Preparation method of novel CXCR4 antagonist

Abstract

The novel CXCR4 antagonist designed by the invention is composed of a novel CXCR4 antagonist 1, a novel CXCR4 antagonist 2 and a novel CXCR4 antagonist 3, which can be used in combination or independently, the three newly designed CXCR4 antagonists form a molecular architecture different from the existing CXCR4 antagonist, the three structural designs are the basis for realizing high affinity and specific antagonism on CXCR4, and the novel CXCR4 antagonist can be used as a novel CXCR4 antagonist. According to the present invention, the combination of the CXCR4 and the ligand thereof is highly specifically blocked, the activation of the downstream signal channel is effectively inhibited, and compared with the existing clinical use drugs, the significant advantages of the antagonistic activity, the synthesis difficulty and the safety are provided, and the key embodiment of the core treatment value is provided.

Description

Technical Field

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

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

[0003] Currently, although some CXCR4 antagonists have been developed (such as AMD3100), they have the following limitations: insufficient chemical stability (such as easy oxidation or hydrolysis), poor pharmacokinetic properties, side effects caused by off-target effects (such as cardiotoxicity), complex synthesis processes, and high costs. Therefore, developing novel CXCR4 antagonists with high efficiency, safety, and good pharmacokinetic properties has important clinical significance and market demand. Summary of the Invention

[0004] To solve the above technical problems, the present application designs a preparation method of a novel CXCR4 antagonist. The novel CXCR4 antagonist prepared by the present application has excellent CXCR4 antagonistic activity and can effectively inhibit CXCR4-related physiological and pathological processes in vivo and in vitro.

[0005] A preparation method of a novel CXCR4 antagonist, comprising the following steps: Step S1: React quinoline-6-carboxylic acid, dichloromethane, N,N-dimethylformamide, and oxalyl chloride to obtain the initial compound 1; Step S2: React the initial compound 1 with tert-butyl (4-(aminomethyl)benzyl)carbamate, triethylamine, dichloromethane, trifluoroacetic acid, and sodium bicarbonate, and perform extraction, drying, and filtration, and then purification to obtain the intermediate product 1; Step S3: React the intermediate product 1 with triethylamine and benzenesulfonyl chloride, dry, filter, and evaporate, and then separate and purify to obtain the novel CXCR4 antagonist 1; Step S4: React piperazine-1-carboxylic acid tert-butyl ester, triethylamine, 3-bromobenzoyl chloride, dichloromethane, trifluoroacetic acid, sodium bicarbonate, and anhydrous sodium sulfate to obtain the initial compound 2; Step S5: React the initial compound 2 with triethylamine and phenylacetyl chloride, carry out extraction and drying, and then carry out purification to obtain intermediate product 2; Step S6: Dissolve 3-methoxyphenylboronic acid, tetrakis(triphenylphosphine)palladium, potassium carbonate, and intermediate product 2 in a 1:1 mixed solution of tetrahydrofuran and water; under nitrogen protection, reflux and react at 100 °C for 18 hours, and obtain the novel CXCR4 antagonist 2 through column chromatography; Step S7: Dissolve 2-(4-(methoxycarbonyl)phenyl)acetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, and triethylamine in dichloromethane, and stir at room temperature for 1 hour; then add 2-(naphthalen-1-yl)ethan-1-amine, and the mixture continues to react for 12 hours; after the reaction is completed, wash with saturated sodium bicarbonate solution, 1M hydrochloric acid solution, and saturated brine in sequence, add anhydrous sodium sulfate and then filter, dry to remove the solvent, and then carry out purification with a silica gel column to obtain the initial compound 3; Step S8: Dissolve the initial compound 3 in a 1:1 mixed solution of tetrahydrofuran and water, add sodium hydroxide solution, and react at room temperature for 12 hours; after the reaction is completed, acidify to pH 1 with 1M hydrochloric acid, at this time a precipitate appears, carry out filtration to obtain intermediate product 3; Step S9: Dissolve 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 the mixture continues to react for 12 hours; after the reaction is completed, wash with saturated sodium bicarbonate solution, 1M hydrochloric acid solution, and saturated brine in sequence, add anhydrous sodium sulfate and then filter, remove the solvent by vacuum drying, and then carry out purification with high performance liquid chromatography to obtain the novel CXCR4 antagonist 3; Step S10: Mix the novel CXCR4 antagonist 1 obtained in Step S3, the novel CXCR4 antagonist 2 obtained in Step S6, and the novel CXCR4 antagonist 3 obtained in Step S9 to obtain the final novel CXCR4 antagonist.

[0006] Preferably, Step S1 specifically includes: Step S11: Dissolve quinoline-6-carboxylic acid in a mixed solvent of dichloromethane and N,N-dimethylformamide; Step S12: Slowly add oxalyl chloride and stir at room temperature for reaction; Step S13: After the reaction is completed, obtain the initial compound 1 by evaporating the solvent to dryness.

