Synthesis method of acridinium ester compounds

Abstract

The application relates to a synthesis method of an acridine ester compound, which comprises the following steps: under the action of a first alkaline reagent, CTC resin is reacted with 3,5-dimethyl-p-hydroxybenzoic acid to prepare compound 2; compound 2 is reacted with 9-acridine-formyl chloride to prepare compound 3; under the action of an ionic solvent and a second alkaline reagent, compound 3 is reacted with 1,3-propane sulfolactone to prepare compound 4; compound 4 is reacted with an acid reagent to remove 2-chloro-triphenylmethyl chloride resin and prepare compound 5; under the action of a third alkaline reagent and a condensing agent, compound 5 is reacted with compound 6 to prepare the acridine ester compound; wherein the compound 6 comprises any one or both of TSTU and NHS. The synthesis method is simple in process, easy in purification and short in time consumption.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of organic synthesis, in particular to a synthesis method of acridinium ester compounds. BACKGROUND

[0002] Chemiluminescence Immunoassay (CLIA) is a new type of luminescent labeling technology that combines chemiluminescence or bioluminescence system with immune reaction for detecting trace amounts of antigens or antibodies. Its detection principle of immune reaction is basically the same as that of radioimmunoassay and enzyme immunoassay, except that it uses a luminescent substance instead of a color-developing substance, and then the content of the measured substance is deduced quantitatively by the relative intensity of luminescence. After rapid development for nearly ten years, there are various types of chemiluminescent substrates on the market, the common types mainly include acridinium esters, luminol and its derivatives, peroxodisuccinates and adamantane, etc. Among them, acridinium esters and acridinium ester derivatives have many advantages as immune analysis tracers, such as: no need for catalyst, high specificity, high luminescence quantum yield, good stability, etc., and thus are widely used.

[0003] At present, the most widely used acridinium ester substance in the market is NSP-DMAE-NHS, and its chemical structure is as follows: In the chemical structure, two methyl groups are introduced at the ortho position of the aromatic ring of the easily hydrolyzed phenolic ester, which increases the steric hindrance and delays the hydrolysis rate in aqueous solution, thereby improving the stability.

[0004] At present, NSP-DMAE-NHS is synthesized by a liquid phase method, using 4-hydroxy-3,5-dimethylbenzoic acid as the initial raw material, and going through esterification reaction, condensation reaction, reduction reaction, substitution reaction, oxidation reaction, hydrolysis reaction and activation of carboxyl group reaction, a total of seven steps, to finally obtain the target product (reference literature Green Chemistry Letters and Reviews, 6:3, 237-248). On the one hand, the reactant equivalent needs to be strictly controlled, otherwise it will affect the purification. On the other hand, column chromatography purification or high performance liquid chromatography purification needs to be carried out for each step, and a freeze-drying process is required after each purification, which is extremely time-consuming. In the last three steps, due to the introduction of hydrophilic sulfonic acid chains, only C18 column and high performance liquid chromatography can be used for purification. In addition, due to the need for purification, the reaction amount cannot be scaled up. SUMMARY

[0005] Therefore, it is necessary to provide a synthesis method of acridinium ester compounds, which is simple in synthesis, easy in purification and short in time consumption.

[0006] A synthesis method of acridinium ester compounds, comprising the following steps:

[0007] reacting 2-chlorotrityl chloride resin with 3,5-dimethyl-p-hydroxybenzoic acid in the presence of a first basic reagent to produce compound 2 having a structure of wherein, 2-chlorotrityl chloride resin is represented by

[0008] reacting compound 2 with 9-acridine-carbonyl chloride to produce compound 3 having a structure of

[0009] reacting compound 3 with 1,3-propane sultone in the presence of an ionic solvent and a second basic reagent to produce compound 4 having a structure of

[0010] reacting compound 4 with an acidic reagent to remove 2-chlorotrityl chloride resin to produce compound 5 having a structure of

[0011] reacting compound 5 with compound 6 in the presence of a third basic reagent to produce acridine ester compound having a structure of wherein, compound 6 includes any one or both of 2-succinimidyl-1,1,3,3-tetramethyl uronium tetrafluoroborate and N-hydroxysuccinimide.

[0012] In one embodiment, the step of reacting 2-chlorotrityl chloride resin with 3,5-dimethyl-p-hydroxybenzoic acid in the presence of a first basic reagent satisfies any one or several of the following conditions:

[0013] (1) the molar ratio of 2-chlorotrityl chloride resin to 3,5-dimethyl-p-hydroxybenzoic acid is 1:(2-5);

[0014] (2) the molar ratio of 2-chlorotrityl chloride resin to the first basic reagent is 1:(2-5);

[0015] (3) the first basic reagent includes any one or combination of several of N,N-diisopropyl ethylamine and triethylamine.

[0016] In one embodiment, the step of producing compound 2 further includes, after the reaction is completed, washing with a methanol solution having a mass percentage concentration of 5-10% of the first basic reagent, then washing with dichloromethane, and then filtering.

[0017] ​​​​In one of the embodiments, the step of reacting the compound 2 with 9-acridine-formyl chloride comprises: first, reacting 9-acridine carboxylic acid with p-toluenesulfonyl chloride at 20-30°C for 20-40 min to prepare the 9-acridine-formyl chloride, and then adding the compound 2 to continue the reaction at 20-30°C for 8-12 h.

[0018] In one of the embodiments, the molar ratio of the 9-acridine carboxylic acid, the p-toluenesulfonyl chloride and the compound 2 is 1:(2-4):(0.2-0.5).

