N-benzylimidazopyridine derivative as well as preparation method and application thereof

Abstract

The invention discloses an N-benzylimidazopyridine derivative as well as a preparation method and application thereof. According to the derivative, a structural fragment of lauric acid serving as a catalytic substrate of CYP4Z1 is introduced to N-benzylimidazopyridine, and the length, a connecting chain and an ending group of the introduced fragment are changed, so that the compound disclosed by the invention is prepared; as a CYP450 enzyme inhibitor, the N-benzylimidazopyridine derivative can specifically inhibit CYP4Z1 enzyme, further reduces or inhibits the activity of the CYP4Z1 enzyme in breast cancer cells, achieves the purpose of treating and / or preventing the problems of poor prognosis, invasion and metastasis and the like of breast cancer mediated by the CYP4Z1 enzyme, and has great application prospects in the field of tumor treatment drugs.

Description

Technical Field

[0001] The invention relates to an N-benzyl imidazopyridine derivative and also relates to a preparation method and application of the derivative. Background Art

[0002] CYP4 enzymes are a class of enzymes that are widely present in the human body and other organisms and are mainly responsible for the metabolism of endogenous substances. Their metabolic substrates include steroids, endogenous fatty acids, etc. Under physiological conditions, CYP4 enzymes work together to complete the metabolism of different substrates and help the body maintain normal physiological functions. Under pathological conditions, CYP4 enzymes mediate the activation and inactivation of many eicosane signaling pathways, affecting the occurrence and development of diseases. Therefore, it provides a potential drug target for the treatment of various diseases including inflammation and tumors. CYP4Z1 is a new member of the CYP4 enzyme family. In 2004, it was found that its expression level was significantly increased in human breast cancer, which was associated with tumor progression and poor prognosis. In vivo and in vitro, CYP4Z1 affects angiogenesis, tumor growth and metastasis by catalyzing the metabolites 20-hydroxyeicosatrienoic acid (20-HETE) and 1,4-epoxyeicosatrienoic acid (14,15-EET) generated from the substrate arachidonic acid. The specific expression of CYP4 in the breast provides a new approach and drug target for the treatment of breast cancer. CYP4 inhibitors have been successfully developed and used in the treatment of related cancers.

[0003] As research reveals the role of CYP4 enzymes in the pathophysiology of diseases, the development and use of small molecule inhibitors that inhibit CYP4 enzymes has gradually become a treatment strategy for a variety of related diseases, providing new ideas for cancer treatment. However, due to the lack of CYP4Z1 protein crystal structure and applicable lead compounds, the CYP4Z1 protein co-crystal structure can only be simulated by homologous protein model construction technology, so the development of effective and highly selective CYP4Z1 inhibitors remains a daunting task. At present, the development of CYP4 enzyme inhibitors mainly relies on strategies such as simulating the endogenous substrate structure of CYP4 enzymes. Studies have found that N-benzyl imidazopyridine is an effective CYP4Z1 inhibitor. At a concentration of 10μM, the inhibition rate of N-benzyl imidazopyridine on CYP4Z1 is 95±2%. However, since the inhibitory effect of N-benzyl imidazopyridine on CYP4Z1 is poor at low concentrations, and its inhibitory effect on CYP4F12 is equivalent to that on CYP4Z1, and its selectivity for CYP4Z1 is poor, there is an urgent need to obtain CYP4Z1 inhibitors with better activity and selectivity. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide an N-benzyl imidazopyridine derivative having a significant inhibitory effect on CYP4Z1 protease, and also provide a preparation method and application of the above derivative.

[0005] Technical solution: The N-benzyl imidazopyridine derivatives of the present invention are shown in Formula I:

[0006]

[0007] Wherein, R1 is selected from: hydrogen, halogen, C1-C8 alkyl, C1-C8 alkoxy, C1-C8 haloalkyl, amino or C1-C8 alkyl substituted amino;

[0008] n is 1-8;

[0009] R2 is selected from: an ester group, a C1-C8 alkyl group, an oxygen atom or an amide group;

[0010] R3 is selected from the group consisting of hydrogen, carboxyl, C1-C7 alkoxycarbonyl, C1-C7 alkyl or amide.

[0011] Preferably, R1 is selected from: hydrogen, halogen, C1-C4 alkyl, C1-C4 alkoxy;

[0012] n is 1-7;

[0013] R2 is selected from: an ester group, a C1-C4 alkyl group, an oxygen atom or an amide group;

[0014] R3 is selected from the group consisting of hydrogen, carboxyl, methoxycarbonyl, ethoxycarbonyl, and C1-C7 alkyl.

[0015] Preferably, R1 is selected from: hydrogen, fluorine, chlorine, bromine, methyl, trifluoromethyl;

[0016] n is 1-7;

[0017] R2 is selected from: methylene, ester group, oxygen atom or amide group;

[0018] The R3 is selected from the group consisting of hydrogen, hydroxyl, carboxyl, and C1-C7 alkyl.

[0019] Preferably, the derivative or its pharmaceutically acceptable salt or racemate is selected from the following compounds:

[0020]

[0021]

[0022] The derivatives also include optically active forms or racemates, diastereoisomer mixtures, solvates, prodrugs, pharmaceutically acceptable salts or cocrystals thereof.

[0023] The preparation method of the above-mentioned N-benzyl imidazopyridine derivatives is as follows:

[0024] When the N-benzyl imidazopyridine derivative is an ester or an amine, the preparation method is:

[0025] Get raw materials Under the action of SOCl2, an ester intermediate is formed with an alcohol; the ester intermediate is reacted with a long-chain or branched alcohol or amine to obtain an ester or amine intermediate, which is then reacted with 1H-imidazole [4,5-C] pyridine under the action of sodium hydride to obtain the ester or amine N-benzyl imidazopyridine derivative;

[0026] When the N-benzyl imidazopyridine derivative is an ester, the preparation method is:

[0027] Get raw materials Under the action of SOCl2, an ester intermediate is formed with an alcohol; the ester intermediate is reacted with 1H-imidazole [4,5-C] pyridine under the action of sodium hydride to obtain N-benzyl imidazopyridine, which is reduced to an alcohol with LiAlH4 and then reacted with a long-chain acid to obtain the ester N-benzyl imidazopyridine derivative;

[0028] When the N-benzyl imidazopyridine derivative is an ether, the preparation method is:

[0029] Get raw materials Under the action of SOCl2, an ester intermediate is formed with an alcohol; the ester intermediate is reacted with 1H-imidazole [4,5-C] pyridine under the action of sodium hydride to obtain N-benzyl imidazopyridine, which is reduced to an alcohol with LiAlH4 and then reacted with a long-chain 1-bromoalkane to obtain the ether N-benzyl imidazopyridine derivative;

[0030] When the N-benzyl imidazopyridine derivative is an alkyl derivative, the preparation method is:

[0031] The raw material 4-n-pentylbenzoic acid, 4-n-hexylbenzoic acid, 4-n-heptylbenzoic acid or 4-n-octylbenzoic acid is reduced to alcohol by LiAlH4 and then reacted with PBr3 to generate a benzyl bromide intermediate; the benzyl bromide intermediate is reacted with 1H-imidazole [4,5-C] pyridine under the action of sodium hydride to obtain an alkyl N-benzyl imidazopyridine derivative.

