CXCR4 antagonist and its preparation method and application

Abstract

This invention discloses 20 compounds that can act as CXCR4 antagonists, their pharmaceutically acceptable salts, as well as methods for preparing these compounds, synthetic routes, and their use in preparing drugs for preventing and treating related diseases by inhibiting CXCR4. The compounds disclosed in this invention are excellent CXCR4 antagonists for the prevention and treatment of diseases such as inflammatory bowel disease.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and in particular to a class of CXCR4 antagonists and a preparation method and application thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory bowel disease that poses a global public health challenge and has serious negative impacts on the physical and mental health of IBD patients.

[0003] IBD is believed to be caused by inappropriate and persistent activation of the mucosal immune system triggered by the presence of normal intestinal flora. IBD includes two major diseases: Crohn's disease (CD) and ulcerative colitis (UC). These two forms of idiopathic inflammatory bowel disease are empirically defined as chronic and uncontrolled inflammation of the digestive tract based on clinical, pathological, endoscopic and radiological characteristics. Although researchers have made great efforts to treat IBD, the currently available drugs are still limited. Therefore, there is an urgent need to develop new drugs for the treatment of IBD with better efficacy and lower side effects.

[0004] Chemokine (CXC motif) receptor 4 (CXCR4) is a transmembrane receptor in the chemokine family. It binds to the chemokine ligand CXCL12 (also known as SDF-1) and is involved in regulating the migration of various immune cells.

[0005] The CXCL12 / CXCR4 pathway plays an important role in various physiological and pathological conditions, including immune responses, hematopoiesis, tumor metastasis, and HIV infection. Studies have shown that CXCR4 also plays a crucial role in the development and progression of IBD. In the intestinal tissues of IBD patients, CXCL12 expression is significantly elevated, which in turn leads to excessive chemotaxis of inflammatory cells (such as inflammatory mediators and immune cells) that overexpress CXCR4, causing them to migrate and aggregate in the intestinal tissues, resulting in persistent inflammation and intestinal mucosal damage.

[0006] Research and drug development targeting CXCR4 have also become a hot topic in the treatment of IBD. Inhibiting CXCR4 can reduce the accumulation and activation of inflammatory cells, thereby alleviating intestinal inflammation and improving disease symptoms. Therefore, developing CXCR4 antagonists to control and reduce inflammation is an effective strategy for treating IBD.

[0007] There are several reports on CXCR4 antagonists in the prior art. For example, Patent Specification No. CN113845439A discloses a bisbenzylamide CXCR4 antagonist. Another example is Patent Specification No. CN101678213A discloses a cyclic peptide CXCR4 antagonist.

[0008] Traditional CXCR4 antagonists are discovered through a process that includes target identification, lead compound screening, activity evaluation, and lead compound optimization. However, antagonists discovered through this process have shortcomings such as insufficient specificity, immunogenicity, pharmacokinetic issues, and drug resistance. Summary of the Invention

[0009] In a first aspect, the present invention provides 20 compounds that can serve as CXCR4 antagonists and pharmaceutically acceptable salts thereof.

[0010] The CXCR4 antagonist is selected from any one of the following compounds of Formula I to Formula XX or a pharmaceutically acceptable salt thereof:

[0011]

[0012] In a second aspect, the present invention provides a method for preparing the CXCR4 antagonist.

[0013] The synthetic routes and preparation methods of the above 20 compounds are described below.

[0014] 1. 1-(2-furoyl)piperazine 1 is subjected to an amination reduction reaction with 1-phenyl-4-piperidone to obtain a compound represented by the following formula I;

[0015] Synthesis route:

[0016]

[0017] 2. The compound represented by formula II, and its synthetic route:

[0018]

[0019] The preparation method comprises the steps of:

[0020] (1) 3-Chloromethylpyridine 2 reacts with 4-Boc aminopiperidine to give intermediate 3;

[0021] (2) Removal of the Boc protecting group from intermediate 3 to obtain intermediate 4;

[0022] (3) Intermediate 4 undergoes amide condensation reaction with quinoline-3-carboxylic acid to obtain the compound represented by formula II.

[0023] 3. 4-Pyrrolidin-1-yl-piperidine 5 is subjected to an amide condensation reaction with quinoline-3-carboxylic acid to obtain the compound represented by the following formula III; Synthesis route:

[0024]

[0025] 4. The compound represented by formula IV, and its synthetic route:

[0026]

[0027] The preparation method comprises the steps of:

[0028] (1) 2-Chloroquinoline 6 reacts with N-Boc-piperazine to form intermediate 7;

[0029] (2) Removal of the Boc protecting group from intermediate 7 to obtain intermediate 8;

[0030] (3) Intermediate 8 is reacted with 3,4-difluorobenzene-1-sulfonyl chloride to undergo sulfonamide condensation reaction to obtain the compound represented by formula IV.

[0031] 5. 1-(4-Fluorophenyl)piperazine 9 and 3-hydroxy-5-methylbenzoic acid undergo amide condensation reaction to obtain the compound represented by the following formula V;

[0032] Synthesis route:

[0033]

[0034] 6. 4-Amino-1-benzylpiperidine 10 is reacted with 2,4-dichlorobenzoic acid to undergo an amide condensation reaction to obtain a compound represented by the following formula VI;

[0035] Synthesis route:

[0036]

[0037] 7. The compound represented by formula VII, and its synthetic route:

[0038]

[0039] The preparation method comprises the steps of:

[0040] (1) 1-Chloroisoquinoline 11 reacts with N-tert-butyloxycarbonyl (Boc)-piperazine to form intermediate 12;

[0041] (2) Removal of the Boc protecting group from intermediate 12 yields intermediate 13;

[0042] (3) Phenyl chloroformate 14 reacts with 3-methylaniline to produce intermediate 15;

[0043] (4) Intermediate 13 and intermediate 15 undergo amine ester exchange reaction to obtain the compound represented by formula VII.

[0044] 8. The compound represented by formula VIII, and its synthetic route:

[0045]

[0046] The preparation method comprises the steps of:

[0047] (1) 1-Chloroisoquinoline 11 reacts with N-Boc-piperazine to form intermediate 12;

[0048] (2) intermediate 12 is deprotected by removing the Boc protecting group to obtain intermediate 13; (3) 2,5-difluoroaniline 16 is reacted with chloroacetyl chloride to undergo amide condensation reaction to obtain intermediate 17;

[0049] (4) Intermediate 13 reacts with intermediate 17 to produce intermediate 18 through nucleophilic substitution reaction;

[0050] (5) The carbonyl group of the amide bond in intermediate 18 is reduced to give the compound of formula VIII.

[0051] 9. The compound represented by formula IX, and its synthetic route:

[0052]

[0053] The preparation method comprises the steps of:

[0054] (1) Methyl 4-aminobenzoate 19 reacts with 2-trifluoromethylbenzoyl chloride to form intermediate 20;

[0055] (2) Intermediate 20 undergoes an amine transesterification reaction with an aqueous solution of hydroxylamine to obtain a compound represented by Formula IX.

[0056] 10. The compound represented by formula X, and its synthetic route:

[0057]

[0058] The preparation method comprises the steps of:

[0059] (1) Ethyl 2-methylacetoacetate 21 reacts with resorcinol to form intermediate 22;

[0060] (2) Intermediate 22 is reacted with 3,4-difluorobenzyl alcohol to obtain the compound represented by formula X.

[0061] 11. The compound represented by formula XI, and its synthetic route:

[0062]

[0063] The preparation method comprises the steps of:

[0064] (1) 4-Methylbenzyl chloride 23 reacts with N-Boc-piperazine to form intermediate 24;

[0065] (2) Removal of the Boc protecting group from intermediate 24 yields intermediate 25;

[0066] (3) 4-Amino-2-chlorothienyl[3,2-D]pyrimidine 26 was protected with di-tert-butyl dicarbonate to give intermediate 27;

[0067] (4) Intermediate 25 reacts with intermediate 27 to produce intermediate 28;

[0068] (5) The Boc protecting group of intermediate 28 is removed to obtain the compound represented by formula XI.

[0069] 12. 4-Phenylbenzylamine 29 and 2,4,5-trifluorobenzenesulfonyl chloride undergo sulfonamide condensation reaction to obtain the compound represented by the following formula XII;

[0070] Synthesis route:

[0071]

[0072] 13. 2-Quinoxalinecarboxylic acid 30 and 2-(1-pyrazolyl)benzylamine undergo amide condensation reaction to obtain the compound represented by the following formula XIII;

[0073] Synthesis route:

[0074]

[0075] 14. Quinoline-3-carboxylic acid 31 and 1-(2-fluorophenyl)piperazine undergo amide condensation reaction to obtain the compound represented by the following formula XIV:

[0076] Synthesis route:

[0077]

[0078] 15. 2,5-Dimethylphenylacetic acid 32 reacts with N-phenylpiperazine to form compound XV:

[0079] Synthesis route:

[0080]

[0081] 16. The compound represented by formula XVI, and its synthetic route:

[0082]

[0083] The preparation method comprises the steps of:

[0084] (1) 2-Hydroxy-3-methylbenzaldehyde 33 undergoes amination reduction reaction with 1-Boc-4-aminopiperidine to give intermediate 34;

[0085] (2) Intermediate 34 undergoes amide condensation reaction with 1-naphthoic acid to obtain intermediate 35;

[0086] (3) Removal of the Boc protecting group from intermediate 35 yields intermediate 36;

[0087] (4) Intermediate 36 is reacted with glacial acetic acid to undergo amide condensation reaction to obtain the compound represented by formula XVI.