[0019] In one of the embodiments, the step of preparing the compound 3 further comprises: after the reaction is completed, first washing with N,N-dimethylformamide, then washing with dichloromethane, and then filtering.

[0020] In one of the embodiments, the step of reacting the compound 3 with 1,3-propane sultone under the action of an ionic solvent and a second basic reagent meets any one or several of the following conditions:

[0021] (1) the molar ratio of the compound 3, the 1,3-propane sultone and the second basic reagent is 1:(2-4):(10-30);

[0022] (2) the second basic reagent comprises any one or several of 2,6-di-tert-butylpyridine and potassium carbonate;

[0023] (3) the ionic solvent comprises any one or several of 1-butyl-3-methylimidazolium hexafluorophosphate and 1-butyl-3-methylimidazolium tetrafluoroborate;

[0024] (4) the temperature for the reaction of the compound 3 with the 1,3-propane sultone is 155-165°C, and the reaction time is 8-12 h.

[0025] In one of the embodiments, the step of preparing the compound 4 further comprises: after the reaction is completed, first washing with N,N-dimethylformamide, then washing with dichloromethane, and then filtering.

[0026] In one of the embodiments, the step of preparing the compound 4 further comprises: after the reaction is completed, before washing and filtering, continuously adding the ionic liquid, the second basic reagent and the 1,3-propane sultone to the reaction system.

[0027] In one of the embodiments, the step of reacting the compound 4 with an acidic reagent meets any one or several of the following conditions:

[0028] (1) the molar ratio of the compound 4 and the acidic reagent is 1:(40-80);

[0029] (2) the reaction time of the compound 4 with the acidic reagent is 30 min to 60 min, and the reaction temperature is 20 °C to 30 °C;

[0030] (3) the acidic reagent includes any one or several of trifluoroacetic acid, hydrochloric acid, and hexafluoroisopropanol.

[0031] In one of the embodiments, the step of reacting the compound 5 with the compound 6 under the action of the third basic reagent satisfies any one or several of the following conditions:

[0032] (1) the molar ratio of the compound 5 to the compound 6 is 1: (2-5) ;

[0033] (2) the molar ratio of the compound 5 to the third basic reagent is 1: (2-4) ;

[0034] (3) the third basic reagent includes any one or several of N, N-diisopropyl ethylamine and triethylamine;

[0035] (4) the reaction time of the compound 5 with the compound 6 is 4 h to 10 h, and the temperature is 20 °C to 30 °C.

[0036] In one of the embodiments, the compound 6 includes 2-succinimidyl-1, 1, 3, 3-tetramethyl uronium tetrafluoroborate, and the third basic reagent includes N, N-diisopropyl ethylamine; or,

[0037] the compound 6 is N-hydroxy succinimide, and a condensing agent is added during the reaction of the compound 5 with the compound 6, and the condensing agent includes any one or several of dicyclohexyl carbodiimide, 1- (3-dimethyl aminopropyl) -3-ethyl carbodiimide hydrochloride, and N, N-diisopropyl carbodiimide.

[0038] The synthesis method of the acridine ester compound ingeniously uses the solid-phase synthesis method of polypeptide synthesis in the synthesis of the acridine ester compound NSP-DMAE-NHS, fully utilizes the advantages of solid-phase synthesis, optimizes the synthesis route by selecting a suitable resin carrier, reduces two-step reactions by eliminating the protection and deprotection reactions of the carboxyl group, realizes the synthesis of the acridine ester compound NSP-DMAE-NHS in a total of five steps, and does not need to use column chromatography for separation and purification in the intermediate process, but only needs simple washing and filtration, and only needs to use high-performance liquid chromatography for separation and purification in the last step, thereby greatly saving time and cost. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 a schematic diagram of the light-emitting principle of the acridine ester compound;

[0040] Figure 2 Process flow chart for the synthesis method of acridinium ester compound of an embodiment;

[0041] Figure 3 Mass spectrum of intermediate compound 2 prepared in Example 1;

[0042] Figure 4 Mass spectrum of acridinium ester compound prepared in Example 1;

[0043] Figure 5 HPLC purity test of acridinium ester compound prepared in Example 1;

[0044] Figure 6 HPLC purity test of commercially available standard acridinium ester compound. DETAILED DESCRIPTION

[0045] In order to facilitate the understanding of the present application, the present application will be described in more detail below with specific embodiments. The preferred embodiments of the present application are given in the specific embodiments. However, the present application can be realized in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the description of the present application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application.

[0047] Unless otherwise indicated or contradictory, the terms or phrases used in the present application have the following meanings:

[0048] In the present application, "first", "second", and the like are only for the purpose of description, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined with "first", "second" can explicitly or implicitly include at least one of the features.

[0049] In the present application, "one or more" refers to any one, any two or any two or more of the listed items. Among them, "several" refers to any two or more.

[0050] In the present application, the percentage concentration involved, unless otherwise specified, refers to the final concentration. The final concentration refers to the proportion of the added component in the system after the component is added.

[0051] The terms "preferably", "more preferred", "most preferred" and the like in the application refer to embodiments of the application which can provide certain benefits under certain circumstances. However, alternative embodiments can also be preferred under the same or other circumstances. Furthermore, the representation of one or more preferences is not to be construed as excluding embodiments falling within the scope of the application from the scope of the application.

