KR102027693B1 - A amphiphatic compound containing heterocyclic structure with benzene and nitrogen-atom rings, the preparation method and a surfactant composition containing the compound - Google Patents

Abstract

The present invention relates to a novel compound having amphiphilic properties including a benzene ring and a nitrogen ring in a chemical formula, and a method for manufacturing the same. The novel compound of the present invention maintains a sample form for a long time in nucleic acid analysis or blood analysis, thereby being very effective as an active component of a surfactant.

Description

Amphiphilic compound containing heterocyclic structure with benzene and nitrogen-atom rings, the preparation method and a surfactant composition containing the compound}

The present invention relates to a novel compound having an amphiphilic property including a benzene ring and a nitrogen ring in a chemical formula, and a method for preparing the same. The novel compound of the present invention maintains a sample form for a long time in nucleic acid analysis or blood analysis. It is very effective as an active ingredient of a surfactant.

In order to analyze an organism such as blood using a fluorescent probe, the shape, size, shape, maturity of the blood, etc. must be maintained for a long time, and in particular, to minimize noise to selectively analyze an organism to be analyzed. Except for the organisms that are the hazard, other organisms need to be dissolved or decomposed.

In particular, when analyzing whole blood using flow cytometry, a plurality of reaction chambers, which are areas in which blood is divided and reacted with each analytical reagent before analysis, are arranged, and dozens of blood cells in the blood are contained in one reactor. Since information is difficult to obtain at once, near-infrared fluorescence for nucleic acid staining to give different fluorescence wavelengths to the same 633 nm light source with various detergents that can selectively induce red blood cell hemolysis, leukocyte selection damage, and hemolysis in each reactor Using dyes, a technique of measuring complex components of blood cells obtained in each reactor, comparing them, and correcting them is mainly used.

Although there are differences among manufacturers in blood cell analysis, special reagents are used to analyze blood samples in an appropriate manner.These reagents include DNA, RNA and fluorescence staining using fluorescent dyes, and hemolysis of red blood cells. ), Hemoglobin oxidation reagents, dilution of blood samples, protein and hemolytic components cleaning, attaching, and sheath reagents for arranging blood cells in line at the measurement site.

For example, when erythrocytes are dissolved with reagents of acidic hemoglobin, basophils are separated from leukocytes with selective suppression of degranulation, and the measurement of leukocyte count is also possible without being affected by platelet aggregation. In case of analyzing by using a laser, noise is minimized in the information of forward scattered light and side scattered light so that blood cells can be specifically detected and interpreted.

In addition, when measuring granulocytes of blood, various information can be confirmed by improving the fluorescence efficiency of fluorescent reagents for staining DNA / RNA using leukocyte hemolysis reagents together with leukocyte hemolysis reagents. There is a trend to develop a commercially available surfactant reagent and the like.

The novel amphiphilic compound of the present invention is particularly excellent in the hemolysis of blood cells in the analysis of whole blood, and when the fluorescent compound is applied simultaneously, the noise of the fluorescence wavelength can be minimized to measure and confirm various information through the wavelength analysis. It has excellent properties as a surfactant for whole blood analysis.

The present invention stably maintains only the target object to be analyzed in the analysis of cells and organisms, in particular whole blood analysis, and the other organisms and cells present in the sample are dissolved or hemolyzed to analyze the target object using a fluorescent dye or the like. It is an object of the present invention to provide novel compounds that can minimize contamination and noise.

Another object of the present invention is to provide a surfactant composition that is applied to the preparation method of the novel compound and the analysis of the sample containing the novel compound.

Prior art literature

Patent Literature

Patent Document 1: Korea Patent Publication No. KR 10-1996-0042056

(Patent Document 2) Korean Patent Publication No. KR 10-2006-0103902

In order to solve the above problems, the present invention provides a novel compound represented by the following [Formula 1] and salts thereof.

[Formula 1]

In Chemical Formula 1

m is an integer from 1 to 20, n is an integer from 1 to 25, R ₁ is a substituted or unsubstituted alkyl of 1 to 12 carbons, a substituted or unsubstituted amine of 1 to 20 carbons, 1 to 6 carbons Carboxylic acid, sulfonic acid, imido ester, maleimide, succinimidyloxyl, ethenesulfonyl, alkylaminyl having 1 to 6 carbons. The compound of Formula 1 may form a salt with halogen or sulfuric acid.