[0088] 17. p-Anisic acid 37 is reacted with (R)-3-amino-1,2,3,4-tetrahydrocarbazole to undergo amide condensation reaction to obtain the compound represented by the following formula XVII:

[0089] Synthesis route:

[0090]

[0091] 18. The compound represented by formula XVIII, and its synthetic route:

[0092]

[0093] The preparation method comprises the steps of:

[0094] (1) Aniline 38 reacts with monomethyl phthalate to produce intermediate 39;

[0095] (2) Intermediate 39 is subjected to ester hydrolysis to obtain intermediate 40;

[0096] (3) amide condensation reaction of 3,4-difluorobenzoyl chloride 41 with 4-Boc-2-methylpiperazine gave intermediate 42;

[0097] (4) Removal of the Boc protecting group from intermediate 42 yields intermediate 43;

[0098] (5) Intermediate 40 and intermediate 43 undergo amide condensation reaction to obtain the compound represented by formula XVIII.

[0099] 19. 4-(4-Piperidinyl)morpholine 44 and quinoline-3-carboxylic acid undergo amide condensation reaction to obtain the compound shown in the following formula XIX;

[0100] Synthesis route:

[0101]

[0102] 20. 2-Indolecarboxylic acid 45 and 1-(5-chloropiperidinyl)piperazine undergo amide condensation reaction to obtain the compound represented by the following formula XX;

[0103] Synthesis route:

[0104]

[0105] In a third aspect, the present invention provides the use of the CXCR4 antagonist in the preparation of a drug for preventing and treating related diseases by inhibiting CXCR4, including inflammatory bowel disease.

[0106] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides 20 compounds and pharmaceutically acceptable salts thereof having excellent CXCR4 antagonistic effects and being useful as CXCR4 antagonists; these compounds as CXCR4 antagonists have excellent effects in preventing and treating diseases such as inflammatory bowel disease. DETAILED DESCRIPTION

[0107] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The operating methods in the following examples where no specific conditions are specified are generally performed under conventional conditions or as recommended by the manufacturer.

[0108] Example 1: Preparation of furan-2-yl(4-(1-phenylpiperidin-4-yl)piperazin-1-yl)methanone (I)

[0109]

[0110] To a 50 mL round-bottom flask equipped with a magnetic stirrer were added 1-(2-furoyl)piperazine (740 mg, 4.1 mmol), 1-phenyl-4-piperidone (600 mg, 3.4 mmol), glacial acetic acid (656 mg, 10.3 mmol), and 1,2-dichloroethane (DCE, 10 mL). The mixture was reacted at room temperature. Two hours later, sodium triacetoxyborohydride (1090 mg, 5.2 mmol) was added, followed by another two hours later. After completion of the reaction, the organic solvent was removed by concentration under reduced pressure, the mixture was neutralized with saturated sodium bicarbonate solution, and the mixture was extracted with dichloromethane 3-4 times. The organic layer was collected and dried over anhydrous sodium sulfate. The organic layer was concentrated under reduced pressure to remove the organic solvent, and then purified by thin-layer chromatography (TLC) using dichloromethane:methanol (volume ratio 80:1-50:1) as the developing solvent to obtain Compound I (618 mg, 53%).

[0111] mp148.8-149.5℃; 1H NMR(500MHz,DMSO-d6)δ7.83(s,1H),7.21–7.16(m,2H),6.97(d,J=3.2Hz,1H),6.9 2(d,J=8.1Hz,2H),6.74(t,J=7.2Hz,1H),6.61(dd,J=3.4,1.8Hz,1H),3.71(d,J=12 .4Hz,2H),3.64(s,3H),3.32(s,1H),2.64(td,J=12.2,2.4Hz,2H),2.54(t,J=5.0Hz ,4H),2.38(t,J=11.4Hz,1H),1.83(d,J=11.7Hz,2H),1.51(qd,J=12.1,4.0Hz,2H); 13 C NMR (125MHz, DMSO-d6) δ158.1,151.0,147.1,144.6,128.9,118.5,115.7 115.4,111.3,60.8,48.9,48.1,34.0,27.5.LC-MS: m / z 340.3[M+H] + .

[0112] Example 2: Preparation of N-(1-(pyridin-3-ylmethyl)piperidin-4-yl)quinoline-3-carboxamide (II)

[0113]

[0114] To a 50 mL round-bottom flask equipped with a magnetic stirrer were added 3-chloromethylpyridine (700 mg, 4.3 mmol), 4-Boc-aminopiperidine (1110 mg, 5.6 mmol), N,N-diisopropylethylamine (DIPEA, 2203 mg, 17.1 mmol), and acetonitrile (10 mL) in sequence and allowed to react at room temperature. After completion of the reaction, the organic solvent was removed by concentration under reduced pressure, the mixture was dissolved in dichloromethane (10 mL), washed 3-4 times with water (10 mL), and once with saturated brine (10 mL). The organic layer was collected and dried over anhydrous sodium sulfate, concentrated under reduced pressure to remove the organic solvent, and purified by column chromatography using dichloromethane to methanol (volume ratio 60:1-40:1) as the developing solvent to obtain Intermediate 3 (2152 mg, 86%) as a yellow solid.

[0115] To a 50 mL round-bottom flask equipped with a magnetic stirrer, intermediate 3 (1200 mg, 4.12 mmol) and methanol (10 mL) were added sequentially. A hydrochloric acid solution in dioxane (12 mL) was added under ice-cooling and allowed to react. After completion of the reaction, the organic solvent and excess hydrochloric acid were removed by concentration under reduced pressure to yield intermediate 4 (1300 mg, 94%) as a white solid.

[0116] To a 50 mL round-bottom flask equipped with a magnetic stirrer were added intermediate 4 (500 mg, 1.5 mmol), quinoline-3-carboxylic acid (334 mg, 1.9 mmol), N,N-diisopropylethylamine (DIPEA, 1148 mg, 8.9 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 567 mg, 3 mmol), 1-hydroxybenzotriazole (HOBT, 400 mg, 3 mmol), and DCM (10 mL). The reaction was allowed to react at room temperature. The reaction progress was monitored by TLC. After completion, the product was washed 3-4 times with water (10 mL) and once with saturated brine (10 mL). The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the organic solvent. The product was purified by thin-layer chromatography (TLC) using dichloromethane to methanol (60:1 by volume) as the developing solvent to obtain Compound II (167 mg, 33%) as a white solid.

[0117] mp177.7-178.2℃; 1 H NMR (500MHz, DMSO-d6) δ9.27 (d, J=2.2Hz, 1H), 8.81

[0118] (d,J=2.2Hz,1H),8.66(d,J=7.6Hz,1H),8.55–8.46(m,2H),8.08(t,J=7.5Hz,2H),7.85(ddd,J=8.4,6.8,1.5Hz,1H),7.76–7.70(m,1H),7.68(t ,J=7.6Hz,1H),7.37(dd,J=7.8,4.7Hz,1H),3.87(s,1H),3.55(s,2H),2 .86(s,2H),2.11(s,2H),1.87(d,J=10.3Hz,2H),1.64(d,J=12.2Hz,2H); 13 C NMR(125MHz,DMSO-d6)δ164.3,150.1,149.0,148.4,136.6,135.4,131.0,1 29.1,128.7,127.3,127.3,126.5,123.4,59.0,52.0,46.9,31.3.LC-MS:m / z 347.2[M+H] + .

[0119] Example 3: Preparation of (4-(pyrrolidin-1-yl)piperidin-1-yl)(quinolin-3-yl)methanone (III)

[0120]

[0121] To a 50 mL round-bottom flask equipped with a magnetic stirrer were added 4-pyrrolidin-1-ylpiperidine (185 mg, 1.2 mmol), quinoline-3-carboxylic acid (173 mg, 1 mmol), N,N-diisopropylethylamine (DIPEA, 256 mg, 2 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 230 mg, 1.2 mmol), 1-hydroxybenzotriazole (HOBT, 162 mg, 1.2 mmol), and DCM (10 mL) in sequence. The reaction was allowed to react at room temperature. The reaction progress was monitored by TLC. After completion, the mixture was washed 3-4 times with water (10 mL) and once with saturated brine (10 mL). The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the organic solvent. The product was then purified by thin-layer chromatography (TLC) using dichloromethane to methanol (volume ratio 15:1) as the developing solvent to obtain Compound III (280 mg, 90%) as a yellow liquid.