[0052] When a numerical range is disclosed herein, such range is inclusive of the minimum and maximum values and of each discrete value between the minimum and the maximum values. Further, when a range is provided, it is intended to include every subrange between and including the minimum value and the maximum value of the range. In other words, unless specifically stated otherwise, all ranges disclosed herein are to be understood to be inclusive of all sub-ranges subsumed therein.

[0053] In the application, the open-ended technical features include both the closed technical solution consisting of the listed features and the open technical solution containing the listed features.

[0054] The terms "comprising", "having", "including", and "containing" and any variations thereof in the application are intended to cover a non-exclusive inclusion. For example, a process, method, system, product, or apparatus that comprises a list of steps or elements is not necessarily limited to those listed steps or elements but can include other not-listed steps or elements. In other words, unless specifically set forth otherwise, the process, method, system, product, or apparatus that comprises a list of steps or elements does not necessarily preclude the inclusion of other steps or elements.

[0055] Reference in the application to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the application. The appearances of the phrase "in an embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are they necessarily mutually exclusive of one another. It is expressly understood that any of the described embodiments of the application are combinable with each other.

[0056] Reference in the application to "room temperature" means a temperature of 20°C to 30°C.

[0057] The principle of luminescence of acridinium ester compounds is as follows: in alkaline hydrogen peroxide solution, the molecule of acridinium ester is attacked by hydrogen peroxide ion, and the substituent group on the acridine ring can form unstable dioxetane with C-9 on the acridine ring and hydrogen peroxide. The dioxetane is decomposed into carbon dioxide and an excited state of N-methyl acridinium ketone, which emits photons with a maximum emission wavelength of 440 nm when it returns to the excited state. Please refer to Figure 1 . Figure 1 Blue Emission refers to blue light emission.

[0058] As described in the background, the synthesis of traditional NSP-DMAE-NHS usually adopts a seven-step liquid-phase synthesis method, which has many reaction steps and each step needs to be purified by column chromatography or high-performance liquid chromatography, which is troublesome and time-consuming. Based on this, the present application provides a synthesis method of acridine ester compounds, please refer to Figure 2 , which comprises the following steps:

[0059] Step S110: reacting 2-chlorotrityl chloride resin and 3,5-dimethyl-p-hydroxybenzoic acid under the action of a first basic reagent to prepare compound 2.

[0060] wherein the structural formula of compound 2 is represents 2-chlorotrityl chloride resin.

[0061] In some embodiments, the first basic reagent can include but is not limited to any one or several of N,N-diisopropylethylamine (DIPEA) and triethylamine.

[0062] In some embodiments, the molar ratio of 2-chlorotrityl chloride resin (CTC resin) to 3,5-dimethyl-p-hydroxybenzoic acid is 1:(1-5). In a specific example, the molar ratio of CTC resin to 3,5-dimethyl-p-hydroxybenzoic acid can be but not limited to 1:2, 1:2.5, 1:3, 1:3.2, 1:3.4, 1:3.5, 1:3.6, 1:3.8, 1:4, 1:4.2, 1:4.4, 1:4.5, 1:4.6, 1:4.8, 1:5 or a range composed of any two of these values.

[0063] In some embodiments, the molar ratio of 2-chlorotrityl chloride resin to the first basic reagent is 1:(2-5). In a specific example, the molar ratio of 2-chlorotrityl chloride resin to the first basic reagent can be but not limited to 1:2, 1:2.5, 1:3, 1:3.2, 1:3.4, 1:3.5, 1:3.6, 1:3.8, 1:4, 1:4.2, 1:4.4, 1:4.5, 1:4.6, 1:4.8, 1:5 or a range composed of any two of these values.

[0064] In some embodiments, the reaction time is 4h-6h. In a specific example, the reaction time can be but not limited to 4h, 4.5h, 5h, 5.5h, 6h or a range composed of any two of these values.

[0065] In some embodiments, the reaction temperature is room temperature. The room temperature can be, but is not limited to, 20-30°C, for example, 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, or a range between any two of these values.

[0066] In some embodiments, step S110 further comprises a purification step. Specifically, the purification step comprises: after the reaction is completed, first washing with a methanol solution with a mass percentage of 5-10% of the first basic reagent, then washing with dichloromethane, and then filtering.

[0067] In some embodiments, step S110 comprises: swelling the CTC resin in a first organic solvent, then adding 3,5-dimethyl-p-hydroxybenzoic acid and the first basic reagent, reacting at room temperature for 4-6 hours, filtering out the unreacted CTC resin after the reaction is completed, then washing with a methanol solution with a mass percentage of 5-10% of the first basic reagent, then washing with dichloromethane, filtering, and obtaining compound 2. In a specific example, the first organic solvent can be, but is not limited to, N,N-dimethylformamide (DMF).

[0068] Solid-phase synthesis refers to a method of using insoluble polymers as carriers to fix one of the reactants on the polymer carrier through active groups, so that organic synthesis is carried out on the same phase. At present, solid-phase synthesis is mostly used for polypeptide synthesis. In the present embodiment, the inventors apply solid-phase synthesis to the synthesis of acridine ester compounds, use 2-chlorotrityl chloride resin (CTC resin) as a carrier, optimize the reaction route, and finally obtain the target product through a five-step synthesis method. The whole process only needs simple washing and filtering, and only needs to use high-performance liquid chromatography for purification when the final target product is obtained, which is convenient and fast, and has high yield.