[0007] Preferably, Step S2 specifically includes: Step S21: Dissolve the initial compound 1 in dichloromethane, successively add tert-butyl (4-(aminomethyl)benzyl)carbamate and triethylamine, and react at room temperature; Step S22: After the reaction in Step S21 is completed, evaporate the solvent, add dichloromethane and trifluoroacetic acid, and stir at room temperature; Step S23: Adjust the pH to neutral with sodium bicarbonate solution, extract with ethyl acetate 2 - 5 times, combine the organic phases, dry with anhydrous sodium sulfate, filter, evaporate the solvent, and purify by silica gel column chromatography to obtain the intermediate product 1.

[0008] Preferably, the specific steps of Step S3 are as follows: Step S31: Dissolve the intermediate product 1 in dichloromethane, add triethylamine and benzenesulfonyl chloride, and react at room temperature; Step S32: After the reaction is completed, wash with water, retain the organic phase, add anhydrous sodium sulfate for drying, filter, evaporate the solvent, and obtain the crude product; Step S33: Separate and purify the crude product obtained in Step S32 by high performance liquid chromatography to finally obtain the novel CXCR4 antagonist 1.

[0009] Preferably, the specific steps of Step S4 are as follows: Step S41: Dissolve tert-butyl piperazine-1-carboxylate and triethylamine in dichloromethane, slowly add 3-bromobenzoyl chloride under ice bath conditions, and then transfer the reaction system to room temperature and react 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 overnight at room temperature; Step S43: After the reaction is completed, slowly add the above solution to sodium bicarbonate solution for alkalization, adjust the pH to neutral, extract and wash three times; then add anhydrous sodium sulfate to the organic phase, filter to remove the organic phase, and purify by silica gel column to obtain the initial compound 2.

[0010] Preferably, the specific steps of Step S5 are as follows: Step S51: Add triethylamine to the initial compound 2 and dissolve it in dichloromethane; Step S52: Add phenylacetyl chloride under ice bath conditions, transfer to room temperature for reaction; after extraction, directly dry and remove the organic phase, and then purify by silica gel column to obtain the intermediate product 2.

[0011] Preferably, the specific steps of Step S6 are as follows: Dissolve the intermediate product 2, (3-methoxyphenyl)boronic acid, tetrakis(triphenylphosphine)palladium, and potassium carbonate in a 1:1 mixed solution of tetrahydrofuran and water; under nitrogen protection, reflux at 100 °C for reaction, and obtain the novel CXCR4 antagonist 2 through column chromatography.

[0012] Preferably, step S7 specifically includes: Step S71: Dissolve 2-(4-(methoxycarbonyl)phenyl)acetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, and triethylamine in dichloromethane, and stir at room temperature for 1 hour; Step S72: Then add 2-(naphthalen-1-yl)ethan-1-amine, and continue to react the mixture for 12 hours; Step S73: After the reaction is completed, wash with saturated sodium bicarbonate solution, 1M hydrochloric acid solution, and saturated brine in sequence, add anhydrous sodium sulfate and then filter, dry to remove the solvent, and then purify by silica gel column to obtain the initial compound 3.

[0013] Preferably, step S8 specifically includes: Step S81: Dissolve the initial compound 3 in a mixed solution of tetrahydrofuran and water at a ratio of 1:1, add sodium hydroxide solution, and react at room temperature for 12 hours; Step S82: After the reaction is completed, acidify with 1M hydrochloric acid to pH 1, at this time a precipitate will form, filter to obtain the intermediate product 3.

[0014] Preferably, step S9 specifically includes: Step S91: Dissolve the intermediate product 3, EDCI, HOBT, and triethylamine in dichloromethane, and stir at room temperature for 1 hour; Step S92: Then add methylamine hydrochloride, and continue to react the mixture for 12 hours; Step S93: After the reaction is completed, wash with saturated sodium bicarbonate solution, 1M hydrochloric acid solution, and saturated brine in sequence, add anhydrous sodium sulfate and then filter, remove the solvent by vacuum drying, and then purify by high performance liquid chromatography to obtain the novel CXCR4 antagonist 3.

[0015] The advantages and effects of this application are as follows: The novel CXCR4 antagonists designed in the present invention are composed of novel CXCR4 antagonist 1, novel CXCR4 antagonist 2, and novel CXCR4 antagonist 3. They can be used in combination or alone. The three newly designed CXCR4 antagonists constitute a molecular architecture different from the existing CXCR4 antagonists. These three structural designs are the basis for achieving high affinity and specific antagonistic effects on CXCR4, thereby achieving highly specific blockade of the binding of CXCR4 to its ligand and effectively inhibiting the activation of downstream signaling pathways. Their significant advantages over existing clinically used drugs in terms of antagonistic activity, synthesis difficulty, and safety are the key manifestations of their core therapeutic value.

[0016] The above description is only an overview of the technical solution of the present application. In order to better understand the technical means of the present application, it can be implemented according to the content of the specification. In addition, in order to make the above and other purposes, features and advantages of the present application more obvious and understandable, the following describes the preferred embodiments of the present application in detail in conjunction with the accompanying drawings.

