A bis-amidine-benzimidazole-phenyl derivative, and a synthesis method and application thereof

Abstract

The application discloses a kind of double amidine-benzimidazole-benzene derivatives and synthesis method and application thereof, it is related to the field of biological medicine, the dihydroxy ether of hydroxybenzaldehyde and dihalogenated hydrocarbon occurs alkylation reaction by the present application, again with 4-amidino-1,2-benzene diamine hydrochloride in 1,4-benzoquinone is oxidant under the condition of cyclization successfully synthesized double amidine-benzimidazole-benzene derivative.The present application simultaneously investigates the pharmacological activity of double amidine-benzimidazole-benzene derivative to RA and AD, and the result shows that the derivative inhibits the cell viability and migration ability of RA-FLS;The swelling number of arthritic paw of high-dose administration group, arthritis index reduces more significantly;Intra-articular blood flow signal reduces more obviously, and is related to administration amount;Improve mouse working memory disorder and image discrimination memory disorder caused by lateral ventricle injection of Aβ 1‑42 , and there is no significant influence on cerebral cortex excitability and motor function;Improve the cerebral cortex and hippocampus nerve injury of mouse caused by lateral ventricle injection of Aβ 1‑42 .

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a diamidinium-benzimidazole-benzene derivative, its synthesis method, and its application. Background Technology

[0002] Transcription factors (TFs) are central to many cellular processes, accounting for 5-10% of eukaryotic genes. Their pivotal role in many cellular pathways provides a strong theoretical basis for their attractive drug targets. Specifically, small-molecule inhibitors that block the binding of transcription factors to their regulatory sites could offer novel treatments for various human diseases. PU.1, a member of the E26 transformation-specific family of transcription factors, plays a crucial role in the development of various tissues. Studies have found that PU.1 not only functions in the determination and differentiation of hematopoietic lineages but also in immunity, adipogenesis, tissue fibrosis, and neurodevelopment. Recent research has also explored the role of PU.1 in autoimmune diseases such as rheumatoid arthritis (RA), experimental autoimmune encephalomyelitis (EAE), systemic lupus erythematosus (SLE), and Alzheimer's disease (AD).

[0003] Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease. The basic pathological changes include synovitis, pannus formation, and gradual destruction of articular cartilage and bone, ultimately leading to joint deformity and loss of function. Currently, RA treatment primarily involves medication, including nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, antirheumatic drugs (ART), and biologics. While these treatments alleviate joint inflammation to some extent, these drugs generally suffer from short circulation times, wide distribution, and significant side effects, severely limiting their further clinical application.

[0004] The ETS transcription factor family controls a range of physiological processes in many tissues. Sequence-specific binding is an essential step in ETS-mediated gene activation; therefore, inhibiting appropriate ETS-DNA complexes with small molecules offers significant potential for treating a variety of diseases. All ETS proteins possess a conserved DNA-binding domain that recognizes sites containing the 5'-GGAA / T-3' consensus. The protein inserts an α-helical contact in the large groove of its core sequence, while the loop interacts with flanking bases through backbone contacts in the minor groove. These flanking sequences tend to be conserved for specific ETS members, allowing for the development of compounds with high affinity for DNA minor grooves as potent isomeric inhibitors of the ETS-DNA complex. The applicant team discovered that PU.1 not only promotes excessive activation and inflammation of macrophages and synovial fibroblasts (FLS) in RA, but also directly binds to its promoter region to inhibit FLT3 (FMS-like tyrosine kinase) transcription. Inhibiting PU.1 activity or knocking out PU.1 significantly reduces the secretion of inflammatory factors and bone damage in a collagen antibody-induced arthritis model (CAIA), greatly delaying the progression of arthritis. Besides RA, PU.1 is also closely related to Alzheimer's disease. Analysis of patient tissue samples showed that downregulation of PU.1 expression reduced the expression of myeloid trigger receptor 2, tyrosine kinase-binding protein, and β-amyloid protein, effectively inhibiting the development of Alzheimer's disease. Based on this, this invention synthesizes a series of amidine-benzimidazole-benzene derivatives that, by binding to the DNA small groove AT sequence and inhibiting PU.1 activity, achieve the treatment of RA and AD. Summary of the Invention

[0005] The present invention aims to provide a diamidinium-benzimidazole-benzene derivative, its synthesis method, and its application. The method involves the alkylation reaction of p-hydroxybenzaldehyde with a dihaloalkane to obtain a diphenol ether, which is then cyclized with 4-amidinyl-1,2-phenylenediamine hydrochloride under 1,4-benzoquinone as an oxidant to obtain the diamidinium-benzimidazole-benzene derivative. The pharmacological activities of the diamidinium-benzimidazole-benzene derivative against rheumatoid arthritis (RA) and atopic dermatitis (AD) are also investigated. To achieve the above objective, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a diamidinium-benzimidazole-benzene derivative, the structural formula of which is as follows:

[0007]

[0008] In the formula, n is an integer from 1 to 10.

[0009] Furthermore, n is an integer from 1 to 4.

[0010] In a second aspect, the present invention provides a method for synthesizing a diamidinium-benzimidazole-benzene derivative, the method comprising the following steps:

[0011] Step S1: Weigh p-hydroxybenzaldehyde and anhydrous potassium carbonate into a round-bottom flask, add anhydrous DMF as solvent, stir at room temperature under nitrogen protection, and add dibromoalkane dropwise; after the addition is complete, heat the reaction under nitrogen. After the reaction is complete, cool to room temperature, pour the mixture into cold water, the product precipitates, filter, recrystallize the filter cake with anhydrous ethanol, and dry under reduced pressure.

[0012] Step S2: Weigh the product from step S1, 4-amidinyl-1,2-phenylenediamine hydrochloride and 1,4-benzoquinone into a round-bottom flask, add anhydrous ethanol, heat under nitrogen and reflux until the reaction is complete, stop the reaction, cool to room temperature, filter and collect the dark solid, wash with dry acetone, ice-cold anhydrous ethanol and ice-cold diethyl ether, and dry.

[0013] Step S3: Slowly dissolve the product from step S2 in hot anhydrous ethanol and filter while hot. Concentrate the filtrate by volume, acidify with hydrochloric acid saturated ethanol solution and stir overnight, evaporate to dryness, dilute with anhydrous diethyl ether, filter to collect the solid, and vacuum dry.