The novel compound according to the present invention can be used as an active ingredient of a surfactant used in the analysis of biomolecules, and can maintain the form of the target for a long time when observing a biomolecule such as a protein or an antibody. The noise generated in the organic material can be minimized.

The present technology relates to the development of fluorescence, hemolysis, dilution, washing and fixation reagents for blood analysis equipment for comprehensive blood analysis. Specifically, fluorescence, hemolysis, dilution, washing and fixation reagent technologies for blood analysis equipment for comprehensive blood analysis. 5 leukocytes, delusions of neutrophils (NEUT), eosinophils (eosinphil, EO), basophil (BASO), lymphocytes (lymphocyte, LYMPH) and monocytes (monocyte, MONO) The present invention relates to a novel compound for analysis optimized for equipment for analyzing red blood cells and platelets, such as red blood cells (Reticulocyte, RET), nucleated red blood cells (NRBC), hemoglobin (HGB), and a surfactant composition comprising the same. .

The target market of surfactant composition for blood cell analysis is the whole blood cell test market. Diagnosis and progress of blood diseases, differentiation of blood cell counts and morphological abnormalities are discriminated against infection status, blood coagulation ability, anemia and its cause, urinary system. It may be a variety of medical examinations or the most common symptomatic examination prescribed by a doctor for the purpose of examining abnormalities and abnormalities in blood components or circulatory system.

In the blood analysis market, most of the consumers are concentrated in medium and large hospitals, and for this purpose, it is essential to develop a reagent for blood / blood cell analysis that can generate not only the supply of equipment but also the majority of profits and become a continuous source of revenue. In particular, there is an urgent need to secure a surfactant that can be effectively used for the analysis of red blood cells, white blood cells, platelets, etc., in contrast to an expired patent or a method for developing blood cell analysis, which is easy to access as a washing and washing reagent.

The present invention provides a compound represented by the following formula (1) and salts thereof.

[Formula 1]

In Chemical Formula 1

m is an integer from 1 to 20, n is an integer from 1 to 25, R ₁ is hydrogen, substituted or unsubstituted alkyl of carbon 1 to 12, substituted or unsubstituted amine of carbon 1 to 20, carbon 1 to 6 carboxylic acid, sulfonic acid, imido ester, maleimide, succinimidyloxyl, ethensulfonyl, alkylaminyl having 1 to 6 carbons. The compound may form a salt with halogen or sulfuric acid.

Specific compounds belonging to the present invention may be a compound of Formula 2 and Formula 3 below.

[Formula 2]

N in Formula 2 is 8 or 9.

[Formula 3]

N in Formula 3 is 8 or 9.

Blood analysis reagent composition comprising a compound of the present invention for blood analysis using the compound represented by the formula (1) as an active ingredient and also myristyltrimethylammonium bromide (Sigma aldrich) and citric acid (Citric acid, Sigma aldrich) can be prepared. The reagent composition for blood analysis of the present invention can be used by titrating to pH 3.0 to 4.0 using 1M sodium hydroxide (NaOH, Sigma aldrich) after mixing the above compounds. The blood assay reagent composition of the present invention has been shown to exert excellent efficacy in hemolyzing red blood cells in blood.

Hereinafter, preferred examples are provided to help understanding of the present invention, but the following examples are merely to illustrate the present invention, and the scope of the present invention is not limited to the following examples.

Example

Hereinafter, the present invention will be described in more detail through examples and comparative experiments. However, the examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

First, experimental apparatus, analytical equipment, and reagents used in Examples and Comparative Experiments will be described.

Instruments for FT-NMR spectroscopy were measured using Avance 300 and 500 from Bruker and LC / MSD (G-1956B) from Agilent for LC / MS.

Column chromatography for the separation and purification of organic compounds used Merck's kieselgel 60 (230-400 mesh) as a silica gel in the normal phase, silica gel 60 GF254 for thin layer chromatography (TLC) (0.25 mm, Merck) was used, and the compound identification on TLC was performed using ultraviolet rays of 254 nm and 365 nm, or 20-30% ethanol solution of phosphomolybdic acid (PMA) as a colorant or KMnO ₄ was used.