[0032] Wherein, when R3 is carboxylic acid,

[0033] (1-1) p-bromomethylbenzene alkyl acid is reacted with thionyl chloride to generate intermediate 1; the alkyl acid in p-bromomethylbenzene alkyl acid is a C1-C7 alkyl acid;

[0034] (1-2) The intermediate 1 described in step (1-1) reacts with 1H-imidazole [4,5-C] pyridine under the action of sodium hydride to generate intermediate 1;

[0035] (1-3) Intermediate 1 and alkyl alcohol undergo condensation reaction to generate intermediate 2; the alkyl alcohol is C1-C7 alkyl alcohol;

[0036] (1-4) The intermediate 2 described in step (1-3) undergoes a hydrolysis reaction under the action of sodium hydroxide to obtain an N-benzyl imidazopyridine derivative;

[0037] When R2 is an ester group and R3 is a C1-C7 alkyl group,

[0038] (2-1) condensation reaction of p-bromomethylbenzene alkyl acid and alkyl alcohol to generate an intermediate; the alkyl alcohol is C1-C7 alkyl alcohol;

[0039] (2-2) reacting the intermediate in step (2-1) with 1H-imidazole [4,5-C] pyridine to produce the N-benzyl imidazopyridine derivative;

[0040] When R2 is an oxygen atom and R3 is a C1-C7 alkyl group,

[0041] (3-1) p-bromomethyl alkyl acid is reacted with thionyl chloride to generate intermediate 1; the alkyl acid in p-bromomethyl alkyl acid is C1-C7 alkyl acid;

[0042] (3-2) The intermediate 1 described in step (3-1) reacts with 1H-imidazole [4,5-C] pyridine under the action of sodium hydride to generate benzyl imidazopyridine;

[0043] (3-3) The N-benzyl imidazopyridine described in step (3-2) is reduced to an alcohol to obtain intermediate 2;

[0044] (3-4) The intermediate 2 described in step (3-3) reacts with 1-bromoalkane to generate the corresponding N-benzyl imidazopyridine derivative; the alkane in the 1-bromoalkane is a C1-C7 alkane;

[0045] When R2 is amide and R3 is C1-C7 alkyl:

[0046] (4-1) a condensation reaction of p-bromomethylbenzene alkyl acid and an alkylamine to generate an intermediate; the alkylamine is a C1-C7 alkylamine;

[0047] (4-2) reacting the intermediate obtained in step (4-1) with 1H-imidazole [4,5-C] pyridine to produce the N-benzyl imidazopyridine derivative;

[0048] When R2 is C1-C7 alkyl and R3 is hydrogen,

[0049] (5-1) p-Alkylbenzoic acid is reduced with LiAlH4 to intermediate 1;

[0050] (5-2) The intermediate 1 described in step (5-1) undergoes a substitution reaction with PBr3 to obtain intermediate 2;

[0051] (5-3) The intermediate 2 described in step (5-2) reacts with 1H-imidazole [4,5-C] pyridine under the action of sodium hydride to generate the corresponding N-benzyl imidazopyridine derivative.

[0052] The present invention also discloses a pharmaceutical composition comprising the above-mentioned N-benzyl imidazopyridine derivative, its optically active form or racemic form, diastereoisomer mixture, solvate, prodrug, metabolite pharmaceutically acceptable salt or cocrystal and a pharmaceutically acceptable carrier.

[0053] The above-mentioned N-benzyl imidazopyridine derivative or its pharmaceutically acceptable salt or the above-mentioned pharmaceutical composition can also be used in the preparation of CYP4Z1 inhibitor drugs.

[0054] The above-mentioned N-benzyl imidazopyridine derivative or its pharmaceutically acceptable salt or the above-mentioned pharmaceutical composition can also be used in the preparation of drugs for preventing and / or treating breast cancer.

[0055] Principle of the invention: The present invention introduces a structural fragment of lauric acid, a catalytic substrate of CYP4Z1, into the lead compound N-benzylimidazolopyridine, and changes the length, connecting chain and ending group of the introduced fragment to obtain a novel compound molecule with a better structure capable of specifically inhibiting the activity of CYP4Z1 enzyme.

[0056] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The N-benzyl imidazopyridine derivatives of the present invention have a significant inhibitory effect on CYP4Z1 protease, and have good enzyme selectivity and liver microsomal stability, and have better drugability; (2) As tumor therapeutic drugs, they have great application prospects and can reduce or inhibit the activity of CYP4Z1 enzyme in breast cancer cells, thereby being used to treat and / or prevent problems such as poor prognosis and invasion and metastasis of breast cancer mediated by CYP4Z1 enzyme, thereby achieving the purpose of radical cure of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 is the general structural formula of the N-benzyl imidazopyridine derivative of the present invention;

[0058] Figure 2Statistical graphs of the in vitro antiproliferative activity results of compound 11 and 1-benzyl-1H-imidazo[4,5-c]pyridine, wherein A is the effect of different concentrations of compound 11 and lead compound 1-benzyl-1H-imidazo[4,5-c]pyridine on human breast ductal carcinoma cells (T47D); B is the effect of different concentrations of compound 11 and lead compound 1-benzyl-1H-imidazo[4,5-c]pyridine on human normal breast epithelial cells (MCF-10A); C is the effect of different concentrations of compound 11 and lead compound 1-benzyl-1H-imidazo[4,5-c]pyridine on human breast cancer cells (MCF-7); D is the effect of different concentrations of compound 11 and lead compound 1-benzyl-1H-imidazo[4,5-c]pyridine on human breast cancer cells (MDA-MB-231);

[0059] Figure 3 The results of the Transwell experiment of compound 11 and 1-benzyl-1H-imidazo[4,5-c]pyridine are shown in Figure A, where Figure B shows the effects of different concentrations of compound 11 and lead compound 1-benzyl-1H-imidazo[4,5-c]pyridine on human breast cancer cells (MCF-7), and Figure 11 shows the effects of different concentrations of compound 11 and lead compound 1-benzyl-1H-imidazo[4,5-c]pyridine on human breast cancer cells (MDA-MB-231).

[0060] Figure 4 The results of the Transwell experiment are statistically shown in Figure A, where Figure A shows the effects of different concentrations of compound 11 and the lead compound 1-benzyl-1H-imidazo[4,5-c]pyridine on human breast cancer cells (MCF-71), and Figure B shows the effects of different concentrations of compound 11 and the lead compound 1-benzyl-1H-imidazo[4,5-c]pyridine on human breast cancer cells (MDA-MB-231);

[0061] Figure 5Figures are the results of the scratch test, wherein Figure A is the result of the scratch test of compound 11 and the lead compound 1-benzyl-1H-imidazo[4,5-c]pyridine in human breast cancer cells (MCF-7), and Figure B is the result of the scratch test of compound 11 and the lead compound 1-benzyl-1H-imidazo[4,5-c]pyridine in human breast cancer cells (MDA-MB-231);

[0062] Figure 6 Figure 1 is a statistical graph of the scratch experiment results, where Figure A shows the effects of different concentrations of compound 11 and the lead compound 1-benzyl-1H-imidazo[4,5-c]pyridine on the scratches of human breast cancer cells (MCF-7), and Figure B shows the effects of different concentrations of compound 11 and the lead compound 1-benzyl-1H-imidazo[4,5-c]pyridine on the scratches of human breast cancer cells (MDA-MB-231). DETAILED DESCRIPTION