[0014] Step S4: Purify the product from step S3 to obtain a diamidinium-benzimidazole-benzene derivative.

[0015] Further, in step S1, the molar ratio of p-hydroxybenzaldehyde, anhydrous potassium carbonate, and dibromoalkane is 1:1 to 1.2:0.5 to 0.6;

[0016] Further, in step S1, the solvent volume is 10.0–30.0 mL; the stirring time at room temperature is 2–4 h; the heating reaction temperature is 80–130 °C and the reaction time is 2–12 h; the cold water volume is 50–100 mL; the vacuum drying temperature is 40–60 °C and the time is 8–12 h.

[0017] Further, in step S2, the molar ratio of the product of step S1, 4-amidinyl-1,2-phenylenediamine hydrochloride, and 1,4-benzoquinone is 1:2 to 2.2:2 to 2.2.

[0018] Furthermore, in step S2, the volume of anhydrous ethanol is 10–60 mL; the reflux reaction temperature is 80–100 °C; and the reaction time is 8–12 h.

[0019] Further, in step S3, the volume of hot anhydrous ethanol is 200–600 mL; the concentration volume is 40–100 mL; the vacuum drying temperature is 40–60 °C; and the time is 8–12 h.

[0020] Furthermore, in step S4, methanol is the mobile phase.

[0021] Thirdly, the present invention provides the use of a diamidinium-benzimidazole-benzene derivative in the preparation of a medicament for the prevention and / or treatment of rheumatoid arthritis and / or Alzheimer's disease.

[0022] The technical effects and advantages of this invention are as follows:

[0023] This invention successfully synthesized a diamidine-benzimidazole-benzene derivative. First, the in vitro therapeutic effect of the diamidine-benzimidazole-benzene derivative on rheumatoid arthritis (RA) was investigated. Cytotoxicity experiments showed that the diamidine-benzimidazole-benzene derivative exhibited the best inhibitory effect on the proliferation of RA-FLS (rheumatoid arthritis-associated sclerosis). Further, pharmacological results showed that the diamidine-benzimidazole-benzene derivative inhibited the cell viability and migration ability of RA-FLS. The high-dose group showed a more significant reduction in the number of swollen toe edema and the arthritis index; the reduction in intra-articular blood flow signal was more pronounced and correlated with the dosage. Simultaneously, the therapeutic activity of the diamidine-benzimidazole-benzene derivative on aplastic anemia (AD) was investigated. Results showed that the diamidine-benzimidazole-benzene derivative improved the effect of intraventricular injection of Aβ. 1-42 The induced working memory and image discrimination memory impairment in mice had no significant effect on cortical excitability or motor function; it improved the effect of lateral ventricle injection of Aβ. 1-42 The induced damage to the cerebral cortex and hippocampus of mice.

[0024] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention may be realized and obtained by means of the structures pointed out in the description, claims and drawings. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Compound 3a synthesized in Example 1 of this invention 1 H NMR spectrum;

[0027] Figure 2 The HRMS spectrum of compound 3a synthesized in Example 1 of this invention;

[0028] Figure 3 The compound PD4232 synthesized in Example 1 of this invention 1 H NMR spectrum;

[0029] Figure 4 The compound PD4232 synthesized in Example 1 of this invention 13 C NMR spectrum;

[0030] Figure 5 The HRMS spectrum of compound PD4232 synthesized in Example 1 of this invention;

[0031] Figure 6 Compound 3b synthesized in Example 2 of this invention 1 H NMR spectrum;

[0032] Figure 7 The HRMS spectrum of compound 3b synthesized in Example 2 of this invention;

[0033] Figure 8 The compound PD4234 synthesized in Example 2 of this invention 1 H NMR spectrum;

[0034] Figure 9 The compound PD4234 synthesized in Example 2 of this invention 13 C NMR spectrum;

[0035] Figure 10 The HRMS spectrum of compound PD4234 synthesized in Example 2 of this invention;

[0036] Figure 11 Compound 3c synthesized in Example 3 of this invention 1 HNMR spectrum;

[0037] Figure 12 The HRMS spectrum of compound 3c synthesized in Example 3 of this invention;

[0038] Figure 13 The compound PD4236 synthesized in Example 3 of this invention 1 H NMR spectrum;

[0039] Figure 14 The compound PD4236 synthesized in Example 3 of this invention 13 C NMR spectrum;

[0040] Figure 15 The HRMS spectrum of compound PD4236 synthesized in Example 3 of this invention;

[0041] Figure 16 Compound 3d synthesized in Example 4 of this invention 1 H NMR spectrum;

[0042] Figure 17The HRMS spectrum of compound 3d synthesized in Example 4 of this invention;

[0043] Figure 18 The compound PD4238 synthesized in Example 4 of this invention 1 H NMR spectrum;

[0044] Figure 19 The compound PD4238 synthesized in Example 4 of this invention 13 C NMR spectrum;

[0045] Figure 20 The HRMS spectrum of compound PD4238 synthesized in Example 4 of this invention;

[0046] Figure 21 This is a bar chart showing the effect of the diamidinium-benzimidazole-benzene derivative of the present invention on the in vitro proliferation level of RA-FLS;

[0047] Figure 22 This is a graph showing the number of swollen paws due to arthritis in mice of the Normal, Model, and each drug administration group according to the present invention.

[0048] Figure 23 This is a graph showing the arthritis index of mice in the Normal, Model, and each drug administration group according to the present invention.

[0049] Figure 24 This is a schematic diagram of arthritis, paw swelling, and arthritis in mice of the Normal, Model, and each drug administration group according to the present invention.

[0050] Figure 25 This is a schematic diagram of synovial Doppler blood flow in mice of the Normal, Model, and each drug administration group according to the present invention;

[0051] Figure 26 This is a schematic diagram illustrating the effect of the Normal, Model, and each drug-treated group mice on the migration ability of RA-FLS cells in this invention.

[0052] Figure 27 This is a schematic diagram illustrating the effects of the Normal, Model, and each drug-treated group mice on RA-FLS cell viability in this invention.