[0017] Those skilled in the art will better understand the above and other purposes, advantages and features of the present application according to the following detailed description of the specific embodiments of the present application in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or in the prior art, the following briefly introduces the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained according to these drawings. In all the drawings, similar elements or parts are generally denoted by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0019] Figure 1 Chemical reaction formula for the first-step preparation method of CXCR4 antagonist 1 designed for the present application; Figure 2 Chemical reaction formula for the second-step preparation method of CXCR4 antagonist 1 designed for the present application; Figure 3 Chemical reaction formula for the third-step preparation method of CXCR4 antagonist 1 designed for the present application; Figure 4 Chemical reaction formula for the first-step preparation method of CXCR4 antagonist 2 designed for the present application; Figure 5 Chemical reaction formula for the second-step preparation method of CXCR4 antagonist 2 designed for the present application; Figure 6 Chemical reaction formula for the third-step preparation method of CXCR4 antagonist 2 designed for the present application; Figure 7 Chemical reaction formula for the first-step preparation method of CXCR4 antagonist 3 designed for the present application; Figure 8 Chemical reaction formula for the second-step preparation method of CXCR4 antagonist 3 designed for the present application; Figure 9 Chemical reaction formula for the third-step preparation method of CXCR4 antagonist 3 designed for the present application; Figure 10 Imaging diagram obtained by incubating with rhodamine-labeled streptavidin (3 ug / ml) designed for the present application, washing three times, and then imaging with a confocal microscope; Figure 11 Migration response diagram of tumor cells designed for this application; Figure 12 Diagram of tumor cells crossing Matrigel by invasion designed for this application; Figure 13 Diagram of inhibition of xylene-induced acute inflammation designed for this application; Figure 14 Lung metastasis effect diagram of 4T1 cells designed for this application; Figure 15 Diagram of cell viability of antagonist at various concentration gradients designed for this application; Figure 16 1H NMR spectrum of CXCR4 antagonist 1 designed for this application; Figure 17 13C NMR spectrum of CXCR4 antagonist 1 designed for this application; Figure 18 Mass spectrum of CXCR4 antagonist 1 designed for this application; Figure 19 1H NMR spectrum of CXCR4 antagonist 2 designed for this application; Figure 20 13C NMR spectrum of CXCR4 antagonist 2 designed for this application; Figure 21 Mass spectrum of CXCR4 antagonist 2 designed for this application; Figure 22 1H NMR spectrum of CXCR4 antagonist 3 designed for this application; Figure 23 13C NMR spectrum of CXCR4 antagonist 3 designed for this application; Figure 24 Mass spectrum of CXCR4 antagonist 3 designed for this application; Figure 25 Molecular formula of CXCR4 antagonist 1 designed for this application; Figure 26 Molecular formula of CXCR4 antagonist 2 designed for this application; Figure 27 Molecular formula of CXCR4 antagonist 3 designed for this application. Detailed implementation manners

[0020] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are some, but not all, of the embodiments of this application. In the following description, specific details such as specific configurations and components are provided only to assist in a comprehensive understanding of the embodiments of this application. Therefore, those skilled in the art should understand that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of this application. Additionally, descriptions of known functions and structures are omitted for clarity and conciseness in the embodiments.

[0021] It should be understood that the phrase "one embodiment" or "this embodiment" mentioned throughout the specification means that a specific feature, structure, or characteristic related to the embodiment is included in at least one embodiment of this application. Therefore, the phrase "one embodiment" or "this embodiment" appearing throughout the specification does not necessarily refer to the same embodiment. Furthermore, these specific features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0022] In addition, this application may repeat reference numerals and / or letters in different instances. This repetition is for the purpose of simplicity and clarity and does not in itself indicate the relationship between the various embodiments and / or arrangements discussed.

[0023] The term "and / or" in this document is merely a description of the associated relationship of the associated objects, indicating that three relationships can exist. For example, A and / or B can represent: A exists alone, B exists alone, and both A and B exist simultaneously. The term " / and" in this document is a description of another associated object relationship, indicating that two relationships can exist. For example, A / and B can represent: A exists alone, and both A and B exist. Additionally, the character " / " in this document generally indicates that the associated objects before and after are in an "or" relationship.

[0024] The term "at least one" in this document is merely a description of the associated relationship of the associated objects, indicating that three relationships can exist. For example, at least one of A and B can represent: A exists alone, both A and B exist simultaneously, and B exists alone.

[0025] It should also be noted that in this document, relational terms such as first and second are only used 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 "comprise," "include," or any other variant thereof are intended to cover non-exclusive inclusion. Embodiment

[0026] This embodiment mainly introduces an optimized preparation method of a novel CXCR4 antagonist 1, including the following steps: Step S1: React quinoline-6-carboxylic acid, dichloromethane, N,N-dimethylformamide, and oxalyl chloride to obtain the initial compound 1; Step S2: React the initial compound 1 with tert-butyl (4-(aminomethyl)benzyl)carbamate, triethylamine, dichloromethane, trifluoroacetic acid, and sodium bicarbonate, perform extraction and drying filtration, and then purify to obtain the intermediate product 1; Step S3: React the intermediate product 1 with triethylamine and benzenesulfonyl chloride, dry, filter, and evaporate, then separate and purify to obtain the novel CXCR4 antagonist 1; for its molecular formula R8, please refer to Figure 25 ; for its hydrogen spectrum, carbon spectrum, and mass spectrum, please refer to Figure 16 , Figure 17 , Figure 18 .