Example 1: Preparation of Compound 2-1

(1) Synthesis of Compound a

Nonaethylene glycol (1 g, 2.4 mmol, 1 eq, Leap Labchem) and 5N sodium hydroxide (0.72 ml, 3.6 mmol, 0.75 eq) are placed in a round flask and dissolved in 12 ml of Tetrahydrofuran. Then p-toluenesulfonyl chloride (p-Toluenesulfonyl chloride) (0.342 mg, 1.8 mmol, 0.75 eq, TCI) was slowly added at room temperature and stirred for 12 hours. Thereafter, the mixture was extracted with dichloromethane and distilled water, and then the dichloromethane layer was dehydrated with magnesium sulfate and filtered to obtain transparent liquid particles through distillation under reduced pressure (0.941 g, 69%).

R _f = 0.43 (Silicagel, dichloromethane / methanol 9: 1 v / v)

LC / MS, calculated 568.26, found 591.3 (+ Na ⁺ )

(2) Synthesis of Compound b

Compound a (0.941 g, 1.66 mmol, 1 eq) and potassium carbonate (275 mg, 1.99 mmol, 1.2 eq, Deoksan) were placed in a round flask, dissolved in 8 ml of acetonitrile and 4-Iodophenol (4-io Dophenol) (383 mg, 1.74 mmol, 1.05 eq, TCI) was added slowly at room temperature and stirred for 1 hour. Thereafter, the mixture was extracted with dichloromethane and distilled water, and then the dichloromethane layer was dried over magnesium sulfate, filtered, and dried through vacuum distillation. The dichloromethane / methanol 9: 1 was purified by silica gel normal chromatography with a developing solution to obtain a pure compound. b was obtained. (0.707 g, 69.2%)

R _f = 0.6 (Silicagel, dichloromethane / methanol 9: 1 v / v)

LC / MS, calculated 616.17, measured 639.8 (+ Na ⁺ )

(3) Synthesis of Compound c

Compound b (0.409 g, 0.664 mmol, 1 eq) with Trimethylsilylacetylene (0130 g, 1.33 mmol, 2 eq, TCI), bis (triphenylphosphine) palladium (II) dichloride (Bis (triphenylphosphine) palladium (II) Dichloride) (2.3 mg, 0.003 mmol, 0.005eq), Triphenylphosphine (3.5 mg, 0.013 mmol, 0.02eq), copper iodide (4 mg, 0.02 mmol, 0.03 eq) was dissolved in 4 ml of triethylamine and stirred for 20 minutes in a sonicator. Thereafter, potassium hydroxide (75 mg, 1.33 mmol, 2 eq) was dissolved in 8 ml of ethanol and stirred for 20 minutes in a sonicator. Thereafter, the mixture was extracted with ethyl ether and distilled water, and then the ethyl ether layer was dried over magnesium sulfate, and then filtered to obtain transparent liquid particles through distillation under reduced pressure (0.562 g, 91.1%).

R _f = 0.46 (Silicagel, Dichloromethane / Methanol 9: 1 v / v)

LC / MS, calculated 514.28, found 553.4 (+ K ⁺ )

(4) Synthesis of Compound d

To a round flask, 1-iodooctane (5 g, 20 mmol, 1 eq, TCI) and sodium azide (2.7 g, 41 mmol, 2 eq, TCI) were added, followed by di 5 ml of methylformamide was added and stirred at room temperature for 12-24 hours. Thereafter, the mixture was extracted with dichloromethane and saturated ammonium chloride solution, and the dichloromethane layer was dried with magnesium sulfate, and then filtered to obtain yellow liquid particles through distillation under reduced pressure (2.7 g, 84.3%).

R _f = 0.2 (Silicagel, dichloromethane / methanol 10: 1 v / v)

(5) Synthesis of Compound 2-1

Compound c (0.259 g, 0.504 mmol, 1 eq), compound d (0.156 g, 1 mmol, 2 eq) and a small amount of copper acetate were added to a round flask, dissolved in 5 ml of methanol, and then sonicated for 20 minutes in a sonicator. Stirred. Thereafter, the mixture was extracted with dichloromethane and distilled water, and then the dichloromethane layer was dried over magnesium sulfate, filtered, and dried through vacuum distillation. The dichloromethane / methanol 9: 1 was purified by silica gel normal chromatography with a developing solution to obtain a pure compound. 2-1 was obtained (0.120 g, 35.5%).