[0122] 1 H NMR (500MHz, DMSO-d6) δ8.89(d,J=2.1Hz,1H),8.45(d,J=2.1Hz,1H),8.08–8.04(m,2H),7.83(ddd,J=8.4,6.8,1.4Hz,1H),7.67(ddd,J=8.0,6.8,1.2H z,1H),4.30(s,1H),3.61(s,1H),3.20–3.03(m,2H),2.48(s,4H),2.26(t,J =9.5Hz,1H),1.93(s,1H),1.78(s,1H),1.66(s,4H),1.45(d,J=10.1Hz,2H); 13 C NMR (125MHz, DMSO-d6) δ166.6,148.5,147.5,134.2,130.6,129.3,128.8,128.7,127.3,126.6,60.4,50.8,45.8,31.3,30.7,23.0.LC-MS: m / z 310.2[M+H] + .

[0123] Example 4: Preparation of 2-(4-((3,4-difluorophenyl)sulfonyl)piperazin-1-yl)quinoline (IV)

[0124]

[0125] To a 50 mL round-bottom flask equipped with a magnetic stirrer, 2-chloroquinoline (327.2 mg, 2 mmol), N-Boc-piperazine (1863 mg, 10 mmol), potassium carbonate (414 mg, 3 mmol), and DMF (8 mL) were added sequentially and reacted at 140°C. The reaction progress was monitored by TLC. After completion, water (40 mL) was added to the reaction solution, filtered, and the filter cake was washed with water to obtain a white solid, Intermediate 7.

[0126] To a 100 mL round-bottom flask equipped with a magnetic stirrer, intermediate 7 (402.5 mg, 1.3 mmol) and methanol (5 mL) were added sequentially. A hydrochloric acid solution in dioxane (3 mL) was added under ice-cooling and allowed to react at 0°C. After completion of the reaction, the organic solvent and excess hydrochloric acid were removed by concentration under reduced pressure to obtain intermediate 8, a white solid.

[0127] To a 50 mL round-bottom flask equipped with a magnetic stirrer, intermediate 8 (300 mg, 1.3 mmol), 3,4-difluorobenzene-1-sulfonyl chloride (327 mg, 1.5 mmol), triethylamine (0.53 mL, 3.9 mmol), 4-dimethylaminopyridine (DMAP, 30 mg, 0.13 mmol), and DMF (5 mL) were added sequentially and the reaction was allowed to proceed from 0°C to room temperature. After completion of the reaction, water (40 mL) was added to the reaction solution, which was then filtered and the filter cake was washed with water to obtain a white solid, Compound IV (368 mg, 74%).

[0128] mp211.1-211.7℃; 1 H NMR(500MHz,Chloroform-d)δ7.90(d,J=9.1Hz,1H),

[0129] 7.68(d,J=8.4Hz,1H),7.64–7.58(m,2H),7.58–7.52(m,2H),7.32(td,J=9.1,7.3Hz,1H), 7.25(d,J=7.1Hz,1H), 6.91(d,J=9.1Hz,1H), 3.87(t,J=5.1Hz,4H), 3.17(t,J=5.0Hz,4H); 13 C NMR(125MHz,DMSO-d6)δ156.4,153.6(d, 1 J=252.5Hz),147.0,144.3(d, 1 J=285Hz),137.6,132.1,129.5,127.4,126.0,125.6(t, 4 J=3.8Hz),122.9,122.4,118.8(d, 2J=17.5Hz),117.6(d, 2 J=20Hz),110.2,45.7,43.9.LC-MS:m / z 390.2[M+H] + .

[0130] Example 5: Preparation of (4-(4-fluorophenyl)piperazin-1-yl)(3-hydroxy-5-methylphenyl)methanone (V)

[0131]

[0132] To a 50 mL round-bottom flask equipped with a magnetic stirrer were added 1-(4-fluorophenyl)piperazine (431 mg, 1.7 mmol), 3-hydroxy-5-methylbenzoic acid (200 mg, 1.3 mmol), N,N-diisopropylethylamine (DIPEA, 677 mg, 5.2 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 502 mg, 2.6 mmol), 1-hydroxybenzotriazole (HOBT, 354 mg, 2.6 mmol), and DCM (10 mL) in sequence and allowed to react at room temperature. After completion of the reaction, the mixture was washed 3-4 times with 10 mL of water and once with 10 mL of saturated brine. The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the organic solvent. The mixture was then purified by thin-layer chromatography (TLC) using dichloromethane to methanol (volume ratio 80:1-60:1) as the developing solvent to obtain a light brown solid, Compound V (233 mg, 57%).

[0133] mp186.4-186.9℃; 1 H NMR (500MHz, DMSO-d6) δ9.57 (s, 1H), 7.06 (t, J = 8.9Hz,

[0134] 2H),6.97(dd,J=9.2,4.7Hz,2H),6.65(dt,J=15.7,1.8Hz,2H),6.57(t,J=1.9Hz,1H),3.71(s,2H),3.47(s,2H),3.08(s,4H),2.25(s,3H); 13 C NMR(125MHz,DMSO-d6)δ169.0,157.3(d, 3 J=6.3Hz),155.4,147.7(d, 4 J=2.5Hz),139.2,136.9,118.0,117.8(d, 3 J=7.5Hz),117.0,115.4(d, 2J=21.3Hz),110.9,49.4,46.8,41.3,26.5,21.0.LC-MS:m / z315.2[M+H] + .

[0135] Example 6: Preparation of N-(1-benzylpiperidin-4-yl)-2,4-dichlorobenzamide (VI)

[0136]

[0137] To a 50 mL round-bottom flask equipped with a magnetic stirrer was added 4-amino-1-benzylpiperidine (382 mg, 2 mmol), 2,4-dichlorobenzoic acid (380 mg, 2 mmol), 4-dimethylaminopyridine (DMAP, 49 mg, 0.4 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 768 mg, 4 mmol), potassium carbonate (414 mg, 3 mmol), and DCM (10 mL) in sequence and allowed to react at room temperature. After completion of the reaction, the mixture was washed 3-4 times with water (10 mL) and once with saturated brine. The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the organic solvent. The mixture was dissolved in DMF (5 mL) and then added with water (40 mL). The precipitated solid was filtered, and the filter cake was washed with water to obtain a white solid, Compound VI (392 mg, 54%).

[0138] mp145.0-145.6℃; 1 H NMR(500MHz,Chloroform-d)δ7.61(d,J=8.2Hz,1H),

[0139] 7.40(d,J=1.9Hz,1H),7.33–7.29(m,5H),7.28–7.21(m,1H),6.11(s,1H),4.11–3.98(m,1H),3. 52(s,2H),2.83(d,J=10.4Hz,2H),2.20(t,J=10.7Hz,2H),2.06–2.00(m,2H),1.64–1.55(m,2H); 13 C NMR (125MHz, DMSO-d6) δ164.7,138.6,136.1,134.2,131.1,130.1,129.0,128.7,128.1,127.2,126.8,62.1,51.9,46.7,31.3.LC-MS:m / z 363.2[M+H] + .

[0140] Example 7: Preparation of 4-(isoquinolin-1-yl)-N-(m-tolyl)piperazine-1-carboxamide (VII)

[0141]

[0142] To a 50 mL round-bottom flask equipped with a magnetic stirrer were added 1-chloroisoquinoline (982 mg, 6 mmol), N-Boc-piperazine (5588 mg, 30 mmol), potassium carbonate (1242 mg, 9 mmol), and N,N-dimethylformamide (DMF, 20 mL) in sequence and stirred at 140°C. After completion of the reaction, the reaction solution was poured into water (100 mL) and filtered. The filter cake was rinsed with water 3-4 times and the filter cake was collected to obtain a light yellow solid, Intermediate 12 (1743 mg, 93%).

[0143] To a 50 mL round-bottom flask equipped with a magnetic stirrer, intermediate 12 (1743 mg, 5.6 mmol), methanol (10 mL), and a dioxane hydrochloride solution (15 mL) were added sequentially and reacted at 0°C. The reaction progress was monitored by TLC. After completion of the reaction, the reaction solution was concentrated under reduced pressure to remove the reaction solvent and hydrochloric acid, ultimately obtaining intermediate 13 (700 mg, 59%) as a white solid.

[0144] 3-Methylaniline (412 mg, 3.8 mmol) was dissolved in DMF (5 mL) in a 50 mL reaction flask equipped with a magnetic stirrer. A DMF solution of phenyl chloroformate (5 mL, 3.2 mmol) was added dropwise to the reaction flask via a dropping funnel. After the reaction, water (30 mL) was added to precipitate a solid, which was filtered and washed with water to afford a white solid, Intermediate 15 (330 mg, 45%).