[0027] Furthermore, the specific steps of step S1 include: Step S11: Dissolve quinoline-6-carboxylic acid in a mixed solvent of dichloromethane and N,N-dimethylformamide; Step S12: Slowly add oxalyl chloride and stir at room temperature for reaction; Step S13: After the reaction is completed, obtain the initial compound 1 by evaporating the solvent to dryness.

[0028] Furthermore, dissolve quinoline-6-carboxylic acid (1 g) in a mixed solvent of dichloromethane (4 ml) and a small amount of N,N-dimethylformamide, slowly add oxalyl chloride (0.6 ml), and stir at room temperature for 3 hours. After the reaction is completed, obtain the initial compound 1 by evaporating the solvent under reduced pressure (directly used for the next step without further purification). For its chemical reaction formula, please refer to Figure 1 .

[0029] Furthermore, the specific steps of step S2 include: Step S21: Dissolve the initial compound 1 in dichloromethane, and successively add tert-butyl (4-(aminomethyl)benzyl)carbamate and triethylamine, and react at room temperature; Step S22: After the reaction in step S21 is completed, evaporate the solvent, add dichloromethane and trifluoroacetic acid, and stir at room temperature; Step S23: Adjust the pH to neutral with sodium bicarbonate solution, extract 2 - 5 times with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, filter, evaporate the solvent, and purify by silica gel column chromatography to obtain the intermediate product 1.

[0030] Further, weigh the initial compound 1 (1.1 g) obtained in the first step and dissolve it in dichloromethane. Then, successively add tert-butyl (4-(aminomethyl)benzyl)carbamate (1.37 g) and triethylamine (966 μl), and react at room temperature for 7 hours. After the reaction is completed, evaporate the solvent, add dichloromethane (6 ml) and trifluoroacetic acid (2 ml), and stir at room temperature for 1 hour. Subsequently, adjust the pH to neutral with a sodium bicarbonate solution, extract three times with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, filter, and then evaporate the solvent. Purify by silica gel column chromatography to obtain intermediate 1. Please refer to the chemical reaction formula Figure 2 。

[0031] Further, step S3 specifically includes: Step S31: Dissolve intermediate 1 in dichloromethane, add triethylamine and benzenesulfonyl chloride, and react at room temperature; Step S32: After the reaction is completed, wash with water, retain the organic phase, dry with anhydrous sodium sulfate, filter, and then evaporate the solvent to obtain a crude product; Step S33: Purify the crude product obtained in step S32 by high performance liquid chromatography to finally obtain the novel CXCR4 antagonist 1.

[0032] Further, weigh intermediate 1 (1 g) obtained in the above step and dissolve it in dichloromethane. Add triethylamine (573 μl) and benzenesulfonyl chloride (482 μl), and react at room temperature for 5 hours. After the reaction is completed, wash with water, retain the organic phase, dry with anhydrous sodium sulfate, filter, and then evaporate the solvent. The obtained crude product is purified by high performance liquid chromatography (HPLC) to finally obtain the novel CXCR4 antagonist 1. Please refer to the chemical reaction formula Figure 3 。 Example

[0033] Based on Example 1, this example mainly introduces the optimized preparation method of the novel CXCR4 antagonist 2, including the following steps: Step S4: React piperazine-1-carboxylic acid tert-butyl ester, triethylamine, 3-bromobenzoyl chloride, dichloromethane, trifluoroacetic acid, sodium bicarbonate, and anhydrous sodium sulfate to obtain the initial compound 2; Step S5: React the initial compound 2 with triethylamine and phenylacetyl chloride, perform extraction and drying, and then purification to obtain intermediate 2; Step S6: Dissolve 3-methoxyphenylboronic acid, tetrakis(triphenylphosphine)palladium, potassium carbonate, and intermediate 2 in a 1:1 mixed solution of tetrahydrofuran and water; under nitrogen protection, reflux at 100 °C for 18 hours, and obtain the novel CXCR4 antagonist 2 through column chromatography; Please refer to the molecular formula R6 Figure 26 , and please refer to the hydrogen spectrum, carbon spectrum, and mass spectrum Figure 19 、Figure 20 , Figure 21 。

[0034] Further, step S4 specifically includes: Step S41: Dissolve tert-butyl piperazine-1-carboxylate and triethylamine in dichloromethane, slowly add 3-bromobenzoyl chloride under ice bath conditions, and then transfer the reaction system to room temperature and react for 16 hours; Step S42: After the reaction is completed, wash three times with water, extract and then dry the organic phase; then add 6 ml of dichloromethane and 2 ml of trifluoroacetic acid and react overnight at room temperature; Step S43: After the reaction is completed, slowly add the above solution to sodium bicarbonate solution for alkalization, adjust the pH to neutral, extract and wash three times; then add anhydrous sodium sulfate to the organic phase, filter to remove the organic phase, and purify through a silica gel column to obtain the initial compound 2.