R _f = 0.32 (Silicagel, dichloromethane / methanol 9: 1 v / v)

LC / MS, calculated 671.44, found 695.2 (+ Na ⁺ )

Example 2: Preparation of Compound 2-2

(1) Synthesis of Compound e

Decaethylene glycol (1 g, 2.1 mmol, 1 eq, Leap Labchem) and 5N sodium hydroxide (0.87 ml, 1.6 mmol, 0.75 eq) are placed in a round flask and dissolved in 10 ml of Tetrahydrofuran. Then p-toluenesulfonyl chloride (p-Toluenesulfonyl chloride) (0.311 mg, 1.6 mmol, 1.5 eq, TCI) was slowly added at room temperature and stirred for 12 hours. Thereafter, the mixture was extracted with dichloromethane and distilled water, and then the dichloromethane layer was dried with magnesium sulfate, and then filtered to obtain transparent liquid particles through distillation under reduced pressure (0.821 g, 63.8%).

R _f = 0.43 (Silicagel, dichloromethane / methanol 9: 1 v / v)

LC / MS, calculated 612.28, measured 635.6 (+ Na ⁺ )

(2) Synthesis of Compound f

Compound e (0.235 g, 1 mmol, 1 eq) and potassium carbonate (63 mg, 1.6 mmol, 1.2 eq, Deoksan) were added to a round flask, dissolved in 5 ml of acetonitrile and 4-Iodophenol (4-io). Dophenol) (88 mg, 0.8 mmol, 1.05 eq, TCI) was added slowly at room temperature and stirred for 1 hour. Thereafter, the mixture was extracted with dichloromethane and distilled water, and then the dichloromethane layer was dried over magnesium sulfate, filtered, and dried through vacuum distillation. The dichloromethane / methanol 9: 1 was purified by silica gel normal chromatography with a developing solution to obtain a pure compound. f was obtained (0.353 g, 53.4%).

R _f = 0.59 (Silicagel, Dichloromethane / Methanol 9: 1 v / v)

LC / MS, calculated 660.20, found 699.6 (+ K ⁺ )

(3) Synthesis of Compound g

Compound f (0.353 g, 0.535 mmol, 1 eq) with Trimethylsilylacetylene (0.105 g, 1.07 mmol, 2 eq, TCI), bis (triphenylphosphine) palladium (II) dichloride (Bis (triphenylphosphine) palladium (II) Dichloride) (187 mg, 0.267 mmol, 0.5eq), Triphenylphosphine (7 mg, 0.267 mmol, 0.5eq), Copper iodide (5 mg, 0.267 mmol, 0.5 eq) was dissolved in 4 ml of triethylamine and stirred for 20 minutes in a sonicator. Thereafter, potassium hydroxide (60 mg, 1.07 mmol, 2 eq) was dissolved in 8 ml of ethanol and stirred in a sonicator for 20 minutes. Thereafter, the mixture was extracted with ethyl ether and distilled water, and then the ethyl ether layer was dried over magnesium sulfate, and then filtered to obtain transparent liquid particles through distillation under reduced pressure (0.301 g, 78.8%).

R _f = 0.48 (Silicagel, Dichloromethane / Methanol 9: 1 v / v)

LC / MS, calculated 558.67, measured 597.3 (+ K ⁺ )

(4) Synthesis of Compound 2-2

Compound g (0.301 g, 0.538 mmol, 1 eq), compound d (0.159 g, 1.1 mmol, 2 eq) and a small amount of copper acetate were added to a round flask, dissolved in 5 ml of methanol, and then sonicated for 20 minutes in a sonicator. Stirred. Thereafter, the mixture was extracted with dichloromethane and distilled water, and then the dichloromethane layer was dried over magnesium sulfate, filtered, and dried through vacuum distillation. The dichloromethane / methanol 9: 1 was purified by silica gel normal chromatography with a developing solution to obtain a pure compound. 2-2 was obtained. (0.102 g, 26.5%)

R _f = 0.32 (Silicagel, dichloromethane / methanol 9: 1 v / v)

LC / MS, calculated 715.46, found 736.9 (+ Na ⁺ )

Example 3: Preparation of Compound 3-1

(1) Synthesis of Compound h

In a round flask, 2,2,4-trimethyl-2-pentanone (2,2,4-Trimethyl-2-pentanone) (2 g, 15.3 mmol, 1 eq, TCI) and trimethylsilyl bromide (2.22) ml, 16.8 mmol, 1.1 eq, TCI) was added and stirred at room temperature for 1-2 hours. Thereafter, transparent liquid particles were obtained by distillation under reduced pressure (2.96 g, 100%).

