Acridine sulfonamide derivative, preparation method, application thereof and kit

Abstract

The present application relates to the field of chemiluminescent immunoassay, and specifically to acridine sulfonamide derivatives, preparation methods, applications thereof, and kits. In some embodiments, the acridine sulfonamide derivatives can label sulfhydryl groups under weakly alkaline or acidic conditions, expanding the labeling of proteins by acridine salts under acidic conditions.

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 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 acridine sulfonamide derivatives, preparation methods, applications and kits thereof, which can label sulfhydryl groups under weakly alkaline or acidic conditions, expanding the labeling of proteins by acridine 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 acridine sulfonamide derivative having 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, comprising the following steps:

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

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

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

[0012] Wherein, the acridine sulfonamide derivative has the structural formula shown in Formula 1, the acridine sulfonamide 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, DIEA is added to the organic solvent.

[0016] In one or more feasible embodiments, after the reaction is completed, separating and purifying 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 acridine sulfonamide derivative described in the first aspect or the stereoisomer or tautomer described in the third aspect.

[0024] According to the examples of this application, the acridinium sulfonamide derivatives of this application are prepared by a one-step coupling reaction of acridinium sulfonamide and a maleimide intermediate hydrochloride. The raw materials are readily available, the synthesis is simple, and the yield is high. Furthermore, the synthesized product has an extended linker arm and less steric hindrance. Furthermore, this application utilizes the specific reaction of maleimide with sulfhydryl groups to label sulfhydryl groups under weakly alkaline or acidic conditions, expanding the application of acridinium salts for protein labeling 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 with reference to 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 1 is a concentration-luminescence value curve of an acridinium sulfonamide marker in T3 hapten detection as shown in one 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 an 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 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 maleimide intermediate hydrochloride in an organic solvent;

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

[0049] Wherein, the acridine sulfonamide derivative has the structural formula shown in Formula 1, the acridine sulfonamide 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 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 labeling substance, which has a simple synthesis and 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 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 acridinium sulfonamide derivative with a measured yield of 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 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 acridinium sulfonamide derivative with a measured yield of 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 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 lyophilized in a freeze dryer (at -50 to -80°C, with the pressure reduced to below 0.1 mbar) to obtain a dry acridinium sulfonamide derivative in a yield of 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, the compound of formula 1 is obtained, and its M=706.8.

[0064] Currently, 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) tests 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) that 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 the turbidity of the sample); haptens, which can be detected through 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, it 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 discovered 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 microparticle chemiluminescence immunoassay kit is used to detect small molecules. The sample and antibody marker are first incubated to allow the hapten to be detected in the sample to fully react with the antibody. Then, the competitive hapten is reacted with the excess antibody, and then combined with the magnetic microparticles. After washing to remove the excess unreacted competitive hapten, the hapten is excited to emit photons, and the strength of the light signal is detected, thereby detecting the high and low concentrations of the antigen in the sample.

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

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

[0069] S1. Carrier protein BSA is thiolated and labeled with an acridine sulfonamide derivative (Formula 1)

[0070] A certain amount of BSA was weighed and dissolved in a 0.1M PB pH 6.0 buffer solution. EDTA was added to make the EDTA concentration 2mM / L. An appropriate amount of 5mM / L tris(2-chloroethyl) phosphate (TCEP) aqueous solution was added to open the disulfide bonds. The reaction was carried out at room temperature for 15 minutes. Without purification, an appropriate amount of acridine sulfonamide maleimide label was directly added. The reaction was carried out at room temperature for 1 hour. The acridine sulfonamide-labeled BSA was obtained by dialysis purification using 0.1M pH 7.4 buffer.

[0071] S2 and T3 haptens label the BSA acridine sulfonamide marker labeled 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 label buffer. React for 1.5 hours and purify by dialyzing with 0.1M pH 6.8 buffer to obtain the competitive hapten label used in the kit - T3 BSA acridine sulfonamide label.

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

[0074] Dilute the T3 BSA acridinium sulfonamide label 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] A certain amount of BSA was weighed and dissolved in 0.1M PB pH 6.0 buffer solution to make the BSA concentration and volume the same as in the previous example. An equal molar amount of acridine sulfonamide active ester labeling substance was added, and the mixture was reacted at room temperature for 1 hour. The mixture was dialyzed and purified with 0.1M pH 7.4 buffer to obtain acridine sulfonamide-labeled BSA.

[0078] S2 and T3 haptens label the BSA acridine sulfonamide marker labeled 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 label buffer. React for 1.5 hours and purify by dialyzing with 0.1M pH 6.8 buffer to obtain the competitive hapten label used in the kit - T3 BSA acridine sulfonamide label.

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

[0081] Dilute the T3 BSA acridinium sulfonamide label with reagent buffer to obtain reagent R2 component.

[0082] The calibration material was tested on the chemiluminescence instrument according to the method for detecting T3 hapten, and the results were as follows: 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 (luminescence index) reached 0.98, with a high overall luminescence value, good gradient, and high sensitivity. This indicates that the synthesized acridine sulfonamide derivative label is very suitable as a luminescent label 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 based on the disclosure of this application.

[0087] By way of example and not limitation, the acridine sulfonamide derivatives are used to label thiol-containing polypeptides or proteins. For example, they can be used to detect thyroid hormones, including thyroxine and triiodothyronine, or 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 stereoisomers / tautomers.

[0089] In summary, the acridinium sulfonamide derivatives described herein are synthesized by coupling acridinium sulfonamide with a maleimide intermediate hydrochloride in a one-step reaction, resulting in simple synthesis and high yield. Furthermore, the resulting product has an extended linker arm and reduced steric hindrance. Furthermore, by utilizing the specific reactivity of maleimide toward sulfhydryl groups, the present invention enables the labeling of sulfhydryl groups under weakly alkaline or acidic conditions, expanding the application of acridinium salts for protein labeling under acidic conditions.

[0090] 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.

[0091] 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. An acridine sulfonamide derivative, characterized in that: The acridine sulfonamide derivative has the 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 maleimide intermediate hydrochloride in an organic solvent; After the reaction is completed, separating and purifying to obtain the acridine sulfonamide derivative; Wherein, the acridine sulfonamide derivative has the structural formula shown in Formula 1, the acridine sulfonamide 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 DIEA is added to the organic solvent.

5. The preparation method according to claim 2 or 3, wherein After the reaction is completed, the acridine 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. Use of the acridine sulfonamide derivative according to claim 1 in the preparation of a small molecule hapten detection reagent for detecting thyroid hormones, wherein the thyroid hormones are selected from thyroxine and triiodothyronine.

7. The use according to claim 6, characterized in that Used to label peptides or proteins containing sulfhydryl groups.

8. A kit for detecting small molecule haptens, characterized in that: Including the acridinium sulfonamide derivative according to claim 1.

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