[0035] Further, dissolve tert-butyl piperazine-1-carboxylate (1.44 g) and triethylamine (1.29 ml) in dichloromethane, slowly add 3-bromobenzoyl chloride under ice bath conditions, and then transfer the reaction system to room temperature and react for 16 hours. After the reaction is completed, wash three times with water, extract and then dry the organic phase. Then add 6 ml of dichloromethane and 2 ml of trifluoroacetic acid and react overnight at room temperature. After the reaction is completed, slowly add the above solution to sodium bicarbonate solution for alkalization, adjust the pH to neutral, extract and wash three times. Add anhydrous sodium sulfate to the organic phase, filter to remove the organic phase, and purify through a silica gel column to obtain the initial compound 2; for its chemical reaction formula, please refer to Figure 4 。

[0036] Further, step S5 specifically includes: Step S51: Add triethylamine to the initial compound 2 and dissolve it in dichloromethane; Step S52: Add phenylacetyl chloride under ice bath conditions, transfer to room temperature for reaction; after extraction, directly dry and remove the organic phase, and then purify through a silica gel column to obtain the intermediate product 2.

[0037] Weigh 500 mg of the initial compound 2, add 310 μl of triethylamine, and dissolve it in dichloromethane. Add 245 μl of phenylacetyl chloride under ice bath conditions, transfer to room temperature and react for 5 hours. After extraction, directly dry and remove the organic phase, and then purify through a silica gel column to obtain the intermediate product 2; for its chemical reaction formula, please refer to Figure 5 。

[0038] Further, step S6 specifically includes: dissolving intermediate 2, (3-methoxyphenyl)boronic acid, tetrakis(triphenylphosphine)palladium, and potassium carbonate in a 1:1 mixed solution of tetrahydrofuran and water; under nitrogen protection, refluxing at 100 °C for reaction, and obtaining novel CXCR4 antagonist 2 through column chromatography.

[0039] Further, weigh intermediate 2 (737 mg), (3-methoxyphenyl)boronic acid (347 mg), tetrakis(triphenylphosphine)palladium (66 mg), and potassium carbonate (526 mg), and dissolve them in a 1:1 mixed solution of tetrahydrofuran and water. Under nitrogen protection, reflux at 100 °C for 18 hours, and obtain novel CXCR4 antagonist 2 through column chromatography; for its chemical reaction formula, please refer to Figure 6 。

[0040] Example 3 Based on Example 1, this example mainly introduces the optimized preparation method of novel CXCR4 antagonist 3, including the following steps: Step S7: Dissolve 2-(4-(methoxycarbonyl)phenyl)acetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, and triethylamine in dichloromethane, and stir at room temperature for 1 hour; then add 2-(naphthalen-1-yl)ethan-1-amine, and continue to react the mixture for 12 hours; after the reaction is completed, wash with saturated sodium bicarbonate solution, 1M hydrochloric acid solution, and saturated brine once each, add anhydrous sodium sulfate and then filter, dry to remove the solvent, and then purify with a silica gel column to obtain the initial compound 3; Step S8: Dissolve the initial compound 3 in a 1:1 mixed solution of tetrahydrofuran and water, add sodium hydroxide solution, and react at room temperature for 12 hours; after the reaction is completed, acidify with 1M hydrochloric acid to pH 1, at this time a precipitate will form, filter to obtain intermediate 3; Step S9: Dissolve intermediate 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 the mixture for 12 hours; after the reaction is completed, wash with saturated sodium bicarbonate solution, 1M hydrochloric acid solution, and saturated brine once each, add anhydrous sodium sulfate and then filter, remove the solvent by vacuum drying, and then purify with high performance liquid chromatography to obtain novel CXCR4 antagonist 3. For its molecular formula R10, please refer to Figure 27 , and for its 1H NMR spectrum, 13C NMR spectrum, and mass spectrum, please refer to Figure 22 、 Figure 23 、 Figure 24 。

[0041] Further, step S7 specifically includes: Step S71: Dissolve 2-(4-(methoxycarbonyl)phenyl)acetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, and triethylamine in dichloromethane and stir at room temperature for 1 hour; Step S72: Then add 2-(naphthalen-1-yl)ethan-1-amine, and the mixture continues to react for 12 hours; Step S73: After the reaction is completed, wash successively with saturated sodium bicarbonate solution, 1M hydrochloric acid solution, and saturated brine once each. Add anhydrous sodium sulfate and then filter. Dry to remove the solvent, and then purify by silica gel column to obtain the initial compound 3.

[0042] Furthermore, dissolve 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) in dichloromethane and stir at room temperature for 1 hour. Then add 2-(naphthalen-1-yl)ethan-1-amine (88 mg), and the mixture continues to react for 12 hours. After the reaction is completed, wash successively with saturated sodium bicarbonate solution, 1M hydrochloric acid solution, and saturated brine once each. Add anhydrous sodium sulfate and then filter. Dry to remove the solvent, and then purify by silica gel column to obtain the initial compound 3; for the chemical reaction formula, please refer to Figure 7 .

