Acridine sulfonamide derivative, preparation method, application thereof and kit

Abstract

The invention relates to the field of chemiluminescence immunodetection, in particular to an acridine sulfonamide derivative, a preparation method, application of the acridine sulfonamide derivative and a kit, in some embodiments, the acridine sulfonamide derivative can label sulfydryl under the weakly alkaline or acidic condition, and protein labeling of acridine salt under the acidic condition is expanded.

Description

Technical Field

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

[0002] Chemiluminescence immunoassay (CLIA) combines highly sensitive chemiluminescence assay technology with highly specific immune reactions. It can be divided into three categories according to the different markers, namely direct chemiluminescence immunoassay, chemiluminescence enzyme immunoassay and electrochemiluminescence immunoassay. Chemiluminescence immunoassay is mainly 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 the present application is to provide an acridinium sulfonamide derivative, a preparation method, an application thereof and a kit, which can label sulfhydryl groups under weakly alkaline or acidic conditions, thereby 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 an acridinium sulfonamide derivative, wherein the acridinium 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 an acridinium sulfonamide derivative, which comprises the following steps:

[0009] Providing hydrochloride salts of acridine sulfonamide and maleimide intermediates;

[0010] dissolving the acridine sulfonamide and the maleimide intermediate hydrochloride in an organic solvent;

[0011] After the reaction is completed, separating and purifying to obtain the acridinium sulfonamide derivative;

[0012] Wherein, the acridine sulfonamide derivative has a structural formula shown in Formula 1, the acridine sulfonamide has a structural formula shown in Formula 2, and the maleimide intermediate hydrochloride has a 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, DIEA is added to the organic solvent.

[0016] In one or more feasible embodiments, after the reaction is completed, separating and purifying to obtain the acridinium sulfonamide derivative comprises:

[0017] After the reaction is completed, liquid phase separation and purification are performed, and then most of the solvent is evaporated and freeze-dried to obtain the acridine sulfonamide derivative.

[0018] In a third aspect, the present application provides a stereoisomer or tautomer of an acridinium sulfonamide derivative prepared according to the 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 derivative according to the first aspect or the stereoisomer or tautomer according to the third aspect in the detection of small molecule haptens.

[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, the method is used to detect thyroid hormones, wherein the thyroid hormones include thyroxine and triiodothyronine.

[0022] In one or more possible embodiments, the method is used to detect sex hormones, including progesterone, testosterone and estradiol.

[0023] In a fifth aspect, the present application provides a kit for detecting small molecule haptens, which comprises the acridinium sulfonamide derivative described in the first aspect or the stereoisomer or tautomer described in the third aspect.

[0024] According to the embodiments of the present application, the acridinium sulfonamide derivative of the present application is prepared by coupling acridinium sulfonamide and maleimide intermediate hydrochloride in a one-step reaction, and the raw materials are readily available, the synthesis is simple, and the yield is high; and the synthesized product has an extended connecting arm and less steric hindrance. At the same time, the present application utilizes the specific reaction of maleimide to sulfhydryl groups, and can label sulfhydryl groups under weakly alkaline or acidic conditions, expanding the labeling of proteins by acridinium salts under acidic conditions.

[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 in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0028] Figure 3 This is a concentration-luminescence value curve of an acridinium sulfonamide derivative marker in T3 hapten detection as shown in one embodiment of the present application.

[0029] Figure 4 This is a concentration-luminescence value curve of an acridine sulfonamide marker in T3 hapten detection as shown in an embodiment of the present application. DETAILED DESCRIPTION

[0030] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways 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 without conflict.

[0031] The terms "include", "comprises", "have", etc. are used interchangeably and have the same meaning. Similarly, "includes", "includes", "have", etc. are used interchangeably and have the same meaning. Specifically, the definition of each term is consistent with the definition of "include" under ordinary U.S. patent law, so each term can be understood as an open term, which means "at least the following" and can also be interpreted as not excluding additional features, limitations, aspects, etc. Therefore, 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. In addition, although the steps and processes may be outlined in a particular order herein, those skilled in the art will recognize that the order steps and processes may vary.

