Water-soluble acridine sulfonamide derivative, preparation method, application thereof and kit

Abstract

The present application relates to the field of chemiluminescent immunoassay, and specifically to water-soluble acridine sulfonamide derivatives, preparation methods, applications thereof, and kits. In some embodiments, the water-soluble acridine sulfonamide derivatives can label sulfhydryl groups under weakly alkaline or acidic conditions and are water-soluble, thereby being applicable to carbohydrate antigen detection.

Description

Technical Field

[0001] The present application relates to water-soluble acridine sulfonamide derivatives, preparation methods, applications and kits thereof, and belongs to chemiluminescent immunoassay. Background Art

[0002] Chemiluminescence immunoassays (CLIA) combine highly sensitive chemiluminescence technology with highly specific immune responses. Based on the specific markers used, they can be divided into three categories: direct chemiluminescence immunoassay, chemiluminescent enzyme immunoassay, and electrochemiluminescent immunoassay. CLIA is primarily used for the detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, and drugs. It is the latest immunoassay technology developed after radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, and time-resolved fluorescence immunoassay. Summary of the Invention

[0003] The purpose of this application is to provide a water-soluble acridinium sulfonamide derivative, a preparation method, its application and a kit, which can label sulfhydryl groups under weak alkaline or acidic conditions, expanding the labeling of proteins by acridinium salts under acidic conditions.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] In a first aspect, the present application provides a water-soluble acridine sulfonamide derivative, characterized in that the water-soluble acridine sulfonamide derivative has a structural formula shown in Formula 1:

[0006] (Formula 1)

[0007]

[0008] In a second aspect, the present application provides a method for preparing a water-soluble acridine sulfonamide derivative, comprising the following steps:

[0009] Providing acridine sulfonamide carboxylic acid and maleimide intermediate hydrochloride;

[0010] dissolving the acridine sulfonamide carboxylic acid, maleimide intermediate hydrochloride and coupling agent in an organic solvent;

[0011] After cooling, a catalyst is added to carry out a reaction, and after the reaction is completed, the water-soluble acridine sulfonamide derivative is separated and purified to obtain the water-soluble acridine sulfonamide derivative;

[0012] The water-soluble acridine sulfonamide derivative has the structural formula shown in Formula 1, the acridine sulfonamide carboxylic acid has the structural formula shown in Formula 2, and the maleimide intermediate hydrochloride has the structural formula shown in Formula 3:

[0013]

[0014] In one or more feasible embodiments, the organic solvent is selected from any one or more of DMF, DCM, ethyl acetate or DMSO.

[0015] In one or more feasible embodiments, the coupling agent is HBTU; and / or the catalyst is DIEA.

[0016] In one or more feasible embodiments, the step of adding a catalyst to react after cooling, and separating and purifying the water-soluble acridine sulfonamide derivative after the reaction is completed comprises:

[0017] The mixture was cooled in an ice-water bath, DIEA was added and the mixture was reacted at room temperature overnight. After the reaction was completed, liquid phase separation and purification were performed, and then most of the solvent was evaporated and freeze-dried to obtain the water-soluble acridine sulfonamide derivative.

[0018] In a third aspect, the present application provides a stereoisomer or tautomer of a water-soluble acridinium sulfonamide derivative prepared according to the water-soluble acridinium sulfonamide derivative described in the first aspect or the preparation method described in the second aspect.

[0019] In a fourth aspect, the present application provides a use of the acridinium sulfonamide according to the first aspect or the stereoisomer or tautomer according to the third aspect in the detection of carbohydrate antigens.

[0020] In one or more feasible embodiments, the method is used to label polypeptides or proteins containing thiol groups.

[0021] In one or more feasible embodiments, it is used to detect carbohydrate antigen 199 under weakly alkaline or acidic conditions.

[0022] In a fifth aspect, the present application provides a kit for detecting carbohydrate antigens, which comprises the water-soluble acridine sulfonamide derivative described in the first aspect or the stereoisomer or tautomer described in the third aspect.