[0043] Furthermore, step S8 specifically includes: Step S81: Dissolve the initial compound 3 in a mixed solution of tetrahydrofuran and water at a ratio of 1:1, add sodium hydroxide solution, and react at room temperature for 12 hours; Step S82: After the reaction is completed, acidify with 1M hydrochloric acid to pH 1. At this time, a precipitate will form, and then filter to obtain the intermediate product 3.

[0044] Furthermore, dissolve the initial compound 3 in a mixed solution of tetrahydrofuran and water at a ratio of 1:1, add an appropriate amount of sodium hydroxide, and react at room temperature for 12 hours. After the reaction is completed, acidify with 1M hydrochloric acid to pH 1. At this time, a precipitate will form, and then filter to obtain the intermediate product 3; for the chemical reaction formula, please refer to Figure 8 .

[0045] Furthermore, step S9 specifically includes: Step S91: Dissolve the intermediate product 3, EDCI, HOBT, and triethylamine in dichloromethane and stir at room temperature for 1 hour; Step S92: Then add methylamine hydrochloride, and the mixture continues to react for 12 hours; Step S93: After the reaction is completed, wash it successively with saturated sodium bicarbonate solution, 1M hydrochloric acid solution and saturated brine once each, add anhydrous sodium sulfate and then filter, remove the solvent by vacuum drying, and then purify it by high performance liquid chromatography to obtain the novel CXCR4 antagonist 3.

[0046] Further, dissolve the intermediate 3 (100 mg), EDCI (69 mg), HOBT (49 mg) and triethylamine (164 μl) in dichloromethane, stir at room temperature for 1 hour. Then add methylamine hydrochloride (21 mg), and the mixture continues to react for 12 hours. After the reaction is completed, wash it successively with saturated sodium bicarbonate solution, 1M hydrochloric acid solution and saturated brine once each, add anhydrous sodium sulfate and then filter, remove the solvent by vacuum drying, and then purify it by high performance liquid chromatography (HPLC) to obtain the novel CXCR4 antagonist 3. The reaction formula is as Figure 9 shown.

[0047] Example 4 1. Competitive affinity assay: Based on Examples 1 - 3, use MDA - MB - 231 cells to perform the affinity assay of the compound with the target. Take the FDA - approved CXCR4 antagonist AMD3100 as the positive control. The TN14003 peptide has a high binding ability to CXCR4 and is often used as a tool for the competitive affinity assay of drugs for this target. Prepare various test compounds into a 1 mM stock solution with DMSO and finally dilute them to 1, 10, 100, 1000 nM for testing. Treat with different concentration combinations of the above various compounds for 15 min, then fix with 4% paraformaldehyde at room temperature, and wash three times with PBS. Then add 0.05 ug / ml biotinylated TN14003 peptide, incubate for 30 min, after washing three times with PBS, incubate with rhodamine - labeled streptavidin (3 ug / ml), and after washing three times, image with a confocal microscope. The results are as Figure 10 , the fluorescence intensity decreased significantly after treatment with the three compounds, indicating that each compound has a high affinity for the target CXCR4, thus reducing the binding of TN14003 to the target. In addition, the affinity of the three compounds is significantly higher than that of the positive control AMD3100.

[0048] 2. Tumor cell migration and invasion experiments: After cell seeding, culture until 85% confluence, use a pipette tip to draw a straight line at the bottom of the well plate, wash three times with PBS and then treat with 100 nM of the test compound for 24 hours. The results are as Figure 11, The bottom line width of the orifice plate reflects the migration ability of tumor cells, and the treatment with the three compounds significantly inhibits the migration of tumor cells. In addition, a Matrigel invasion assay was designed. Cells were seeded on the upper layer of a transwell chamber pre-coated with Matrigel and treated with the test compounds, while CXCL12 was added to the lower layer to stimulate the cells to invade downward. After 24 hours of treatment, the cells at the bottom of the chamber were stained and counted. This number represents the situation of tumor cells crossing Matrigel through invasion. The results are shown in Figure 12 , The three compounds provided by the present invention all effectively reduce the invasion of tumor cells.

[0049] 3. In vivo acute inflammation experiment: The test compounds were formulated into stock solutions, and three mice in each group were injected 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 the right ear of each mouse. The animals were sacrificed 1 hour later, ear pieces were taken with an 8 mm punch, weighed, fixed with paraformaldehyde, and stained with HE. The results are shown in Figure 13 , The three compounds provided in this article all effectively inhibit xylene-induced acute inflammation.

[0050] 4. In vivo anti-tumor metastasis experiment: An anti-metastasis experiment of any one of the compounds provided in this article (compound R10 was selected in this experiment) was carried out. Twelve 5-week-old Balb / c mice were randomly divided into two groups. Then, each mouse was injected with about 1 million 4T1-Luc cells via the tail vein. Compound R10 was injected every day for 18 days thereafter at a dose of 20 mg / kg. On the last day, 150 mg / kg of sodium fluorescein was injected 5 min before imaging for bioluminescence imaging. The results are shown in Figure 14 , The luminescence intensity in the mice treated with compound R10 was significantly reduced, indicating that compound R10 effectively reduced the lung metastasis of 4T1 cells.

