A preparation method of CXCR4 antagonist

Abstract

The novel CXCR4 antagonist designed in the present invention consists of novel CXCR4 antagonist 1, novel CXCR4 antagonist 2, and novel CXCR4 antagonist 3, which can be used in combination or individually. The three newly designed CXCR4 antagonists constitute a molecular architecture different from existing CXCR4 antagonists. These three structural designs are the basis for achieving high-affinity and specific antagonism against CXCR4, thereby achieving highly specific blocking of the binding of CXCR4 to its ligand, effectively inhibiting the activation of downstream signaling pathways. Compared with existing clinically used drugs, their significant advantages in antagonistic activity, ease of synthesis and safety are the key manifestation of their core therapeutic value.

Description

Technical Field

[0001] The present invention belongs to the field of drug preparation, and particularly relates to a method for preparing a CXCR4 antagonist. Background Art

[0002] The chemokine receptor CXCR4 plays a key role in various physiological and pathological processes. Under normal physiological conditions, it binds to its ligand CXCL12 and participates in processes such as hematopoietic stem cell homing and lymphocyte migration. However, abnormal activation or overexpression of CXCR4 plays a significant role in the development and progression of many diseases, such as cancer metastasis, autoimmune diseases, and viral infections (such as HIV). Inhibiting the CXCR4 signaling pathway is an important strategy to prevent the development of these diseases.

[0003] Although some CXCR4 antagonists have been developed (such as AMD3100), they have the following limitations: insufficient chemical stability (e.g., susceptibility to oxidation or hydrolysis), poor pharmacokinetic properties, off-target effects leading to side effects (e.g., cardiotoxicity), complex synthesis processes, and high costs. Therefore, the development of highly effective, safe, and pharmacokinetic CXCR4 antagonists is of great clinical significance and market demand. Summary of the Invention

[0004] In order to solve the above technical problems, the present application designs a method for preparing a CXCR4 antagonist. The CXCR4 antagonist prepared by the present application has excellent CXCR4 antagonistic activity and can effectively inhibit physiological and pathological processes related to CXCR4 in vitro and in vivo.

[0005] A method for preparing a CXCR4 antagonist comprises the following steps:

[0006] Step S1, using quinoline-6-carboxylic acid, dichloromethane, N,N-dimethylformamide, and oxalyl chloride to react to obtain an initial compound 1;

[0007] Step S2, reacting the initial compound 1 with tert-butyl (4-(aminomethyl)benzyl)carbamate, triethylamine, dichloromethane, trifluoroacetic acid, and sodium bicarbonate, extracting, drying, filtering, and then purifying to obtain the intermediate product 1;

[0008] Step S3, reacting the intermediate product 1 with triethylamine and benzenesulfonyl chloride, drying, filtering, evaporating, and separating and purifying to obtain the CXCR4 antagonist 1;

[0009] Step S4, using piperazine-1-carboxylic acid tert-butyl ester, triethylamine, 3-bromobenzoyl chloride, dichloromethane, trifluoroacetic acid, sodium bicarbonate, and anhydrous sodium sulfate to react to obtain the initial compound 2;

[0010] Step S5, reacting the initial compound 2 with triethylamine and phenylacetyl chloride, extracting and drying, and then purifying to obtain an intermediate product 2;

[0011] Step S6, dissolving 3-methoxyphenylboronic acid, tetrakis(triphenylphosphine)palladium, potassium carbonate, and intermediate 2 in a 1:1 mixture of tetrahydrofuran and water; reflux the mixture at 100° C. for 18 hours under nitrogen protection, and obtaining CXCR4 antagonist 2 by column chromatography;

[0012] Step S7, dissolving 2-(4-(methoxycarbonyl)phenyl)acetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, and triethylamine in dichloromethane, and stirring at room temperature for 1 hour; then adding 2-(naphthalen-1-yl)ethan-1-amine, and continuing to react for 12 hours; after completion of the reaction, washing with saturated sodium bicarbonate solution, 1M hydrochloric acid solution, and saturated brine, respectively, adding anhydrous sodium sulfate, filtering, drying to remove the solvent, and then purifying with a silica gel column to obtain the initial compound 3;

[0013] Step S8, dissolving the initial compound 3 in a 1:1 mixture of tetrahydrofuran and water, adding sodium hydroxide solution, and reacting at room temperature for 12 hours; after the reaction is complete, acidifying with 1M hydrochloric acid to a pH of 1, at which time a precipitate is precipitated, which is filtered to obtain an intermediate product 3;

[0014] Step S9, dissolving the intermediate product 3, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, and triethylamine in dichloromethane, and stirring at room temperature for 1 hour; then adding methylamine hydrochloride, and continuing to react for 12 hours; after the reaction is completed, washing with a saturated sodium bicarbonate solution, a 1M hydrochloric acid solution, and a saturated sodium chloride solution, respectively, adding anhydrous sodium sulfate, filtering, and removing the solvent by vacuum drying, and then purifying by high performance liquid chromatography to obtain a CXCR4 antagonist 3;

[0015] Step S10: Mix the CXCR4 antagonist 1 obtained in step S3, the CXCR4 antagonist 2 obtained in step S6, and the CXCR4 antagonist 3 obtained in step S9 to obtain a final CXCR4 antagonist.