[0023] According to the embodiments of the present application, the water-soluble acridinium sulfonamide derivative of the present application is prepared by coupling water-soluble acridinium sulfonamide carboxylic acid and maleimide intermediate hydrochloride in a one-step reaction. The synthesis is simple and the yield is high. In addition, the synthesized product has an extended connecting arm, good water solubility, and can be directly dissolved in water.

[0024] At the same time, the water-soluble acridinium sulfonamide derivatives of the present application can label sulfhydryl groups under weakly alkaline or acidic conditions, and the labeled antibodies will not have good water solubility and will not precipitate from the solution.

[0025] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a H NMR spectrum of a water-soluble acridinium sulfonamide derivative shown in an embodiment of the present application;

[0027] Figure 2 is a mass spectrum of a water-soluble acridinium sulfonamide derivative shown in an embodiment of the present application;

[0028] Figure 3 This is a concentration-luminescence value curve of a water-soluble acridine sulfonamide derivative marker in antigen CA199 detection as shown in an embodiment of the present application;

[0029] Figure 4 This is a concentration-luminescence value curve of a common acridinium sulfonamide derivative marker in antigen CA199 detection shown in an embodiment of the present application. DETAILED DESCRIPTION

[0030] The following describes the embodiments of the present invention through specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. The details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the following embodiments and features in the embodiments can be combined with each other unless they conflict.

[0031] The terms "including," "comprising," and "having" are used interchangeably and have the same meaning. Likewise, "including," "comprising," and "having" are used interchangeably and have the same meaning. Specifically, the definition of each term is consistent with the definition of "including" under ordinary U.S. patent law, so that each term can be understood as an open term meaning "at least the following" and can also be interpreted as not excluding additional features, limitations, aspects, etc. Thus, for example, "a device having components a, b, and c" means that the device includes at least components a, b, and c. Similarly, the phrase: "a method involving steps a, b, and c" means that the method includes at least steps a, b, and c. Furthermore, although steps and processes may be outlined herein in a particular order, those skilled in the art will recognize that the order steps and processes may vary.

[0032] The term "derivative" is used in its ordinary sense in chemistry and biology to refer to a compound that is structurally similar to another compound (the so-called "reference" compound) but has a different composition (e.g., an atom is replaced by an atom of a different element, or a specific functional group is present, or one functional group is replaced by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound is different). Thus, an analog is a compound that is similar or equivalent in function and appearance to a reference compound but has a different structure or origin.

[0033] As used herein, the term "antibody" refers to immunoglobulins or immunoglobulin-like molecules, including (for example) but not limited to: IgA, IgD, IgE, IgG and IgM and combinations thereof; similar molecules produced during the immune response of any vertebrate (for example, in mammals such as humans, goats, rabbits and mice); and antibody fragments that specifically bind to a target molecule (or a group of highly similar target molecules) and substantially exclude binding to other molecules. "Antibody" further refers to a polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region, which specifically recognizes and binds to an epitope of an antigen. An antibody can be composed of a heavy chain and a light chain, each of which has a variable region, referred to as a variable heavy chain (VH) region and a variable light chain (VL) region. The VH region and the VL region are jointly responsible for binding to the antigen recognized by the antibody. The term "antibody" also includes complete immunoglobulins and variants and portions thereof well known in the art.

[0034] The titles of this application are for convenience only and are not used to interpret the scope or meaning of the disclosed embodiments of the invention.

[0035] Direct chemiluminescent immunoassay uses acridinium ester to directly label antibodies (antigens). After an immune reaction occurs with the corresponding antigen (antibody) in the test specimen, a solid-phase coated antibody-test antigen-acridinium ester-labeled antibody complex is formed. At this time, only oxidants (H2O2) and NaOH need to be added to create an alkaline environment. When the molecules are attacked by hydrogen peroxide, unstable ethylene dioxide is generated. At this time, ethylene dioxide decomposes into CO2 and electronically excited N-methylacridone. When it returns to the ground state, it emits photons with a maximum emission wavelength of 430nm. The acridinium ester decomposes and emits light without the need for a catalyst.