[0051] 5. Cell-level safety test: Human normal liver cells L-02 were used for the safety evaluation of the compounds. The cells were plated on 96-well plates and cultured. After adherent growth, the drugs were added, and the test concentrations of several test compounds were 100 nM, 1 μM, and 10 μM respectively. They were cultured at 37 °C and 5% carbon dioxide concentration for 24 hours. Then, 10 μl of CCK8 reagent was added to each well, and after culturing for another 2 hours, the absorbance value of each well was measured with an enzyme-linked immunosorbent assay reader. The test wavelength was 450 nm, and the reference wavelength was 600 nm. The results are shown in Figure 15 , The three compounds provided in this article have little effect on cell viability at various concentration gradients, especially at high concentrations, and their safety is much higher than that of the control group AMD3100.

[0052] The CXCR4 antagonist provided by the present invention has a brand-new chemical structure such as Figure 25 , Figure 26 and Figure 27 , constituting a molecular architecture different from that of existing CXCR4 antagonists. This structural design is the basis for achieving high affinity and specific antagonistic effects on CXCR4, thereby achieving highly specific blockade of the binding of CXCR4 to its ligand and effectively inhibiting the activation of downstream signaling pathways. Its significant advantages over existing clinically used drugs in terms of antagonistic activity, ease of synthesis, and safety are the key manifestations of its core therapeutic value.

[0053] The above are only the preferred embodiments of the present invention, and it does not thereby limit the protection scope of the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any changes, modifications, substitutions, integrations, and parameter changes made to these embodiments by means of conventional substitutions or capable of achieving the same functions without departing from the principle and spirit of the present invention fall within the protection scope of the present invention.

Claims

1. A preparation method of a novel CXCR4 antagonist, characterized in that, It includes the following steps: Step S1: React quinoline-6-carboxylic acid, dichloromethane, N,N-dimethylformamide, and oxalyl chloride to obtain the initial compound 1; Step S2: React the initial compound 1 with tert-butyl (4-(aminomethyl)benzyl)carbamate, triethylamine, dichloromethane, trifluoroacetic acid, and sodium bicarbonate, perform extraction and drying filtration, and then purification to obtain the intermediate product 1; Step S3: React the intermediate product 1 with triethylamine and benzenesulfonyl chloride, dry, filter, and evaporate, then separate and purify to obtain the novel CXCR4 antagonist 1; Step S4: React piperazine-1-carboxylic acid tert-butyl ester, triethylamine, 3-bromobenzoyl chloride, dichloromethane, trifluoroacetic acid, sodium bicarbonate, and anhydrous sodium sulfate to obtain the initial compound 2; Step S5: React the initial compound 2 with triethylamine and phenylacetyl chloride, perform extraction and drying, and then purification to obtain the intermediate product 2; Step S6: Dissolve 3-methoxyphenylboronic acid, tetrakis(triphenylphosphine)palladium, potassium carbonate, and the intermediate product 2 in a 1:1 mixed solution of tetrahydrofuran and water; under nitrogen protection, reflux at 100 °C for 18 hours, and obtain the novel CXCR4 antagonist 2 through column chromatography; Step S7: Dissolve 2-(4-(methoxycarbonyl)phenyl)acetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, and triethylamine in dichloromethane, stir at room temperature for 1 hour; then add 2-(naphthalen-1-yl)ethan-1-amine, and the mixture continues to react for 12 hours; after the reaction is completed, wash with saturated sodium bicarbonate solution, 1M hydrochloric acid solution, and saturated brine in sequence, add anhydrous sodium sulfate and filter, dry to remove the solvent, and then purify with a silica gel column to obtain the initial compound 3; Step S8: Dissolve the initial compound 3 in a 1:1 mixed solution of tetrahydrofuran and water, add sodium hydroxide solution, and react at room temperature for 12 hours; after the reaction is completed, acidify with 1M hydrochloric acid to pH 1, at this time a precipitate appears, filter to obtain the intermediate product 3; Step S9: Dissolve the intermediate product 3, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, and triethylamine in dichloromethane, stir at room temperature for 1 hour; then add methylamine hydrochloride, and the mixture continues to react for 12 hours; after the reaction is completed, wash with saturated sodium bicarbonate solution, 1M hydrochloric acid solution, and saturated brine in sequence, add anhydrous sodium sulfate and filter, remove the solvent by vacuum drying, and then purify with high performance liquid chromatography to obtain the novel CXCR4 antagonist 3; Step S10: Mix the novel CXCR4 antagonist 1 obtained in Step S3, the novel CXCR4 antagonist 2 obtained in Step S6, and the novel CXCR4 antagonist 3 obtained in Step S9 to obtain the final novel CXCR4 antagonist.