[0016] Preferably, the step S1 specifically includes:

[0017] Step S11, dissolving quinoline-6-carboxylic acid in a mixed solvent of dichloromethane and N,N-dimethylformamide;

[0018] Step S12, slowly adding oxalyl chloride and stirring the mixture at room temperature;

[0019] Step S13: After the reaction is completed, the solvent is evaporated to obtain the initial compound 1.

[0020] Preferably, the step S2 specifically includes:

[0021] Step S21, dissolving the initial compound 1 in dichloromethane, adding tert-butyl (4-(aminomethyl)benzyl)carbamate and triethylamine in sequence, and reacting at room temperature;

[0022] Step S22: After the reaction in step S21 is completed, the solvent is evaporated, dichloromethane and trifluoroacetic acid are added, and the mixture is stirred at room temperature;

[0023] Step S23: Adjust the pH to neutral with sodium bicarbonate solution, extract with ethyl acetate 2 to 5 times, combine the organic phases and dry with anhydrous sodium sulfate, filter and evaporate the solvent, and purify by silica gel column chromatography to obtain intermediate 1.

[0024] Preferably, the step S3 specifically includes:

[0025] Step S31, dissolving the intermediate product 1 in dichloromethane, adding triethylamine and benzenesulfonyl chloride, and reacting at room temperature;

[0026] Step S32: After the reaction is completed, the organic phase is washed with water, dried over anhydrous sodium sulfate, filtered, and the solvent is evaporated to obtain a crude product;

[0027] Step S33: Separate and purify the crude product obtained in step S32 by high performance liquid chromatography to finally obtain CXCR4 antagonist 1.

[0028] Preferably, the step S4 specifically includes:

[0029] Step S41, dissolving tert-butyl piperazine-1-carboxylate and triethylamine in dichloromethane, slowly adding 3-bromobenzoyl chloride in an ice bath, then bringing the reaction system to room temperature and reacting for 16 hours;

[0030] Step S42: After the reaction is completed, wash with water three times, extract and dry the organic phase; then add 6 ml of dichloromethane and 2 ml of trifluoroacetic acid, and react at room temperature overnight;

[0031] Step S43: After the reaction is completed, the above solution is slowly added to a sodium bicarbonate solution for alkalization, the pH is adjusted to neutral, and extraction and washing are performed three times; then anhydrous sodium sulfate is added to the organic phase, the organic phase is removed after filtration, and purification is performed through a silica gel column to obtain the initial compound 2.

[0032] Preferably, the step S5 specifically includes:

[0033] Step S51, adding triethylamine to the initial compound 2 and dissolving it with dichloromethane;

[0034] Step S52: adding phenylacetyl chloride under ice bath conditions, transferring the mixture to room temperature for reaction; performing extraction, directly drying and removing the organic phase, and then purifying the mixture through a silica gel column to obtain intermediate product 2.

[0035] Preferably, step S6 specifically comprises: dissolving the intermediate product 2, (3-methoxyphenyl)boric acid, tetrakis(triphenylphosphine)palladium and potassium carbonate in a 1:1 mixed solution of tetrahydrofuran and water; reacting under reflux at 100° C. under nitrogen protection, and obtaining the CXCR4 antagonist 2 by column chromatography.

[0036] Preferably, the step S7 specifically includes:

[0037] Step S71, dissolving 2-(4-(methoxycarbonyl)phenyl)acetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole and triethylamine in dichloromethane, and stirring at room temperature for 1 hour;

[0038] Step S72: 2-(naphthalen-1-yl)ethan-1-amine was then added, and the mixture was reacted for 12 hours;

[0039] Step S73: After the reaction is completed, the mixture is washed with saturated sodium bicarbonate solution, 1M hydrochloric acid solution and saturated brine in sequence, anhydrous sodium sulfate is added, and the mixture is filtered, dried to remove the solvent, and then purified with a silica gel column to obtain the initial compound 3.

[0040] Preferably, the step S8 specifically includes:

[0041] Step S81, dissolving the initial compound 3 in a 1:1 mixture of tetrahydrofuran and water, adding sodium hydroxide solution, and reacting at room temperature for 12 hours;

[0042] Step S82: After the reaction is completed, the mixture is acidified with 1M hydrochloric acid to a pH of 1. A precipitate is formed and filtered to obtain an intermediate product 3.

[0043] Preferably, the step S9 specifically includes:

[0044] Step S91, dissolving the intermediate product 3, EDCI, HOBT and triethylamine in dichloromethane, and stirring at room temperature for 1 hour;

[0045] Step S92: then adding methylamine hydrochloride, and the mixture continues to react for 12 hours;

[0046] Step S93: After the reaction is completed, wash with saturated sodium bicarbonate solution, 1M hydrochloric acid solution and saturated brine respectively, add anhydrous sodium sulfate and filter, remove the solvent by vacuum drying, and purify by high performance liquid chromatography to obtain CXCR4 antagonist 3.

[0047] The advantages and effects of this application are as follows:

[0048] The CXCR4 antagonists designed in the present invention are composed of CXCR4 antagonist 1, CXCR4 antagonist 2, and CXCR4 antagonist 3, which can be used in combination or individually. The three newly designed CXCR4 antagonists constitute a molecular architecture different from existing CXCR4 antagonists. These three structural designs are the basis for achieving high-affinity and specific antagonism against CXCR4, thereby achieving highly specific blocking of the binding of CXCR4 to its ligand, effectively inhibiting the activation of downstream signaling pathways. Compared with existing clinically used drugs, their significant advantages in antagonistic activity, ease of synthesis and safety are the key manifestation of their core therapeutic value.