[0036] For example, the acridinium ester and acridinium sulfonamide shown in the figure below, in which R, R', and R" are alkyl, alkoxy, aryl, and other substituents; X, X', and X" are coupling groups used to couple antigens or antibodies and increase the solubility of the compound.

[0037]

[0038] Its luminescence mechanism is:

[0039]

[0040] Acridinium ester luminescent reagents have mild reaction conditions, simple luminescence systems, and no catalytic process or enhancer is required, thereby reducing background luminescence, improving the signal-to-noise ratio, and reducing interference. At the same time, light release is rapid and concentrated, with high luminescence efficiency and high luminescence intensity. However, the acridinium ester structure is unstable and easily decomposed. Based on this, the present application provides a water-soluble acridinium sulfonamide derivative as a marker based on acridinium sulfonamide. The acridinium sulfonamide maleimide marker has the structural formula shown in Formula 1:

[0041] (Formula 1)

[0042]

[0043] In one embodiment, the present application provides a method for preparing a water-soluble acridine sulfonamide derivative, as shown in the following figure:

[0044]

[0045] It includes the following steps:

[0046] Providing acridine sulfonamide carboxylic acid and maleimide intermediate hydrochloride;

[0047] dissolving the acridine sulfonamide carboxylic acid, maleimide intermediate hydrochloride and coupling agent in an organic solvent;

[0048] After cooling, a catalyst is added to carry out a reaction, and after the reaction is completed, the water-soluble acridine sulfonamide derivative is separated and purified to obtain the water-soluble acridine sulfonamide derivative;

[0049] The water-soluble acridine sulfonamide derivative has the structural formula shown in Formula 1, the acridine sulfonamide carboxylic acid (SM) has the structural formula shown in Formula 2, and the maleimide intermediate hydrochloride has the structural formula shown in Formula 3:

[0050]

[0051] As an example but not a limitation, the organic solvent is selected from any one or more of DMF, DCM, ethyl acetate or DMSO. The coupling agent is HBTU, and the catalyst is DIEA.

[0052] As an example but not a limitation, the catalyst is added after cooling to carry out the reaction. After the reaction is completed, the water-soluble acridine sulfonamide derivative is obtained by separation and purification, including:

[0053] The mixture was cooled in an ice-water bath, DIEA was added and the mixture was reacted at room temperature overnight. After the reaction was completed, liquid phase separation and purification were performed, and then most of the solvent was evaporated and freeze-dried to obtain the water-soluble acridine sulfonamide derivative.

[0054] In the examples, unless otherwise specified, the reagents used were purchased from the market.

[0055] In the examples of the present application, acridine sulfonamide and maleimide intermediate hydrochloride are coupled through a one-step reaction to obtain the above-mentioned water-soluble acridine sulfonamide derivative marker, which has a simple synthesis and high yield.

[0056] Example 1: Specifically, in one embodiment, the raw material SM (10 mg, 1 eq), maleimide intermediate hydrochloride (2.8 mg, 1.2 eq) and HBTU (6.1 mg, 1.2 eq) were added to DMF (0.2 ml), followed by the addition of DIEA (6 mg, 3.5 eq), and the reaction mixture was stirred at room temperature using a magnetic stirrer and maintained at room temperature for overnight reaction.

[0057] After the reaction is complete, the reaction mixture is mixed with water or another solvent and separated and purified. A rotary evaporator is used to evaporate most of the solvent at a relatively low temperature and pressure, leaving a concentrated product solution. The concentrated product solution is then freeze-dried in a freeze dryer (at -50 to -80°C, with the pressure reduced to below 0.1 mbar) to obtain a dry, water-soluble acridine sulfonamide derivative with a measured yield of 74.1%.

[0058] Example 2: Specifically, in one embodiment, the raw material SM (10 mg, 1 eq), maleimide intermediate hydrochloride (2.8 mg, 1.2 eq) and HBTU (6.1 mg, 1.2 eq) were added to DMF (0.2 ml), followed by the addition of DIEA (6 mg, 3.5 eq), and the reaction mixture was stirred at room temperature using a magnetic stirrer and maintained at room temperature for overnight reaction.