[0145] To a 50 mL round-bottom flask equipped with a magnetic stirrer were added intermediate 13 (250 mg, 1 mmol), intermediate 15 (273 mg, 1.2 mmol), acetonitrile (10 mL), and triethylamine (303.6 mg, 3 mmol) in sequence, and the mixture was reacted at 60°C. The reaction progress was monitored by TLC. After completion of the reaction, the acetonitrile was removed by concentration under reduced pressure, and the product was dissolved in dichloromethane (15 mL). The product was washed 3-4 times with water (15 mL) and once with saturated sodium chloride solution (10 mL). The dichloromethane layer was dried over anhydrous sodium sulfate, and the solvent was concentrated under reduced pressure. The product was purified by thin-layer chromatography (dichloromethane:methanol volume ratio 80:1-60:1) to give a white solid, Compound VII (35 mg, 10%).

[0146] mp193.0-193.8℃; 1H NMR(500MHz,Chloroform-d)δ8.16(d,J=5.7Hz,1H),8.11(d,J=8.4Hz,1H),7.79(d,J=8.1Hz,1H),7.66(ddd,J=8.2,6.9,1.2Hz,1H),7.56(ddd,J=8.3,6.9 ,1.3Hz,1H),7.31(d,J=5.8Hz,1H),7.28(s,1H),7.21–7.14(m,2H),6.88(d,J =6.9Hz,1H),6.46(s,1H),3.79–3.75(m,4H),3.53–3.48(m,4H),2.34(s,3H); 13 C NMR(125MHz,DMSO-d6)δ160.6,155.2,140.4,140.4,137.7,137.3,130.0,128.2,12 7.2,126.7,125.2,122.5,120.9,120.3,116.8,115.6,51.0,43.9,21.2.LC-MS:m / z 347.3[M+H] + .

[0147] Example 8: Preparation of 2,5-difluoro-N-(2-(4-(isoquinolin-1-yl)piperazin-1-yl)ethyl)aniline (VIII)

[0148]

[0149] To a 50 mL round-bottom flask equipped with a magnetic stirrer, 1-chloroisoquinoline (982 mg, 6 mmol), N-Boc-piperazine (5588 mg, 30 mmol), potassium carbonate (1242 mg, 9 mmol), and N,N-dimethylformamide (DMF, 20 mL) were added sequentially and stirred at 140°C. The reaction progress was monitored by TLC. After completion, the reaction solution was poured into water (100 mL) to precipitate a solid, which was filtered and the filter cake rinsed with water 3-4 times to afford a light yellow solid, Intermediate 12 (1743 mg, 93%).

[0150] To a 50 mL round-bottom flask equipped with a magnetic stirrer, intermediate 12 (1743 mg, 5.6 mmol), methanol (10 mL), and a dioxane hydrochloride solution (15 mL) were added sequentially and reacted at 0°C. The reaction progress was monitored by TLC. After completion of the reaction, the reaction solution was concentrated under reduced pressure to remove the reaction solvent and hydrochloric acid, yielding intermediate 13 (700 mg, 59%) as a white solid.

[0151] In a 50 mL reaction flask equipped with a magnetic stirrer, 2,5-difluoroaniline (1.29 g, 10 mmol) and triethylamine (1.01 g, 10 mmol) were dissolved in DMF (10 mL). A solution of chloroacetyl chloride in DMF (10 mL, 12 mmol) was added dropwise to the flask via a dropping funnel. After completion of the reaction, the reaction mixture was added to water (60 mL). The precipitated solid was filtered, and the filter cake was washed with water to obtain a white solid, Intermediate 17 (1521 mg, 74%).

[0152] To a 50 mL round-bottom flask equipped with a magnetic stirrer were added Intermediate 13 (213 mg, 1 mmol), Intermediate 17 (308 mg, 1.5 mmol), potassium carbonate (414 mg, 3 mmol), potassium iodide (17 mg, 0.1 mmol), and acetonitrile (10 mL) in sequence, and the mixture was refluxed at 80°C. The reaction progress was monitored by TLC. After completion of the reaction, the acetonitrile was removed by concentration under reduced pressure, the product was dissolved in dichloromethane (15 mL), washed 3-4 times with water (15 mL), and once with saturated sodium chloride solution (10 mL), the dichloromethane was dried over anhydrous sodium sulfate, and the solvent was removed by concentration under reduced pressure. The product was purified by thin-layer chromatography (dichloromethane:methanol volume ratio 100:1-50:1) to afford Intermediate 18 (50 mg, 13%) as a white solid.

[0153] To a 50 mL round-bottom flask equipped with a magnetic stirrer, a tetrahydrofuran solution of lithium aluminum tetrahydride (15 mg, 0.39 mmol) was added under ice-bath, followed by the addition of intermediate 18 (50 mg, 0.13 mmol). The mixture was gradually warmed to room temperature and then refluxed at 70°C. The reaction progress was monitored by TLC. After completion of the reaction, sodium hydroxide solution (15%, 0.5 mL), water (0.5 mL), sodium hydroxide solution (15%, 0.5 mL), and water (1.5 mL) were added sequentially under ice-bath conditions. The mixture was filtered, and the filter cake was rinsed with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure to remove the solvent, and purified by thin-layer chromatography (dichloromethane:methanol volume ratio 100:1-50:1) to obtain a yellow solid, compound VIII (28 mg, 58%).

[0154] mp125.9-126.4℃; 1 H NMR(500MHz,Chloroform-d)δ8.15(d,J=5.7Hz,1H),

[0155] 8.10(d,J=8.4Hz,1H),7.75(d,J=8.1Hz,1H),7.65–7.58(m,1H),7.55–7.47(m,1H),7.25(s,1H),6.88(ddd,J=11.0,8.7,5.1Hz,1H),6.39 (ddd,J=10.3,7.0,3.0Hz,1H),6.26(tt,J=8.4,3.2Hz,1H),4.76(s,1H),3.47(s,4H),3.23(q,J=5.6Hz,2H),2.79(dt,J=8.4,5.3Hz,6H); 13 C NMR(125MHz,DMSO-d6)δ160.7,160.3(d, 1 J=236.3Hz),148.0(d, 1 J=232.5Hz),140.4,138.3(dd, 2 J=25Hz, 4 J=2.5Hz),137.6,129.9,127.1,126.5,125.2,120.8,115.6,114.7(q, 3 J=11.3Hz),100.2(dd, 2 J=25Hz, 3 J=7.5Hz),98.9(dd, 2 J=28.8Hz, 4 J=3.8Hz),56.2,52.3,51.1.LC-MS:m / z 369.3[M+H] + .

[0156] Example 9: Preparation of N-(4-(hydroxycarbamoyl)phenyl)-2-(trifluoromethyl)benzamide (IX)

[0157]

[0158] To a 100 mL round-bottom flask equipped with a magnetic stirrer, methyl 4-aminobenzoate (756 mg, 5 mmol), triethylamine (505 mg, 5 mmol), and N,N-dimethylformamide (DMF, 5 mL) were added sequentially. A DMF solution (5 mL) of 2-trifluoromethylbenzoyl chloride (1252 mg, 6 mmol) was then added dropwise using a dropping funnel. The mixture was stirred in an ice bath. The reaction progress was monitored by TLC. After completion of the reaction, water (50 mL) was added to the reaction mixture, which was filtered. The filter cake was rinsed with water 3-4 times and the filter cake was collected to obtain a white solid, Intermediate 20 (856 mg, 53%).

[0159] To a 50 mL round-bottom flask equipped with a magnetic stirrer were added intermediate 20 (500 mg, 1.6 mmol), methanol (20 mL), and dichloromethane (10 mL). Sodium hydroxide (620 mg, 15.5 mmol) and an aqueous hydroxylamine solution (3069 mg, 46.5 mmol) were added sequentially at 0°C and stirred. The reaction progress was monitored by TLC. After completion of the reaction, the pH was adjusted to 8-9 with dilute hydrochloric acid. The mixture was concentrated under reduced pressure to remove dichloromethane and methanol, filtered, and the filter cake was washed three to four times with water. The filter cake was then dried under vacuum to afford a pale yellow solid, Compound IX (212 mg, 42%).

[0160] mp242.5-243.2℃; 1 H NMR (500MHz, DMSO-d6) δ11.14(s,1H),10.77(s,1H),8.97(s,1H),7.86(d,J=7.3Hz,1H),7.81(t,J=7.5Hz,1H),7.77–7.70(m,6H); 13 CNMR(125MHz,DMSO-d6)δ165.8,163.9,141.4,135.9(d, 4 J=2.5Hz),132.7,130.2,128.5,128.0,127.8,126.4(q, 4 J=5Hz),126.0(d, 2 J=31.3Hz),124.9(d, 1 J=272.5Hz),119.0.LC-MS:m / z325.1[M+H] + .

