Acridine sulfonamide carboxylic acid derivative as well as preparation method and application thereof

Abstract

The invention relates to an acridine sulfonamide carboxylic acid derivative as well as a preparation method and application thereof, and the preparation method comprises the following steps: in an alkaline environment, acridine sulfonamide and amino acid or an amino acid derivative are subjected to amidation reaction in an organic solvent, and the acridine sulfonamide carboxylic acid derivative is obtained after purification; wherein the acridine sulfonamide carboxylic acid derivative has two acridine sulfonamide-like groups, and the two acridine sulfonamide-like groups are connected through the amino acid or the amino acid derivative. The yield of the preparation method exceeds 70%, and the luminous efficiency of the prepared acridine sulfonamide carboxylic acid derivative is higher and is about two times that of acridine sulfonamide NSP-SA-NHS.

Description

Technical Field

[0001] The present application relates to an acridine sulfonamide carboxylic acid derivative, a preparation method thereof and an application thereof, belonging to the field of chemiluminescent immunoassay detection. Background Art

[0002] Chemiluminescence immunoassay (CLIA) is a detection and analysis technology that combines the highly sensitive chemiluminescence determination technology with the highly specific immune reaction, and is used for the detection of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, drugs, etc. It is the latest immunoassay technology developed after radioimmunoassay, enzyme immunoassay, fluorescence immunoassay and time-resolved fluorescence immunoassay.

[0003] Chemiluminescence immunoassay can be divided into three categories according to different labels, namely direct chemiluminescence immunoassay, chemiluminescent enzyme immunoassay and electrochemiluminescence immunoassay. In direct chemiluminescence immunoassay, acridinium ester is directly labeled on the antibody (antigen). After an immune reaction occurs with the corresponding antigen (antibody) in the test sample, a solid-phase coated antibody - test antigen - acridinium ester-labeled antibody complex is formed. At this time, only oxidant (H2O2) and NaOH are added to make an alkaline environment. When the molecule is attacked by hydrogen peroxide, unstable dioxethane is generated. At this time, dioxethane decomposes into CO2 and electronically excited N-methylacridone. When it returns to the ground state, photons with a maximum emission wavelength of 430 nm are emitted. The acridinium ester decomposes and emits light without the need for a catalyst.

[0004] Acridinium ester-based luminescent reagents have mild reaction conditions, a simple luminescence system, do not require a catalytic process, and do not require an enhancer, thereby reducing background luminescence, improving the signal-to-noise ratio, and having less interference. At the same time, the light release is rapid and concentrated, with high luminescence efficiency and high luminescence intensity.

[0005] However, among the current acridinium ester-based markers on the market, many are in the form of an ester structure, which has poor stability and is easily decomposed under alkaline conditions. The commonly used one is acridine sulfonamide NSP-SA-NHS, which cleverly replaces the ester structure with a sulfonamide structure, increasing the stability of the marker under weakly alkaline conditions. From the luminescence mechanism, it can be seen that the structure of the acridine ring remains unchanged and the luminescence performance remains unchanged. Summary of the Invention

[0006] The purpose of the present application is to provide a novel acridine sulfonamide carboxylic acid derivative with higher luminescence efficiency, better water solubility and higher stability, a preparation method thereof and an application thereof. The preparation method has simple steps and low cost.

[0007] To achieve the above object, the present application provides the following technical solutions:

[0008] First aspect, a preparation method of an acridine sulfonamide carboxylic acid derivative, the preparation method comprising the following steps:

[0009] Under an alkaline environment, carrying out an amidation reaction on acridine sulfonamide and an amino acid or an amino acid derivative in an organic solvent, and after purification, obtaining the acridine sulfonamide carboxylic acid derivative;

[0010] Wherein, the acridine sulfonamide carboxylic acid derivative has two acridine sulfonamide-like groups, and the two acridine sulfonamide-like groups are connected by the amino acid or the amino acid derivative.

[0011] In one or more feasible embodiments, the amino acid or the amino acid derivative is lysine or a lysine salt.

[0012] In one or more feasible embodiments, the alkaline environment is provided by a carbonate.

[0013] In one or more feasible embodiments, the organic solvent is any one or more of DMF, DMAc or DMSO.

[0014] In one or more feasible embodiments, the preparation method comprises:

[0015] Dissolving lysine and sodium carbonate in water to obtain an alkaline lysine salt solution;

[0016] Dissolving acridine sulfonamide in DMF to obtain an acridine sulfonamide solution;

[0017] Adding the lysine salt solution to the acridine sulfonamide solution for reaction, after the reaction is completed, adjusting the pH to acidic, and after purification, obtaining the acridine sulfonamide carboxylic acid derivative.