[0049] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application so that it can be implemented in accordance with the contents of the specification, and to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the following is a detailed description of the preferred embodiment of the present application in conjunction with the accompanying drawings.

[0050] Based on the detailed description of the specific embodiments of the present application in conjunction with the accompanying drawings below, those skilled in the art will become more aware of the above and other objects, advantages and features of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present application. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without inventive work. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.

[0052] Figure 1 Chemical reaction formula for the first step of the preparation method of the CXCR4 antagonist 1 designed for this application;

[0053] Figure 2 Chemical reaction formula for the second step of the preparation method of the CXCR4 antagonist 1 designed for this application;

[0054] Figure 3 Chemical reaction formula for step 3 of the preparation method of the CXCR4 antagonist 1 designed for this application;

[0055] Figure 4 Chemical reaction formula for the first step of the preparation method of the CXCR4 antagonist 2 designed for this application;

[0056] Figure 5 Chemical reaction formula for the second step of the preparation method of the CXCR4 antagonist 2 designed for this application;

[0057] Figure 6 Chemical reaction formula for the third step of the preparation method of the CXCR4 antagonist 2 designed for this application;

[0058] Figure 7 Chemical reaction formula for the first step of the preparation method of the CXCR4 antagonist 3 designed for this application;

[0059] Figure 8 Chemical reaction formula for the second step of the preparation method of the CXCR4 antagonist 3 designed for this application;

[0060] Figure 9 Chemical reaction formula for step 3 of the preparation method of CXCR4 antagonist 3 designed for this application;

[0061] Figure 10 The cells were incubated with rhodamine-labeled streptavidin (3 μg / ml) designed for this application and imaged using a confocal microscope after washing three times;

[0062] Figure 11 Migration response diagram of tumor cells designed for this application;

[0063] Figure 12 Diagram showing tumor cells invasively crossing Matrigel for this application;

[0064] Figure 13 Figure 2 shows inhibition of acute inflammation induced by xylene designed for this application;

[0065] Figure 14 Diagram of the lung metastasis effect of 4T1 cells designed for this application;

[0066] Figure 15 The effect of various concentration gradients of the antagonists designed for this application on cell viability;

[0067] Figure 16 Proton spectrum of CXCR4 antagonist 1 designed for this application;

[0068] Figure 17 Carbon spectrum of CXCR4 antagonist 1 designed for this application;

[0069] Figure 18 Mass spectrum of CXCR4 antagonist 1 designed for this application;

[0070] Figure 19 Proton spectrum of CXCR4 antagonist 2 designed for this application;

[0071] Figure 20Carbon spectrum of CXCR4 antagonist 2 designed for this application;

[0072] Figure 21 Mass spectrum of CXCR4 antagonist 2 designed for this application;

[0073] Figure 22 Proton spectrum of CXCR4 antagonist 3 designed for this application;

[0074] Figure 23 C-ray spectrum of CXCR4 antagonist 3 designed for this application;

[0075] Figure 24 Mass spectrum of CXCR4 antagonist 3 designed for this application;

[0076] Figure 25 The molecular formula of CXCR4 antagonist 1 designed for this application;

[0077] Figure 26 The molecular formula of CXCR4 antagonist 2 designed for this application;

[0078] Figure 27 The molecular formula of CXCR4 antagonist 3 designed for this application. DETAILED DESCRIPTION

[0079] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. In the following description, specific details such as specific configurations and components are provided only to help fully understand the embodiments of the present application. Therefore, it should be clear to those skilled in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. In addition, for clarity and brevity, the description of known functions and structures has been omitted in the embodiments.

[0080] It should be understood that references throughout this specification to "one embodiment" or "this embodiment" mean that a particular feature, structure, or characteristic associated with the embodiment is included in at least one embodiment of the present application. Therefore, the appearance of "one embodiment" or "this embodiment" throughout this specification does not necessarily refer to the same embodiment. Furthermore, these particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0081] In addition, the present application may repeat reference numerals and / or letters in different examples. This repetition is for the purpose of simplicity and clarity and does not in itself indicate the relationship between the various embodiments and / or settings discussed.

[0082] The term "and / or" in this article is only a description of the association relationship of associated objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist at the same time. The term " / and" in this article describes another type of association object relationship, indicating that two relationships can exist. For example, A / and B can mean: A exists alone, and A and B exist alone. In addition, the character " / " in this article generally indicates that the previous and subsequent associated objects are in an "or" relationship.

[0083] The term "at least one" in this article is merely a description of the association relationship between associated objects, indicating that three relationships may exist. For example, at least one of A and B can mean: A exists alone, A and B exist at the same time, and B exists alone.

[0084] It should also be noted that, in this document, relational terms such as first and second are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "include," "comprises," or any other variations thereof are intended to cover non-exclusive inclusion.