[0161] Example 10: Preparation of 7-((3,4-difluorobenzyl)oxy)-3,4-dimethyl-2H-carbazol-2-one (X)

[0162]

[0163] To a 50 mL round-bottom flask equipped with a magnetic stirrer, resorcinol (330 mg, 3 mmol), 1,4-dioxane solution (10 mL), concentrated sulfuric acid (3-4 drops), and ethyl 2-methylacetoacetate (519 mg, 3.6 mmol) were added sequentially under an ice bath, and then the reaction temperature was increased to 60°C. The reaction progress was monitored by TLC. After completion, the reaction solution was poured into ice water (50 mL), filtered, and the filter cake was rinsed with water 3-4 times to obtain a white solid, Intermediate 22 (80 mg, 14%).

[0164] To a 50 mL round-bottom flask equipped with a magnetic stirrer were added 3,4-difluorobenzyl alcohol (60 mg, 0.42 mmol), intermediate 22 (80 mg, 0.42 mmol), triphenylphosphine (110 mg, 0.42 mmol), and tetrahydrofuran (10 mL). A solution of diisopropyl azodicarboxylate (DIAD, 85 mg, 0.42 mmol) in tetrahydrofuran (5 mL) was added dropwise using a dropping funnel at 0°C. After complete addition, the mixture was allowed to react at room temperature. The reaction progress was monitored by TLC. After completion, the reaction was filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent. The product was then purified by column chromatography (methylene chloride:methanol volume ratio 200:1-100:1) to afford a white solid, Compound X (38 mg, 29%).

[0165] mp151.6-152.4℃; 1 H NMR(500MHz,Chloroform-d)δ7.52(d,J=8.9Hz,1H),

[0166] 7.27(dt,J=12.9,4.4Hz,1H),7.22–7.13(m,2H),6.90(dd,J=8.8,2.6Hz,1H),6.84(d,J=2.5Hz,1H),5.06(s,2H),2.37(s,3H),2.19(s,3H); 13 C NMR(125MHz,DMSO-d6)δ161.2,160.0,153.0,150.3(dd, 2 J=31.3Hz, 3 J=12.5Hz),148.4(dd, 2 J=31.3Hz, 3 J=12.5Hz),146.7,134.2(q, 4 J=3.8Hz),126.2,124.8(q, 4 J=3.8Hz),118.1,117.7(d, 2 J=17.5Hz),117.0(d, 2 J=17.5Hz),113.9,112.5,101.4,68.4,14.8,12.9.LC-MS:m / z 317.1[M+H] + .

[0167] Example 11: Preparation of 2-(4-(4-methylphenylmethyl)piperazin-1-yl)thiophene[3,2-d]pyrimidin-4-amine (XI)

[0168]

[0169] To a 100 mL round-bottom flask equipped with a magnetic stirrer, 4-methylbenzyl chloride (1.4 g, 10 mmol), N-Boc-piperazine (2.3 g, 12 mmol), potassium carbonate (2.8 g, 20 mmol), potassium iodide (166 mg, 1 mmol), and acetonitrile (30 mL) were added sequentially and allowed to react at room temperature. The reaction progress was monitored by TLC. Upon completion, the organic solvent was removed by concentration under reduced pressure, washed with water, and extracted three to four times with dichloromethane. The organic solvent was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the organic solvent. Purification was performed by thin-layer chromatography (TLC) using dichloromethane:methanol (volume ratio 100:1) as the developing solvent to obtain Intermediate 24 as a white solid.

[0170] To a 100 mL round-bottom flask equipped with a magnetic stirrer, intermediate 24 (3 g, 10.3 mmol) and methanol (20 mL) were added sequentially. A hydrochloric acid solution in dioxane (18 mL) was added under an ice bath and allowed to react at 0°C. The reaction progress was monitored by TLC. After completion, the organic solvent and excess hydrochloric acid were removed by concentration under reduced pressure to yield intermediate 25, a white solid.

[0171] To a 50 mL round-bottom flask equipped with a magnetic stirrer, 4-amino-2-chlorothienyl[3,2-D]pyrimidine (372 mg, 2 mmol), dichloromethane (10 mL), triethylamine (606 mg, 6 mmol), 4-dimethylaminopyridine (DMAP, 25 mg, 0.2 mmol), and di-tert-butyl dicarbonate (873 mg, 4 mmol) were added sequentially and allowed to react at 0°C. The reaction progress was monitored by TLC. After completion of the reaction, the reaction solution was evaporated to obtain a white solid, Intermediate 27.

[0172] To a 100 mL round-bottom flask equipped with a magnetic stirrer, intermediate 25 (143 mg, 0.75 mmol), intermediate 27 (143 mg, 0.5 mmol), potassium carbonate (173 mg, 1.25 mmol), and DMF (5 mL) were added sequentially and reacted at 90°C. The reaction progress was monitored by TLC. After completion of the reaction, the reaction solution was evaporated to obtain a white solid, intermediate 28.

[0173] To a 50 mL round-bottom flask equipped with a magnetic stirrer, intermediate 28 (58 mg, 0.13 mmol) and methanol (2 mL) were added sequentially. A hydrochloric acid solution in dioxane (1 mL) was added under ice-cooling, and the reaction was allowed to proceed. The reaction progress was monitored by TLC using dichloromethane:methanol (10:1 by volume) as the developing solvent. Upon completion of the reaction, the organic solvent and excess hydrochloric acid were removed by concentration under reduced pressure. The pH was adjusted to alkaline, and the mixture was filtered to obtain a white solid, Compound XI (20 mg, 45%).

[0174] mp289.0-289.5℃; 1H NMR (500MHz, DMSO-d6) δ7.88 (d, J=5.3Hz, 1H), 7.20

[0175] (d,J=7.9Hz,2H),7.13(d,J=7.8Hz,2H),7.03(d,J=5.3Hz,1H),6.98(s,2H),3.72–3.63(m,4H),3.44(s,2H),2.38(t,J=5.1Hz,4H),2.28(s,3H); 13 C NMR (125MHz, DMSO-d6) δ162.0,160.6,158.2,136.0,135.0,132.6,128.9,128.8,123.4,105.0,62.0,52.6,43.9,20.7.LC-MS: m / z 340.2[M+H] + .

[0176] Example 12: Preparation of N-([1,1'-biphenyl]-4-ylmethyl)-2,4,5-trifluorobenzenesulfonamide (XII)

[0177]

[0178] To a 50 mL round-bottom flask equipped with a magnetic stirrer were added 4-phenylbenzylamine (346 mg, 1.5 mmol), 2,4,5-trifluorobenzenesulfonyl chloride (550 mg, 3 mmol), 4-dimethylaminopyridine (DMAP, 30 mg, 0.13 mmol), and DMF (5 mL) in sequence. The reaction was allowed to warm to room temperature from 0°C. The reaction progress was monitored by TLC. After completion of the reaction, water (40 mL) was added to the reaction solution to precipitate a solid. The solid was filtered and the filter cake was washed with water to obtain a white solid, Compound XII (362 mg, 64%).

[0179] mp141.9-142.5℃; 1 H NMR(500MHz,DMSO-d6)δ8.88(t,J=6.2Hz,1H),7.77–

[0180] 7.67(m,2H),7.66–7.57(m,3H),7.51(d,J=8.0Hz,2H),7.37(d,J=7.7Hz,2H),7.30(d,J=8.0Hz,2H),4.19(d,J=6.2Hz,2H); 13 C NMR(125MHz,DMSO-d6)δ152.8(dt, 1 J=253.8Hz, 3 J=12.5Hz),146.2(ddd,1 J=243.8Hz, 3 J=12.5Hz, 4 J=2.5Hz),139.8,139.2,136.2,128.9 128.3,127.6,127.4,126.6,126.4,126.0(d, 2 J=17.5Hz),118.0(d, 2 J=22.5Hz),108.0(dd, 2 J=22.5,27.5Hz),45.8.LC-MS:m / z 378.0[M+H] + .

[0181] Example 13: Preparation of N-(2-(1H-pyrimidin-1-yl)benzyl)quinoxaline-2-carboxamide (XIII)

[0182]

[0183] To a 50 mL round-bottom flask equipped with a magnetic stirrer, 2-quinoxalinecarboxylic acid (121 mg, 0.7 mmol), 2-(1-pyrazolyl)benzylamine (100 mg, 0.56 mmol), N,N-diisopropylethylamine (DIPEA, 224 mg, 1.7 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 166 mg, 0.87 mmol), 1-hydroxybenzotriazole (HOBT, 117 mg, 0.87 mmol) and DCM (5 mL) were added in sequence and reacted at room temperature. The reaction progress was monitored by TLC. After the reaction was completed, the mixture was washed with water (10 mL) 3-4 times and once with saturated brine. The organic layer was collected and dried over anhydrous sodium sulfate. The organic layer was concentrated under reduced pressure to remove the organic solvent. The mixture was purified by column chromatography using a developing solvent of dichloromethane to methanol (volume ratio of 100:1) and thin layer chromatography using a developing solvent of dichloromethane to methanol (volume ratio of 30:1) to give a white solid, namely compound XIII (117 mg, 64%).