[0063] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0064] Example 1: Synthesis of Compounds 1 to 3

[0065] 1. Synthesis of Compound 1: 1-(4-methylbenzyl)-1H-imidazolyl[4,5-C]pyridine

[0066] Synthesis route:

[0067] 1H-Imidazolyl[4,5-C]pyridine (300.00 mg, 2.52 mmol) was dissolved in DMF, and NaH (72.52 mg, 3.02 mmol) was added under stirring at 0°C, stirred for 1 h, and then 1-(bromomethyl)-4-methylbenzene (512.66 mg, 2.77 mmol) was slowly added, and the reaction was stirred at room temperature for 2 h. 80 ml of water was added to the reaction system, and the mixture was extracted three times with ethyl acetate, and the organic phases were combined, washed with water, washed with saturated brine, and dried over anhydrous sodium sulfate. After concentration, column chromatography (DCM:MeOH=70:1, v / v) was performed to separate compound 1: 1-(4-methylbenzyl)-1H-imidazole[4,5-C]pyridine (52 mg, 9.25%). 1H NMR (400MHz, DMSO) δ8.88(d,J=1.1Hz,1H),8.62(s,1H),8.30(d,J=5.6Hz,1H),7.65(dd ,J=5.6,1.1Hz,1H),7.32-7.25(m,2H),7.16(d,J=7.8Hz,2H),5.54(s,2H),2.25(s,3H). 13 C NMR (75MHz, DMSO) δ148.31,147.02,141.13,137.34,134.40,133.41,131.36,129.34,127.70,114.32,47.96,20.64.

[0068] 2. Synthesis of Compounds 2-3

[0069] The above 1-(bromomethyl)-4-methylbenzene was replaced by 2.52 mmol of 1-(bromomethyl)-4-isopropylbenzene and 2.52 mmol of 1-(bromomethyl)-4-isobutylbenzene, and compounds 2 to 3 shown in Table 1 were synthesized according to the synthesis method in this example.

[0070] Table 1 Structural formula of compounds 1 to 3

[0071]

[0072] Example 2: Compound 4: Synthesis of ethyl 4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)-benzoate

[0073] Synthesis route:

[0074] 1H-imidazolyl[4,5-C]pyridine (1.00 g, 8.39 mmol) was dissolved in DMF, and NaH (302.17 mg, 12.59 mmol) was added under stirring at 0°C, stirred for 1 h, and methyl 4-bromomethylbenzoate (2.24 g, 9.23 mmol) was slowly added, and heated to 65°C for 5 h. After cooling, 80 ml of water was added to the reaction system, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with water, washed with saturated brine, and dried over anhydrous sodium sulfate. After concentration, column chromatography (DCM: MeOH = 70: 1, v / v) was performed to separate compound 4: methyl 4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)-benzoate (0.158 g, 6.69%). 1H NMR (400MHz, DMSO) δ8.97(d,J=1.0Hz,1H),8.58(s,1H),8.31(d,J=5.6Hz,1H),7.97-7.89(m,2H),7.5 6(dd,J=5.6,1.1Hz,1H),7.51-7.39(m,2H),5.65(s,2H),4.28(q,J=7.1Hz,2H),1.28(t,J=7.1Hz,3H). 13 C NMR (101MHz, DMSO) δ165.31,145.91,142.13,141.67,141.64,140.72,138.31,129.63,129.46,127.67,106.25,60.75,47.36,14.10.ESI-MS m / z: 282.1[M+H] + .

[0075] Example 3: Compound 5: Synthesis of ethyl 4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)-phenylacetate

[0076] Synthesis route:

[0077]

[0078] Step (1): 4-(bromomethyl)phenylacetic acid (6.00 g, 26.19 mmol) was dissolved in 50 ml of ethanol, and dichlorothionyl (2.85 ml, 39.29 mmol) was slowly added dropwise under stirring in an ice bath, and the mixture was heated to 65° C. and refluxed for 2 h. After cooling, the solvent was spin-dried, concentrated, and subjected to column chromatography (PE:EA=200:1, v / v) to separate the intermediate ethyl 4-(bromomethyl)phenylacetate (5.377 g, 79.90%).

[0079] Step (2): 1H-imidazolyl[4,5-C]pyridine (2.26 g, 19.00 mmol) was dissolved in DMF, and NaH (547.15 mg, 22.80 mmol) was added under stirring at 0°C, stirred for 1 h, 4-(bromomethyl)phenylacetic acid ethyl ester (5.377 g, 20.91 mmol) was slowly added, and heated to 65°C for reaction for 5 h. After cooling, 80 ml of water was added to the reaction system, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with water, washed with saturated brine, and dried over anhydrous sodium sulfate. After concentration, the mixture was separated by column chromatography (DCM:MeOH=50:1, v / v) to obtain compound 5: 4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)-phenylacetic acid ethyl ester (0.453 g, 8.07%). 1H NMR (300MHz, DMSO) δ8.86 (s, 1H), 8.47 (s, 1H), 8.21 (d, J = 5.2Hz, 1H), 7.51 (s, 1H), 7.17 ( d,J=9.4Hz,5H),5.43(s,2H),3.95(d,J=7.5Hz,2H),3.53(s,2H),1.05(d,J=7.7Hz,3H). 13 C NMR (75MHz, DMSO) δ170.97,145.81,142.06,141.53,140.73,138.29,134.98,134.21,129.76,127.56,106.32,60.26,47.42,39.87,14.02.ESI-MS m / z: 296.1[M+H] + .

[0080] Example 4: Synthesis of Compound 6: 4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)-benzoic acid

[0081] Synthesis route:

[0082] Dissolve 4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)-benzoic acid ethyl ester (100 mg, 0.355 mmol) and NaOH (42.65 mg, 1.07 mmol) in a mixed solvent of tetrahydrofuran and water (THF:H2O=4:1, v / v), and react at room temperature for 3-4 hours. After concentration, adjust the pH value to 6-7 with 1N hydrochloric acid. Filter, wash with water, and collect the filter residue to obtain compound 6: 4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)-benzoic acid (10 mg, 11.11%). 1 H NMR (400MHz, DMSO) δ12.99(s,1H),8.97(d,J=1.1Hz,1H),8.58(s,1H),8.31(d,J=5.5H z,1H),7.94-7.87(m,2H),7.58(dd,J=5.6,1.1Hz,1H),7.43-7.36(m,2H),5.64(s,2H). 13 C NMR (75MHz, DMSO) δ166.95,145.97,142.10,141.66,141.27,140.70,138.37,130.42,129.84,127.53,106.32,47.38.ESI-MS m / z: 254.1[M+H] + .

[0083] Example 5: Compound 7: Synthesis of 4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)-phenylacetic acid

[0084] Synthesis route: Ethyl 4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)-phenylacetic acid (100 mg, 0.339 mmol) and NaOH (40.63 mg, 1.02 mmol) were dissolved in a mixed solvent of tetrahydrofuran and water (THF:H2O=4:1, v / v), and reacted at room temperature for 3-4 hours. After concentration, the pH value was adjusted to 6-7 with 1N hydrochloric acid. Filtered, washed with water, and the filter residue was collected to obtain compound 7: 4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)-phenylacetic acid (19 mg, 20.99%). 1 H NMR (300MHz, DMSO) δ8.78(s,1H),8.39(s,1H),8.13(d,J=5.5Hz,1H),7.44(d,J=5.5Hz,1H),7.08(q,J=8.0Hz,4H),5.34(s,2H). 13 C NMR (75MHz, DMSO) δ172.57,145.84,142.05,141.54,140.74,138.30,134.86,134.75,129.86,127.49,106.38,47.46,40.27.ESI-MS m / z: 268.1[M+H] + .