[0085] Example 1

[0086] This example mainly introduces the optimized preparation method of CXCR4 antagonist 1, which includes the following steps:

[0087] Step S1, using quinoline-6-carboxylic acid, dichloromethane, N,N-dimethylformamide, and oxalyl chloride to react to obtain an initial compound 1;

[0088] Step S2, reacting the initial compound 1 with tert-butyl (4-(aminomethyl)benzyl)carbamate, triethylamine, dichloromethane, trifluoroacetic acid, and sodium bicarbonate, extracting, drying, filtering, and then purifying to obtain the intermediate product 1;

[0089] Step S3: react the intermediate product 1 with triethylamine and benzenesulfonyl chloride, dry, filter, evaporate, and separate and purify to obtain CXCR4 antagonist 1; its molecular formula R8 can be found in Figure 25 For its hydrogen spectrum, carbon spectrum and mass spectrum, please refer to Figure 16 、 Figure 17 、 Figure 18 .

[0090] Furthermore, the step S1 specifically includes:

[0091] Step S11, dissolving quinoline-6-carboxylic acid in a mixed solvent of dichloromethane and N,N-dimethylformamide;

[0092] Step S12, slowly adding oxalyl chloride and stirring the mixture at room temperature;

[0093] Step S13: After the reaction is completed, the solvent is evaporated to obtain the initial compound 1.

[0094] Furthermore, quinoline-6-carboxylic acid (1 g) was dissolved in a mixed solvent of dichloromethane (4 ml) and a small amount of N,N-dimethylformamide, and oxalyl chloride (0.6 ml) was slowly added. The mixture was stirred at room temperature for 3 hours. After the reaction, the solvent was evaporated under reduced pressure to obtain the initial compound 1 (which was used directly in the next reaction without further purification). The chemical reaction formula is shown in FIG. Figure 1 .

[0095] Furthermore, the step S2 specifically includes:

[0096] Step S21, dissolving the initial compound 1 in dichloromethane, adding tert-butyl (4-(aminomethyl)benzyl)carbamate and triethylamine in sequence, and reacting at room temperature;

[0097] Step S22: After the reaction in step S21 is completed, the solvent is evaporated, dichloromethane and trifluoroacetic acid are added, and the mixture is stirred at room temperature;

[0098] Step S23: Adjust the pH to neutral with sodium bicarbonate solution, extract with ethyl acetate 2 to 5 times, combine the organic phases and dry with anhydrous sodium sulfate, filter and evaporate the solvent, and purify by silica gel column chromatography to obtain intermediate 1.

[0099] Furthermore, the initial compound 1 (1.1 g) obtained in the first step was weighed and dissolved in dichloromethane. Tert-butyl (4-(aminomethyl)benzyl)carbamate (1.37 g) and triethylamine (966 μl) were added sequentially and reacted at room temperature for 7 hours. After the reaction was completed, the solvent was evaporated, dichloromethane (6 ml) and trifluoroacetic acid (2 ml) were added, and the mixture was stirred at room temperature for 1 hour. The pH was then adjusted to neutral with sodium bicarbonate solution, and the mixture was extracted three times with ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated, and the intermediate product 1 was purified by silica gel column chromatography. The chemical reaction formula is shown in FIG. Figure 2 .

[0100] Furthermore, the step S3 specifically includes:

[0101] Step S31, dissolving the intermediate product 1 in dichloromethane, adding triethylamine and benzenesulfonyl chloride, and reacting at room temperature;

[0102] Step S32: After the reaction is completed, the organic phase is washed with water, dried over anhydrous sodium sulfate, filtered, and the solvent is evaporated to obtain a crude product;

[0103] Step S33: Separate and purify the crude product obtained in step S32 by high performance liquid chromatography to finally obtain CXCR4 antagonist 1.

[0104] Furthermore, the intermediate product 1 (1 g) obtained in the above step was weighed and dissolved in dichloromethane. Triethylamine (573 μl) and benzenesulfonyl chloride (482 μl) were added and reacted at room temperature for 5 hours. After the reaction, the organic phase was washed with water, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The crude product was separated and purified by high-performance liquid chromatography (HPLC) to obtain CXCR4 antagonist 1. The chemical reaction formula is shown in Figure 2. Figure 3 . Example 2

[0105] Based on Example 1, this example mainly introduces an optimized preparation method of CXCR4 antagonist 2, which includes the following steps:

[0106] Step S4, using piperazine-1-carboxylic acid tert-butyl ester, triethylamine, 3-bromobenzoyl chloride, dichloromethane, trifluoroacetic acid, sodium bicarbonate, and anhydrous sodium sulfate to react to obtain the initial compound 2;

[0107] Step S5, reacting the initial compound 2 with triethylamine and phenylacetyl chloride, extracting and drying, and then purifying to obtain an intermediate product 2;

[0108] Step S6: dissolving 3-methoxyphenylboronic acid, tetrakis(triphenylphosphine)palladium, potassium carbonate, and intermediate 2 in a 1:1 mixture of tetrahydrofuran and water; reflux the mixture at 100° C. for 18 hours under nitrogen protection, and obtain CXCR4 antagonist 2 by column chromatography; its molecular formula R6 is shown in FIG. Figure 26 For its hydrogen spectrum, carbon spectrum and mass spectrum, please refer to Figure 19 、 Figure 20 、 Figure 21 .