[0053] Figure 28 The compound PD4238 of this invention is effective against Aβ. 1-42 Flowchart of the experiment induced learning and memory impairment in dementia mice;

[0054] Figure 29 This is a schematic diagram of the Y-maze device of the present invention;

[0055] Figure 30 Aβ was injected into the contralateral ventricle of the compound PD4238 of the present invention.1-42 Bar chart showing the effect of the total number of Y-maze arm entries on the dementia model mice (n = 7-11, mean ± SEM);

[0056] Figure 31 Aβ was injected into the contralateral ventricle of the compound PD4238 of the present invention. 1-42 Bar chart showing the effect of the induced dementia model mice on the spontaneous alternation response rate in the Y maze (n=7–11, mean±SEM);

[0057] Figure 32 Aβ was injected into the contralateral ventricle of the compound PD4238 of the present invention. 1-42 Bar chart showing the effect of the priority index on the new object recognition test in mice with dementia induced by the disease (n=8-10, mean±SEM);

[0058] Figure 33 Aβ was injected into the contralateral ventricle of the compound PD4238 of the present invention. 1-42 Bar chart showing the effect of discrimination coefficient on the new object discrimination test in mice with induced dementia (n = 8-10, mean ± SEM);

[0059] Figure 34 Aβ was injected into the contralateral ventricle of the compound PD4238 of the present invention. 1-42 Effects of the morphology of neurons in the cerebral cortex, dentate gyrus (DG), CA1 region, and CA3 region of the dementia model mice (n=5, bar=100μm);

[0060] Figure 35 Aβ was injected into the contralateral ventricle of the compound PD4238 of the present invention. 1-42 Schematic diagram showing the effects of the induced dementia model mouse MAP2 protein expression on the cerebral cortex, hippocampal dentate gyrus, CA1 region, and CA3 region.

[0061] Figure 36 Aβ was injected into the contralateral ventricle of the compound PD4238 of the present invention. 1-42 Bar chart showing the effect of the induced dementia model mice on MAP2 protein expression in the cerebral cortex, hippocampal dentate gyrus, CA1 region, and CA3 region (n=4, bar=100μm, mean±SEM);

[0062] Figure 37 This is a schematic diagram illustrating the histopathological detection of the toxicity of compound PD4238 to the heart, liver, spleen, and kidneys of mice (n=5, bar=250μm). Detailed Implementation

[0063] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0064] This invention discloses a method for synthesizing a diamidinium-benzimidazole-benzene derivative, the synthetic route of which is as follows:

[0065]

[0066] Reagents and conditions: (i) anhydrous potassium carbonate, anhydrous DMF solution, 80℃, 12h; (ii) 4-amidinyl-1,2-phenylenediamine hydrochloride, 1,4-benzoquinone, anhydrous ethanol, N2, 80℃, 8h.

[0067] Specifically, the method for synthesizing the diamidinium-benzimidazole-benzene derivative includes the following steps:

[0068] Step S1, Synthesis of intermediate compounds:

[0069] Weigh p-hydroxybenzaldehyde and anhydrous potassium carbonate into a round-bottom flask, add anhydrous DMF as solvent, and stir at room temperature under nitrogen protection. Add dibromoalkane dropwise. After the addition is complete, heat the mixture under nitrogen. After the reaction is complete, cool to room temperature, pour the mixture into cold water, and the product precipitates. Filter the mixture, recrystallize the filter cake from anhydrous ethanol, and dry under reduced pressure to obtain a white solid intermediate compound.

[0070] In step S1 of the present invention, the molar ratio of p-hydroxybenzaldehyde, anhydrous potassium carbonate and dibromoalkane is 1:1 to 1.2:0.5 to 0.6, preferably 1:1:0.5; the solvent volume is 10.0 to 30.0 mL; the stirring time at room temperature is 2 to 4 h; the heating reaction temperature is 80 to 130 °C, and the reaction time is 2 to 12 h; the volume of cold water is 50 to 100 mL; and the drying is carried out under reduced pressure at 40 to 60 °C for 8 to 12 h.

[0071] Step S2, Synthesis of diamidinium-benzimidazole-benzene derivative:

[0072] Weigh the intermediate compound, 4-amidinyl-1,2-phenylenediamine hydrochloride, and 1,4-benzoquinone into a round-bottom flask. Add anhydrous ethanol and heat under nitrogen at reflux until the reaction is complete. After the reaction is complete, cool to room temperature, filter to collect the dark solid, wash with dry acetone, ice-cold anhydrous ethanol, and ice-cold diethyl ether, and dry to obtain dihydrochloride. Slowly dissolve the dihydrochloride solid in hot anhydrous ethanol and filter while hot. Concentrate the filtrate by volume, acidify with saturated hydrochloric acid in ethanol solution and stir overnight (blue color), evaporate to dryness, dilute with anhydrous diethyl ether, filter to collect the solid, and vacuum dry to obtain the final product, purple solid tetrahydrochloride. Then purify using preparative liquid chromatography (methanol as mobile phase) to obtain white solid tetrahydrochloride, namely the diamidinyl-benzimidazole-benzene derivatives, including PD4232, PD4234, PD4236, and PD4238.

[0073] In step S2 of the present invention, the molar ratio of the intermediate compound, 4-amidinyl-1,2-phenylenediamine hydrochloride, and 1,4-benzoquinone is 1:2 to 2.2:2 to 2.2, preferably 1:2:2; the volume of anhydrous ethanol is 10 to 60 mL; the reflux reaction temperature is 80 to 100 °C, and the reaction time is 8 to 12 h; the volume of hot anhydrous ethanol is 200 to 600 mL; the concentration volume is 40 to 100 mL; and the product is dried under reduced pressure at 40 to 60 °C for 8 to 12 h.

[0074] Example 1: Synthesis of compound PD4232

[0075] 1. Synthesis of compound 3a

[0076] Weigh p-hydroxybenzaldehyde (0.4884 g, 4 mmol) and anhydrous potassium carbonate (0.552 g, 4 mmol) into a 50 mL round-bottom flask. Add 10.0 mL of anhydrous DMF as solvent. Stir at room temperature for 2 h under nitrogen protection. Add 1,2-dibromoethane (0.376 g, 2 mmol) dropwise. After addition, react at 80 °C under nitrogen for 12 h. After the reaction is complete, cool to room temperature and pour the mixture into cold water (50 mL). The product precipitates, is filtered, and the filter cake is recrystallized from anhydrous ethanol and dried under reduced pressure to give 0.38 g (70.4% yield) of white solid intermediate compound 3a. Figure 1-2 As shown: 1 HNMR (400MHz, DMSO-d6) δ9.92 (s, 2H), 7.92 (dd, J=8.7, 2.0Hz, 4H), 7.23 (dd, J=8.7, 2.0Hz, 4H), 4.53 (s, 4H).HRMS (TOF) calculated for C 16 H 14 O4[M+H] + :271.0970, found 271.0963.