R _f = 0.09 (Silicagel, dichloromethane / methanol 10: 1 v / v)

(2) Synthesis of Compound i

To the round flask, compound d (2.96 g, 15.3 mmol, 1 eq, TCI) and sodium azide (2.99 g, 46.0 mmol, 3 eq, TCI) were added followed by 3 ml of dimethylformamide. Was added and refluxed at 120 ° C. for 12 hours. Then, after extracting with dichloromethane and saturated ammonium chloride solution, the dichloromethane layer to remove the moisture with magnesium sulfate and filtered to obtain a yellow liquid particles through reduced pressure distillation (1.75 g, 73.8%).

R _f = 0.67 (Silicagel, dichloromethane / methanol 10: 1 v / v)

(3) Synthesis of Compound 3-1

Compound c (0.250 g, 0.486 mmol, 1 eq), Compound i (0.151 g, 0.972 mmol, 2 eq) and a small amount of copper acetate were added to a round flask, dissolved in 5 ml of methanol, and then sonicated for 20 minutes in a sonicator. Stirred. Thereafter, the mixture was extracted with dichloromethane and distilled water, and then the dichloromethane layer was dried over magnesium sulfate, filtered, and dried through vacuum distillation. The dichloromethane / methanol 9: 1 was purified by silica gel normal chromatography with a developing solution to obtain a pure compound. 3-1 was obtained (0.0583 g, 17.9%).

R _f = 0.33 (Silicagel, dichloromethane / methanol 9: 1 v / v)

LC / MS, calculated 669.86, found 692.7 (+ Na ⁺ )

Example 4: Preparation of Compound 3-2

(1) Synthesis of Compound 3-2

Compound g (0.250 g, 0.447 mmol, 1 eq), Compound i (0.138 g, 0.894 mmol, 2 eq) and a small amount of copper acetate were added to a round flask, dissolved in 5 ml of methanol, and then sonicated for 20 minutes in a sonicator. Stirred. Thereafter, the mixture was extracted with dichloromethane and distilled water, and then the dichloromethane layer was dried over magnesium sulfate, filtered, and dried through vacuum distillation. The dichloromethane / methanol 9: 1 was purified by silica gel normal chromatography with a developing solution to obtain a pure compound. 3-2 was obtained (0.031 g, 9.7%).

R _f = 0.32 (Silicagel, dichloromethane / methanol 9: 1 v / v)

LC / MS, calculated 713.91, found 736.6 (+ Na ⁺ )

Example 5: Preparation of blood assay reagents using the compounds of Formulas 2 and 3

A blood assay reagent was prepared by mixing Compound 2-1 with myristyltrimethylammonium bromide (Sigma aldrich) and citric acid (Sigma aldrich). The three compounds were mixed and titrated to pH 3.3 using 1M NaOH (Sigma aldrich).

Blood assay reagents were prepared in the same manner as above using the compounds of Compounds 2-2 and 3-1. Hematological reagents prepared using the compounds 2-1, 2-2 and 3-1 are shown in the figure below.

Test Example 1: Verification of the blood hemolytic efficacy of the surfactant of the present invention

Microscopic analysis

In order to further verify the hemolytic effect of the surfactant, analysis was performed using a microscope. Blood was used as a viscosity control reagent, E-check (low concentration), and Compound 3-1 was used by dissolving 0.4% in distilled water. Analysis was carried out on a Nikon ECLIPSE Ti-U microscope and measured under X200 DIC filter conditions. As a control, a sample treated with PBS in an E-check reagent was used. From the analysis results shown in the figure below, it was confirmed that hemolysis of erythrocytes was better when the compound 3-1 surfactant was treated than the control group.

