Example 3 of the present invention.

In conclusion, the mRNA gene group of the DNA binding pathway item in the KEGG Mapper, that is, hsa04350 TGF-beta signaling pathway detailed mRNA hsa:3397, hsa:3398, hsa04550 Signaling pathways regulating pluripotency of stem cells, hsa04390 Hippo signaling pathway, hsa04015 Rap1 signaling The detailed pathway mRNA hsa:3397, and the gene encoding mRNA gene group, i.e., hsa04120 Ubiquitin mediated proteolysis, hsa05203 Viral carcinogenesis, hsa04114 Oocyte meiosis, hsa05166 Human T-cell leukemia virus 1 infection, hsa04110 Cell cycle, hsa05202 Transcriptional misregulation in cancer are evaluated was excluded, and the mRNA gene encoding the protein was identified.

KEGG Mapper Search Result

hsa04350 TGF-beta signaling pathway - Homo sapiens (human) (3) hsa:3397 ID1; inhibitor of DNA binding 1, HLH protein: hsa:3398 ID2; inhibitor of DNA binding 2 hsa:7057 THBS1; thrombospondin 1: blood clotting hsa05144 Malaria - Homo sapiens (human) (2) hsa:6382 SDC1; syndecan 1 ; blood clot hsa:7057 THBS1; thrombospondin 1: blood clotting hsa04145 Phagosome - Homo sapiens (human) (2) hsa:7057 THBS1; thrombospondin 1: blood clotting hsa:10383 TUBB4B; tubulin beta 4B class IVb: antitumor hsa05016 Huntington disease - Homo sapiens (human) (2) hsa:6648 SOD2; superoxide dismutase 2: antioxidant hsa:10383 TUBB4B; tubulin beta 4B class IVb: antitumor hsa04550 Signaling pathways regulating pluripotency of stem cells - Homo sapiens (human) (2) hsa:3397 ID1; inhibitor of DNA binding 1, HLH protein hsa:3398 ID2; inhibitor of DNA binding 2 hsa04390 Hippo signaling pathway - Homo sapiens (human) (2) hsa:3397 ID1; inhibitor of DNA binding 1, HLH protein hsa:3398 ID2; inhibitor of DNA binding 2 hsa04015 Rap1 signaling pathway - Homo sapiens (human) (2) hsa:3397 ID1; inhibitor of DNA binding 1, HLH protein hsa:7057 THBS1; thrombospondin 1: blood clotting hsa05132 Salmonella infection - Homo sapiens (human) (2) hsa:5420 PODXL; podocalyxin like: hematopoietic action hsa:10383 TUBB4B; tubulin beta 4B class IVb: antitumor hsa05205 Proteoglycans in cancer - Homo sapiens (human) (2) hsa:6382 SDC1; syndecan 1: blood clotting hsa:7057 THBS1; thrombospondin 1: blood clotting hsa04512 ECM-receptor interaction - Homo sapiens (human) (2) hsa:6382 SDC1; syndecan 1 ; blood clot hsa:7057 THBS1; thrombospondin 1: blood clotting hsa05014 Amyotrophic lateral sclerosis - Homo sapiens (human) (1) hsa:10383 TUBB4B; tubulin beta 4B class IVb: antitumor hsa04514 Cell adhesion molecules - Homo sapiens (human) (1) hsa:6382 SDC1; syndecan 1: blood clotting hsa04120 Ubiquitin mediated proteolysis - Homo sapiens (human) (1) hsa:991 CDC20; cell division cycle 20: gene encoding protein hsa05012 Parkinson disease - Homo sapiens (human) (1) hsa:10383 TUBB4B; tubulin beta 4B class IVb: antitumor hsa05418 Fluid shear stress and atherosclerosis - Homo sapiens (human) (1) hsa:6382 SDC1; syndecan 1: blood clotting hsa04151 PI3K-Akt signaling pathway - Homo sapiens (human) (1) hsa:7057 THBS1; thrombospondin 1: blood clotting hsa05203 Viral carcinogenesis - Homo sapiens (human) (1) hsa:991 CDC20; cell division cycle 20: gene encoding protein hsa05219 Bladder cancer - Homo sapiens (human) (1) hsa:7057 THBS1; thrombospondin 1: blood clotting hsa04114 Oocyte meiosis - Homo sapiens (human) (1) hsa:991 CDC20; cell division cycle 20: gene encoding protein hsa04068 FoxO signaling pathway - Homo sapiens (human) (1) hsa:6648 SOD2; superoxide dismutase 2: antioxidant hsa04211 Longevity regulating pathway - Homo sapiens (human) (1) hsa:6648 SOD2; superoxide dismutase 2: antioxidant hsa05165 Human papillomavirus infection - Homo sapiens (human) (1) hsa:7057 THBS1; thrombospondin 1: blood clotting hsa05022 Pathways of neurodegeneration - multiple diseases - Homo sapiens (human) (1) hsa:10383 TUBB4B; tubulin beta 4B class IVb: antitumor hsa04510 Focal adhesion - Homo sapiens (human) (1) hsa:7057 THBS1; thrombospondin 1 blood clotting hsa04213 Longevity regulating pathway - multiple species - Homo sapiens (human) (1) hsa:6648 SOD2; superoxide dismutase 2: antioxidant hsa05020 Prion disease - Homo sapiens (human) (1) hsa:10383 TUBB4B; tubulin beta 4B class IVb: antitumor hsa04540 Gap junction - Homo sapiens (human) (1) hsa:10383 TUBB4B; tubulin beta 4B class IVb: antitumor hsa04115 p53 signaling pathway - Homo sapiens (human) (1) hsa:7057 THBS1; thrombospondin 1: blood clotting hsa05130 Pathogenic Escherichia coli infection - Homo sapiens (human) (1) hsa:10383 TUBB4B; tubulin beta 4B class IVb: antitumor hsa05010 Alzheimer disease - Homo sapiens (human) (1) hsa:10383 TUBB4B; tubulin beta 4B class IVb: antitumor hsa05206 MicroRNAs in cancer - Homo sapiens (human) (1) hsa:7057 THBS1; thrombospondin 1: blood clotting hsa05166 Human T-cell leukemia virus 1 infection - Homo sapiens (human) (1) hsa:991 CDC20; cell division cycle 20: gene encoding protein hsa04146 Peroxisome - Homo sapiens (human) (1) hsa:6648 SOD2; superoxide dismutase 2: antioxidant hsa04110 Cell cycle - Homo sapiens (human) (1) hsa:991 CDC20; cell division cycle 20: gene encoding protein hsa05202 Transcriptional misregulation in cancer - Homo sapiens (human) (1) hsa:3398 ID2; inhibitor of DNA binding 2