[0109] Furthermore, the step S4 specifically includes:

[0110] Step S41, dissolving tert-butyl piperazine-1-carboxylate and triethylamine in dichloromethane, slowly adding 3-bromobenzoyl chloride in an ice bath, then bringing the reaction system to room temperature and reacting for 16 hours;

[0111] Step S42: After the reaction is completed, wash with water three times, extract and dry the organic phase; then add 6 ml of dichloromethane and 2 ml of trifluoroacetic acid, and react at room temperature overnight;

[0112] Step S43: After the reaction is completed, the above solution is slowly added to a sodium bicarbonate solution for alkalization, the pH is adjusted to neutral, and extraction and washing are performed three times; then anhydrous sodium sulfate is added to the organic phase, the organic phase is removed after filtration, and purification is performed through a silica gel column to obtain the initial compound 2.

[0113] Furthermore, tert-butyl piperazine-1-carboxylate (1.44 g) and triethylamine (1.29 ml) were dissolved in dichloromethane, and 3-bromobenzoyl chloride was slowly added under ice bath conditions. The reaction system was then moved to room temperature and reacted for 16 hours. After the reaction was completed, the mixture was washed three times with water, and the organic phase was dried after extraction. Then 6 ml of dichloromethane and 2 ml of trifluoroacetic acid were added, and the mixture was reacted overnight at room temperature. After the reaction was completed, the above solution was slowly added to a sodium bicarbonate solution for alkalization, the pH was adjusted to neutral, and the mixture was extracted and washed three times. Anhydrous sodium sulfate was added to the organic phase, the organic phase was removed after filtration, and the initial compound 2 was obtained by purification through a silica gel column; its chemical reaction formula can be found in Figure 4 .

[0114] Furthermore, the step S5 specifically includes:

[0115] Step S51, adding triethylamine to the initial compound 2 and dissolving it with dichloromethane;

[0116] Step S52: adding phenylacetyl chloride under ice bath conditions, transferring the mixture to room temperature for reaction; performing extraction, directly drying and removing the organic phase, and then purifying the mixture through a silica gel column to obtain intermediate product 2.

[0117] Weigh 500 mg of the starting compound 2, add 310 μl of triethylamine, and dissolve in dichloromethane. Add 245 μl of phenylacetyl chloride in an ice bath, and transfer to room temperature for 5 hours. After extraction, directly dry and remove the organic phase. Purify on a silica gel column to obtain intermediate 2. For the chemical reaction formula, refer to Figure 5 .

[0118] Furthermore, the step S6 specifically includes: dissolving the intermediate product 2, (3-methoxyphenyl)boric acid, tetrakis(triphenylphosphine)palladium and potassium carbonate in a 1:1 mixed solution of tetrahydrofuran and water; under nitrogen protection, reflux reaction at 100° C., and obtaining CXCR4 antagonist 2 by column chromatography.

[0119] Furthermore, intermediate product 2 (737 mg), (3-methoxyphenyl)boronic acid (347 mg), tetrakis(triphenylphosphine)palladium (66 mg), and potassium carbonate (526 mg) were weighed and dissolved in a 1:1 mixture of tetrahydrofuran and water. Under nitrogen protection, the mixture was refluxed at 100°C for 18 hours. CXCR4 antagonist 2 was obtained by column chromatography; its chemical reaction formula is shown in the following table. Figure 6 .

[0120] Example 3

[0121] Based on Example 1, this example mainly introduces the optimized preparation method of CXCR4 antagonist 3, which includes the following steps:

[0122] Step S7, dissolving 2-(4-(methoxycarbonyl)phenyl)acetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, and triethylamine in dichloromethane, and stirring at room temperature for 1 hour; then adding 2-(naphthalen-1-yl)ethan-1-amine, and continuing to react for 12 hours; after completion of the reaction, washing with saturated sodium bicarbonate solution, 1M hydrochloric acid solution, and saturated brine, respectively, adding anhydrous sodium sulfate, filtering, drying to remove the solvent, and then purifying with a silica gel column to obtain the initial compound 3;

[0123] Step S8, dissolving the initial compound 3 in a 1:1 mixture of tetrahydrofuran and water, adding sodium hydroxide solution, and reacting at room temperature for 12 hours; after the reaction is complete, acidifying with 1M hydrochloric acid to a pH of 1, at which time a precipitate is precipitated, which is filtered to obtain an intermediate product 3;

[0124] Step S9: Dissolve the intermediate product 3, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, and triethylamine in dichloromethane and stir at room temperature for 1 hour; then add methylamine hydrochloride, and continue to react for 12 hours; after the reaction is completed, wash with saturated sodium bicarbonate solution, 1M hydrochloric acid solution, and saturated brine, respectively, add anhydrous sodium sulfate, filter, remove the solvent by vacuum drying, and purify by high performance liquid chromatography to obtain CXCR4 antagonist 3. Its molecular formula R10 can be found in Figure 27 For its hydrogen spectrum, carbon spectrum and mass spectrum, please refer to Figure 22 、 Figure 23 、 Figure 24 .

[0125] Furthermore, the step S7 specifically includes:

[0126] Step S71, dissolving 2-(4-(methoxycarbonyl)phenyl)acetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole and triethylamine in dichloromethane, and stirring at room temperature for 1 hour;

[0127] Step S72: 2-(naphthalen-1-yl)ethan-1-amine was then added, and the mixture was reacted for 12 hours;

[0128] Step S73: After the reaction is completed, the mixture is washed with saturated sodium bicarbonate solution, 1M hydrochloric acid solution and saturated brine in sequence, anhydrous sodium sulfate is added, and the mixture is filtered, dried to remove the solvent, and then purified with a silica gel column to obtain the initial compound 3.