[0018] In one or more feasible embodiments, after the reaction is completed, adjusting the pH to acidic and after purification to obtain the acridine sulfonamide carboxylic acid derivative comprises:

[0019] Adding dilute hydrochloric acid to adjust the pH to acidic, then evaporating the solvent and purifying by column chromatography to obtain a yellow solid powder.

[0020] In one or more feasible embodiments, the acridine sulfonamide carboxylic acid derivative has the structural formula shown in Formula I:

[0021] (Formula I)

[0022]

[0023] Second aspect, the present application provides an acridine sulfonamide carboxylic acid derivative and / or its isomers prepared by the preparation method according to the first aspect.

[0024] In a third aspect, the present application provides an application of the acridinesulfonamide carboxylic acid derivative and / or its isomers according to the second aspect in the preparation of a chemiluminescent immunoassay product.

[0025] In one or more feasible embodiments, the chemiluminescent immunoassay product is a luminescent reagent, and the acridinesulfonamide carboxylic acid derivative and its isomers are used as luminescent markers.

[0026] In a fourth aspect, the present application provides a detection kit, which includes the acridinesulfonamide carboxylic acid derivative and / or its isomers according to the second aspect.

[0027] Compared with the prior art, the beneficial effects of the present application are as follows:

[0028] 1) Improve luminescence efficiency: The luminescence efficiency of the novel acridinesulfonamide carboxylic acid marker (TM) is twice that of the traditional acridinesulfonamide (NSP-SA-NHS).

[0029] A higher luminescence efficiency means that higher sensitivity can be obtained in chemiluminescent immunoassay, so that lower concentrations of target analytes can be detected.

[0030] 2) Improve water solubility: The acridinesulfonamide carboxylic acid marker (TM) of the present application increases the water solubility of the marker by introducing lysine, which is beneficial to the application of the marker in an aqueous environment.

[0031] 3) Simplify the synthesis steps and reduce costs: The synthesis of this marker adopts a one-step reaction, and its raw materials are easy to obtain and the synthesis is simple, which can reduce production costs and improve production efficiency.

[0032] 4) Improve stability: The acridinesulfonamide structure itself is more stable than the traditional acridinium ester structure, especially under alkaline conditions, which can extend the shelf life of the marker and improve the reliability of detection.

[0033] 5) Increase yield: The preparation method of the present application reacts in an alkaline environment. On the one hand, lysine salt is produced to increase the solubility of lysine in water; on the other hand, the carbonate ions generated by the hydrolysis of sodium carbonate can provide a weakly alkaline reaction environment, which can neutralize the acidic by-products generated by the reaction and maintain the pH stability of the reaction system, which is beneficial to the smooth progress of the reaction; as a result, the yield of the final acridinesulfonamide carboxylic acid derivative exceeds 70%.

[0034] The above description is only an overview of the technical solution of the present application. In order to be able to understand the technical means of the present application more clearly and implement it in accordance with the content of the specification, the following describes in detail with the preferred embodiments of the present application and in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Flow chart of the preparation method of acridinesulfonamide carboxylic acid derivative shown in an embodiment of the present application;

[0036] Figure 2 Mass spectrum of the acridinesulfonamide carboxylic acid derivative prepared by the preparation method shown in an embodiment of the present application. Detailed implementation manners

[0037] The following specific examples illustrate the implementation manners of the present application. Those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in this specification. The present application can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, without conflict, the following embodiments and the features in the embodiments can be combined with each other.

[0038] It should be noted that the diagrams provided in the following embodiments only illustrate the basic concept of the present application in a schematic manner. Therefore, only the components related to the present application are shown in the diagrams, rather than being drawn according to the number, shape, and size of the components in actual implementation. The types, quantities, and proportions of the components in actual implementation can be arbitrarily changed, and the component layout type may also be more complex.

[0039] For the purpose of illustration, some exemplary embodiments of the present application are described. It should be understood that the present application can be implemented in other ways not specifically shown in the drawings.

[0040] As outlined above, the present application provides a preparation method of an acridinesulfonamide carboxylic acid derivative, and the method includes the following steps:

[0041] Reaction step

[0042] Under an alkaline environment, an amidation reaction is carried out between acridinesulfonamide and an amino acid or an amino acid derivative in an organic solvent.

[0043] Wherein, the acridinesulfonamide carboxylic acid derivative has two acridinesulfonamide-like groups, and these two acridinesulfonamide-like groups are connected through the amino acid or the amino acid derivative.

[0044] Through this step, an amidation reaction occurs between acridinesulfonamide and an amino acid or an amino acid derivative to form the target product.