[0069] Commonly used resins for solid-phase synthesis include CTC resin, MBHA resin, and Wang resin, etc. In the present embodiment, CTC resin is used. This is because: MBHA resin is connected with an amino group, and after the reaction, it is cut off, and the final product is an amide bond, not a carboxyl group, so it is not feasible. If Wang resin is used, the first step is to connect the activated carboxyl group with the hydroxyl group of Wang resin, and the raw material for the first step of the above synthesis method is 3,5-dimethyl-p-hydroxybenzoic acid, which itself has a hydroxyl group. Therefore, if Wang resin is used, one step of protection of the hydroxyl group of the raw material is needed. The purpose of the synthesis method of the present embodiment is to reduce the experimental steps and facilitate purification. Therefore, in the present embodiment, CTC resin is used as the carrier for solid-phase synthesis.

[0070] Step S120: reacting compound 2 with 9-acridine-formyl chloride to prepare compound 3.

[0071] The structural formula of compound 3 is

[0072] In some embodiments, the molar ratio of 9-acridine-formyl chloride to compound 2 is 1 : (0.2-0.5). In a specific example, the molar ratio of 9-acridine-formyl chloride to compound 2 can be, but is not limited to, 1 :0.2, 1 :0.22, 1 :0.25, 1 :0.28, 1 :0.3, 1 :0.32, 1 :0.35, 1 :0.38, 1 :0.4, 1 :0.42, 1 :0.45, 1 :0.48, 1 :0.5, or a range formed by any two of these values.

[0073] In some embodiments, the step of reacting compound 2 with 9-acridine-formyl chloride comprises: first reacting 9-acridine carboxylic acid with p-toluenesulfonyl chloride at 20-30 °C for 20-40 min to prepare 9-acridine-formyl chloride, and then adding compound 2 to continue the reaction at 20-30 °C for 8-12 h. Since 9-acridine-formyl chloride is about 10 times more expensive than 9-acridine carboxylic acid, and 9-acridine-formyl chloride is more active and is prone to hydrolysis and deterioration when taken, increasing the difficulty of operation, therefore, in this embodiment, preferably, 9-acridine-formyl chloride is first prepared by reacting 9-acridine carboxylic acid with p-toluenesulfonyl chloride, and then reacted with compound 2.

[0074] In a specific example, the temperature of the reaction can be, but is not limited to, 20 °C, 22 °C, 24 °C, 25 °C, 26 °C, 28 °C, 30 °C, or a range formed by any two of these values.

[0075] In a specific example, the time for reacting 9-acridine carboxylic acid with p-toluenesulfonyl chloride at 20-30 °C can be, but is not limited to, 20 min, 22 min, 24 min, 25 min, 26 min, 28 min, 30 min, 32 min, 34 min, 35 min, 36 min, 38 min, 40 min, or a range formed by any two of these values.

[0076] In a specific example, the time for adding compound 2 to continue the reaction at 20-30 °C can be, but is not limited to, 8 h, 8.5 h, 9 h, 9.5 h, 10 h, 10.5 h, 11 h, 11.5 h, 12 h, or a range formed by any two of these values.

[0077] In some embodiments, the molar ratio of 9-acridinecarboxylic acid to compound 2 is 1:(0.2 to 0.5). In a specific example, the molar ratio of 9-acridinecarboxylic acid to compound 2 may be, but is not limited to, 1:0.2, 1:0.22, 1:0.25, 1:0.28, 1:0.3, 1:0.32, 1:0.35, 1:0.38, 1:0.4, 1:0.42, 1:0.45, 1:0.48, 1:0.5, or a range of any two of these values.

[0078] In some embodiments, the molar ratio of 9-acridinecarboxylic acid to p-toluenesulfonyl chloride is 1:(2 to 4). In a specific example, the molar ratio of 9-acridinecarboxylic acid to p-toluenesulfonyl chloride may be, but is not limited to, 1:2, 1:2.2, 1:2.4, 1:2.5, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.4, 1:3.5, 1:3.6, 1:3.8, 1:4, or a range of any two of these values.

[0079] In some embodiments, step S120 further includes: after the reaction is complete, washing with DMF, then washing with dichloromethane, and then filtering. In a specific example, washing with DMF three times, then washing with dichloromethane three times. The above solvents are commonly used washing solvents in solid-phase synthesis methods. If other solvents are used for washing, it may cause resin shrinkage and affect subsequent reactions. Therefore, in this embodiment, it is preferable to wash with DMF first, and then wash with dichloromethane.

[0080] The above steps S110 and S120, by using excess reactants and allowing for a certain reaction time, can ensure complete reaction and high yield by LC-MS monitoring.

[0081] Step S130: Compound 3 is reacted with 1,3-propanesulfonyl lactone in the presence of an ionic solvent and a second basic reagent to prepare compound 4.

[0082] The structural formula of compound 4 is as follows:

[0083] In some embodiments, the molar ratio of compound 3 to 1,3-propanesulfonyl lactone is 1:(2 to 4). In a specific example, the molar ratio of compound 3 to 1,3-propanesulfonyl lactone may be, but is not limited to, 1:2, 1:2.2, 1:2.4, 1:2.5, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.4, 1:3.5, 1:3.6, 1:3.8, 1:4, or a range of any two of these values.

[0084] In some embodiments, the molar ratio of compound 3 to the second basic reagent is 1:(10-30). In a specific example, the molar ratio of compound 3 to the second basic reagent can be, but is not limited to, 1:10, 1:12, 1:14, 1:15, 1:16, 1:18, 1:20, 1:22, 1:24, 1:25, 1:26, 1:28, 1:30, or a range consisting of any two of these values.