Test Example 2 Verification of Efficacy of Surfactant Using Hematological Reagent

(1) FACS analysis

In order to verify blood assay reagents prepared using Compound 2-1, Compound 2-2, and Compound 3-1, the assay was performed using Fluorescence Activated Cell Sorting (hereinafter referred to as FACS). For the blood sample for analysis, E-check (low concentration), a viscosity management reagent of Sysmex, was used, and FACS analysis was performed 3 seconds after dispensing the blood assay reagent prepared in the E-check reagent. As a control, a sample treated with PBS in an E-check reagent was used. The analysis was performed using Thermofisher's Attune NxT instrument and measured under conditions of FSC 200, SSC 330, BL1 150, RL1 150, YL1 400. As shown in the data below, the sample treated with PBS used as a control did not have red blood cell hemolysis, and it was confirmed that it was measured by spreading as a whole. On the contrary, it was confirmed that red blood cells were hemolyzed to some extent when the blood analysis reagents prepared with Compounds 2-1, 2-2, and 3-1 were treated. Among them, Compound 2-1 was found to have the best performance as a surfactant.

(2) microscopic analysis

Microscopic analysis was performed to visually confirm the blood hemolytic effect of the hematological reagents prepared from Compound 2-1, Compound 2-2, and Compound 3-1. Blood analysis reagents prepared in E-check (low concentration), a viscosity control reagent, were processed and subjected to microscopic analysis. Analysis was carried out on a Nikon ECLIPSE Ti-U microscope and measured under X200 DIC filter conditions. As a control, a sample treated with PBS in an E-check reagent was used. As shown in the figure below, it was confirmed that hemolysis of red blood cells was better when the surfactants were treated with Compound 2-1, Compound 2-2, and Compound 3-1 compared to the control group. The hemolytic effect of Compound 2-1 was confirmed to be the largest.

The present invention is not limited to the embodiments described above, it can be various modifications and alterations by those skilled in the art, in addition can be used in various biological and chemical fields, which are defined in the appended claims It is included in the spirit and scope of the present invention.

Claims (12)

A compound represented by the following formula (1) or a salt thereof
[Formula 1]

In the above formula
m is an integer from 1 to 20, n is an integer from 1 to 25, and R ₁ is methyl. The compound of claim 1, wherein the compound of Formula 1 is represented by Formula 2 below
[Formula 2]

Wherein n is 8 or 9 Compound represented by the formula (3) below
[Formula 3]

Wherein n is 8 or 9 Regarding a reagent composition for blood analysis, the composition is a blood analysis reagent composition, characterized in that using the compound represented by the following formula (1) or a salt thereof as an active ingredient
[Formula 1]

In the above formula
m is an integer from 1 to 20, n is an integer from 1 to 25, and R ₁ is methyl. The blood analysis reagent composition of claim 4, wherein the compound of Formula 1 is represented by the following Formula 2.
[Formula 2]

Wherein n is 8 or 9 Blood analysis reagent composition using a compound represented by the formula (3) below as an active ingredient
[Formula 3]

Wherein n is 8 or 9 The blood assay reagent composition according to any one of claims 4 to 6, wherein the composition is used as a surfactant for hemolysis of red blood cells. The blood assay reagent composition according to any one of claims 4 to 6, wherein the composition further comprises Myristyltrimethylammonium bromide and Citric acid. Regarding the blood analysis method, the method comprises the steps of preparing and obtaining a compound represented by Formula 1 below;
Mixing the compound represented by Formula 1 with Myristyltrimethylammonium bromide and citric acid;
Dropping sodium hydroxide to the mixture to titrate the pH to 3.0 to 4.0; And blood analysis method characterized in that it comprises a step of analyzing by mixing blood to the composition obtained in the step
[Formula 1]

In the above formula
m is an integer from 1 to 20, n is an integer from 1 to 25, and R ₁ is methyl. The method of claim 9, wherein the compound of Formula 1 is represented by Formula 2 below.
[Formula 2]

Wherein n is 8 or 9 Regarding the blood analysis method, the method comprises the steps of preparing and obtaining a compound represented by the following formula (3);
Mixing the compound represented by the formula (3) with myristyltrimethylammonium bromide and citric acid;
Dropping sodium hydroxide to the mixture to titrate the pH to 3.0 to 4.0; And blood analysis method characterized in that it comprises a step of analyzing by mixing blood to the composition obtained in the step
[Formula 3]

Wherein n is 8 or 9
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