2. The preparation method of a novel CXCR4 antagonist according to claim 1, characterized in that, The specific content of Step S1 includes: Step S11: Dissolve quinoline-6-carboxylic acid in a mixed solvent of dichloromethane and N,N-dimethylformamide; Step S12: Slowly add oxalyl chloride and stir at room temperature for reaction; Step S13: After the reaction is completed, the initial compound 1 is obtained by evaporating the solvent to dryness.

3. The preparation method of a novel CXCR4 antagonist according to claim 2, characterized in that, The specific steps of Step S2 are as follows: Step S21: Dissolve the initial compound 1 in dichloromethane, and successively add tert-butyl (4-(aminomethyl)benzyl)carbamate and triethylamine, and react at room temperature. Step S22: After the reaction in Step S21 is completed, evaporate the solvent to dryness, add dichloromethane and trifluoroacetic acid, and stir at room temperature. Step S23: Adjust the pH to neutral with sodium bicarbonate solution, extract with ethyl acetate 2-5 times, combine the organic phases, dry with anhydrous sodium sulfate, filter, evaporate the solvent, and purify by silica gel column chromatography to obtain intermediate 1.

4. The preparation method of a novel CXCR4 antagonist according to claim 3, characterized in that, The specific steps of Step S3 are as follows: Step S31: Dissolve intermediate 1 in dichloromethane, add triethylamine and benzenesulfonyl chloride, and react at room temperature. Step S32: After the reaction is completed, wash with water, retain the organic phase, dry with anhydrous sodium sulfate, filter, evaporate the solvent, and obtain the crude product. Step S33: Purify the crude product obtained in Step S32 by high performance liquid chromatography to finally obtain the novel CXCR4 antagonist 1.

5. The preparation method of a novel CXCR4 antagonist according to claim 1, characterized in that, The specific steps of Step S4 are as follows: Step S41: Dissolve tert-butyl piperazine-1-carboxylate and triethylamine in dichloromethane, slowly add 3-bromobenzoyl chloride under ice bath conditions, and then transfer the reaction system to room temperature and react 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 overnight at room temperature. Step S43: After the reaction is completed, slowly add the above solution to sodium bicarbonate solution for alkalization, adjust the pH to neutral, extract and wash three times; then add anhydrous sodium sulfate to the organic phase, filter to remove the organic phase, and purify by silica gel column to obtain the initial compound 2.

6. The preparation method of a novel CXCR4 antagonist according to claim 5, characterized in that, The specific steps of Step S5 are as follows: Step S51: Add triethylamine to the initial compound 2 and dissolve it in dichloromethane. Step S52: Add phenylacetyl chloride under ice bath conditions, transfer to room temperature for reaction; extract, directly dry and remove the organic phase, and then purify by silica gel column to obtain intermediate 2.

7. The preparation method of a novel CXCR4 antagonist according to claim 6, characterized in that, The specific steps of Step S6 are as follows: Dissolve intermediate 2, (3-methoxyphenyl)boronic acid, tetrakis(triphenylphosphine)palladium and potassium carbonate in a 1:1 mixed solution of tetrahydrofuran and water; under nitrogen protection, reflux and react at 100 °C, and obtain the novel CXCR4 antagonist 2 through column chromatography.

8. The preparation method of a novel CXCR4 antagonist according to claim 1, characterized in that, The specific steps of Step S7 are as follows: Step S71: Dissolve 2-(4-(methoxycarbonyl)phenyl)acetic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole and triethylamine in dichloromethane and stir at room temperature for 1 hour. Step S72: Then add 2-(naphthalen-1-yl)ethan-1-amine, and the mixture continues to react for 12 hours. Step S73: After the reaction is completed, wash once with saturated sodium bicarbonate solution, 1M hydrochloric acid solution and saturated brine in sequence, add anhydrous sodium sulfate, filter, dry to remove the solvent, and then purify by silica gel column to obtain the initial compound 3.

9. The preparation method of a novel CXCR4 antagonist according to claim 8, characterized in that, The specific steps of Step S8 are as follows: Step S81: Dissolve the initial compound 3 in a mixed solution of tetrahydrofuran and water at a ratio of 1:1, add a sodium hydroxide solution, and react at room temperature for 12 hours. Step S82: After the reaction is completed, acidify to pH 1 with 1 M hydrochloric acid. At this time, a precipitate will form. Filter to obtain the intermediate product 3.

10. The preparation method of a novel CXCR4 antagonist according to claim 9, characterized in that, The specific steps of step S9 are as follows: Step S91: Dissolve the intermediate product 3, EDCI, HOBT, and triethylamine in dichloromethane and stir at room temperature for 1 hour. Step S92: Then add methylamine hydrochloride, and the mixture continues to react for 12 hours. Step S93: After the reaction is completed, wash successively with saturated sodium bicarbonate solution, 1 M hydrochloric acid solution, and saturated brine once each. Add anhydrous sodium sulfate and then filter. Remove the solvent by vacuum drying, and then purify by high performance liquid chromatography to obtain the novel CXCR4 antagonist 3.

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