[0085] In some embodiments, the second basic reagent can be, but is not limited to, 2,6-di-tert-butylpyridine. The second basic reagent can also be potassium carbonate.

[0086] In some embodiments, the ionic solvent can be, but is not limited to, 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF6), and can also be 1-butyl-3-methylimidazolium tetrafluoroborate.

[0087] In some embodiments, the temperature for the reaction of compound 3 with 1,3-propane sultone is 155-165°C, and the reaction time is 8-12h. In a specific example, the temperature for the reaction of compound 3 with 1,3-propane sultone can be, but is not limited to, 155°C, 156°C, 157°C, 158°C, 159°C, 160°C, 161°C, 162°C, 163°C, 164°C, 165°C, or a range consisting of any two of these values. In a specific example, the time for the reaction of compound 3 with 1,3-propane sultone can be, but is not limited to, 8h, 8.5h, 9h, 9.5h, 10h, 10.5h, 11h, 11.5h, 12h, or a range consisting of any two of these values.

[0088] In some embodiments, step S130 further comprises, after the reaction is completed, washing with DMF three times, washing with dichloromethane three times, and then filtering. In a specific example, washing with DMF three times, washing with dichloromethane three times.

[0089] In some embodiments, step S130 further comprises, after the reaction is completed, continuing to add ionic liquid, second basic reagent, and 1,3-propane sultone to the reaction system before washing and filtering. In the process of synthesizing compound 4 by traditional liquid phase method, the yield of this step is low. In the present embodiment, by repeatedly adding ionic liquid, second basic reagent, and 1,3-propane sultone, and finally monitoring by LC-MS, the reaction is ensured to be complete.

[0090] Further, the step of repeatedly adding ionic liquid, second basic reagent, and 1,3-propane sultone to the reaction system is repeated three times.

[0091] Step S140: reacting compound 4 with an acidic reagent to remove the 2-chlorotrityl chloride resin and prepare compound 5.

[0092] wherein compound 5 has a structural formula of

[0093] In some embodiments, the molar ratio of compound 4 to the acidic reagent is 1: (40-80). In a specific example, the molar ratio of compound 4 to the acidic reagent can be, but is not limited to, 1:40, 1:42, 1:45, 1:48, 1:50, 1:52, 1:55, 1:58, 1:60, 1:62, 1:65, 1:68, 1:70, 1:72, 1:75, 1:78, 1:80, or a range consisting of any two of these values.

[0094] In some embodiments, the reaction of compound 4 with the acidic reagent is performed for 30-60 min at a temperature of 20-30°C.

[0095] In a specific example, the reaction of compound 4 with the acidic reagent can be, but is not limited to, for 30 min, 32 min, 34 min, 35 min, 36 min, 38 min, 40 min, 42 min, 44 min, 45 min, 46 min, 48 min, 50 min, 52 min, 54 min, 55 min, 56 min, 58 min, 60 min, or a range consisting of any two of these values. In a specific example, the reaction of compound 4 with the acidic reagent can be, but is not limited to, at a temperature of 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, or a range consisting of any two of these values.

[0096] In some embodiments, the acidic reagent includes any one or more of trifluoroacetic acid, hydrochloric acid, and hexafluoroisopropanol. Further, the acidic reagent is mixed with compound 4 in the form of a solution, and the mass percentage concentration of the acidic reagent in the solution containing the acidic reagent is 3-10%. In a specific example, the mass percentage concentration of the acidic reagent can be, but is not limited to, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range consisting of any two of these values.

[0097] Optionally, the solution containing the acidic reagent can be a dichloromethane solution containing the acidic reagent. In a specific example, the solution containing the acidic reagent is a dichloromethane solution containing trifluoroacetic acid, and the mass percentage concentration of trifluoroacetic acid is 3-10%.

[0098] In some embodiments, step S140 comprises: mixing compound 4 with a solution containing an acidic reagent for 30 min to 60 min, after the resin is completely blackened, a second organic solvent is added and left to stand, the upper floating resin is removed, and filtration is performed to obtain compound 5. In this embodiment, the solution containing the acidic reagent is a dichloromethane solution containing trifluoroacetic acid, and the mass percentage concentration of trifluoroacetic acid is 3% to 10%. Alternatively, the second organic solvent comprises dichloromethane.

[0099] Step S150: under the action of a third basic reagent, compound 5 is reacted with compound 6 to prepare an acridinium ester compound; compound 6 comprises any one or both of 2-succinimidyl-1,1,3,3-tetramethyl uronium tetrafluoroborate and N-hydroxysuccinimide.

[0100] In some embodiments, the structure of the acridinium ester compound is

[0101] In some embodiments, the molar ratio of compound 5 to compound 6 is 1:(2-5). In a specific example, the molar ratio of compound 5 to compound 6 can be, but is not limited to, 1:2, 1:2.2, 1:2.4, 1:2.5, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.4, 1:3.5, 1:3.6, 1:3.8, 1:4, 1:4.2, 1:4.4, 1:4.5, 1:4.6, 1:4.8, 1:5, or a range composed of any two of these values.

[0102] In some embodiments, the molar ratio of compound 5 to the third basic reagent is 1:(2-4). In a specific example, the molar ratio of compound 5 to the third basic reagent can be, but is not limited to, 1:2, 1:2.2, 1:2.4, 1:2.5, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.4, 1:3.5, 1:3.6, 1:3.8, 1:4, or a range composed of any two of these values.