[0032] "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 (e.g., in mammals such as humans, goats, rabbits and mice, etc.); 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 is to directly label antibodies (antigens) with acridinium esters. After an immune reaction occurs with the corresponding antigen (antibody) in the sample to be tested, a solid-phase coated antibody-test antigen-acridinium ester labeled antibody complex is formed. At this time, only an oxidant (H 2 O 2 ) and NaOH 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 CO 2 When the N-methylacridone in the electronic excited state returns to the ground state, it emits a photon with a maximum emission wavelength of 430nm. The acridinium ester decomposes and emits light without the need for a catalyst.

[0036] For example, in the acridinium ester and acridinium sulfonamide shown in the figure below, R, R', and R" are other substituents such as alkyl, alkoxy, and aryl; 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 luminescent systems, no catalytic process, and no enhancer, thereby reducing background luminescence, improving 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 has poor stability and is easy to decompose. Based on this, the present application provides an acridinium sulfonamide derivative as a marker based on acridinium sulfonamide, and 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 an acridinium sulfonamide derivative, as shown in the following figure:

[0044]

[0045] It includes the following steps:

[0046] Providing hydrochloride salts of acridine sulfonamide and maleimide intermediates;

[0047] dissolving the acridine sulfonamide and the maleimide intermediate hydrochloride in an organic solvent;

[0048] After the reaction is completed, separating and purifying to obtain the acridinium sulfonamide derivative;

[0049] Wherein, the acridine sulfonamide derivative has a structural formula shown in Formula 1, the acridine sulfonamide has a structural formula shown in Formula 2, and the maleimide intermediate hydrochloride has a structural formula shown in Formula 3:

[0050]

[0051] As an example but not limitation, the organic solvent is selected from any one or more of DMF, DCM, ethyl acetate or DMSO.

[0052] In one embodiment, DIEA is added to the organic solvent as a catalyst and the pH value is adjusted.

[0053] As an example but not a limitation, after the reaction is completed, the acridinium sulfonamide derivative obtained by separation and purification includes:

[0054] After the reaction is completed, liquid phase separation and purification are performed, and then most of the solvent is evaporated and freeze-dried to obtain the acridine sulfonamide derivative.

[0055] In the examples, unless otherwise specified, the reagents used were purchased from the market.

[0056] 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 acridine sulfonamide derivative marker, which has a simple synthesis and a high yield.

[0057] Example 1: Specifically, in one embodiment, NSP-SA-NHS (1 g, 1 eq), maleimide intermediate hydrochloride (0.31 g, 1.2 eq) and DIEA (0.38 g, 2 eq) were added to DMF (100 ml), and the reaction mixture was stirred at room temperature using a magnetic stirrer and kept at room temperature for overnight reaction.

[0058] After the reaction is completed, the reaction mixture is mixed with water or other solvents and separated and purified. Most of the solvent is evaporated at a relatively low temperature and pressure using a rotary evaporator, leaving a concentrated product solution. The concentrated product solution is then placed in a freeze dryer (-50 to -80°C, the pressure is reduced to below 0.1 mbar) for freeze drying to obtain a dry acridinium sulfonamide derivative, and the yield is measured to be 75.1%.

[0059] Example 2: Specifically, in one embodiment, NSP-SA-NHS (1 g, 1 eq), maleimide intermediate hydrochloride (0.31 g, 1.2 eq) and DIEA (0.38 g, 2 eq) were added to DMF (100 ml), and the reaction mixture was stirred at room temperature using a magnetic stirrer and kept at room temperature for overnight reaction.

[0060] After the reaction is completed, the reaction mixture is mixed with water or other solvents and separated and purified. Most of the solvent is evaporated at a relatively low temperature and pressure using a rotary evaporator, leaving a concentrated product solution. The concentrated product solution is then placed in a freeze dryer (-50 to -80°C, the pressure is reduced to below 0.1 mbar) for freeze drying to obtain a dry acridinium sulfonamide derivative, and the yield is measured to be 75.4%.

[0061] Example 3: Specifically, in one embodiment, NSP-SA-NHS (1 g, 1 eq), maleimide intermediate hydrochloride (0.31 g, 1.2 eq) and DIEA (0.38 g, 2 eq) were added to DMF (100 ml), and the reaction mixture was stirred at room temperature using a magnetic stirrer and kept at room temperature for overnight reaction.

[0062] After the reaction is completed, the reaction mixture is mixed with water or other solvents and separated and purified. Most of the solvent is evaporated at a relatively low temperature and pressure using a rotary evaporator, leaving a concentrated product solution. The concentrated product solution is then placed in a freeze dryer (-50 to -80°C, the pressure is reduced to below 0.1 mbar) for freeze drying to obtain a dry acridinium sulfonamide derivative, and the yield is measured to be 75.8%.