[0085] Example 6: Synthesis of Compounds 8 to 10

[0086] 1. Compound 8: Synthesis of 4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)-phenylacetic acid butyl ester

[0087] Synthesis route:

[0088] Step (1): n-Butanol (257.96 ng, 3.48 mmol) and 4-(bromomethyl)phenylacetic acid (2.04 g, 8.91 mmol) were dissolved in THF, and EDCl (2.01 g, 10.44 mmol) and DMAP (42.52 mg, 0.348 mmol) were added under stirring, and stirred at room temperature for 5 h. After concentration, 80 ml of water was added to the reaction system, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with water, washed with saturated brine, and dried over anhydrous sodium sulfate. After concentration, column chromatography (PE:EA=80:1, v / v) was performed to separate the intermediate 4-(bromomethyl)phenylacetic acid butyl ester (535 mg, 53.93%).

[0089] Step (2): 1H-imidazolyl[4,5-C]pyridine (203.17 mg, 1.71 mmol) was dissolved in DMF, and NaH (49.11 mg, 2.05 mmol) was added under stirring at 0°C, stirred for 1 h, and 4-(bromomethyl)phenylacetic acid butyl ester (535 mg, 1.88 mmol) was slowly added, and heated to 65°C for reaction for 5 h. After cooling, 80 ml of water was added to the reaction system, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with water, washed with saturated brine, and dried over anhydrous sodium sulfate. After concentration, the mixture was separated by column chromatography (DCM:MeOH=70:1, v / v) to obtain compound 8: 4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)-phenylacetic acid butyl ester (38 mg, 6.90%). 1 H NMR (300MHz, DMSO) δ8.95(s,1H),8.56(s,1H),8.30(d,J=5.5Hz,1H),7.60(d,J=5.6Hz,1H),7.31-7.23(m,4H),5.5 1(s,2H),3.98(d,J=6.5Hz,2H),3.59(d,J=16.1Hz,2H),1.50(s,2H),1.31(d,J=16.1Hz,2H),0.82(t,J=7.6Hz,3H). 13 C NMR (75MHz, DMSO) δ171.08, 145.84, 142.09, 141.56, 138.40 (d, J = 13.6Hz), 135.00, 134.29, 129.80, 129.10, 128.30 ,127.73(d,J=12.8Hz),106.38,63.94(d,J=5.8Hz),47.48,30.06(d,J=11.6Hz),18.56,13.42(d,J=13.5Hz).ESI-MS m / z: 324.2[M+H] + .

[0090] 2. Synthesis of compounds 9-10

[0091] The above n-butanol was replaced by 3.48 mmol of n-hexanol and 3.48 mmol of n-octanol, respectively, and compounds 9 to 10 shown in Table 2 were synthesized according to the synthesis method of this example.

[0092] Table 2 Structural formula of compounds 8 to 10

[0093]

[0094] Example 7: Synthesis of Compounds 11 to 13

[0095] 1. Synthesis of Compound 11: 2-(4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)phenyl)-N-butylacetamide Synthesis route:

[0096]

[0097] Step (1): n-butylamine (532.14 mg, 7.28 mmol) and 4-(bromomethyl)phenylacetic acid (2.00 g, 8.7 mmol) were dissolved in THF, and EDCl (2.79 g, 14.56 mmol) and DMAP (106.73 mg, 0.87 mmol) were added under stirring, and stirred at room temperature for 5 h. After concentration, 80 ml of water was added to the reaction system, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with water, washed with saturated brine, and dried over anhydrous sodium sulfate. After concentration, column chromatography (PE:EA=2:1, v / v) was performed to separate the intermediate 2-(4-(bromomethyl)phenyl)-N-butylacetamide (987 mg, 47.68%).

[0098] Step (2): 1H-imidazolyl[4,5-C]pyridine (376.12 mg, 3.16 mmol) was dissolved in DMF, and NaH (90.93 mg, 3.79 mmol) was added under stirring at 0°C, stirred for 1 h, and 2-(4-(bromomethyl)phenyl)-N-butylacetamide (987 mg, 3.47 mmol) was slowly added, and heated to 65°C for reaction for 5 h. After cooling, 80 ml of water was added to the reaction system, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with water, washed with saturated brine, and dried over anhydrous sodium sulfate. After concentration, the mixture was separated by column chromatography (DCM:MeOH=30:1, v / v) to obtain compound 11: 2-(4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)phenyl)-N-butylacetamide (70 mg, 13.57%). 1H NMR (400MHz, DMSO) δ8.95(s,1H),8.55(s,1H),8.30(d,J=5.6Hz,1H),7.98(t,J=5.7Hz,1H),7.60(d,J=5.6Hz,1 H),7.28-7.16(m,4H),5.50(s,2H),2.99(q,J=6.5Hz,2H),1.28(dp,J=42.1,7.2Hz,6H),0.82(t,J=7.3Hz,3H). 13 C NMR(101MHz,DMSO)δ169.66,145.77,142.04,141.51,140.74,138.26,136.41,13 4.40,129.34,127.46,106.34,47.48,42.00,38.26,31.15,19.51,13.60.ESI-MS m / z: 323.2[M+H] + .

[0099] 2. Synthesis of compounds 12-13

[0100] The above n-butylamine was replaced by 7.28 mmol of n-hexylamine and 7.28 mmol of n-octylamine, respectively, and compounds 12 to 13 shown in Table 3 were synthesized according to the synthesis method of this example.

[0101] Table 3 Structural formula of compounds 11 to 13

[0102]

[0103] Example 8: Synthesis of Compounds 14 to 16

[0104] 1. Compound 14: Synthesis of 4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)phenethyl butyrate

[0105] Synthesis route:

[0106]

[0107] Step (1): dissolve the compound 4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)-phenylacetic acid ethyl ester (280.00 mg, 0.948 mmol) synthesized in Example 3 in anhydrous THF, replace with Ar gas, slowly add LiAlH4 (89.95 mg, 2.37 mmol) under stirring at 0°C, and stir at room temperature for 4 hours. Add an appropriate amount of saturated aqueous solution of (2R,3R)-2,3-dihydroxysuccinate potassium sodium tetrahydrate to the reaction system under ice bath for quenching, and stir at room temperature for 30 minutes. Extract with ethyl acetate three times, combine the organic phases, wash with water, wash with saturated brine, and dry with anhydrous sodium sulfate. After concentration, column chromatography (DCM:MeOH=20:1, v / v) was performed to separate the intermediate 2-(4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)phenyl)ethyl-1-ol (93 mg, 38.75%).