[0103] In some embodiments, the third basic reagent comprises any one or more of DIPEA and triethylamine. It can be understood that the third basic reagent can be the same as or different from the first basic reagent.

[0104] In some embodiments, the reaction of compound 5 with compound 6 is performed for 4-10 hours at 20-30°C. In a specific example, the reaction of compound 5 with compound 6 can be, but is not limited to, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, or a range defined by any two of these values. In a specific example, the reaction of compound 5 with compound 6 can be, but is not limited to, 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, or a range defined by any two of these values.

[0105] In some embodiments, compound 6 comprises 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU), and the third basic reagent comprises DIPEA, and no condensing agent is added.

[0106] In other embodiments, compound 6 comprises N-hydroxysuccinimide, and a condensing agent is added in step S150, and the condensing agent comprises any one or more of N,N'-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N,N-diisopropylcarbodiimide (DIC).

[0107] Preferably, compound 6 comprises 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU), and the third basic reagent comprises DIPEA. The use of N-hydroxysuccinimide and a condensing agent makes the system sensitive to water, and requires the use of super dry DMF. In addition, HPLC purification is required at the end, and the condensing agent byproduct is also difficult to remove. Therefore, in the present embodiment, compound 6 preferably comprises 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate, and no condensing agent is added.

[0108] In some embodiments, step S150 is performed in a third organic solvent, which can be, but is not limited to, DMF.

[0109] In some embodiments, step S150 further comprises: after the reaction is completed, separating and purifying by high performance liquid chromatography. Since the hydrophilic sulfonic acid chain is introduced in this step, only high performance liquid chromatography can be used for purification.

[0110] The synthesis method of the acridinium ester compound described above has at least the following advantages:

[0111] (1) The synthesis method of the acridine ester compound uses the solid-phase synthesis method of polypeptide synthesis ingeniously for the synthesis of acridine ester (NSP-DMAE-NHS), fully utilizes the advantages of solid-phase synthesis, ingeniously designs the reaction by selecting a suitable resin carrier, reduces two-step reactions by not needing to protect and deprotect the carboxyl group, realizes the synthesis of acridine ester (NSP-DMAE-NHS) in a total of five steps, and does not need to use column chromatography for separation and purification in the intermediate process, but only needs to use the washing and filtering method, and finally needs to use high-performance liquid chromatography for separation and purification, thereby greatly saving time and cost.

[0112] (2) The synthesis method of the acridine ester compound can greatly improve the yield by repeating the reaction for the step that is difficult to react, so that the yield of each step reaction can reach more than 95% except for the last step.

[0113] (3) The synthesis method of the acridine ester compound greatly reduces the use amount of organic solvents, saves cost and reduces pollution by not using column chromatography for purification of the intermediate product.

[0114] In order to make the purpose and advantages of the present application clearer, the synthesis method of the acridine ester compound and its effects will be further described in detail in combination with specific examples below, and it should be understood that the specific examples described herein are only used to explain the present application and should not be used to limit the present application. The following examples do not include other components except for unavoidable impurities if not otherwise specified. The drugs and instruments used in the examples are selected according to the conventional selection in the art if not otherwise specified. The experimental methods not specified in the examples are realized according to the conventional conditions, for example, the conditions described in the literature, books or the methods recommended by the manufacturer.

[0115] Example 1

[0116] The present embodiment provides a synthesis method of an acridine ester compound, and the synthesis route is as follows:

[0117]

[0118] The synthesis steps are as follows:

[0119] (1) Synthesis of compound 2: 2-chlorotrityl chloride resin (1 mmol / g, 1 g) was swelled in 15 mL of DMF for 15 min, then 3,5-dimethyl-p-hydroxybenzoic acid (330 mg, 2 mmol) and DIPEA (0.347 mL, 2 mmol, N,N-diisopropylethylamine) were added, and the reaction was stirred at room temperature for four hours. After the reaction was completed, the resin was filtered, washed with a 5% methanol solution by mass fraction of DIPEA for 10 min, washed with 10 mL of dichloromethane, and suction filtered to obtain compound 2 with a yield of 96%.

[0120] (2) Synthesis of compound 3: 9-Acridinecarboxylic acid (446 mg, 2 mmol) was dissolved in 20 mL of super dry pyridine, and p-toluenesulfonyl chloride (762 mg, 4 mmol) was added, and the reaction was allowed to proceed at room temperature for 20 min, and then the reaction solution was added to compound 2 (with resin, 1 mmol), and the reaction was allowed to proceed at room temperature for 8 hours. After the reaction was completed, it was washed with DMF three times, and then washed with dichloromethane three times, and then filtered and dried, to obtain compound 3, with a yield of 97%.

[0121] (3) Synthesis of compound 4: Compound 3 (with resin, 1 mmol) was added to [BMIM][PF6] (10 mL), followed by the addition of 2,6-di-tert-butylpyridine (2.21 mL, 10 mmol) and 1,3-propane sultone (244 mg, 2 mmol), and the reaction was allowed to proceed at 155 degrees Celsius for 8 hours, and then the temperature was lowered, and it was washed with DMF three times, and then washed with dichloromethane three times, and then filtered. The above step (3) was repeated three times to ensure that the reaction was complete, to obtain compound 4, with a yield of 96%.