[0063] 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, it can be seen that the compound shown in formula 1 is obtained, and its M=706.8.

[0064] At present, some small molecule haptens are often tested in clinical tests, such as T3 and T4 small molecule tests in thyroid function tests, progesterone (P), testosterone (T), estradiol (E2) in sex hormone tests, and other drug small molecule tests.

[0065] In general, detectable labels include: chromogenic, fluorescent, phosphorescent and luminescent molecules and materials; catalysts (such as enzymes), which convert one substance into another to provide a detectable difference (such as by converting a colorless substance into a colored substance, or vice versa, or by producing a precipitate or increasing sample turbidity); haptens, which can be detected by antibody-hapten binding interactions using other detectably labeled antibody conjugates; and paramagnetic and magnetic molecules or materials. Of course, the reporter moiety itself can also be detected indirectly, for example, if the detectable marker is a hapten, the detectable marker can be detected using another antibody specific for the detectable marker, as known to those of ordinary skill in the art.

[0066] The applicant has found that the acridine sulfonamide derivatives of the embodiments of the present application can be used in the detection of small molecule haptens. For example, a magnetic particle chemiluminescent immunoassay kit is used to detect small molecules. The sample and the antibody marker are first incubated to react, allowing the hapten to be detected in the sample to fully react with the antibody. The competitive hapten is then reacted with the excess antibody, and then combined with the magnetic particles. After washing, the excess unreacted competitive hapten is removed, and then it is excited to emit photons, and the strength of the light signal is detected, thereby detecting the concentration of the antigen in the sample.

[0067] Specifically, in one embodiment of detecting T3, the antibody is labeled with biotin to obtain a biotin label of the antibody, and the labeling of the competitive antigen includes:

[0068] Example 4: Labeling proteins with acridine sulfonamide derivatives (Formula 1) under weakly acidic conditions.

[0069] S1. Carrier protein BSA is subjected to thiol treatment and labeled with acridine sulfonamide derivative (Formula 1)

[0070] Weigh a certain amount of BSA and dissolve it in 0.1M PB pH6.0 buffer solution, add EDTA to make the EDTA concentration 2mM / L, add an appropriate amount of 5mM / L tris(2-chloroethyl) phosphate (TCEP) aqueous solution to open the disulfide bond, react at room temperature for 15min, directly add an appropriate amount of acridine sulfonamide maleimide marker without purification, react at room temperature for 1h, and purify by dialyzing with 0.1M pH7.4 buffer to obtain acridine sulfonamide-labeled BSA.

[0071] S2 and T3 hapten label the BSA acridine sulfonamide label in S1

[0072] Dissolve an appropriate amount of T3 derivative NHS active ester in DMF to 2 mg / ml. According to the labeling ratio, add the DMF solution of T3 derivative NHS active ester to BSA acridine sulfonamide marker buffer, react for 1.5 hours, and purify by dialyzing with 0.1M pH 6.8 buffer to obtain the competitive hapten marker used in the kit - T3 BSA acridine sulfonamide marker.

[0073] The biotin label of the antibody is diluted with reagent buffer to obtain reagent R1 component.

[0074] The T3 BSA acridinium sulfonamide marker was diluted with reagent buffer to obtain reagent R2 component.

[0075] Example 5: Comparison of protein labeling with acridine sulfonamide active ester (Formula 2) under weakly acidic conditions.

[0076] S1. Labeling the carrier protein BSA with acridine sulfonamide active ester (Formula 2)

[0077] Weigh a certain amount of BSA and dissolve it in 0.1M PB pH6.0 buffer solution to make the concentration and volume of BSA the same as in the above example. Add an equal molar amount of acridine sulfonamide active ester marker as in the above example, react at room temperature for 1 hour, and purify by dialyzing with 0.1M pH7.4 buffer to obtain acridine sulfonamide-labeled BSA.

[0078] S2 and T3 hapten label the BSA acridine sulfonamide label in S1

[0079] Dissolve an appropriate amount of T3 derivative NHS active ester in DMF to 2 mg / ml. According to the labeling ratio, add the DMF solution of T3 derivative NHS active ester to BSA acridine sulfonamide marker buffer, react for 1.5 hours, and purify by dialyzing with 0.1M pH 6.8 buffer to obtain the competitive hapten marker used in the kit - T3 BSA acridine sulfonamide marker.