[0108] Step (2): 2-(4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)phenyl)ethyl-1-ol (93 mg, 0.367 mmol) and n-butyric acid (82.76 mg, 0.939 mmol) were dissolved in THF, EDCl (211.06 mg, 1.101 mmol) and DMAP (4.48 mg, 0.037 mmol) were added with stirring, and the mixture was stirred at room temperature for 3 h. 80 ml of water was added to the reaction system, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with water, washed with saturated brine, and dried over anhydrous sodium sulfate. After concentration, column chromatography (DCM:MeOH=20:1, v / v) was performed to separate compound 14: 4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)phenylethylbutyrate (113.925 mg, 95.95%). 1 H NMR (300MHz, DMSO) δ8.97 (s, 1H), 8.57 (s, 1H), 8.31 (d, J = 5.6Hz, 1H), 7.65-7.57 (m, 1H), 7.32-7.17 (m, 4H), 5.51 (s, 2H), 4.18(t,J=6.7Hz,2H), 2.83(t,J=6.8Hz,2H), 2.17(t,J=7.2Hz,2H), 1.47(dh,J=22.0,7.3Hz,2H), 0.74(t,J=7.4Hz,3H). 13C NMR(75MHz,DMSO)δ172.65,145.95,141.98,141.46,140.69,138.40,137.91,134.44,1 29.29,127.68,106.46,63.99,47.55,35.34,34.01,17.93(d,J=7.7Hz),13.29.ESI-MS m / z: 324.2[M+H] + .

[0109] 2. Synthesis of compounds 15-16

[0110] The above-mentioned n-butyric acid was replaced by 0.939 mmol of n-hexanoic acid and 0.939 mmol of n-octanoic acid, respectively, and compounds 15 to 16 shown in the table were synthesized according to the synthesis method in this example.

[0111] Table 4 Structural formula of compounds 14 to 16

[0112]

[0113]

[0114] Example 9: Compound 17: Synthesis of 1-(4-(tert-butoxymethyl)phenyl)-1H-imidazolyl[4,5-c]piperidine

[0115] Synthesis route:

[0116]

[0117] Step (1): 1H-imidazolyl[4,5-C]pyridine (538.62 mg, 4.52 mmol) was dissolved in DMF, and NaH (130.20 mg, 5.43 mmol) was added under stirring at 0°C, stirred for 1 h, (4-(bromomethyl)phenyl)methanol (1.00 g, 4.97 mmol) was slowly added, and heated to 65°C for 5 h. After cooling, 80 ml of water was added to the reaction system, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with water, washed with saturated brine, and dried over anhydrous sodium sulfate. After concentration, column chromatography (DCM:MeOH=30:1, v / v) was performed to separate the intermediate 4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)phenyl)methanol (100 mg, 9.26%).

[0118] Step (2): The intermediate 4-((1H-imidazolyl[4,5-c]pyridinyl)-1-methyl)phenyl)methanol (100 mg, 0.418 mmol) obtained in step (1) was dissolved in 2 ml of DMF, and NaH (12.04 mg, 0.502 mmol) was added under stirring at 0°C, stirred for 1 h, and 1-bromobutane (114.53 mg, 0.836 mmol) was slowly added, and the reaction was carried out at room temperature for 3 h. After the reaction was completed, it was extracted three times with ethyl acetate, the organic phases were combined, washed with water, washed with saturated brine, and dried over anhydrous sodium sulfate. After concentration, column chromatography (DCM:MeOH=40:1, v / v) was performed to separate compound 17: 1-(4-(tert-butoxymethyl)phenyl)-1H-imidazolyl[4,5-c]pyridine (100 mg, 9.26%). 1 H NMR (400MHz, DMSO) δ8.95(d,J=1.1Hz,1H),8.56(d,J=1.4Hz,1H),8.30(d,J=5.6Hz,1H),7.60(dt,J=5.6,1.6Hz,1H),7.32-7.28(m ,4H),5.53(d,J=3.5Hz,2H),4.39(s,2H),3.42-3.36(m,2H),1.48(tt,J=8.4,6.3Hz,2H),1.39-1.18(m,3H),0.85(q,J=7.5Hz,3H). 13 C NMR(75MHz,DMSO)δ145.85,142.08,141.57,140.76,138.61,138.31,135.47,127.88(d,J=9.1Hz ),127.48(d,J=11.3Hz),106.39,71.50(d,J=15.6Hz),69.38,47.54,31.29,18.91,13.78.ESI-MS m / z: 296.2[M+H] + .

[0119] Example 10: Synthesis of Compounds 18 to 21

[0120] 1. Compound 18: Synthesis of 1-(4-(2-ethoxyethyl)phenyl)-1H-imidazolyl[4,5-c]pyridine

[0121] Synthesis route:

[0122]

[0123] Step (1): dissolve the compound 4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)-phenylacetic acid ethyl ester (280.00 mg, 0.948 mmol) synthesized in Example 3 in anhydrous THF, replace with Ar gas, slowly add LiAlH4 (89.95 mg, 2.37 mmol) under stirring at 0°C, and stir at room temperature for 4 hours. Add an appropriate amount of saturated aqueous solution of (2R,3R)-2,3-dihydroxysuccinate potassium sodium tetrahydrate to the reaction system under ice bath for quenching, and stir at room temperature for 30 minutes. Extract with ethyl acetate three times, combine the organic phases, wash with water, wash with saturated brine, and dry with anhydrous sodium sulfate. After concentration, column chromatography (DCM:MeOH=20:1, v / v) was performed to separate the intermediate 2-(4-((1H-imidazolyl[4,5-C]pyridinyl)-1-methyl)phenyl)ethyl-1-ol (93 mg, 38.75%).

[0124] Step (2): 2-(4-((1H-imidazolyl[4,5-c]pyridinyl)-1-methyl)phenyl)ethyl-1-ol (93 mg, 0.367 mmol) was dissolved in anhydrous DMF, and NaH (10.55 mg, 0.439 mmol) was added under stirring at 0°C, stirred for 1 h, and 1-bromoethane (80.06 mg, 0.735 mmol) was slowly added, and the reaction was carried out at room temperature for 3 h. 80 ml of water was added to the reaction system, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with water, washed with saturated brine, and dried over anhydrous sodium sulfate. After concentration, the mixture was separated by column chromatography (DCM:MeOH=20:1, v / v) to obtain compound 18: 1-(4-(2-ethoxyethyl)phenyl)-1H-imidazolyl[4,5-c]pyridine (58.12 mg, 56.27%). 1 H NMR (400MHz, DMSO) δ8.96(d,J=1.0Hz,1H),8.57(s,1H),8.31(d,J=5.6Hz,1H),7.61(dd,J=5.6,1.1Hz,1H),7.29 -7.16(m,5H),5.49(s,2H),3.49(t,J=6.9Hz,2H),3.42-3.30(m,9H),2.74(t,J=6.9Hz,2H),1.04(t,J=7.0Hz,3H) 13 C NMR (75MHz, DMSO) δ145.80,142.05,141.53,140.74,139.02,138.30,134.12,129.22,127.51,106.37,70.46,65.20,47.51,35.20,15.05.ESI-MS m / z: 282.2[M+H] + .

[0125] 2. Synthesis of compounds 19-21

[0126] The above 1-bromoethane was replaced by: 0.735 mmol of 1-bromobutane, 0.735 mmol of 1-bromohexane, 0.735 mmol of 1-bromooctane, and the compounds 19 to 21 shown in the table were synthesized according to the synthesis method of this example.