[0122] (4) Synthesis of compound 5: Dried compound 4 (with resin, 1 mmol) was added to a 3% trifluoroacetic acid solution in dichloromethane (15 mL), and the reaction was allowed to proceed for 30 min, and then 50 mL of dichloromethane was added, and it was allowed to stand for 20 min, and then the upper floating resin was removed with a pipette, and then filtered with filter paper, to obtain yellow powder, which was compound 5, without further purification, with a yield of 95%.

[0123] (5) Synthesis of NSP-DMAE-NHS: Compound 5 (493 mg, 1 mmol) was dissolved in 5 mL of DMF, and 2-succinimidyl-1,1,3,3-tetramethyl uronium tetrafluoroborate (602 mg, 2 mmol) and DIPEA (0.347 mL, 2 mmol) were added, and the reaction was allowed to proceed at room temperature for 4 hours. Subsequent separation and purification were performed by high performance liquid chromatography, to obtain the acridine ester compound NSP-DMAE-NHS of the present example, with a yield of 86%.

[0124] Example 2

[0125] The present example provides a synthesis method of an acridine ester compound, and the synthesis route is as follows:

[0126]

[0127] The synthesis steps are as follows:

[0128] (1) Synthesis of compound 2: 2-chlorotrityl chloride resin (1 mmol / g, 1 g) was swelled in 15 mL DMF for 15 min, then 3,5-dimethyl-p-hydroxybenzoic acid (660 mg, 4 mmol) and DIPEA (0.694 mL, 4 mmol, N,N-diisopropylethylamine) were added, and the reaction was stirred at room temperature for four hours. After the reaction was completed, the resin was filtered and washed with a 10% methanol solution of DIPEA for 10 min, then washed with 10 mL dichloromethane, and suction filtered to obtain compound 2 with a yield of 97%.

[0129] (2) Synthesis of compound 3: 9-acridinecarboxylic acid (446 mg, 2 mmol) was dissolved in 20 mL super dry pyridine, and p-toluenesulfonyl chloride (762 mg, 4 mmol) was added, and the reaction was stirred at room temperature for 20 min, then the reaction solution was added to compound 2 (with resin, 1 mmol), and the reaction was stirred at room temperature for 8 hours. After the reaction was completed, it was washed with DMF three times, then washed with dichloromethane three times, and suction filtered and dried to obtain compound 3 with a yield of 95%.

[0130] (3) Synthesis of compound 4: Compound 3 (with resin, 1 mmol) was added to [BMIM][BF4] (10 mL), followed by the addition of potassium carbonate (1.38 g, 10 mmol) and 1,3-propane sultone (244 mg, 2 mmol), and the temperature was raised to 155 degrees Celsius, and the reaction was carried out for 8 hours, then the temperature was lowered, and the reaction was washed with DMF three times, then washed with dichloromethane three times, and suction filtered. The above step (3) was repeated three times to ensure that the reaction was complete, and compound 4 was obtained with a yield of 96%.

[0131] (4) Synthesis of compound 5: Dried compound 4 (with resin, 1 mmol) was added to a 5% hexafluoroisopropanol solution in dichloromethane (15 mL), and the reaction was carried out for 30 min, then 50 mL of dichloromethane was added, and the reaction was allowed to stand for 20 min, then the upper floating resin was removed with a pipette, and the reaction was filtered with filter paper to obtain yellow powder, which was compound 5, and no further purification was required, and the yield was 95%.

[0132] (5) Synthesis of NSP-DMAE-NHS: Compound 5 (493 mg, 1 mmol) was dissolved in 5 mL DMF, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (383 mg, 2 mmol) and DIPEA (0.347 mL, 2 mmol) were added, and the reaction was stirred at room temperature for 4 hours. Subsequent separation and purification were carried out by high performance liquid chromatography to obtain the acridine ester compound NSP-DMAE-NHS of the present example with a yield of 85%.

[0133] Figure 3The mass spectrum of the intermediate compound 2 prepared in Example 1. Figure 4 The mass spectrum of the acridinium ester compound prepared in Example 1. In Figure 3 and Figure 4 In the above, Counts is an arbitrary unit representing relative intensity, Mass-to-charge represents mass-to-charge ratio, ESI scan represents ESI source positive mode scan, and Frag represents input voltage applied to the end of the capillary outlet. Figure 5 The HPLC purity test of the acridinium ester compound prepared in Example 1 was tested, and the tested purity was 95.5%. Figure 6 The HPLC purity test of the acridinium ester compound prepared in Example 1 was tested, and the tested purity was 95.5%. Figure 5 The HPLC purity test of the acridinium ester compound prepared in Example 1 was tested, and the tested purity was 95.5%.

[0134] The technical features of the above-described embodiments can be combined arbitrarily, and for the sake of brevity, all possible combinations of the technical features in the above-described embodiments are not described, but as long as the combinations of the technical features do not contradict, they should be considered as the scope of the present disclosure.

[0135] The above-described embodiments only express several embodiments of the present application, facilitate specific and detailed understanding of the technical solutions of the present application, but should not be understood as a limitation on the scope of patent protection. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of protection of the present application. It should be understood that the technical solutions obtained by the skilled person in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present application are within the scope of protection of the appended claims of the present application. Therefore, the scope of protection of the present application patent should be based on the contents of the appended claims, and the description and drawings can be used to explain the contents of the claims.