[0077] 2. Synthesis of compound PD4232

[0078] Weigh intermediate compound 3a (0.18 g, 0.67 mmol), 4-amidinyl-1,2-phenylenediamine hydrochloride (0.25 g, 1.34 mmol), and 1,4-benzoquinone (0.145 g, 1.34 mmol) into a 50 mL round-bottom flask. Add 30 mL of anhydrous ethanol. Heat under nitrogen and reflux at 80 °C for 8 h. Stop the reaction, cool to room temperature, filter to collect the dark solid, wash with dry acetone, ice-cold anhydrous ethanol, and ice-cold diethyl ether, and dry to give 0.28 g of dihydrochloride (yield 69.1%). Slowly dissolve the solid in hot anhydrous ethanol (300 mL) and filter while hot. Concentrate the filtrate to 70 mL, acidify with saturated hydrochloric acid in ethanol solution and stir overnight (blue color). Rotate to dryness, dilute with anhydrous diethyl ether, filter to collect the solid, and dry under vacuum to give the final product, purple solid tetrahydrochloride. The solution was then purified using a preparative liquid phase (methanol as the mobile phase) to give 0.26 g of a white solid tetrahydrochloride (yield 57.3%). (mp>300℃) Figure 3-5 As shown: HPLC: t R = 8.18 min, 100 area %. 1 H NMR (600MHz, DMSO-d6) δ: 9.33 (s, 4H), 9.21 (s, 4H), 8.24 (d, 4H, J=7.8Hz), 8.15 (s, 2 H), 7.81 (d, 2H, J=7.8Hz), 7.71 (d, 2H, J=7.8Hz), 7.27 (d, 4H, J=7.2Hz), 4.51 (s, 4H). 13 C NMR (150MHz, DMSO-d6) δ: 166.5(C×2), 161.1(C×2), 159.2(C×2), 158.9(C×2), 154.5(C×2), 129.4(C×4), 122. 8(C×2), 122.2(C×2), 121.5(C×2), 117.8(C×2), 115.8(C×2), 115.7(C×4), 67.1(C×2).HRMS(TOF)calculated for C 30 H 26 N8O2[M+H] + 531.2257, found 531.2255.

[0079] Example 2:

[0080] 1. Synthesis of compound 3b

[0081] The method was the same as in Example 1, yielding a white solid compound 3b (yield 73.1%). Figure 6-7 As shown: 1 H NMR (600MHz, DMSO-d6) δ9.87 (s, 2H), 7.87 (dd, J=8.4, 3.0Hz, 4H), 7.13 (dd, J=8.4, 3.0Hz, 4H), 4.18 (t, J=5.4Hz, 4H), 1.92 (m, 4H).HRMS (TOF) calculated for C 18 H 18 O4[M+H] + :299.1283, found 299.1276.

[0082] 2. Synthesis of compound PD4234

[0083] The method was the same as in Example 1, yielding a white solid compound PD4234 (yield 53.8%). mp>300℃. Figure 8-10 As shown: HPLC: t R = 9.03 min, 100 area%. 1 HNMR (600MHz, DMSO-d6) δ9.31 (s, 4H), 9.15 (s, 4H), 8.20 (d, 4H, J=7.8Hz), 8.13 (s, 2H), 7.7 9 (d, 2H, J = 8.4Hz), 7.69 (d, 2H, J = 8.4Hz), 7.20 (d, 4H, J = 8.4Hz), 4.19 (s, 4H), 1.96 (s, 4H). 13 C NMR (150MHz, DMSO-d6) δ: 166.5(C×2), 161.5(C×2), 159.1(C×2), 158.9(C×2), 154.6(C×2), 129.3(C×4), 122.7(C×2 ), 122.1(C×2), 121.2(C×2), 117.8(C×2), 115.8(C×2), 115.6(C×4), 68.0(C×2), 25.8(C×2).HRMS(TOF)calculated for C 32 H 30 N8O2[M+H] + 559.2570, found559.2576.

[0084] Example 3: Synthesis of compound PD4236

[0085] 1. Synthesis of compound 3c

[0086] The method was the same as in Example 1, yielding a white solid compound 3c (yield 69.5%). Figure 11-12 As shown: 1 H NMR (600MHz, DMSO-d6) δ9.86 (s, 2H), 7.85 (dd, J=8.0, 3.0Hz, 4H), 7.12 (dd, J=8.0, 3.0Hz, 4H), 4.10 (t, J=6.6Hz, 4H), 1.78 (m, 4H), 1.50 (m, 4H).HRMS (TOF) calculated for C 20 H 22 O4[M+H] + :327.1596, found 327.1586.

[0087] 2. Synthesis of compound PD4236

[0088] The method was the same as in Example 1, yielding a white solid compound PD4236 (yield 50.6%). mp>300℃. Figure 13-15 As shown: HPLC: t R = 9.74 min, 99.51 area%. 1 H NMR (600MHz, DMSO-d6) δ9.30 (s, 4H), 9.16 (s, 4H), 8.19 (d, 4H, J=8.4Hz), 8.12 (s, 2H), 7.79 (d, 2H, J=8.4 Hz), 7.69 (d, 2H, J=8.4Hz), 7.18 (d, 4H, J=9.0Hz), 4.11 (t, 4H, J=6.0Hz), 1.85-1.78 (m, 4H), 1.54 (s, 4H). 13 C NMR (150MHz, DMSO-d6) δ: 166.5(C×2), 161.5(C×2), 159.1(C×2), 158.9(C×2), 154.6(C×2), 129.3(C×4), 122.7(C×2), 122 .1(C×2), 121.2(C×2), 117.8(C×2), 115.9(C×2), 115.6(C×4), 68.3(C×2), 29.0(C×2), 25.8(C×2).HRMS(TOF)calculated for C 34 H 34 N8O2[M+H] + 587.2883, found 587.2864.