In more detail, THBS1 mRNA binds to fibrinogen, fibronectin, laminin, collagen types V and VII and integrins alpha-V / beta -1 to platelets. It plays a role in aggregation, angiogenesis and tumorigenesis. SDC1 mRNA coding protein (Syndecan 1) is a transmembrane (type I) heparan sulfate proteoglycan.

These two genes are involved in blood coagulation, and showed an increase of 2.848-fold and 3.232-fold, respectively, to 2-fold + 0.848-fold. TUBB4B mRNA is a protein that docks the ciliary basal body-plasma membrane and has antitumor tubulin inhibitory effects. The PODXL gene encodes a member of the CD34 sialomucin protein family and aids in hematopoiesis. The expression of PODXL gene by PATRA applied to EC cells was increased by 2.971, and the change in PODXL2 expression was increased by 2.992 times.

The SOD gene exerts antioxidant effects through the formation of glycosylated homotetramers fixed to the extracellular matrix and cell surface through interaction with heparan sulfate proteoglycan and collagen. That is, it exerts an antioxidant effect through the glucose coating effect from the cells. The increased expression of SOD2 showed a 3.039-fold increase compared to the Control group.

In addition, the change in mRNA values for cholesterol metabolism, inflammatory response, and calcium membrane receptor activity was not higher than this clear difference in ISMC cells of Potassium 9-cis-retinoate (P9CRA) of Example 1, but PATRA was also the same type of mRNA shows a significant change in