[0129] Furthermore, 2-(4-(methoxycarbonyl)phenyl)acetic acid (100 mg), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 119 mg), 1-hydroxybenzotriazole (HOBT, 84 mg) and triethylamine (286 μl) were dissolved in dichloromethane and stirred at room temperature for 1 hour. 2-(naphthalen-1-yl)ethan-1-amine (88 mg) was then added, and the mixture was allowed to react for 12 hours. After the reaction was completed, the mixture was washed with saturated sodium bicarbonate solution, 1M hydrochloric acid solution and saturated brine, respectively, and anhydrous sodium sulfate was added, followed by filtration, drying to remove the solvent, and purification using a silica gel column to obtain the initial compound 3; its chemical reaction formula is shown in FIG. Figure 7 .

[0130] Furthermore, the step S8 specifically includes:

[0131] Step S81, dissolving the initial compound 3 in a 1:1 mixture of tetrahydrofuran and water, adding sodium hydroxide solution, and reacting at room temperature for 12 hours;

[0132] Step S82: After the reaction is completed, the mixture is acidified with 1M hydrochloric acid to a pH of 1. A precipitate is formed and filtered to obtain an intermediate product 3.

[0133] Further, the initial compound 3 was dissolved in a 1:1 mixture of tetrahydrofuran and water, and an appropriate amount of sodium hydroxide was added and reacted at room temperature for 12 hours. After the reaction was completed, it was acidified with 1M hydrochloric acid to pH 1. At this time, a precipitate was precipitated and filtered to obtain the intermediate product 3; its chemical reaction formula is shown in FIG. Figure 8 .

[0134] Furthermore, the step S9 specifically includes:

[0135] Step S91, dissolving the intermediate product 3, EDCI, HOBT and triethylamine in dichloromethane, and stirring at room temperature for 1 hour;

[0136] Step S92: then adding methylamine hydrochloride, and the mixture continues to react for 12 hours;

[0137] Step S93: After the reaction is completed, wash with saturated sodium bicarbonate solution, 1M hydrochloric acid solution and saturated brine respectively, add anhydrous sodium sulfate and filter, remove the solvent by vacuum drying, and purify by high performance liquid chromatography to obtain CXCR4 antagonist 3.

[0138] Furthermore, intermediate 3 (100 mg), EDCI (69 mg), HOBT (49 mg) and triethylamine (164 μl) were dissolved in dichloromethane and stirred at room temperature for 1 hour. Methylamine hydrochloride (21 mg) was then added, and the mixture was allowed to react for 12 hours. After the reaction was completed, the mixture was washed with saturated sodium bicarbonate solution, 1M hydrochloric acid solution and saturated brine, respectively, and filtered after adding anhydrous sodium sulfate. The solvent was removed by vacuum drying and then purified by high performance liquid chromatography (HPLC) to obtain CXCR4 antagonist 3. The reaction formula is as follows: Figure 9 shown.

[0139] Example 4

[0140] 1. Competitive affinity determination:

[0141] Based on Examples 1 to 3, MDA-MB-231 cells were used to determine the affinity between the compound and the target. The FDA-approved CXCR4 antagonist AMD3100 was used as a positive control. The TN14003 peptide has a high binding ability to CXCR4 and is often used as a tool for drug competition affinity determination of this target. Various test compounds were prepared into 1 mM stock solution with DMSO and finally diluted to 1, 10, 100, and 1000 nM for testing. The above-mentioned various compounds were treated with different concentration combinations for 15 min, then fixed with 4% paraformaldehyde at room temperature and washed three times with PBS. Then, 0.05 ug / ml of biotinylated TN14003 peptide was added, incubated for 30 min, washed three times with PBS, and incubated with rhodamine-labeled streptavidin (3 ug / ml). After washing three times, imaging was performed using a confocal microscope. The results are shown in FIG. Figure 10 After treatment with the three compounds, the fluorescence intensity was significantly reduced, reflecting that each compound had a high affinity for the target CXCR4, thereby reducing the binding of TN14003 to the target. In addition, the affinity of the three compounds was significantly higher than that of the positive control AMD3100.

[0142] 2. Tumor cell migration and invasion assay:

[0143] After plating the cells, culture them to 85% confluence, draw a straight line on the bottom of the well plate with a pipette tip, wash three times with PBS and treat with 100 nM of the test compound for 24 hours. Figure 11The line width at the bottom of the well plate reflects the migration ability of tumor cells. The treatment with the three compounds significantly inhibited the migration of tumor cells. In addition, a matrigel invasion experiment was designed. Cells were seeded on the upper layer of a transwell chamber pre-placed with matrigel and treated with the test compound. CXCL12 was added to the lower layer to stimulate the cells to invade downwards. After 24 hours of treatment, the number of cells at the bottom of the chamber was stained and counted. This number represents the situation of tumor cells crossing the matrigel by invasion. The results are shown in Figure 2. Figure 12 The three compounds provided by the present invention all effectively reduced the invasion of tumor cells.

[0144] 3. In vivo acute inflammation experiment:

[0145] Each test compound was prepared into a stock solution and injected into three mice per group at a dose of 10 mg / kg. Thirty minutes after intraperitoneal injection, 30 μl of xylene was evenly applied to the inner and outer surfaces of each mouse's right ear. One hour later, the animals were sacrificed, and ear pieces were removed using an 8 mm hole punch, weighed, fixed with paraformaldehyde, and stained with HE. The results are shown in Figure 2. Figure 13 , the three compounds provided herein all effectively inhibited xylene-induced acute inflammation.