[0059] After the reaction is complete, the reaction mixture is mixed with water or another solvent and separated and purified. A rotary evaporator is used to evaporate most of the solvent at a relatively low temperature and pressure, leaving a concentrated product solution. The concentrated product solution is then freeze-dried in a freeze dryer (at -50 to -80°C, with the pressure reduced to below 0.1 mbar) to obtain a dry, water-soluble acridine sulfonamide derivative in a yield of 74.3%.

[0060] Example 3: Specifically, in one embodiment, the raw material SM (10 mg, 1 eq), maleimide intermediate hydrochloride (2.8 mg, 1.2 eq) and HBTU (6.1 mg, 1.2 eq) were added to DMF (0.2 ml), followed by the addition of DIEA (6 mg, 3.5 eq), and the reaction mixture was stirred at room temperature using a magnetic stirrer and maintained at room temperature for overnight reaction.

[0061] After the reaction is complete, the reaction mixture is mixed with water or another solvent and separated and purified. A rotary evaporator is used to evaporate most of the solvent at a relatively low temperature and pressure, leaving a concentrated product solution. The concentrated product solution is then freeze-dried in a freeze dryer (at -50 to -80°C, with the pressure reduced to below 0.1 mbar) to obtain a dry, water-soluble acridinium sulfonamide derivative in a yield of 74.5%.

[0062] The H NMR spectrum and mass spectrum of the obtained product are shown as follows: Figure 1 and Figure 2 As shown in the figure, the compound of formula 1 was obtained, and its mass spectrum was 878.10 (M+1).

[0063] Currently, in clinical testing, carbohydrate antigens are tested for auxiliary diagnosis of cancer. For example, CA199, or carbohydrate antigen 199, is a carbohydrate antigen related to gastrointestinal cancer. Clinically, the detection of CA199 serum concentration can be used as an auxiliary diagnostic indicator for malignant tumors such as pancreatic cancer and gallbladder cancer.

[0064] The applicant has discovered that the water-soluble acridine sulfonamide derivatives of the embodiments of the present application can be used in the magnetic microparticle chemiluminescence immunoassay for CA199. The chemiluminescence detection method is a sandwich detection method, in which the capture antibody is labeled with biotin and the detection antibody is labeled with acridine sulfonamide, and then they are incubated together with the antigen CA199 to be detected. The excess detection antibody is washed away, and the acridine sulfonamide undergoes a chemical reaction in hydrogen peroxide under alkaline NaOH conditions to emit fluorescence. The strength of the fluorescence signal is detected to detect the concentration of the antigen to be detected.

[0065] The marking principle is:

[0066]

[0067] The detection antibody labeling process is as follows: the antibody (1 eq) is dissolved in 0.1M pH 7.2 PB buffer at a concentration of 1 mg / ml, then Trauts' reagent (30 eq) is added, and then a water-soluble acridinium sulfonamide maleimide label (30 eq) is added. The reaction is shaken at room temperature for 1.5 hours, and then dialyzed for purification to obtain a water-soluble acridinium salt-labeled detection antibody with good labeling effect.

[0068] Comparative experiment 1: Use ordinary acridine sulfonamide (NSP-SA-NHS) to carry out the detection antibody labeling experiment. Dissolve NSP-SA-NHS in DMF, take the same molar amount of solution as the water-soluble acridine sulfonamide maleimide label in the previous example, add the same amount of detection antibody as the previous example, and react at room temperature for more than 1.5 hours. It is found that antibody precipitates in the solution. The reason is that after labeling the antibody, the water solubility of the antibody deteriorates, thereby causing precipitation.