Claims

1. A method for synthesizing acridinium ester compounds, characterized by, The method comprises the following steps: The compound 2 having the structural formula is prepared by reacting a 2-chlorotrityl chloride resin with 3,5-dimethyl-p-hydroxybenzoic acid in the presence of a first basic reagent, wherein represents a 2-chlorotrityl chloride resin. The compound 2 is reacted with 9-acridine-carbonyl chloride to produce a compound 3 having a structure of ​ The compound 3 is reacted with 1,3-propane sultone under the action of an ionic solvent and a second basic agent to prepare a compound 4 with a structural formula of ; wherein the molar ratio of the compound 3 to the 1,3-propane sultone is 1: (2-3), and after the reaction is completed, the step of adding the ionic solvent, the second basic agent and the 1,3-propane sultone to the reaction system is repeated three times. The compound 4 is reacted with an acidic reagent to remove the 2-chlorotrityl chloride resin to produce compound 5 of the structure ​ In the presence of a third basic reagent, the compound 5 is reacted with a compound 6 to produce an acridinium ester compound having a structure of ; wherein the compound 6 is selected from any one or both of 2-succinimidyl-1,1,3,3-tetramethyl uronium tetrafluoroborate and N-hydroxysuccinimide.

2. The method for synthesizing acridine ester compounds according to claim 1, characterized in that, The step of reacting the 2-chlorotrityl chloride resin with 3,5-dimethyl-p-hydroxybenzoic acid under the action of a first basic reagent meets any one or several of the following conditions: (1) the molar ratio of the 2-chlorotrityl chloride resin to the 3,5-dimethyl-p-hydroxybenzoic acid is 1:(2-5); (2) the molar ratio of the 2-chlorotrityl chloride resin to the first basic reagent is 1:(2-5); (3) the first basic reagent is selected from any one or combination of N,N-diisopropylethylamine and triethylamine.

3. The method for synthesizing acridine ester compounds according to claim 1 or 2, characterized in that, The step of preparing the compound 2 further comprises, after the reaction is completed, washing with a methanol solution having a mass percentage concentration of 5-10% of the first basic reagent, washing with dichloromethane, and then filtering.

4. The method for synthesizing acridine ester compounds according to claim 1, characterized in that, The step of reacting the compound 2 with 9-acridine-formyl chloride comprises, first, preparing the 9-acridine-formyl chloride by reacting 9-acridine carboxylic acid with p-toluenesulfonyl chloride at 20-30°C for 20-40 min, and then adding the compound 2 and continuing the reaction at 20-30°C for 8-12 h.

5. The method for synthesizing acridine ester compounds according to claim 4, characterized in that, The molar ratio of the 9-acridine carboxylic acid, the p-toluenesulfonyl chloride, and the compound 2 is 1:(2-4):(0.2-0.5).

6. The method for synthesizing acridine ester compounds according to claim 1, 4, or 5, characterized in that, The step of preparing the compound 3 further comprises, after the reaction is completed, washing with N,N-dimethylformamide, washing with dichloromethane, and then filtering.

7. The method for synthesizing acridine ester compounds according to claim 1, characterized in that, The step of reacting the compound 3 with 1,3-propane sultone under the action of an ionic solvent and a second basic reagent meets any one or several of the following conditions: (1) the molar ratio of the compound 3 to the second basic reagent is 1:(10-30); (2) the second basic reagent is selected from any one or several of 2,6-di-tert-butylpyridine and potassium carbonate; (3) the ionic solvent is selected from any one or several of 1-butyl-3-methylimidazolium hexafluorophosphate and 1-butyl-3-methylimidazolium tetrafluoroborate; (4) the temperature for the reaction of the compound 3 with 1,3-propane sultone is 155-165°C, and the reaction time is 8-12 h.

8. The method for synthesizing acridine ester compounds according to claim 1 or 7, characterized in that, The step of preparing the compound 4 further comprises, after the reaction is completed, washing with N,N-dimethylformamide, washing with dichloromethane, and then filtering.

9. The method of claim 8, wherein the acridinium ester compound is synthesized by the method of claim 1. After the reaction is completed, the compound 4 is washed with N,N-dimethylformamide for 3 times and then washed with dichloromethane for 3 times.

10. The method for synthesizing acridine ester compounds according to claim 1, characterized in that, The step of reacting the compound 4 with an acidic reagent meets any one or several of the following conditions: (1) the molar ratio of the compound 4 to the acidic reagent is 1:(40-80); (2) the reaction time for the reaction of the compound 4 with the acidic reagent is 30-60 min, and the reaction temperature is 20-30°C; (3) the acidic reagent is selected from any one or several of trifluoroacetic acid, hydrochloric acid, and hexafluoroisopropanol.

11. The method for synthesizing acridine ester compounds according to claim 1, characterized in that, The step of reacting the compound 5 with compound 6 under the action of a third basic reagent meets any one or several of the following conditions: (1) the molar ratio of the compound 5 to the compound 6 is 1:(2-5); (2) the molar ratio of the compound 5 to the third basic reagent is 1:(2-4); (3) the third basic reagent is selected from any one or several of N,N-diisopropylethylamine and triethylamine; (4) the reaction time of the compound 5 with the compound 6 is 4-10 hours, and the temperature is 20-30℃.

12. The method for synthesizing acridine ester compounds according to claim 11, characterized in that, The compound 6 is 2-succinimidyl-1,1,3,3-tetramethyl uronium tetrafluoroborate, and the third basic reagent is N,N-diisopropylethylamine; or, The compound 6 is N-hydroxysuccinimide, and a condensing agent is further added in the reaction of the compound 5 with the compound 6, and the condensing agent is selected from any one or a combination of several of dicyclohexyl carbodiimide, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and N,N-diisopropyl carbodiimide.

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