[0184] mp129.1-130.0℃; 1 H NMR(500MHz,DMSO-d6)δ9.51(t,J=6.4Hz,1H),9.47(s,

[0185] 1H),8.22–8.16(m,3H),8.01–7.95(m,2H),7.86(d,J=1.5Hz,1H),7.58–7. 54(m,1H),7.46–7.40(m,3H),6.58(t,J=2.1Hz,1H),4.56(d,J=6.4Hz,2H);13 C NMR(125MHz,DMSO-d6)δ163.0,144.0,143.7,143.0,140.7,139.7,138.8,133.4,131 .9,131.5,131.3,129.5,129.1,129.0,128.2,128.0,125.2,106.9,39.2.LC-MS:m / z 330.2[M+H] + .

[0186] Example 14: Preparation of (4-(2-fluorophenyl)piperazin-1-yl)(quinolin-3-yl)methanone (XIV)

[0187]

[0188] To a 50 mL round-bottom flask equipped with a magnetic stirrer were added quinoline-3-carboxylic acid (217 mg, 1 mmol), 1-(2-fluorophenyl)piperazine (208 mg, 1.2 mmol), N,N-diisopropylethylamine (DIPEA, 388 mg, 3 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 131 mg, 1.3 mmol), 1-hydroxybenzotriazole (HOBT, 176 mg, 1.3 mmol), and DCM (10 mL) in sequence. The reaction was allowed to react at room temperature. The reaction progress was monitored by TLC. After completion of the reaction, the mixture was washed 3-4 times with water (10 mL) and once with saturated brine (10 mL). The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the organic solvent. The product was then purified by thin-layer chromatography using dichloromethane to methanol (60:1 by volume) as the developing solvent to obtain Compound XIV (135 mg, 40%) as a white solid.

[0189] mp121.8-122.5℃; 1 H NMR (500MHz, DMSO-d6) δ8.96 (d, J = 2.1Hz, 1H), 8.52

[0190] (d,J=2.0Hz,1H),8.08(d,J=9.5Hz,2H),7.85(ddd,J=8.4,6.9,1.5Hz,1H),7.69(ddd,J=8.0,6.9,1.2Hz,1H),7.19– 7.10(m,2H),7.06(td,J=8.4,7.9,1.7Hz,1H),7.03–6.97(m,1H),3.87(s,2H),3.61(s,2H),3.08(d,J=41.1Hz,4H); 13C NMR(125MHz,DMSO-d6)δ166.9,156.0,154.0,148.7,147.6,139.4(d, 3 J=8.8Hz),134.7,130.7,128.8(d, 3 J=7.5Hz),128.7,127.4,126.6,124.9(d, 4 J=3.8Hz),122.9(d, 3 J=7.5Hz),119.6(d, 4 J=2.5Hz),116.1(d, 2 J=20.0Hz),50.4,50.0,47.4,41.8.LC-MS:m / z 336.2[M+H] + .

[0191] Example 15: Preparation of 2-(2,5-dimethylphenyl)-1-(4-phenylpiperazin-1-yl)ethanone (XV)

[0192]

[0193] To a 50 mL round-bottom flask equipped with a magnetic stirrer were added 2,5-dimethylphenylacetic acid (487 mg, 3 mmol), N-phenylpiperazine (400 mg, 2.5 mmol), N,N-diisopropylethylamine (DIPEA, 958 mg, 7.4 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 947 mg, 4.9 mmol), 1-hydroxybenzotriazole (HOBT, 667 mg, 4.9 mmol), and DCM (10 mL) in sequence. The reaction was allowed to react at room temperature. TLC monitored the reaction progress. After completion, the mixture was washed 3-4 times with water (10 mL) and once with saturated brine (10 mL). The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the organic solvent. The mixture was then purified by thin-layer chromatography (TLC) using dichloromethane to methanol (volume ratio: 80:1-140:1) as the developing solvent to obtain Compound XV (380 mg, 65%) as a white solid.

[0194] mp144.0-144.5℃; 1 H NMR(500MHz,Chloroform-d)δ7.28(dd,J=8.8,7.1Hz,

[0195] 2H),7.07(d,J=7.5Hz,1H),6.98(d,J=10.8Hz,2H),6.92(d,J=7.6Hz,3H),3.85(t,J=5.2Hz,2H),3.69 (s,2H),3.58(t,J=5.2Hz,2H),3.19(t,J=5.2Hz,2H),3.05(t,J=5.2Hz,2H),2.28(s,3H),2.25(s,3H); 13 C NMR(125MHz,Chloroform-d)δ169.9,151.0,135.8,133.4,133.1,130.4,129.5, 129.4,127.8,120.7,116.7,49.7,49.6,46.0,41.8,38.6,21.1,19.4.LC-MS:m / z 309.3[M+H] + .

[0196] Example 16: Preparation of N-(1-acetylpiperidin-4-yl)-N-(2-hydroxy-3-methylphenyl)-1-naphthamide (XVI)

[0197]

[0198] To a 50 mL round-bottom flask equipped with a magnetic stirrer, 2-hydroxy-3-methylbenzaldehyde (409 mg, 3 mmol), 1-Boc-4-aminopiperidine (301 mg, 1.5 mmol), glacial acetic acid (360 mg, 6 mmol) and 1,2-dichloroethane (DCE, 10 mL) were added in sequence and reacted at room temperature. After two hours, half of the sodium triacetoxyborohydride (891 mg, 4.2 mmol) was added, and after another two hours, the remaining half of the sodium triacetoxyborohydride (891 mg, 4.2 mmol) was added. The reaction progress was monitored by TLC. After the reaction, the organic solvent was concentrated under reduced pressure, neutralized with saturated sodium bicarbonate solution until the bubbles disappeared, and extracted with dichloromethane 3-4 times. The organic layer was collected and dried over anhydrous sodium sulfate. The organic solvent was removed by concentration under reduced pressure, and purified by TLC thin-layer chromatography using dichloromethane:methanol (volume ratio 30:1-50:1) as the developing solvent to obtain a white solid, namely, intermediate 34 (358 mg, 74%).

[0199] To a 50 mL round-bottom flask equipped with a magnetic stirrer were added intermediate 34 (358 mg, 1.1 mmol), 1-naphthoic acid (193 mg, 1.1 mmol), potassium carbonate (310 mg, 2.2 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 431 mg, 2.2 mmol), 4-dimethylaminopyridine (DMAP, 28 mg, 0.2 mmol), and DCM (10 mL) in sequence. The reaction was allowed to react at room temperature. The reaction progress was monitored by TLC. After completion of the reaction, the mixture was washed 3-4 times with water (10 mL) and once with saturated brine (10 mL). The organic layer was collected and dried over anhydrous sodium sulfate, concentrated under reduced pressure to remove the organic solvent, and purified by thin-layer chromatography (TLC) using dichloromethane to methanol (volume ratio 80:1-50:1) as the developing solvent to obtain intermediate 35 (474 ​​mg, 89%) as a white solid.

[0200] To a 50 mL round-bottom flask equipped with a magnetic stirrer, intermediate 35 (474 ​​mg, 1 mmol) and methanol (1 mL) were added sequentially. Hydrochloric acid in dioxane (2 mL) was added under ice-cooling, and the reaction was allowed to proceed. The reaction progress was monitored by TLC. After completion of the reaction, the organic solvent and excess hydrochloric acid were removed by concentration under reduced pressure to yield intermediate 36 (200 mg, 53%) as a white solid.

[0201] To a 50 mL round-bottom flask equipped with a magnetic stirrer were added intermediate 36 (200 mg, 0.54 mmol), glacial acetic acid (33 mg, 0.54 mmol), potassium carbonate (150 mg, 1.08 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 208 mg, 1.08 mmol), 4-dimethylaminopyridine (DMAP, 14 mg, 0.11 mmol), and DCM (10 mL). The reaction was allowed to react at room temperature. The reaction progress was monitored by TLC. After completion, the reaction was washed 3-4 times with water (10 mL) and once with saturated brine (10 mL). The organic layer was collected and dried over anhydrous sodium sulfate. The organic layer was concentrated under reduced pressure to remove the organic solvent and purified by thin-layer chromatography (TLC) using dichloromethane to methanol (volume ratio: 50:1-30:1-40:1) as the developing solvent to obtain compound XVI (53 mg, 24%) as a white solid.

[0202] mp117.5-118.3℃; 1H NMR(500MHz,Chloroform-d)δ9.63(d,J=12.1Hz,1H),7.95–7.87(m,2H),7.72(dd,J=13.4,8.4H z,1H),7.55–7.46(m,3H),7.40(d,J=6.7Hz,1H),7.15(d,J=7.3Hz,1H),7.05(d,J=7.4Hz,1H),6 .77(t,J=7.5Hz,1H),4.94–4.49(m,3H),3.71–3.54(m,2H),2.47(dt,J=65.7,12.2Hz,1H),2.32 (d,J=3.0Hz,3H),1.99(d,J=11.4Hz,3H),1.90(d,J=9.6Hz,1H),1.73(s,2H),1.68–1.44(m,2H); 13 CNMR(125MHz,DMSO-d6)δ167.2,165.8,150.9,149.0,139.2,136.6,133.5,130.6,128.7,128.0,126.9,12 6.6,123.7,120.0,119.7,117.9,117.8,116.5,50.6,46.7,45.0,41.1,29.0,15.4.LC-MS:m / z417.3[M+H] + .