[0069] The antibody reagent labeled with water-soluble acridine sulfonamide maleimide was tested on a chemiluminescence instrument. Figure 3 And the results shown in Table 1:

[0070] Table 1

[0071] Concentration (ng / ml) Luminescence value (RLU) C0 0IU / ml 2067 C1 6.95 IU / ml 151837 C2 27.43 IU / ml 560721 C3 52.93 IU / ml 1033740 C4 232.27 IU / ml 4224199 C5 976.69 IU / ml 15462191

[0072] Depend on Figure 3 As shown in Table 1, the concentration and luminescence value have a good correlation, and the correlation coefficient R 2 It reaches 0.9988, indicating that the synthesized water-soluble acridine sulfonamide derivative label is very suitable as a luminescent label for magnetic microparticle chemiluminescence immunoassay.

[0073] Comparison of the results of the common acridine sulfonamide (NSP-SA-NHS) labeled antibody reagent on the chemiluminescence instrument Figure 4 As shown in Table 2:

[0074] Table 2

[0075] Concentration (ng / ml) Luminescence value (RLU) C0 0IU / ml 2543 C1 6.95 IU / ml 3653 C2 27.43 IU / ml 3982 C3 52.93 IU / ml 4100 C4 232.27 IU / ml 4508 C5 976.69 IU / ml 4646

[0076] Depend on Figure 3 As shown in Table 1, the linearity is poor, and the luminescence value does not change much with the increase of the concentration of antigen CA199. The correlation coefficient R 2 Only 0.3633,

[0077] Of course, in other embodiments for detecting carbohydrate antigens, stereoisomers or tautomers of the water-soluble acridine sulfonamide derivatives shown in Formula 1 also have the above-mentioned effects, and those skilled in the art can perform corresponding tests based on the disclosure of this application.

[0078] As an example and not a limitation, the water-soluble acridine sulfonamide derivatives are used to label polypeptides or proteins containing sulfhydryl groups, particularly under weakly alkaline or acidic conditions.

[0079] The present application also discloses a chemiluminescent immunoassay kit, which comprises the above-mentioned water-soluble acridine sulfonamide derivative or its stereoisomers / tautomers.

[0080] In summary, the water-soluble acridinium sulfonamide derivative of the present application is prepared by coupling water-soluble acridinium sulfonamide carboxylic acid and maleimide intermediate hydrochloride in a one-step reaction, which has a simple synthesis and a high yield. In addition, the synthesized product has an extended linker arm, good water solubility, and can be directly dissolved in water.

[0081] At the same time, the water-soluble acridinium sulfonamide derivatives of the present application can label sulfhydryl groups under weakly alkaline or acidic conditions, and the labeled antibodies will not have good water solubility and will not precipitate from the solution.

[0082] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0083] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A water-soluble acridine sulfonamide derivative, characterized in that: The water-soluble acridine sulfonamide derivative has the structural formula shown in Formula 1: (Formula 1) 。 2. A method for preparing a water-soluble acridine sulfonamide derivative, characterized in that: The following steps are involved: Providing acridine sulfonamide carboxylic acid and maleimide intermediate hydrochloride; dissolving the acridine sulfonamide carboxylic acid, maleimide intermediate hydrochloride and coupling agent in an organic solvent; After cooling, a catalyst is added to carry out a reaction, and after the reaction is completed, the water-soluble acridine sulfonamide derivative is separated and purified to obtain the water-soluble acridine sulfonamide derivative; The water-soluble acridine sulfonamide derivative has the structural formula shown in Formula 1, the acridine sulfonamide carboxylic acid has the structural formula shown in Formula 2, and the maleimide intermediate hydrochloride has the structural formula shown in Formula 3: 。 3. The preparation method according to claim 2, wherein The organic solvent is selected from any one or more of DMF, DCM, ethyl acetate or DMSO.

4. The preparation method according to claim 2 or 3, wherein The coupling agent is HBTU; and / or the catalyst is DIEA.

5. The preparation method according to claim 2 or 3, wherein After cooling, a catalyst is added to react. After the reaction is completed, the water-soluble acridine sulfonamide derivative is obtained by separation and purification, including: The mixture was cooled in an ice-water bath, DIEA was added and the mixture was reacted at room temperature overnight. After the reaction was completed, liquid phase separation and purification were performed, and then most of the solvent was evaporated and freeze-dried to obtain the water-soluble acridine sulfonamide derivative.

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