[0089] Example 4: Synthesis of compound PD4238

[0090] 1. Synthesis of compound 3d

[0091] The method was the same as in Example 1, yielding a white solid compound 3d (yield 72.3%). Figure 16-17 As shown: 1 H NMR (600MHz, DMSO-d6) δ9.86 (s, 2H), 7.85 (dd, J=8.0, 2.4Hz, 4H), 7.11 (dd, J=8.0, 2.4Hz, 4 H), 4.10 (t, J=6.6Hz, 4H), 1.74 (m, 4H), 1.43 (m, 4H), 1.36 (m, 4H).HRMS (TOF) calculated for C 22 H 26 O4[M+H] + :355.1909, found 355.1898.

[0092] 2. Synthesis of compound PD4238

[0093] The method was the same as in Example 1, yielding a white solid compound PD4238 (yield 49.1%). mp>300℃. Figure 18-20 As shown: HPLC: t R = 10.39 min, 100 area%. 1 H NMR (600MHz, DMSO-d6) δ9.31 (s, 4H), 9.15 (s, 4H), 8.18 (d, 4H, J=9.0Hz), 8.13 (s, 2H), 7.79 (d, 2H, J=8.4Hz), 7.69 (dd, 2 H, J=8.4Hz, J=1.8Hz), 7.18 (d, 4H, J=9.0Hz), 4.09 (t, 4H, J=6.6Hz), 1.80-1.74 (m, 4H), 1.50-1.43 (m, 4H), 1.39 (s, 4H). 13 C NMR (150MHz, DMSO-d6) δ: 166.5(C×2), 161.5(C×2), 159.1(C×2), 158.9(C×2), 154.6(C×2), 129.3(C×4), 122.7(C×2), 122.1(C× 2), 121.1(C×2), 117.7(C×2), 115.8(C×2), 115.6(C×4), 68.3(C×2), 29.2(C×2), 29.0(C×2), 25.9(C×2).HRMS(TOF)calculated for C 36 H 38 N8O2[M+H] +615.3196, found615.3192.

[0094] Example 5: Cytotoxicity test of diamidinium-benzimidazole-benzene derivative

[0095] Example 5 of this invention uses the CCK-8 assay to compare the effects of PD4232, PD4234, PD4236, and PD4238 on the in vitro proliferation level of RA-FLS. The results are as follows: Figure 21 As shown, PD4238 exhibited the best inhibitory effect on the proliferation of RA-FLS under the condition of treatment with a concentration of 0.5 μM.

[0096] Example 6: Evaluation of the therapeutic efficacy and safety of PD4238 series derivatives

[0097] 1. Effects of PD4238 on the migration ability of RA-FLS cells

[0098] (1) Transwell assay of the effects of DB2115 and PD4238 on the migration ability of RA-FLS cells

[0099] Synovial membranes were isolated from the joints of RA patients, and RA-FLS cells were extracted. After culturing in six-well plates, the cells were divided into Normal, Model, DB2115, low-dose PD4238, and high-dose PD4238 groups. Twenty-four hours after drug administration, the cells were digested, resuspended, and seeded in the upper chamber of a Transwell chamber (0.4 μm). Images were taken and analyzed after 24 hours. Results are as follows: Figure 22 As shown, DB2115 and PD4238 inhibited the migration ability of RA-FLS.

[0100] (2) Effects of DB2115 and PD4238 on RA-FLS cell viability as detected by CCK-8 assay

[0101] Synovial membrane was isolated from the joints of RA patients, and RA-FLS cells were extracted. After culturing in six-well plates, the cells were digested, centrifuged, and seeded into 96-well plates, divided into Normal, Model, DB2115, low-dose PD4238, and high-dose PD4238 groups. Cell viability was assessed 24 hours after drug administration. Results are as follows: Figure 23 The results showed that DB2115 and PD4238 inhibited the cell viability of RA-FLS.

[0102] 2. Scoring criteria for the CIA mouse model

[0103] (1) Number of paw swellings in arthritis-related feet

[0104] Before the first administration of the CIA model, the number of swollen paws in each group of mice was counted every three days. Each paw was counted as one ankle joint and five toe joints, with each joint being scored as 1 point. The maximum score for each mouse was 24 points.

[0105] (2) Arthritis Index (AI) score

[0106] From the time the CIA model was successfully established until the first administration, the arthritis index of mice in each modeling group was scored every three days. The scoring details are shown in Table 1 below, with a maximum score of 16 points per mouse.

[0107] Table 1. Arthritis Index Scoring Rules

[0108]

[0109] 3. Dosing regimen

[0110] After successful CIA model establishment, mice were randomly divided into five groups based on their overall body score, paw swelling score, and arthritis index score: Normal, Model, DB2115 (17 mg / kg), PD4238 (8.5 mg / kg), and PD4238 (17 mg / kg), with five mice in each group. On day 29, after successful model establishment, the first intraperitoneal injection was administered. Administered every three days for a total of eighteen days. On the last day, the mice were sacrificed, and all relevant indicators were measured.

[0111] 3. Treatment efficacy of RA

[0112] (1) Overall evaluation of CIA mice

[0113] Based on the measurement results, the number of swollen paws and arthritis index in mice of the Normal, Model, and each treatment group are shown in Figures (24-26). Compared with the Model group, the arthritis index and the number of swollen paws were significantly reduced after treatment with DB2115 (17 mg / kg), PD4238 (8.5 mg / kg), and PD4238 (17 mg / kg). The experimental results indicate that DB2115 and low-dose PD4238 have a certain degree of therapeutic effect on RA, but the reduction in the number of swollen paws and the arthritis index is more significant in the high-dose PD4238 group.

[0114] (2) Synovial Doppler flow grading

[0115] After the CIA model was established and the drugs were administered until the day before sacrifice, synovial Doppler blood flow grading was performed on mice in each modeling group. The grading details are shown in Table 2 below. The results are as follows: Figure 27As shown, compared with Model, blood flow signals decreased in all groups after drug treatment, but the decrease in blood flow signals was more significant in the PD4238 group and was related to the dosage.

[0116] Table 2. Detailed Rules for Synovial Doppler Blood Flow Grading

[0117]

[0118] Example 7: Compound PD4238's resistance to Aβ 1-42 Results of experiments on cognitive impairment in mice

[0119] 1. Laboratory mice

[0120] Ninety-two male ICR mice (17-22g each), SPF grade, were purchased from Liaoning Changsheng Biotechnology Co., Ltd., Certificate No.: NO.210726231101565653, License No.: SCXK(Liaoning)2020-0001. They were housed at the SPF-grade Animal Center of Shenyang Pharmaceutical University under 12-hour continuous lighting, at an ambient temperature of 20-24℃, and with free access to food and water.