Evaluation results

No.	Gene Category	Target Effect / NGS Fold Change	evaluation target
			Protein Coding (Analysis after on-chip test)
efficacy
One	Blood coagulation	Induce / 0.848 Increase	Syndecan 1	(SDC1)
		Induce / 1.232 Increase	THBS1	(THBS1) Thrombospondin 1
2	Inflammatory response	inhibitor / 0.870 decrease	Tissue factor X (factor X)	lysophosphatidic acid receptor 2 (LPAR2)
3	Antineoplastic	inhibitor / 0.982 decrese	Tubulin beta-4B chain	(TUBB4B) Tubulin beta-4A chain
4	Hematopoietic action	Induce / 0.232 Increase	Podocalyxin-like protein 1	PODXL
5	Antioxidant	Induce / 1.039 Increase	Extracellular superoxide dismutase [Cu-Zn]	(SOD) superoxide dismutase
3	calcium transmembrane transporter activity	inhibitor / 1.245 Increase	ATPase plasma membrane Ca 2+ transporting 1	(ATP2B1) ATPase plasma membrane Ca2+ transporting 1
4	cholesterol metabolic process	inhibitor / 0.848 decrease	cholesterol efflux regulatory protein (CERP)_ABCA1	(ABCA1) ATP binding cassette subfamily A member 1
		inhibitor / 0.984 decrease	Apolipoprotein B	(APOBR) apolipoprotein B receptor
		inhibitor / 1.855 Increase	Apolipoprotein E	(APOE) apolipoprotein E
		inhibitor / 0.971 decrease	C/EBP	(CEBPA) CCAAT enhancer binding protein alpha
		inhibitor / 1.620 Increase	Insulin-induced gene 1 protein	(INSIGI) insulin induced gene 1
		inhibitor / 0.684 decrease	Transcription factor MafB	MafB
		inhibitor / 1.694 Increase	squalene epoxidase	(SQLE) Squalene monooxygenase

Considering the human body absorption and biologic synthesis route, the absorption rate is excellent in the human body, and it is a powerful antioxidant that can lower the risk of cancer and cardiovascular disease. There is a possibility.

Claims (5)

1. Synthesizing and preparing all-trans retinal;

   dissolving the prepared All-trans retinal in methanol, mixing a solution of Na2PO4 and KMnO4 in sterile distilled water (DW), stirring, and cooling to crystallize all-trans retinoic acid;

   Potassium retinoate synthesis method, characterized in that after adding hexane to the all-trans retinoic acid, KOH dissolved in sterile distilled water is added dropwise to synthesize Potassium all-trans retinoate
2. Methyl β-formylcrotonate was added to EtOH, KOH aqueous solution was added, 9-cis phosphonium chloride ethanol solution was added, and then 50% KOH aqueous solution was added to maintain the reaction, sterile distilled water (DW) was added, stirred, and then filtered to crystallize 9-cis retinoic acid;

   Potassium retinoate synthesis method, characterized in that after adding hexane to the 9-cis retinoic acid, KOH dissolved in sterile distilled water is added dropwise to synthesize potassium 9-cis retinoate
3. The method according to claim 1, wherein the synthesis of all-trans retinal is

   [Step A] 2.2.6 reacting trimethyl cyclohexanone with (Z)-3-Methylpent-2-en-4-yn-1-ol;

   [Step B] After reducing the acetylene completed in [Step A] to E-ethylene using lithium aluminum hydride (LiAlH4),

   [Step C] Selective oxidation reaction of the alcohol group at the end of the chemical synthesized in [Step B] produces aldehyde,

   [Step D] Synthesizing C ₁₈ hydroxy ester with Phosphonium ylides in C ₁₅ Aldaehyde synthesized in [Step C];

   [Step E] After dehydrating the alcohol group at the C ₁₈ terminal synthesized in [Step D] with HCOOH acid to form ethylene, the OH of the cyclohexanol group is oxidized;

   [Step F] After ethylene is formed in [Step E], Rochelle salt is mixed and reduced to the alcohol group of OH of esters, carboxylic acids and amides;

   [Step G] The C ₁₇ alcohol produced in [Step F] undergoes selective oxidation to produce Aldehyde;

   [Step H] Inducing a reaction of alkylating a ketone with a methyl group through the Grignard reaction and [Step I] Inducing a Hydro Oxidation reaction;

   [Step J] Inducing a C = C coupling reaction with Aldehyde or ketone as an RO group;

   [Step K] After reducing the ester group with a LiAlH4 reducing agent to form an alcohol group;

   [Step L] Inducing a reaction to replace the alcohol group with 9-cis Phosphonium salt,

   [Step M] Potassium retinoate synthesis method comprising the step of synthesizing all-trans retinal by inducing a reaction of Knoevenagel condensation between β-C15 aldehyde and 3-Methyl-2-butenal
4. A pharmaceutical composition for inhibiting blood coagulation comprising a compound represented by Formula 1 or 2 as an active ingredient;

   [Formula 1]

   

   [Formula 2]

   
5. A pharmaceutical composition for cardiovascular treatment comprising a compound represented by Formula 1 or 2 as an active ingredient;

   [Formula 1]

   

   [Formula 2]

   

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