[0080] The biotin label of the antibody is diluted with reagent buffer to obtain reagent R1 component.

[0081] The T3 BSA acridinium sulfonamide marker was diluted with reagent buffer to obtain reagent R2 component.

[0082] The calibration material was tested on a chemiluminescence instrument according to the method for detecting T3 hapten, and the following was obtained: Figure 3 And the results shown in Table 1:

[0083] Table 1

[0084] Concentration (ng / ml) Example 4 Luminescence value (RLU) Example 5 Luminescence value (RLU) S0 0 1104370 204327 S1 2.5 944237 190542 S2 6 683522 167632 S3 19.8 186891 108726 S4 27.5 101870 36585 S5 55 23217 13573

[0085] Depend on Figure 3 , Figure 4 As shown in Table 1, compared with Example 5, the experimental data of Example 4 shows a better correlation between its concentration and luminescence value, and the correlation coefficient R 2 The value of the luminescence index was 0.98, with a high overall luminescence value, good gradient and high sensitivity, indicating that the synthesized acridine sulfonamide derivative marker is very suitable as a luminescent marker for magnetic microparticle chemiluminescence immunoassay.

[0086] Of course, in other embodiments, the stereoisomers or tautomers of the acridinium sulfonamide derivatives shown in Formula 1 also have the above-mentioned effects, and those skilled in the art can perform corresponding tests according to the disclosure of this application.

[0087] As an example but not a limitation, the acridine sulfonamide derivatives are used to label polypeptides or proteins containing thiol groups, for example, to detect thyroid hormones, including thyroxine and triiodothyronine, or to detect sex hormones, including progesterone, testosterone, and estradiol.

[0088] The present application also discloses a chemiluminescent immunoassay kit, which comprises the above-mentioned acridine sulfonamide derivative or its stereoisomer / tautomer.

[0089] In summary, the acridinium sulfonamide derivative of the present application is prepared by coupling acridinium sulfonamide and maleimide intermediate hydrochloride in a one-step reaction, and the synthesis is simple and the yield is high; and the synthesized product has an extended connecting arm and less steric hindrance. At the same time, the present application utilizes the specific reaction of maleimide to sulfhydryl groups, and can label sulfhydryl groups under weakly alkaline or acidic conditions, expanding the labeling of proteins by acridinium salts under acidic conditions.

[0090] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, 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, they should be considered to be within the scope of this specification.

[0091] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An acridine sulfonamide derivative, characterized in that: The acridine sulfonamide derivative has a structural formula shown in Formula 1: (Formula 1) 2. A method for preparing an acridine sulfonamide derivative, characterized in that: The following steps are involved: Providing hydrochloride salts of acridine sulfonamide and maleimide intermediates; dissolving the acridine sulfonamide and the maleimide intermediate hydrochloride in an organic solvent; After the reaction is completed, separating and purifying to obtain the acridinium sulfonamide derivative; Wherein, the acridine sulfonamide derivative has a structural formula shown in Formula 1, the acridine sulfonamide has a structural formula shown in Formula 2, and the maleimide intermediate hydrochloride has a structural formula shown in Formula 3:

3. The preparation method according to claim 2, characterized in that: 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, characterized in that: DIEA is added into the organic solvent.

5. The preparation method according to claim 2 or 3, characterized in that: After the reaction is completed, the acridinium sulfonamide derivative obtained by separation and purification includes: After the reaction is completed, liquid phase separation and purification are performed, and then most of the solvent is evaporated and freeze-dried to obtain the acridine sulfonamide derivative.

6. The stereoisomer or tautomer of the acridinium sulfonamide derivative according to claim 1 or the acridinium sulfonamide derivative prepared by the preparation method according to any one of claims 2 to 5.

7. Use of the acridinium sulfonamide according to claim 1 or the stereoisomer or tautomer according to claim 6 in the detection of small molecule haptens.

8. The use according to claim 7, characterized in that Used to label peptides or proteins containing thiol groups.

9. The use according to claim 7, characterized in that Used to detect thyroid hormones, including thyroxine and triiodothyronine; and / or, used to detect sex hormones, including progesterone, testosterone and estradiol.

10. A kit for detecting small molecule haptens, characterized in that: The invention comprises the acridinium sulfonamide derivative according to claim 1 or the stereoisomer or tautomer according to claim 6.

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