[0121] 2. Experimental Grouping and Experimental Design

[0122] like Figure 28 As shown, the experiment divided mice into 8 groups: sham-operated group, model group, positive control group 1 (rivastigmine 1.25 mg / kg), positive control group 2 (donepezil 1.25 mg / kg), and four dosage groups of compound PD4238 (10, 5, 2.5, and 1.25 mg / kg), with 11-14 mice in each group. The drug was dissolved in 1% CMC-Na. Each group started receiving the drug via gavage on the day of modeling and continued administration for 7 days. After that, the Y-maze test, new object recognition test, spontaneous activity test, and Morris water maze test were performed. The drug was continued during the behavioral tests until the animals were sacrificed.

[0123] 3. Injection of Aβ into the lateral ventricle 1-42 Establishment of an AD mouse model

[0124] Aβ 1-42 0.5 mg (molecular weight 4514.04) was dissolved in 6 μL DMSO, added to 805 μL physiological saline, and incubated at 37°C for 120 h. Mice were anesthetized by intraperitoneal injection of 2.5% Avertin. Hair was clipped from the cranial incision area, and after disinfection with alcohol, the skin of the cranial vault was incised and fixed on a stereotaxic instrument. Based on the location of the lateral ventricle (left lateral ventricle, 0.5 mm posterior to the anterior fontanelle, 1.1 mm lateral to the left of the sagittal line, and 3.0 mm below the surface of the skull) determined by a mouse stereotaxic atlas, 3 μL (containing 410 pmol) of Aβ was injected vertically using a microsyringe. 1-42Slowly inject the solution for 3 minutes, leave the needle in for 3 minutes, and inject an equal volume of physiological saline in the sham surgery group using the same method. Apply sodium penicillin, suture the incision, disinfect the upper edge of the incision with iodine, and then apply collodion to prevent the wound from opening. Place the animals in cages for rearing. After the operation, administer an intramuscular injection of sodium penicillin (1.6 million U, 0.1 ml / animal) to fight infection.

[0125] 4. Y-maze Experiment Method

[0126] like Figure 29 As shown, the experimental setup consists of three wooden arms, A, B, and C, with an included angle of 120° (see schematic diagram). The dimensions are 40 × 12 × 10 cm (length × height × width). During the experiment, the animal is placed at the end of arm A and allowed to freely enter and exit the three arms. The total number of arm entries (N) and the order in which the mouse enters the arms within 5 minutes are recorded. After the experiment, each mouse is removed from the field, and its excrement is quickly cleaned up. The field is then wiped clean with 10% alcohol. A successful alternation is defined as consecutive entry into three different arms. The number of successful alternations is recorded. The alternation behavior (%) reflects spatial working memory ability. The calculation formula is as follows:

[0127] Alternationbehavior(%)=number ofalternation / (N-2)×100%.

[0128] 5. Experimental methods for identifying new objects

[0129] The experimental setup was a square, open wooden enclosure, measuring 50×50×15cm. Two days prior to the test, mice were placed in the experimental area for 5 minutes to acclimatize, with 2-3 mice per session, twice daily. On the day of the test, the animals were first placed in the apparatus to explore freely for 3 minutes to adapt. The animals were then removed, and two identical objects (A1 and A2) were placed inside the apparatus parallel to the walls and equidistant from the surrounding area. The mice were placed on the opposite side of the apparatus, equidistant from the two objects, facing away from them, and the time spent exploring the two objects within 5 minutes was recorded (tA1, tA2). One hour later, A2 was replaced with a new object (B), and the mice were placed back in, recording the time spent exploring the two objects (tA1, tB). The preference index and discrimination index of the mice towards the new object were calculated.

[0130] The formula for calculating the priority index is as follows: Preferential index(1h) = tB / (tA1+tB) × 100%;

[0131] The formula for calculating the discrimination index is as follows: Discrimination index(1h)=(tB-tA1) / (tA1+tB)×100%.

[0132] 6. Spontaneous Activity Experiment Method

[0133] The experimental setup consists of four rectangular activity boxes. Before the experiment, connect the computer host and the activity boxes, plug in the spontaneous activity dongle, and turn on all the switches in the spontaneous activity device. Turn on the computer connected to the device and double-click the "Spontaneous Activity" icon on the desktop. Select the automatic video acquisition settings, confirm the Digbev animal behavior analysis system, and fill in the "Experiment Number," "Experiment Name," "Experiment Personnel," and "Animal Species." During the experiment, place the animals in the four activity boxes respectively, and collect their spontaneous activity data for 5 minutes. The data collection will stop automatically after completion. Identify the animals' spontaneous activity trajectories and export the trajectory diagram, total distance traveled, activity time, and average speed.

[0134] 7. Obtain materials

[0135] Half of the mice in each group were perfused to extract their brains, while the other half were decapitated and their brains extracted directly.

[0136] (1) Brain harvesting via perfusion: Each mouse was anesthetized by intraperitoneal injection of 2.5% Avertin. After anesthesia, the thoracic cavity was opened at the xiphoid process, the right auricle was removed, and an IV needle was immediately inserted through the left apex of the heart. Pre-cooled saline was injected to flush out the blood. After injecting about 50 mL, the tail tip was removed to check the blood flushing. Once the tail tip was confirmed to be clean, pre-cooled 4% paraformaldehyde was used for perfusion until the mouse became rigid. The head was then decapitated and the brain was harvested. After removing the olfactory bulb, the brain was post-fixed in 4% paraformaldehyde. After fixation at 4°C for 48 h, the brain was dehydrated with different concentrations of alcohol, cleared with xylene, impregnated with paraffin, and embedded.

[0137] (2) Direct brain removal: The mouse was decapitated directly, and the brain tissue was removed. After the bloodstains were absorbed with filter paper on the ice box, the hippocampus and cortex were quickly separated into small EP tubes, flash-frozen with liquid nitrogen, and then stored in a -80℃ freezer.