[0127] Table 5 Structural formula of compounds 18 to 21

[0128]

[0129] Example 11: Synthesis of Compounds 22 to 25

[0130] 1. Compound 22: Synthesis of 1-(4-pentylphenyl)-1H-imidazolyl[4,5-c]pyridine

[0131] Synthesis route:

[0132]

[0133] Step (1): 4-n-pentylbenzoic acid (3.00 g, 15.60 mmol) was dissolved in anhydrous THF, replaced with Ar gas, and LiAlH4 (710.67 mg, 18.72 mmol) was slowly added under stirring at 0°C, and stirred at room temperature for 4 h. Under an ice bath, an appropriate amount of saturated aqueous solution of (2R, 3R)-2,3-dihydroxysuccinate potassium sodium tetrahydrate was added to the reaction system for quenching, and stirred at room temperature for 30 min. Extracted three times with ethyl acetate, the organic phases were combined, washed with water, washed with saturated brine, and dried over anhydrous sodium sulfate. After concentration, column chromatography (PE: EA = 20: 1, v / v) was performed to separate the intermediate 4-n-pentylbenzyl alcohol (2.657 g, 95.58%).

[0134] Step (2): Dissolve the intermediate 4-n-pentylbenzoic acid (2.657 g, 14.90 mmol) in DCM, slowly add PBr3 (1.611 g, 5.952 mmol) at 0°C, and react at room temperature for 4 hours after adding PBr3. Add 80 ml of water to the reaction system, extract with dichloromethane three times, combine the organic phases, wash with water, wash with saturated brine, and dry with anhydrous sodium sulfate. After concentration, column chromatography (PE:EA=200:1, v / v) was performed to separate the intermediate 1-(bromomethyl)-4-pentylbenzene (3.31 g, 92.11%).

[0135] Step (3): 1H-imidazolyl[4,5-c]pyridine (3.01 g, 12.47 mmol) was dissolved in DMF, and NaH (359.04 mg, 14.96 mmol) was added under stirring at 0°C, stirred for 1 h, (4-(bromomethyl)phenyl)methanol (3.31 g, 13.72 mmol) was slowly added, and heated to 65°C for 4 h. After cooling, 80 ml of water was added to the reaction system, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with water, washed with saturated brine, and dried over anhydrous sodium sulfate. After concentration, the mixture was separated by column chromatography (DCM:MeOH=50:1, v / v) to obtain compound 22: 1-(4-pentylphenyl)-1H-imidazolyl[4,5-c]pyridine (777.54 mg, 22.32%). 1 HNMR (300MHz, DMSO) δ8.98(s,1H),8.58(s,1H),8.33(d,J=5.6Hz,1H),7.62(dd,J=5.6,1.1Hz,1H),7.26(d,J=7.9Hz,2H),7.15 (d,J=8.1Hz,2H),5.50(s,2H),2.50(t,J=7.6Hz,3H),1.49(p,J=7.4Hz,2H),1.24(qt,J=8.0,3.6Hz,5H),0.81(t,J=6.8Hz,4H). 13 C NMR(75MHz,DMSO)δ145.77,142.15,142.05,141.50,140.75,138.29,133.69 ,128.64,127.54,106.33,47.51,34.72,30.84,30.54,21.89,13.84.ESI-MS m / z: 280.2[M+H] + .

[0136] 2. Synthesis of Compounds 23-25

[0137] The above 1-bromoethane was replaced by: 0.735 mmol of 1-bromobutane, 0.735 mmol of 1-bromohexane, 0.735 mmol of 1-bromooctane, and the compounds 19 to 21 shown in the table were synthesized according to the synthesis method of this example.

[0138] Table 6 Structural formula of compounds 18 to 21

[0139]

[0140]

[0141] Example 12 Evaluation of pharmacological activity of compounds

[0142] Determination of CYP4Z1 and CYP4F12 enzyme inhibitory activity of N-benzyl imidazopyridine derivatives:

[0143] (1) Add 12.5 μL of test compounds 1 to 66 (1 μM) to each well of a 96-well plate.

[0144] (2) CYP reaction group: Add 12.5 μL of 4×CYP (CYP4Z1 enzyme or CYP4F12 enzyme) to each well and mix gently.

[0145] Negative control group: add 12.5 μL of water to each well.

[0146] Positive control group: 12.5 μL of 4×CYP (CYP4Z1 enzyme or CYP4F12 enzyme) was added to each well.

[0147] (3) Preincubate at 37°C for 10 minutes.

[0148] (4) Add 25 μL of 2×NADPH (Progema, catalog number: V9510) to the CYP reaction group and negative control group respectively to initiate the CYP reaction.

[0149] (5) Incubate at the same temperature as the preincubation for 30 minutes.

[0150] (6) Add 50 μL of recombinant luciferin detection reagent (Promega, catalog number: V8752) to the CYP reaction group, negative control group, and positive control group, respectively.

[0151] (7) Oscillate the 96-well plate on a shaker for 10 seconds.

[0152] (8) Incubate at room temperature for 20 minutes to stabilize the luminescent signal.

[0153] (9) Use a photometer to measure the luminescence.

[0154] Note: "2×" and "4×" refer to reagents prepared at 2 or 4 times the final reagent concentration, respectively.

[0155] Table 7 Inhibitory activity of compounds on CYP4Z1 enzyme and CYP4F12 (1 μM inhibition rate)

[0156]

[0157]

[0158]

[0159]

[0160] Example 13 In vitro antiproliferative activity test

[0161] In vitro antiproliferative activity test of the representative compound 2-1 (compound 11) of the N-benzyl imidazopyridine derivative and the lead compound 1-benzyl-1H-imidazo[4,5-c]pyridine (compound 0, N-benzyl imidazopyridine) by CCK-8 method:

[0162] (1) Human breast ductal carcinoma cells (T47D), normal human breast epithelial cells (MCF-10A), breast cancer cells (MCF-7), and MDA-MB-231 cells stored in the laboratory were cultured at 5×10 3 Cells / well were implanted into a 96-well plate. After incubation for 12 hours, 100 μL of the test compound solution (compound 11 or lead compound 1-Ben) with concentrations of 0.01, 0.1, 1, 10, and 100 μM were added, respectively, and then cultured for another 48 hours.

[0163] (2) Add 200 μL of DMEM culture medium (Prunosai, catalog number: PM150220) containing 10% CCK-8 to each well and continue incubating in a cell culture incubator for 4 hours.

[0164] (3) Measure the absorbance at 450nm. Figure 2 As shown, compound 11 and lead compound 1-Ben had no cytotoxicity to human breast ductal carcinoma cells (T47D), human normal breast epithelial cells (MCF-10A), and human breast cancer cells (MCF-7, MDA-MB-231).

[0165] Example 14 Effects of N-benzylimidazolopyridine derivatives on cell invasion and migration

[0166] The representative compound 2-1 (compound 11) of N-benzyl imidazopyridine derivative and the lead compound 1-benzyl-1H-imidazo[4,5-c]pyridine (compound 0) were used to detect cell invasion and migration using the Transwell method:

[0167] (1) Place the Matrigel (Corning, Catalog No.: 356234) in a 4°C refrigerator one day in advance to thaw it, and bury the Matrigel in a foam box filled with crushed ice; since the concentration of Matrigel required for the invasion experiment is 0.3 mg / mL and the product concentration is 12 mg / mL, it is diluted 40 times. It can only be diluted directly, not multiple dilutions (gradient dilutions), and diluted with serum-free DMED medium (Pnosel, Catalog No.: PM150220), all of which need to be pre-cooled. All the above operations must be performed on ice.