[0203] Example 17: Preparation of (R)-4-methoxy-N-(2,3,4,9-tetrahydro-1H-carbazol-3-yl)benzamide (XVII)

[0204]

[0205] To a 50 mL round-bottom flask equipped with a magnetic stirrer were added p-anisic acid (304 mg, 2 mmol), (R)-3-amino-1,2,3,4-tetrahydrocarbazole (186 mg, 1 mmol), N,N-diisopropylethylamine (DIPEA, 387 mg, 3 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 384 mg, 2 mmol), 1-hydroxybenzotriazole (HOBT, 270 mg, 2 mmol), and DCM (10 mL) in sequence. The reaction was allowed to react at room temperature. The reaction progress was monitored by TLC. After completion, the reaction was washed 3-4 times with water (10 mL) and once with saturated brine (10 mL). The organic layer was collected and dried over anhydrous sodium sulfate. The organic layer was concentrated under reduced pressure to remove the organic solvent and purified by thin-layer chromatography (TLC) using dichloromethane:methanol (60:1 by volume) as the developing solvent to obtain Compound XVII (212 mg, 66%) as a white solid.

[0206] mp253.5-254.3℃; 1 H NMR(500MHz,DMSO-d6)δ10.71(s,1H),8.34(d,J=7.8Hz,1H),7.93–7.87(m,2 H),7.34(d,J=7.6Hz,1H),7.26(d,J=7.9Hz,1H),7.03–6.98(m,3H),6.96–6. 91(m,1H),4.32–4.23(m,1H),3.81(s,3H),3.00(dd,J=14.8,5.5Hz,1H),2.8 7(d,J=11.0Hz,2H),2.71–2.63(m,1H),2.14–2.07(m,1H),1.98–1.88(m,1H); 13 C NMR (125MHz, DMSO-d6) δ165.3,161.5,136.2,133.6,129.2,127.1,127.0,120. 2,118.1,117.2,113.4,110.6,106.9,55.3,46.3,29.1,27.3,21.9.LC-MS:m / z 321.2[M+H] + .

[0207] Example 18: Preparation of 2-(4-(3,4-difluorobenzoyl)-3-methylpiperazine-1-carboxyl)-N-phenylbenzamide (XVIII)

[0208]

[0209] To a 50 mL round-bottom flask equipped with a magnetic stirrer were added aniline (698 mg, 7.5 mmol), monomethyl phthalate (900 mg, 5 mmol), potassium carbonate (2070 mg, 15 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 1440 mg, 7.5 mmol), 1-hydroxybenzotriazole (HOBT, 1012 mg, 7.5 mmol), and DCM (10 mL) in sequence. The reaction was allowed to react at room temperature. The reaction progress was monitored by TLC. After completion, the reaction was washed 3-4 times with water (10 mL) and once with saturated brine (10 mL). The organic layer was collected and dried over anhydrous sodium sulfate, concentrated under reduced pressure to remove the organic solvent, and purified by thin-layer chromatography (TLC) using dichloromethane to methanol (60:1 by volume) as the developing solvent to obtain Intermediate 39 (300 mg, 24%) as a white solid.

[0210] To a 50 mL round-bottom flask equipped with a magnetic stirrer, intermediate 39 (300 mg, 1.18 mmol) and methanol (5 mL) were added sequentially. 4 M aqueous sodium hydroxide solution (1.5 mL) was added dropwise at room temperature. The reaction progress was monitored by TLC. After completion of the reaction, the organic solvent was removed by concentration under reduced pressure. The pH was adjusted to 5-6 with dilute hydrochloric acid, and the filter cake was filtered to obtain a white solid, intermediate 40 (200 mg, 83%).

[0211] To a 100 mL round-bottom flask equipped with a magnetic stirrer were added 4-Boc-2-methylpiperazine (720 mg, 3.6 mmol), triethylamine (303 mg, 3 mmol), and N,N-dimethylformamide (DMF, 5 mL) in sequence. A solution of 3,4-difluorobenzoyl chloride (528 mg, 3 mmol) in DMF (5 mL) was then added dropwise using a dropping funnel and stirred in an ice bath. The reaction progress was monitored by TLC. After completion, water (50 mL) was poured into the reaction mixture to precipitate a solid, which was filtered and the filter cake rinsed with water 3-4 times to afford a white solid, Intermediate 42 (720 mg, 71%).

[0212] To a 50 mL round-bottom flask equipped with a magnetic stirrer, intermediate 42 (720 mg, 2.1 mmol) and methanol (10 mL) were added sequentially. A hydrochloric acid solution in dioxane (9 mL) was added under ice-cooling, and the reaction was allowed to proceed. TLC was used to monitor the reaction progress. After completion of the reaction, the organic solvent and excess hydrochloric acid were removed by concentration under reduced pressure to yield intermediate 43, a white solid.

[0213] To a 50 mL round-bottom flask equipped with a magnetic stirrer were added intermediate 40 (698 mg, 7.5 mmol), intermediate 43 (200 mg, 0.83 mmol), potassium carbonate (344 mg, 2.5 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 239 mg, 1.2 mmol), 1-hydroxybenzotriazole (HOBT, 168 mg, 1.2 mmol), and DCM (10 mL) in sequence. The reaction was allowed to react at room temperature. The reaction was monitored by TLC. After completion of the reaction, the mixture was washed 3-4 times with water (10 mL) and once with saturated brine (10 mL). The organic layer was collected and dried over anhydrous sodium sulfate, concentrated to remove the organic solvent, and purified by thin-layer chromatography (TLC) using dichloromethane to methanol (50:1 volume ratio) as the developing solvent to obtain compound XVIII (18 mg, 24%) as a white solid.

[0214] mp206.9-207.8℃; 1H NMR (500MHz, DMSO-d6) δ10.44(d,J=44.4Hz,1H),7.75(ddd,J=33.9,15.2,8.1Hz,3H),7.61–7.48(m,4H),7.36(dt,J=15. 5,7.3Hz,3H),7.28(s,1H),7.09(t,J=7.3Hz,1H),3.35(s,3H),3.29–3.09(m,2H),3.07–2.96(m,1H),1.32–1.13(m,4H); 13 C NMR(125MHz,DMSO-d6)δ167.2,166.2,165.8,159.9(dd, 1 J=246.3Hz, 3 J=11.3Hz),150.3(dd, 1 J=245Hz, 3 J=13.8Hz),139.2,139.1,136.6,133.5(d, 4 J=5Hz),130.6,128.6(d, 4 J=2.5Hz),128.0,126.9,126.6,124.0(d, 3 J=11.3Hz),123.7,119.8(d, 2 J=32.5Hz),117.9(d, 2 J=17.5Hz),116.4(d, 2 J=16.3Hz),50.6,46.7,45.0,41.1,15.5.LC-MS:m / z 464.2[M+H] + .

[0215] Example 19: Preparation of (4-morpholinylpiperidin-1-yl)(quinolin-3-yl)methanone (XIX)

[0216]

[0217] To a 50 mL round-bottom flask equipped with a magnetic stirrer were added quinoline-3-carboxylic acid (300 mg, 1.7 mmol), 4-(4-piperidinyl)morpholine (353 mg, 2.1 mmol), N,N-diisopropylethylamine (DIPEA, 671 mg, 5.2 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 663 mg, 3.5 mmol), 1-hydroxybenzotriazole (HOBT, 468 mg, 3.5 mmol), and DCM (10 mL) in sequence. The reaction was allowed to react at room temperature. The reaction progress was monitored by TLC. After completion of the reaction, the mixture was washed 3-4 times with water (10 mL) and once with saturated brine (10 mL). The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the organic solvent. The mixture was then purified by thin-layer chromatography (TLC) using dichloromethane to methanol (60:1 volume ratio) as the developing solvent to obtain Compound XIX (193 mg, 35%) as a colorless liquid.

[0218] 1 H NMR(500MHz,DMSO-d6)δ8.90(d,J=1.8Hz,1H),8.45(d,J=2.1Hz,1H),8.07(dt,J=8.4,1.7Hz, 2H),7.83(ddd,J=8.5,6.8,1.4Hz,1H),7.71–7.64(m,1H),4.51(d,J=12.8Hz,1H),3.66(d,J= 13.0Hz,1H),3.56(t,J=4.6Hz,4H),3.00(d,J=121.9Hz,2H),2.44-2.40(m,5H),1.89(d,J=12 .7Hz,1H),1.72(d,J=11.9Hz,1H),1.46(dd,J=12.1,4.2Hz,1H),1.41(dd,J=12.1,4.2Hz,1H); 13 C NMR (125MHz, DMSO-d6) δ166.6,148.5,147.5,134.3,130.6,129.2,128.8,128.7,127.3,126.6,66.5,60.9,49.4,46.5,40.9,28.4,27.6.LC-MS: m / z 326.2[M+H] + .