[0138] 8. HE staining method

[0139] Dewaxing and hydration of paraffin sections: Place the sections in a constant temperature oven at 58-60℃ and bake for 3 hours. Dewax in the following order: Immerse in xylene I and II for 15 minutes each, immerse in anhydrous ethanol I and II for 5 minutes each, immerse in 95%, 85% and 75% ethanol for 5 minutes each, and rinse with PBS 3 times for 3 minutes each time.

[0140] Hematoxylin counterstaining: Lightly counterstain with hematoxylin solution for 3 minutes, then immediately rinse with tap water for 5 minutes.

[0141] Hydrochloric acid-ethanol differentiation: The sections were immersed in 1% hydrochloric acid-ethanol for 2-3 extractions, and then rinsed under tap water for 8 minutes.

[0142] Dehydration and mounting: Dehydrate with 75%, 85% and 95% ethanol for 3 min, soak in anhydrous ethanol I and II for 5 min each, soak in xylene I and II for 10-15 min each, mount with neutral resin, and observe under a microscope.

[0143] 9. Immunohistochemistry

[0144] Dewaxing and hydration of paraffin sections: Place the sections in a constant temperature oven at 58-60℃ and bake for 3 hours. Dewax in the following order: Immerse in xylene I and II for 15 minutes each, immerse in anhydrous ethanol I and II for 5 minutes each, immerse in 95%, 85%, and 75% ethanol for 5 minutes each, and rinse with PBS 3 times for 3 minutes each time.

[0145] Citrate heat antigen retrieval: Place the slide in a beaker containing pH 6.0 citrate buffer, place the beaker in a microwave oven, and heat on high until the solution boils. Immediately stop heating. Allow the solution to cool to room temperature, then gently wash with PBS for 3 minutes each time.

[0146] Blocking endogenous peroxidase with 3% hydrogen peroxide: Add 50 μL of 3% H2O2 to each brain slice, incubate in a humidified chamber at room temperature for 10 min, and wash gently with PBS for 3 min × 3 times.

[0147] 5% BSA blocking: Add 50 μL of 5% BSA to each brain slice to prevent subsequent antibody additions from binding to non-specific sites and reduce background. Block in a humidified chamber at room temperature for 30 min. Discard excess liquid; do not wash.

[0148] Primary antibody incubation: Add 50 μL of primary antibody (dilution ratio: GFAP: 1:1000, Iba-1: 1:500, diluted with PBS) to each brain slice and incubate overnight in a humidified chamber at 4°C. Wash gently with PBST for 5 min × 3 times.

[0149] Secondary antibody incubation: Add 50 μL of polymerized HRP-labeled IgG to each brain slice and incubate in a humidified chamber at 37°C for 30 min. Gently shake with PBST for 5 min × 3 times.

[0150] DAB staining: Add 50 μL of DAB working solution to each brain slice (staining times are GFAP: 40s and Iba-1: 1min, respectively) until the brain slice turns brownish-yellow, and rinse with tap water for 8min.

[0151] Hematoxylin counterstaining: Lightly counterstain with hematoxylin solution for 3 minutes, then immediately rinse with tap water for 5 minutes.

[0152] Hydrochloric acid-ethanol differentiation: The sections were immersed in 1% hydrochloric acid-ethanol for 2-3 extractions, and then rinsed under tap water for 8 minutes.

[0153] Dehydration and mounting: Dehydrate with 75%, 85% and 95% ethanol for 3 min, soak in anhydrous ethanol I and II for 5 min each, soak in xylene I and II for 10-15 min each, mount with neutral resin, and observe under a microscope.

[0154] Imaging and statistics: The target area was observed under a microscope, and the average optical density of the positive area was calculated using ImageJ image analysis software.

[0155] 10. Statistical methods

[0156] SPSS 21.0 was used for data analysis. One-way ANOVA was used for significance testing. When variances were homogeneous, the LSD method was used for inter-group comparisons; when variances were unequal, Dunnett's T3 method was used. Experimental data are expressed as mean ± SEM, and p < 0.05 was considered statistically significant.

[0157] 11. Experimental Results

[0158] (1) Compound PD4238 improves Aβ 1-42 Cognitive impairment in mice caused by

[0159] The Y-maze utilizes the innate exploratory behavior of rodents in their search for new environments and serves as an indicator of working memory in rodents. Under normal conditions, animals remember previously explored directions and do not repeat the exploration; therefore, the spatial recognition and working memory abilities of mice can be indirectly tested by calculating the spontaneous alternation response rate. No significant difference was found in the total number of Y-maze entries among the groups of mice, suggesting that Aβ injection and drug treatment did not affect the motor function or cerebral cortex excitability of mice. Figure 30 The spontaneous alternation response rate results showed that, compared with the model group, the spontaneous alternation response rates of mice in the 5 mg / kg and 10 mg / kg dose groups of PD4238 and the donepezil group were significantly increased, suggesting that PD4238 can improve the working memory ability of Aβ-injected mice. Figure 31 Compared with the model group, *p<0.05, **p<0.01).

[0160] Novel object recognition is a learning assessment method based on the principle that rodents have a tendency to explore new objects. In this experiment, animals were first allowed to freely explore two similar objects for a period of time, and then one of them was replaced with a completely different object. If the animal's memory is normal, it should spend more time exploring the new object than the original similar object. Therefore, calculating the animal's priority index and discrimination coefficient for the new object can examine its non-spatial image recognition memory ability. Compared with the sham-operated group, the priority index and discrimination coefficient of the new object in the model group mice were significantly reduced at the 1-hour test stage; compared with the model group, the priority index and discrimination coefficient of the new object in the PD4238 dose groups and the rivastigmine group mice were significantly increased at the 1-hour test stage, suggesting that PD4238 can significantly improve image recognition memory impairment in Aβ-induced dementia mice. Figure 32 (Compared with the sham surgery group, ##p<0.01; compared with the model group, *p<0.05, **p<0.01) Figure 33 (Compared with the sham surgery group, ##p<0.01; compared with the model group, *p<0.05, **p<0.01)).

[0161] The spontaneous activity experiment primarily examines changes in the excitability of the cerebral cortex in mice and whether their motor function is normal. Studies have found that central nervous system stimulants can increase spontaneous activity in animals, and caffeine can also improve work efficiency and promote learning and memory functions to some extent. To rule out the association between the effect of drugs in improving cognitive impairment and increasing central nervous system excitability, or to rule out the possibility that cognitive changes are caused by abnormal motor function and to exclude non-specific mechanisms, this experiment used a spontaneous activity box to detect the spontaneous activity of rats and mice. The experimental results showed that there were no significant differences in the total distance of spontaneous activity, the average speed of spontaneous activity, and the duration of spontaneous activity among the groups of mice, suggesting that drugs and Aβ injection had no significant effect on the excitability of the cerebral cortex and motor function (Table 3).