[0168] (2) Add 100 μL of Matrigel to each well of the Transwell chamber (Corning, Catalog No.: 3422), and then carefully and slowly place it in a cell culture incubator (37°C) for 2 h.

[0169] (3) Human breast cancer cells in the logarithmic growth phase: MCF-7 cells and MDA-MB-231 cells were digested with trypsin, and the trypsin was neutralized with 2 ml of serum-free DMED medium. The cells were collected by centrifugation, the supernatant was removed, and 1 ml of serum-free DMEM medium was added to resuspend the cells. The cell density in the cell suspension was adjusted to 1×10 6 cell / mL, so that the number of cells in each sample is the same and reduce experimental errors.

[0170] (4) Take 600ul of culture medium containing 20% ​​FBS and add it to the lower chamber. Set up 3 replicate wells for each group.

[0171] (5) Three replicate wells were set in each group. 100 μL of cell suspension was added to the upper chamber of each replicate well, and then 100 μL of drug solution diluted in serum-free DMEM was added and blown evenly. The drugs were compound 11 and lead compound 1-Ben, and the concentrations of compound 11 in MCF-7 cells were 2.5 μM and 10 μM, respectively, and the concentration of lead compound 1-Ben was 10 μM; the concentrations of compound 11 in MDA-MB-231 cells were 0.25 μM and 1.0 μM, respectively, and the concentration of lead compound 1-Ben was 1.0 μM.

[0172] (6) After the Transwell migration chamber was placed in a 37°C, 5% CO2 incubator for 36 h, the chamber was removed, the upper culture medium of the chamber was aspirated, and the cells were washed once with PBS. The cells on the polycarbonate microporous membrane at the bottom of the chamber were fixed with 800 μL 70% ethanol at room temperature for 5 min, and then washed once with PBS. Then, the MCF-7 cells and MDA-MB-231 cells were immersed in 200 μL 0.1% crystal violet for staining for 30 min (added to the lower chamber).

[0173] (7) Wash with PBS to remove excess dye, gently wipe off the cells in the upper chamber that have not passed through the hole with a cotton swab, and wipe off the excess crystal violet to prevent it from affecting the subsequent quantitative results, and let it air dry naturally. Observe and take pictures under a 200x microscope. The results are as follows: Figure 3 As shown. Solvent in the figure represents the control group without drug addition, 1-Ben in Figure A represents the addition of 10μM lead compound 1-Ben, 2.5μM represents the addition of 2.5μM compound 11, and 10μM represents the addition of 10μM compound 11; 1-Ben in Figure B represents the addition of 1.0μM lead compound 1-Ben, 0.25μM represents the addition of 0.25μM compound 11, and 1.0μM represents the addition of 1.0μM compound 11.

[0174] (8) Decolorize with 200 μL of 95% ethanol to completely elute the crystal violet, and read the OD value of the eluate at 570 nm on an ELISA reader to avoid errors caused by uneven cell penetration under the field of view. The corresponding OD value results are statistically shown as follows Figure 4 The statistical results are based on the control group solvent as the base 1, and the solvent in the figure is the control group without drug addition.

[0175] The results showed that both compound 11 and lead compound 1-Ben could significantly inhibit the invasion and migration of human breast cancer cells (MCF-7, MDA-MB-231), and compound 11 had a better inhibitory effect on both cells than lead compound 1-Ben. In Figure A, 1-Ben represents the addition of 10μM lead compound 1-Ben, 2.5μM represents the addition of 2.5μM compound 11, and 10μM represents the addition of 10μM compound 11; in Figure B, 1-Ben represents the addition of 1.0μM lead compound 1-Ben, 0.25μM represents the addition of 0.25μM compound 11, and 1.0μM represents the addition of 1.0μM compound 11.

[0176] Example 13 Scratch assay to detect cell invasion and migration

[0177] The scratch assay of representative compound 2-1 (compound 11) of N-benzylimidazole derivatives and lead compound 1-Ben was used to detect cell invasion and migration:

[0178] (1) Human breast cancer cells (5×10 5 MCF-7 and 5×10 5 The cells were inoculated into six-well plates and waited for the cells to adhere. After the negative control group was established, 2.5 mL of culture medium was added to the attached cells according to the group settings, wherein the culture medium of the negative control (solvent) did not contain drugs; the culture medium of the MCF-7 cell experimental group contained 2.5 μM compound 11, 10 μM compound 11 or 10 μM lead compound 1-Ben; the culture medium of the MDA-MB-231 cell experimental group contained 0.25 μM compound 11, 1.0 μM compound 11 or 1.0 μM lead compound 1-Ben.

[0179] (2) 24 h after drug exposure, cells were digested with 0.25% trypsin and collected;

[0180] (3) Take a new six-well plate, re-inoculate the collected cells, measure the cell aggregation to 60%, and then use a sterile pipette to evenly draw a line in the six-well plate;

[0181] (4) Wash the floating cells with PBS, then replace with fresh culture medium and continue to culture in a cell culture incubator; after culturing MCF-7 cells for 24 hours, remove the cells, take photos (magnification 100×) and measure the cell migration distance; after culturing MDA-MB-231 cells for 48 hours, remove the cells, take photos (magnification 100×) and measure the cell migration distance. The results are shown in the figure below. Figure 5 As shown, through measurement and statistics, the results are as follows Figure 6 shown.

[0182] The experiment proved that both compound 11 and lead compound 1-Ben can significantly inhibit the migration of human breast cancer cells, and the inhibitory effect of compound 11 is better than that of lead compound 1-Ben. In Figure A, 1-Ben represents the addition of 10μM lead compound 1-Ben, 2.5μM represents the addition of 2.5μM compound 11, and 10μM represents the addition of 10μM compound 11; in Figure B, 1-Ben represents the addition of 1.0μM lead compound 1-Ben, 0.25μM represents the addition of 0.25μM compound 11, and 1.0μM represents the addition of 1.0μM compound 11.

[0183] Other compounds were also tested according to the methods of Examples 11 to 13, and the results were the same. Therefore, pharmacological experiments have shown that the compounds in general formula (I) can significantly inhibit the activity of the target enzyme CYP4Z1, and the inhibitory effect on the CYP4F12 enzyme is poor. Since the inhibition of CYP4Z1 only affects the invasion and migration of breast cancer cells, the CCK-8 experiment proves that the representative compound has no cytotoxicity, and the Transwell experiment and the scratch experiment prove that the representative compound can significantly inhibit the invasion and migration of breast cancer cells. Therefore, the N-benzyl imidazopyridine derivatives of the present invention have a significant inhibitory effect on CYP4Z1 protease, and have good enzyme selectivity and liver microsomal stability; as tumor therapeutic drugs, they have great application prospects, can reduce or inhibit the activity of CYP4Z1 enzymes in breast cancer cells, and are thus used to treat and / or prevent problems such as poor prognosis and invasion and metastasis of breast cancer mediated by CYP4Z1 enzymes, thereby achieving the purpose of radical cure of breast cancer.