[0045] Purification step

[0046] The reaction product is purified to obtain the acridinesulfonamide carboxylic acid derivative.

[0047] The purification method can include column chromatography, recrystallization, extraction, etc. to ensure the high purity of the product.

[0048] In at least one embodiment of the present application, the amino acid or amino acid derivative is preferably lysine or lysine salt. Since the side-chain amino group of lysine can react with two acridine sulfonamide molecules, and lysine salt can increase the solubility of lysine in water, which is beneficial to the progress of the reaction.

[0049] In at least one embodiment of the present application, the alkaline environment is preferably provided by carbonates (such as sodium carbonate, sodium bicarbonate). Carbonates can provide a mild alkaline environment, which is beneficial to the progress of the amidation reaction and neutralize the acid generated in the reaction.

[0050] In at least one embodiment of the present application, the organic solvent is preferably DMF, DMAc or DMSO, etc. These organic solvents have good solubility, can dissolve the reactants, and promote the progress of the reaction.

[0051] Specifically, by way of illustration and not limitation, the preparation steps are as Figure 1 shown:

[0052] Dissolve lysine and sodium carbonate in water to obtain an alkaline lysine salt solution;

[0053] Dissolve acridine sulfonamide in DMF to obtain an acridine sulfonamide solution;

[0054] Add the lysine salt solution to the acridine sulfonamide solution for reaction;

[0055] After the reaction is completed, add dilute hydrochloric acid to adjust the pH to acidic;

[0056] Evaporate the solvent and purify by column chromatography to obtain a yellow solid powder.

[0057] Specifically, in one embodiment, the acridine sulfonamide carboxylic acid derivative has a specific structural formula (Formula I), and the acridine sulfonamide (NSP-SA-NHS) has the structural formula shown in Formula II: (Formula I)

[0058]

[0059] (Formula II)

[0060]

[0061] The present application will be further described in detail below with reference to specific embodiments.

[0062] Example 1

[0063] Raw materials:

[0064] 1 mmol of lysine (Lys)

[0065] Sodium carbonate (Na2CO3) 2.5 mmol

[0066] NSP-SA-NHS (acridinesulfonamide) 2.2 mmol, N,N-dimethylformamide (DMF) 20 mL

[0067] Deionized water 10 mL

[0068] Appropriate amount of dilute hydrochloric acid

[0069] Steps:

[0070] Add lysine (1 mmol) and sodium carbonate (2.5 mmol) to deionized water (10 mL), stir to dissolve to obtain an alkaline lysine salt solution.

[0071] Add NSP-SA-NHS (2.2 mmol) to DMF (20 mL), stir to dissolve to obtain an acridinesulfonamide solution.

[0072] Slowly add the alkaline lysine salt solution to the acridinesulfonamide solution, and stir and react at room temperature (about 25 °C) overnight (about 16 hours).

[0073] After the reaction is completed, adjust the pH of the reaction solution to 6 with dilute hydrochloric acid.

[0074] Evaporate the solvent from the reaction solution on a rotary evaporator to obtain a crude product.

[0075] Purify the crude product by silica gel column chromatography, using dichloromethane / methanol (volume ratio 9:1) as the eluent.

[0076] Collect the target product, evaporate the solvent by rotary evaporation to obtain a yellow solid powder, namely acridinesulfonamide carboxylic acid derivative (TM), and the yield is measured to be 71.7%.

[0077] Figure 2 The mass spectrum of the yellow solid powder is shown, from Figure 2 From the mass spectrometry test results, its mass spectrum data is 1279.80 (M+1), 640.50 (1 / 2M+1).

[0078] Example 2

[0079] Dissolve lysine (9.7 mg, 1 eq) and sodium carbonate (17.67 mg, 2.5 eq) in water (2 ml), add NSP-SA-NHS (117 mg, 2.5 eq) to DMF (8 ml), then add the DMF solution of NSP-SA-NHS to the lysine solution, react at room temperature overnight, then adjust the pH to 5.5 with dilute hydrochloric acid, evaporate the solvent, purify by column chromatography to obtain a yellow solid powder (target product), and the yield is 74%.

[0080] Example 3

[0081] Provide raw materials:

[0082] 1 mmol of L-lysine (Lys)

[0083] 2.5 mmol of sodium carbonate (Na2CO3)

[0084] 2.2 mmol of NSP-SA-NHS (acridinesulfonamide)

[0085] 20 mL of N,N-dimethylacetamide (DMAc)

[0086] 10 mL of deionized water

[0087] Appropriate amount of dilute hydrochloric acid

[0088] According to the steps of Example 1 or 2, carry out the preparation and purification of the compound, and the measured yield is 72.3%.