[0162] Table 3 PD4238 vs Aβ 1-42 The effect of induced dementia model mice on spontaneous activity (n=11–14, mean±SEM)

[0163]

[0164] (2) Compound PD4238 improves Aβ 1-42 Morphology of neurons in mouse cerebral cortex and hippocampus

[0165] In the sham-operated group, the cerebral cortex, dentate gyrus (DG), CA1 region, and CA3 region of mice showed normal cell morphology, with layered arrangement, uniform distribution, intact cell morphology, and tight cell arrangement. In the model group, all brain regions showed decreased neuronal volume, eosinophilic degeneration, and deeply stained and pyknotted nuclei, particularly severe in the cerebral cortex, dentate gyrus, and CA3 region. In the PD4238 1.25 mg / kg and 2.5 mg / kg dose groups, some cells still showed disordered arrangement, reduced cell number, and deeply stained and pyknotted nuclei. The PD4238 5 mg / kg and 10 mg / kg dose groups and the rivastigmine group showed significantly improved neuronal arrangement and morphology in all brain regions compared to the model group. The donepezil group showed significantly improved hippocampal neuronal morphology compared to the model group. Figure 34 ).

[0166] (3) Compound PD4238 on Aβ 1-42 Effects of injection on MAP2 protein expression in mouse cerebral cortex and hippocampus

[0167] Microtubule-associated protein 2 (MAP2) is one of the main proteins constituting microtubules and plays an important role in maintaining microtubule dynamics. MAP2 is mainly distributed in the dendrites of neurons and in the microtubules within the cell body. After brain injury, MAP2 expression in the brain shows a decreasing trend. Experimental results show that, compared with the sham-operated group, MAP2 protein expression in the cerebral cortex, dentate gyrus of the hippocampus, CA1 region, and CA3 region of model mice was significantly reduced; compared with the model group, the PD4238 group mice showed significantly increased MAP2 protein expression levels in the cerebral cortex, dentate gyrus of the hippocampus, and CA3 region. Figures 35-36 (Compared with the sham surgery group, #p<0.05, ##p<0.01; compared with the model group, *p<0.05,

[0168] **p<0.01)).

[0169] (4) Compound PD4238 on Aβ 1-42 Effects of injection on the morphology of heart, liver, spleen, and kidney tissues in mice

[0170] Mice were administered PD4238 at doses of 1.25, 2.5, 5, and 10 mg / kg via gavage for 18 days. The mice were then sacrificed, and the heart, liver, spleen, and kidneys were paraffin-embedded for further HE staining. No significant damage was observed in these organs. Figure 37 This suggests that an oral dose of 10 mg / kg in mice may be safe.

[0171] In summary, the results of Example 7 of this invention show that compound PD4238 improves the effects of intraventricular injection of Aβ. 1-42The induced working memory and image discrimination memory impairment in mice had no significant effect on cortical excitability or motor function; compound PD4238 improved the effect of intraventricular injection of Aβ. 1-42 The induced damage to the cerebral cortex and hippocampus of mice.

[0172] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of a diamidinium-benzimidazole-benzene derivative in the preparation of a medicament for the prevention and / or treatment of rheumatoid arthritis, wherein the structural formula of the diamidinium-benzimidazole-benzene derivative is as follows: ； In the formula, n is 4.

2. The application according to claim 1, characterized in that, The method for synthesizing the diamidinium-benzimidazole-benzene derivative includes the following steps: Step S1: Weigh p-hydroxybenzaldehyde and anhydrous potassium carbonate into a round-bottom flask, add anhydrous DMF as solvent, stir at room temperature under nitrogen protection, and add dibromoalkane dropwise; after the addition is complete, heat the reaction under nitrogen. After the reaction is complete, cool to room temperature, pour the mixture into cold water, the product precipitates, filter, recrystallize the filter cake with anhydrous ethanol, and dry under reduced pressure. Step S2: Weigh the product from step S1, 4-amidinyl-1,2-phenylenediamine hydrochloride and 1,4-benzoquinone into a round-bottom flask, add anhydrous ethanol, heat under nitrogen and reflux until the reaction is complete, stop the reaction, cool to room temperature, filter and collect the dark solid, wash with dry acetone, ice-cold anhydrous ethanol and ice-cold diethyl ether, and dry. Step S3: Slowly dissolve the product from step S2 in hot anhydrous ethanol and filter while hot. Concentrate the filtrate by volume, acidify with hydrochloric acid saturated ethanol solution and stir overnight, evaporate to dryness, dilute with anhydrous diethyl ether, filter to collect the solid, and vacuum dry. Step S4: Purify the product from step S3 to obtain a diamidinium-benzimidazole-benzene derivative.

3. The application according to claim 2, characterized in that, In step S1, the molar ratio of p-hydroxybenzaldehyde, anhydrous potassium carbonate, and dibromoalkane is 1:1~1.2:0.5~0.

6.

4. The application according to claim 3, characterized in that, In step S1, the solvent volume is 10.0~30.0 mL; the stirring time at room temperature is 2~4 h; the heating reaction temperature is 80~130℃ and the reaction time is 2~12 h; the cold water volume is 50~100 mL; the vacuum drying temperature is 40~60 ℃ and the time is 8~12 h.

5. The application according to claim 2, characterized in that, In step S2, the molar ratio of the product from step S1, 4-amidinyl-1,2-phenylenediamine hydrochloride, and 1,4-benzoquinone is 1:2~2.2:2~2.

2.

6. The application according to claim 5, characterized in that, In step S2, the volume of anhydrous ethanol is 10-60 mL; the reflux reaction temperature is 80-100 °C; and the reaction time is 8-12 h.

7. The application according to claim 2, characterized in that, In step S3, the volume of hot anhydrous ethanol is 200-600 mL; the volume of concentration is 40-100 mL; the vacuum drying temperature is 40-60 °C, and the time is 8-12 h.

8. The application according to claim 2, characterized in that, In step S4, methanol is the mobile phase.

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