[0146] 4. In vivo anti-tumor metastasis experiment:

[0147] Randomly select one of the compounds provided in this article for anti-metastasis experiments (R10 was selected in this experiment). Twelve 5-week-old Balb / c mice were randomly divided into two groups. Each mouse was injected with approximately 1 million 4T1-Luc cells through the tail vein. For the next 18 days, compound R10 was injected every day at a dose of 20 mg / kg. On the last day, 150 mg / kg of luciferin sodium salt was injected 5 minutes before imaging for bioluminescence imaging. The results are shown in the figure below. Figure 14 As shown, the luminescence intensity in mice was significantly reduced after treatment with compound R10, indicating that compound R10 effectively reduced the lung metastasis of 4T1 cells.

[0148] 5. Cellular level safety testing:

[0149] The safety of the compounds was evaluated using normal human liver cells L-02. The cells were plated on a 96-well plate and cultured. After the cells adhered to the plate, the drug was added to the test compounds at concentrations of 100 nM, 1 μM, and 10 μM, respectively. The cells were cultured at 37°C and 5% carbon dioxide for 24 hours. 10 μl of CCK8 reagent was added to each well. After another 2 hours of culture, the absorbance of each well was measured using a microplate reader. The test wavelength was 450 nm and the reference wavelength was 600 nm. The results are shown in Figure 2. Figure 15 The three compounds provided in this article have little effect on cell survival rate under various concentration gradients, especially under high concentration conditions, and their safety is much higher than that of the control group AMD3100.

[0150] The present invention provides a CXCR4 antagonist having the following characteristics: Figure 25 , Figure 26 and Figure 27 The novel chemical structure of this drug forms a molecular architecture distinct from existing CXCR4 antagonists. This structural design underpins its high-affinity and specific antagonistic effect on CXCR4, thereby enabling highly specific blocking of the binding of CXCR4 to its ligand, effectively inhibiting activation of downstream signaling pathways. Its significant advantages over existing clinically used drugs in antagonistic activity, ease of synthesis, and safety are key to its core therapeutic value.

[0151] The foregoing description is merely a preferred embodiment of the present invention and does not limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any variation, modification, replacement, integration, or parameter change to these embodiments, which is within the spirit and principles of the present invention and which achieves the same functionality through conventional substitutions, without departing from the principles and spirit of the present invention, falls within the scope of protection of the present invention.

Claims

1. A method for preparing a CXCR4 antagonist, characterized in that: The following steps are involved: Step S1: using quinoline-6-carboxylic acid, dichloromethane, N,N-dimethylformamide, and oxalyl chloride to react to obtain the initial compound 1 ; Step S2: reacting the initial compound 1 with tert-butyl (4-(aminomethyl)benzyl)carbamate, triethylamine, dichloromethane, trifluoroacetic acid, and sodium bicarbonate, extracting, drying, filtering, and then purifying to obtain the intermediate product 1 ; Step S3: react the intermediate product 1 with triethylamine and benzenesulfonyl chloride, dry, filter, evaporate, and separate and purify to obtain CXCR4 antagonist 1. ； Step S4: using piperazine-1-carboxylic acid tert-butyl ester, triethylamine, 3-bromobenzoyl chloride, dichloromethane, trifluoroacetic acid, sodium bicarbonate, and anhydrous sodium sulfate to react to obtain the initial compound 2 ； Step S5: react the initial compound 2 with triethylamine and phenylacetyl chloride, extract and dry, and then purify to obtain the intermediate product 2. ； Step S6: dissolving 3-methoxyphenylboronic acid, tetrakis(triphenylphosphine)palladium, potassium carbonate and intermediate 2 in a 1:1 mixture of tetrahydrofuran and water; reflux the mixture at 100° C. for 18 hours under nitrogen protection, and obtain CXCR4 antagonist 2 by column chromatography. ; Step S7, dissolving 2-(4-(methoxycarbonyl)phenyl)acetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole and triethylamine in dichloromethane, stirring at room temperature for 1 hour; then adding 2-(naphthalen-1-yl)ethan-1-amine, and continuing to react the mixture for 12 hours; after completion of the reaction, washing with saturated sodium bicarbonate solution, 1M hydrochloric acid solution and saturated brine, respectively, adding anhydrous sodium sulfate, filtering, drying to remove the solvent, and then purifying with a silica gel column to obtain the initial compound 3 ; Step S8: Dissolve the initial compound 3 in a 1:1 mixture of tetrahydrofuran and water, add sodium hydroxide solution, and react at room temperature for 12 hours; after the reaction is complete, acidify with 1M hydrochloric acid to pH 1, at which time a precipitate is precipitated, which is filtered to obtain the intermediate product 3. ; Step S9, dissolving the intermediate product 3, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole and triethylamine in dichloromethane, stirring at room temperature for 1 hour; then adding methylamine hydrochloride, and continuing to react for 12 hours; after the reaction is completed, washing with saturated sodium bicarbonate solution, 1M hydrochloric acid solution and saturated brine respectively, adding anhydrous sodium sulfate and filtering, removing the solvent by vacuum drying, and then purifying by high performance liquid chromatography to obtain CXCR4 antagonist 3 ; Step S10: Mix the CXCR4 antagonist 1 obtained in step S3, the CXCR4 antagonist 2 obtained in step S6, and the CXCR4 antagonist 3 obtained in step S9 to obtain a final CXCR4 antagonist.