[0089] Example 4

[0090] Provide raw materials:

[0091] 1 mmol of L-lysine (Lys)

[0092] 2.5 mmol of sodium carbonate (Na2CO3)

[0093] 2.2 mmol of NSP-SA-NHS (acridinesulfonamide)

[0094] 20 mL of dimethyl sulfoxide (DMSO)

[0095] 10 mL of deionized water

[0096] Appropriate amount of dilute hydrochloric acid

[0097] According to the steps of Example 1 or 2, carry out the preparation and purification of the compound, and the measured yield is 73.1%.

[0098] Example 5

[0099] Dissolve NSP-SA and TM in DMSO first, and then dilute to a certain concentration with 0.1M PBS buffer solution at PH6.8 to make a suitable high value that can be tested by the chemiluminescence instrument. At this time, the concentration is set as C5, and then buffer and serial dilution are used to obtain C4. Similarly, serial dilution is used to obtain C3, C2, C1, and C0, and a series of the same molar concentrations are configured to test the luminescence value on the chemiluminescence instrument.

[0100] Among them, NSP-SA has the structural formula shown in Formula III below:

[0101]

[0102] The test results of the buffer solution prepared with two kinds of acridinesulfonamides on the chemiluminescence analyzer are as follows:

[0103] Molar concentration NSP-SA luminescence value TM luminescence value C0 228791 464038 C1 460532 927753 C2 925687 1854108 C3 1849652 3709632 C4 3716303 7413374 C5 7408520 14828740

[0104] The test results show that:

[0105] 1. With the serial dilution of the molar concentration, the luminescence values of the two kinds of acridinesulfonamides also show a trend of serial decrease, which is in line with linear dilution.

[0106] 2. At the same molar concentration, the luminescence value of the acridinesulfonamide carboxylic acid derivative (TM) is twice that of the acridinesulfonamide (NSP-SA), which is in line with the luminescence mechanism of acridinesulfonamide.

[0107] In summary, for the acridinesulfonamide carboxylic acid derivative and its preparation method of the present application, through a simple and efficient synthesis method, the luminescence efficiency, water solubility and stability are significantly improved. It has significant technical advantages and broad application prospects, and can be applied in the field of immunoluminescence detection, and play an important role in the fields of biological detection, medical diagnosis, drug research and development, etc.

[0108] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0109] The above-described embodiments only represent several implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A method for preparing an acridine sulfonamide carboxylic acid derivative, characterized in that: The preparation method comprises the following steps: Under alkaline conditions, subjecting acridine sulfonamide to an amidation reaction with an amino acid or an amino acid derivative in an organic solvent, and obtaining the acridine sulfonamide carboxylic acid derivative after purification; The acridinium sulfonamide carboxylic acid derivative has two acridinium sulfonamide-like groups, and the two acridinium sulfonamide-like groups are connected by the amino acid or amino acid derivative.

2. The preparation method according to claim 1, characterized in that The amino acid or amino acid derivative is lysine or a lysine salt.

3. The preparation method according to claim 1, characterized in that: The alkaline environment is provided by carbonate; and / or, The organic solvent is any one or more of DMF, DMAc or DMSO.

4. The preparation method according to any one of claims 1 to 3, characterized in that The preparation method comprises: Dissolve lysine and sodium carbonate in water to obtain an alkaline lysine salt solution; dissolving acridine sulfonamide in DMF to obtain an acridine sulfonamide solution; The lysine salt solution is added to the acridine sulfonamide solution for reaction. After the reaction is completed, the pH is adjusted to acidic, and the acridine sulfonamide carboxylic acid derivative is obtained after purification.

5. The preparation method according to claim 4, characterized in that: After the reaction is completed, the pH is adjusted to acidic, and the acridinium sulfonamide carboxylic acid derivative obtained after purification includes: Dilute hydrochloric acid was added to adjust the pH to acidic, and then the solvent was evaporated and purified by column to obtain a yellow solid powder.

6. The preparation method according to any one of claims 1 to 3, characterized in that: The acridine sulfonamide carboxylic acid derivative has a structural formula shown in Formula 1: (Formula 1) 7. An acridinium sulfonamide carboxylic acid derivative and / or its isomers obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the acridinium sulfonamide carboxylic acid derivative and / or its isomers according to claim 7 in the preparation of chemiluminescent immunoassay products.

9. The use according to claim 8, characterized in that The chemiluminescent immunoassay product is a luminescent reagent, and the acridine sulfonamide carboxylic acid derivative and its isomers are used as luminescent markers.

10. A detection kit, characterized in that: The detection kit comprises the acridinium sulfonamide carboxylic acid derivative and / or its isomers obtained by the preparation method according to claim 7.

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