[0219] Example 20: Preparation of (4-(5-chloropyridin-2-yl)piperazin-1-yl)(1H-indol-2-yl)methanone (XX)

[0220]

[0221] To a 50 mL round-bottom flask equipped with a magnetic stirrer were added 2-indolecarboxylic acid (195 mg, 1.2 mmol), 1-(5-chloropiperidinyl)piperazine (200 mg, 1 mmol), N,N-diisopropylethylamine (DIPEA, 392 mg, 3 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 387 mg, 2 mmol), 1-hydroxybenzotriazole (HOBT, 273 mg, 2 mmol), and DCM (10 mL) in sequence. The reaction was allowed to react at room temperature. The reaction progress was monitored by TLC. Upon completion, the product was washed with water (10 mL) 3-4 times and once with saturated brine (10 mL). The organic layer was collected and dried over anhydrous sodium sulfate. The organic layer was concentrated under reduced pressure to remove the organic solvent and purified by thin-layer chromatography (TLC) using dichloromethane to methanol (50:1 by volume) as the developing solvent to obtain Compound XX (90 mg, 26%) as a white solid.

[0222] mp205.4-206.0℃; 1 H NMR (500MHz, DMSO-d6) δ11.62(s,1H),8.15(d,J=2.7Hz,1H),7.71–7.58(m,2H),7.44(dd,J=8.2,1.1Hz,1H),7.20(ddd,J=8.1,6.9 ,1.1Hz,1H),7.06(ddd,J=8.0,6.9,1.0Hz,1H),6.89(d,J=9.1Hz,1H),6.86(dd,J=2.2,0.9Hz,1H),3.88(s,4H),3.72–3.56(m,4H); 13 C NMR(125MHz,DMSO-d6)δ162.2,157.2,145.6,137.3,136.0,129.8,126.8,12 3.3,121.4,119.8,119.1,112.1,108.5,104.2,44.6.LC-MS:m / z341.2[M+H] + .

[0223] Example 21: Pharmacological activity test and experimental data

[0224] 1. Determination of compound affinity for CXCR4

[0225] Biotinylated TN14003 (an effective CXCR4 peptide antagonist) is used to compete with the compound for CXCR4 binding, and the affinity of the compound for CXCR4 is tested by measuring the inhibition rate of the compound in inhibiting the binding of TN14003 to CXCR4.

[0226] Experimental method: MDA-MB-231 cells were digested and seeded into 96-well plates, with 1×10 cells per well. 5 cells. The next day, the corresponding concentration of compound (10nM) was added according to the group settings and incubated for 10 minutes. Fix the cells with 4% paraformaldehyde for more than 30 minutes and wash 3 times with PBS. Add 0.05μg / mL of peptide TN14003 and incubate at room temperature for 30 minutes, and wash 3 times with PBS. Add Streptavidin-Rhodamine (TRITC) (stock concentration 1mg / mL) 1:500 dilution, incubate at room temperature for 30 minutes in the dark, and wash 3 times with PBS. Add DAPI staining solution and incubate at room temperature in the dark for 5 minutes, and wash 3 times with PBS. Observe the expression under a confocal microscope, and take pictures of 3 high expression areas for preservation.

[0227] As shown in the experimental results (Table 1), sixteen of the twenty examples exhibited binding affinity inhibition rates of greater than 50% against CXCR4 at concentrations as low as 10 nM. Compounds I, II, III, IV, and V all exhibited superior inhibitory effects compared to the positive drug plerixafor, with compound I exhibiting the strongest inhibitory effect, achieving the highest inhibition rate of 78.85%.

[0228] Table 1. Binding affinity inhibition rate of CXCR4

[0229]

[0230] 2. Inhibition test of inflammatory cell drive in preferred embodiments

[0231] Transwell matrix gel was used for the experiment, and compounds with a CXCR4 binding affinity inhibition rate higher than 50% were selected for further testing of their ability to block CXCR4 / CXCL12-mediated inflammatory cell activation at 10 nM.

[0232] Experimental Methods: RAW264.7 cells were serum-depleted and cultured in incomplete medium for 24 hours. Matrigel was allowed to thaw overnight at 4°C. The melted Matrigel was diluted to half with incomplete medium. 30 μL of the diluted Matrigel was added to the upper chamber of the Transwell and incubated at 37°C for 120 minutes to allow the Matrigel to polymerize. The cells were digested and counted, and the cell density was adjusted to 1×10 using incomplete medium. 5Cells were fixed in 4% paraformaldehyde for at least 30 minutes. 500 μL of 0.1% crystal violet was added to the 24-well plate, immersing the plate in the dye. After 30 minutes at 37°C, cells were removed from the plate. The cells were washed with PBS, inverted, and air-dried. Three fields of view were taken on the 24-well plate, photographed (200× magnification), and counted.

[0233] As can be seen from the experimental results (Table 2), all preferred embodiments have a strong inhibitory effect on RAW264.7 cell transformation. Examples I, II, III, IV, V, VI, IX, XI, XII, XV, XVI and XVIII have an inhibition rate of more than 50% on RAW264.7 cell transformation at a concentration of 10 nM. Among them, Examples I, II, III, IV, V and XVIII have an inhibitory effect on RAW264.7 cell transformation that is better than that of the positive drug, and Examples I and II show very significant inhibitory effects, with inhibition rates of 79.19% and 75.63% on RAW264.7 cell transformation, respectively.

[0234] Table 2. Inhibition rate of RAW264.7 inflammatory cell lineage

[0235]

[0236] 3. In vivo anti-enteritis activity study of the preferred embodiment

[0237] The two compounds of Example 1 (Compound I) and Example 2 (Compound II) with the strongest inhibition rates in the cell-mediated chemotaxis experiment were selected to further study their in vivo anti-enteritis activities in mice with ulcerative colitis induced by dextran sulfate sodium salt (DSS).

[0238] Experimental Methods: Eight-week-old male C57BL / 6 mice were randomly divided into five groups (blank, model, positive drug Plerixafor, Compound I, and Compound II), with eight mice in each group. Except for the blank group, mice in all other groups received 3% dextran sulfate sodium salt (DSS) in their drinking water for seven consecutive days. Simultaneously with model establishment, mice in the positive drug, Compound I, and Compound II groups were gavaged twice daily for 14 days. Drug administration continued for another seven days after seven days of model establishment. Animal body weight, stool characteristics, and fecal occult blood were recorded, and the disease activity index (DAI) was scored. At the end of the experiment, the intestines were photographed, colon length was calculated, and the mice were longitudinally dissected, washed, and observed for bleeding spots. The area of ​​bleeding spots was calculated using Image-J software. Myeloperoxidase (MPO) activity and the expression of inflammatory factors IL-6, TNF-α, and IL-1β in intestinal tissue were measured using a myeloperoxidase (MPO) assay kit and immunofluorescence. Inflammatory pathology of the colorectal mucosa was scored using a hematoxylin-eosin staining kit and the histological activity index (HAI).

[0239] The experimental results (Tables 3 to 6) show that the weight of mice in the model group gradually decreased, the colon length shortened, and the stool quality score, colon bleeding points, percentage of bleeding area, MPO activity (U / g), and HAI score significantly increased. Compared with the model group, compound groups I and II were able to steadily increase the weight of mice, normalize colon length, and significantly reduce stool quality score, colon bleeding points, percentage of bleeding area, MPO activity (U / g), and HAI score. The expression of inflammatory factors IL-6, TNF-α, and IL-1β was also significantly reduced, and was slightly better than that of positive drugs, showing excellent anti-enteritis activity.

[0240] Table 3. Effects on mouse body weight

[0241]

[0242] Table 4. Effects of stool characteristics scores on mice

[0243]

[0244] Note: The higher the score, the more severe the enteritis symptoms.

[0245] Table 5. Impact results of DAI score

[0246]

[0247] Note: The higher the score, the more severe the enteritis symptoms.

[0248] Table 6. Results of the effect of enteritis on the degree of enteritis in mice

[0249]

[0250] In addition, it should be understood that after reading the above description of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A CXCR4 antagonist, characterized in that Selected from the following compounds of formula I or pharmaceutically acceptable salts thereof:

2. A method for preparing a CXCR4 antagonist, characterized in that: 1-(2-furoyl)piperazine 1 and 1-phenyl-4-piperidone undergo an amination reduction reaction to obtain the CXCR4 antagonist, namely the compound shown in the following formula I; Synthesis route:

3. Use of the CXCR4 antagonist according to claim 1 in the preparation of a drug for preventing and treating related diseases by inhibiting CXCR4, characterized in that: The disease is inflammatory bowel disease.

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