2. The method for preparing a CXCR4 antagonist according to claim 1, characterized in that: The step S1 specifically includes: Step S11, dissolving quinoline-6-carboxylic acid in a mixed solvent of dichloromethane and N,N-dimethylformamide; Step S12, slowly adding oxalyl chloride and stirring the mixture at room temperature; Step S13: After the reaction is completed, the solvent is evaporated to obtain the initial compound 1.

3. The method for preparing a CXCR4 antagonist according to claim 2, characterized in that: The step S2 specifically includes: Step S21, dissolving the initial compound 1 in dichloromethane, adding tert-butyl (4-(aminomethyl)benzyl)carbamate and triethylamine in sequence, and reacting at room temperature; Step S22: After the reaction in step S21 is completed, the solvent is evaporated, dichloromethane and trifluoroacetic acid are added, and the mixture is stirred at room temperature; Step S23: Adjust the pH to neutral with sodium bicarbonate solution, extract with ethyl acetate 2 to 5 times, combine the organic phases and dry with anhydrous sodium sulfate, filter and evaporate the solvent, and purify by silica gel column chromatography to obtain intermediate 1.

4. The method for preparing a CXCR4 antagonist according to claim 3, characterized in that: The step S3 specifically includes: Step S31, dissolving the intermediate product 1 in dichloromethane, adding triethylamine and benzenesulfonyl chloride, and reacting at room temperature; Step S32: After the reaction is completed, the organic phase is washed with water, dried over anhydrous sodium sulfate, filtered, and the solvent is evaporated to obtain a crude product; Step S33: Separate and purify the crude product obtained in step S32 by high performance liquid chromatography to finally obtain CXCR4 antagonist 1.

5. The method for preparing a CXCR4 antagonist according to claim 1, characterized in that: The step S4 specifically includes: Step S41, dissolving tert-butyl piperazine-1-carboxylate and triethylamine in dichloromethane, slowly adding 3-bromobenzoyl chloride in an ice bath, then bringing the reaction system to room temperature and reacting for 16 hours; Step S42: After the reaction is completed, wash with water three times, extract and dry the organic phase; then add 6 ml of dichloromethane and 2 ml of trifluoroacetic acid, and react at room temperature overnight; Step S43: After the reaction is completed, the above solution is slowly added to a sodium bicarbonate solution for alkalization, the pH is adjusted to neutral, and extraction and washing are performed three times; then anhydrous sodium sulfate is added to the organic phase, the organic phase is removed after filtration, and purification is performed through a silica gel column to obtain the initial compound 2.

6. The method for preparing a CXCR4 antagonist according to claim 5, characterized in that: The step S5 specifically includes: Step S51, adding triethylamine to the initial compound 2 and dissolving it with dichloromethane; Step S52: adding phenylacetyl chloride under ice bath conditions, transferring the mixture to room temperature for reaction; performing extraction, directly drying and removing the organic phase, and then purifying the mixture through a silica gel column to obtain intermediate product 2.

7. The method for preparing a CXCR4 antagonist according to claim 6, characterized in that: The step S6 specifically comprises: dissolving the intermediate product 2, (3-methoxyphenyl)boric acid, tetrakis(triphenylphosphine)palladium and potassium carbonate in a 1:1 mixed solution of tetrahydrofuran and water; reacting under reflux at 100° C. under nitrogen protection, and obtaining the CXCR4 antagonist 2 by column chromatography.

8. The method for preparing a CXCR4 antagonist according to claim 1, characterized in that: The step S7 specifically includes: Step S71, dissolving 2-(4-(methoxycarbonyl)phenyl)acetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole and triethylamine in dichloromethane, and stirring at room temperature for 1 hour; Step S72: 2-(naphthalen-1-yl)ethan-1-amine was then added, and the mixture was reacted for 12 hours; Step S73: After the reaction is completed, the mixture is washed with saturated sodium bicarbonate solution, 1M hydrochloric acid solution and saturated brine in sequence, anhydrous sodium sulfate is added, and the mixture is filtered, dried to remove the solvent, and then purified with a silica gel column to obtain the initial compound 3.

9. The method for preparing a CXCR4 antagonist according to claim 8, characterized in that: The step S8 specifically includes: Step S81, dissolving the initial compound 3 in a 1:1 mixture of tetrahydrofuran and water, adding sodium hydroxide solution, and reacting at room temperature for 12 hours; Step S82: After the reaction is completed, the mixture is acidified with 1M hydrochloric acid to a pH of 1. A precipitate is formed and filtered to obtain an intermediate product 3.

10. The method for preparing a CXCR4 antagonist according to claim 9, characterized in that: The step S9 specifically includes: Step S91, dissolving the intermediate product 3, EDCI, HOBT and triethylamine in dichloromethane, and stirring at room temperature for 1 hour; Step S92: then adding methylamine hydrochloride, and the mixture continues to react for 12 hours; Step S93: After the reaction is completed, wash with saturated sodium bicarbonate solution, 1M hydrochloric acid solution and saturated brine respectively, add anhydrous sodium sulfate and filter, remove the solvent by vacuum drying, and purify by high performance liquid chromatography to obtain CXCR4 antagonist 3.

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