Claims

1. An N-benzyl imidazopyridine derivative, characterized in that: The derivative is shown in Formula I: Wherein, R1 is selected from: hydrogen, halogen, C1-C8 alkyl, C1-C8 alkoxy, C1-C8 haloalkyl, amino or C1-C8 alkyl substituted amino; n is 1-8; R2 is selected from: an ester group, a C1-C8 alkyl group, an oxygen atom or an amide group; R3 is selected from the group consisting of hydrogen, carboxyl, C1-C7 alkoxycarbonyl, C1-C7 alkyl or amide.

2. The N-benzyl imidazopyridine derivative according to claim 1, characterized in that: The R1 is selected from: hydrogen, halogen, C1-C4 alkyl, C1-C4 alkoxy; n is 1-7; R2 is selected from: an ester group, a C1-C4 alkyl group, an oxygen atom or an amide group; R3 is selected from the group consisting of hydrogen, carboxyl, methoxycarbonyl, ethoxycarbonyl, and C1-C7 alkyl.

3. The N-benzyl imidazopyridine derivative according to claim 1, characterized in that: The R1 is selected from: hydrogen, fluorine, chlorine, bromine, methyl, trifluoromethyl; n is 1-7; R2 is selected from: methylene, ester group, oxygen atom or amide group; The R3 is selected from the group consisting of hydrogen, hydroxyl, carboxyl, and C1-C7 alkyl.

4. The N-benzyl imidazopyridine derivative according to claim 1, characterized in that: The derivative or its pharmaceutically acceptable salt or racemate is selected from the following compounds:

5. The N-benzyl imidazopyridine derivative according to claim 1, characterized in that: The derivatives also include optically active forms or racemates, diastereoisomer mixtures, solvates, prodrugs, pharmaceutically acceptable salts or co-crystals thereof.

6. A method for preparing the N-benzyl imidazopyridine derivatives according to any one of claims 1 to 5, characterized in that: The preparation method of the derivative is: When the N-benzyl imidazopyridine derivative is an ester or an amine, the preparation method is: Get raw materials Under the action of SOCl2, an ester intermediate is formed with an alcohol; the ester intermediate is reacted with a long-chain or branched alcohol or amine to obtain an ester or amine intermediate, which is then reacted with 1H-imidazole [4,5-C] pyridine under the action of sodium hydride to obtain the ester or amine N-benzyl imidazopyridine derivative; When the N-benzyl imidazopyridine derivative is an ester, the preparation method is: Get raw materials Under the action of SOCl2, an ester intermediate is formed with an alcohol; the ester intermediate is reacted with 1H-imidazole [4,5-C] pyridine under the action of sodium hydride to obtain N-benzyl imidazopyridine, which is reduced to an alcohol with LiAlH4 and then reacted with a long-chain acid to obtain the ester N-benzyl imidazopyridine derivative; When the N-benzyl imidazopyridine derivative is an ether, the preparation method is: Get raw materials Under the action of SOCl2, an ester intermediate is formed with an alcohol; the ester intermediate is reacted with 1H-imidazole [4,5-C] pyridine under the action of sodium hydride to obtain N-benzyl imidazopyridine, which is reduced to an alcohol with LiAlH4 and then reacted with a long-chain 1-bromoalkane to obtain the ether N-benzyl imidazopyridine derivative; When the N-benzyl imidazopyridine derivative is an alkyl derivative, the preparation method is: The raw material 4-n-pentylbenzoic acid, 4-n-hexylbenzoic acid, 4-n-heptylbenzoic acid or 4-n-octylbenzoic acid is reduced to alcohol by LiAlH4 and then reacted with PBr3 to generate a benzyl bromide intermediate; the benzyl bromide intermediate is reacted with 1H-imidazole [4,5-C] pyridine under the action of sodium hydride to obtain an alkyl N-benzyl imidazopyridine derivative.

7. The preparation method according to claim 6, characterized in that: The preparation method is specifically: When R3 is a carboxylic acid, (1-1) p-bromomethylbenzene alkyl acid is reacted with thionyl chloride to generate intermediate 1; the alkyl acid in p-bromomethylbenzene alkyl acid is a C1-C7 alkyl acid; (1-2) The intermediate 1 described in step (1-1) reacts with 1H-imidazole [4,5-C] pyridine under the action of sodium hydride to generate intermediate 1; (1-3) Intermediate 1 and alkyl alcohol undergo condensation reaction to generate intermediate 2; the alkyl alcohol is C1-C7 alkyl alcohol; (1-4) The intermediate 2 described in step (1-3) undergoes a hydrolysis reaction under the action of sodium hydroxide to obtain an N-benzyl imidazopyridine derivative; When R2 is an ester group and R3 is a C1-C7 alkyl group, (2-1) condensation reaction of p-bromomethylbenzene alkyl acid and alkyl alcohol to generate an intermediate; the alkyl alcohol is C1-C7 alkyl alcohol; (2-2) reacting the intermediate in step (2-1) with 1H-imidazole [4,5-C] pyridine to produce the N-benzyl imidazopyridine derivative; When R2 is an oxygen atom and R3 is a C1-C7 alkyl group, (3-1) p-bromomethyl alkyl acid is reacted with thionyl chloride to generate intermediate 1; the alkyl acid in p-bromomethyl alkyl acid is C1-C7 alkyl acid; (3-2) The intermediate 1 described in step (3-1) reacts with 1H-imidazole [4,5-C] pyridine under the action of sodium hydride to generate benzyl imidazopyridine; (3-3) The N-benzyl imidazopyridine described in step (3-2) is reduced to an alcohol to obtain intermediate 2; (3-4) The intermediate 2 described in step (3-3) reacts with 1-bromoalkane to generate the corresponding N-benzyl imidazopyridine derivative; the alkane in the 1-bromoalkane is a C1-C7 alkane; When R2 is amide and R3 is C1-C7 alkyl: (4-1) a condensation reaction of p-bromomethylbenzene alkyl acid and an alkylamine to generate an intermediate; the alkylamine is a C1-C7 alkylamine; (4-2) reacting the intermediate obtained in step (4-1) with 1H-imidazole [4,5-C] pyridine to produce the N-benzyl imidazopyridine derivative; When R2 is C1-C7 alkyl and R3 is hydrogen, (5-1) p-Alkylbenzoic acid is reduced with LiAlH4 to intermediate 1; (5-2) The intermediate 1 described in step (5-1) undergoes a substitution reaction with PBr3 to obtain intermediate 2; (5-3) The intermediate 2 described in step (5-2) reacts with 1H-imidazole [4,5-C] pyridine under the action of sodium hydride to generate the corresponding N-benzyl imidazopyridine derivative.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the N-benzyl imidazopyridine derivative according to claim 1, its optically active form or racemate, diastereoisomer mixture, solvate, prodrug, metabolite pharmaceutically acceptable salt or cocrystal and a pharmaceutically acceptable carrier.

9. Use of the N-benzyl imidazopyridine derivative or a pharmaceutically acceptable salt thereof according to claim 1 or the pharmaceutical composition according to claim 8 in the preparation of a CYP4Z1 inhibitor drug.

10. Use of the N-benzyl imidazopyridine derivative or a pharmaceutically acceptable salt thereof according to claim 1 or the pharmaceutical composition according to claim 8 in the preparation of a drug for preventing and / or treating breast cancer.

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