Novel amidino-containing iNOS inhibitor with neuroprotection function as well as preparation method and application of novel amidino-containing iNOS inhibitor

Abstract

The invention discloses a novel amidino-containing iNOS inhibitor with a neuroprotection function as well as a preparation method and application of the novel amidino-containing iNOS inhibitor. The structural formula of the novel amidino-containing iNOS inhibitor is I, II or III. The compound takes an iNOS inhibitor 1400W, a neuroprotective agent NBP or a free radical scavenger Eda fragment as a skeleton, combines the advantages of NBP and Eda, fuses the characteristics of a selective iNOS inhibitor 1400W, can selectively inhibit iNOS, has a neuroprotective capability, plays a synergistic role, and better treats ischemic stroke. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a compound, in particular to a novel amidine-containing iNOS inhibitor with neuroprotective function and a preparation method and application thereof. Background Art

[0002] Stroke, also known as a stroke (Mol. Basis. Dis., 2020, 1866(4): 165-260.), is caused by blocked or ruptured cerebral blood vessels, which lead to insufficient or interrupted cerebral blood flow, thus causing brain tissue damage (J. Clin. Neurosci., 2021, 93: 174-182.). The symptoms of stroke may vary from patient to patient. Common symptoms include sudden weakness, numbness or fatigue in the limbs, sudden difficulty speaking, difficulty understanding, loss of vision or blurring, as well as severe headache, dizziness, loss of balance, etc. (Neurol. India, 2021, 69(2): 272-283). Stroke is a serious condition that usually requires emergency treatment.

[0003] Stroke is divided into ischemic stroke and hemorrhagic stroke (Lancet, 2020, 396 (10258): 1223-1249.). Ischemic stroke refers to an acute cerebrovascular disease in which vascular damage and occlusion damage the blood oxygen supply to the brain due to multiple causes, ischemic hypoxic necrosis occurs at the ischemic part of the brain tissue, and the patient suffers from neurological deficits (Circulation, 2018, 137 (12): e67-e492.). With the rapid increase of population aging and the continuous increase of cardiovascular diseases, the incidence of acute ischemic stroke continues to rise, causing a significant burden on health (J. Stroke Cerebrovasc., 2022, 31 (4): 106274.). In Western countries, its incidence is about 10 times that of hemorrhagic stroke. Stroke is the second most common cause of death and the third most common cause of disability in the world. Globally, the absolute number of deaths and disability caused by stroke has been increasing, placing a growing burden on low- and middle-income countries (Int. J. Mol. Med., 2022, 49(2):1-9).

[0004] Currently used drugs in clinical practice, such as the neuroprotectant butylphthalide (NBP) and the free radical scavenger edaravone (Eda), show certain efficacy in anti-cerebral ischemia, but each has significant limitations. Although NBP has multi-target anti-cerebral ischemia activity, its therapeutic effect is not outstanding (Neurosci. Lett., 2012, 516 (2): 247-252); Eda, due to its poor water solubility and low oral bioavailability, often needs to be administered by intravenous injection, which increases the cost and inconvenience of treatment. 1400W, as a highly efficient and selective iNOS inhibitor, has shown good iNOS inhibitory activity in cell experiments, but its ability to improve cerebral blood flow is weak and fails to achieve ideal anti-cerebral ischemia efficacy (Neurochem. Res., 2011, 36: 476-486.).

[0005] Summary of the invention

[0006] Purpose of the invention: The first purpose of the present invention is to provide a novel amidine-containing iNOS inhibitor with neuroprotective function, which selectively inhibits iNOS and simultaneously has neuroprotective ability; the second purpose of the present invention is to provide a method for preparing the novel amidine-containing iNOS inhibitor with neuroprotective function; the third purpose of the present invention is the application of the novel amidine-containing iNOS inhibitor with neuroprotective function.

[0007] The novel iNOS inhibitor containing an amidine group and having a neuroprotective function described in the present invention is a compound represented by Formula I or II or a pharmaceutically acceptable salt thereof;

[0008]

[0009] Among them, R 1 Selected from

[0010] R 2 Selected from -CH2- or R 3 Selected from R 4 Selected from

[0011] Preferably, the compound represented by formula I is selected from the following compounds:

[0012] 3-(Acetimidomethyl)-N-(1-butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)benzamide

[0013]

[0014] N-(1-Butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-3-((N'-hydroxyacetimidyl)methyl)benzamide

[0015]

[0016] 3-Acetimido-N-(1-butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)benzamide

[0017]

[0018] N-(1-Butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-3-(N'-hydroxyacetimidyl)benzamide

[0019]

[0020] N-(3-(((1-butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)amino)methyl)phenyl)acetamidine

[0021]

[0022] N-(3-(((1-butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)amino)methyl)phenyl)-N'-hydroxyacetamidine

[0023]

[0024] N-(3-(((1-butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)amino)methyl)benzyl)acetamidine

[0025]

[0026] N-(3-(((1-butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)amino)methyl)benzyl)-N'-hydroxyacetamidine

[0027]

[0028] 4-(3-(((1-butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)amino)methyl)benzyl)-3-methyl-1,2,4-oxadiazol-5(4H)-one

[0029]

[0030] N-(1-Butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-3-((3-methyl-5-oxo-1,2,4-oxadiazol-4(5H)-yl)methyl)benzamide

[0031]

[0032] Preferably, the compound represented by formula II is selected from the following compounds:

[0033] N-(1-Butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)acetamidine

[0034]

[0035] N-(1-Butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-N'-hydroxyacetamidine

[0036]

[0037] Preferably, the compound represented by formula III is selected from the following compounds:

[0038] N-(4-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzyl)acetamidine

[0039]

[0040] N-(3-(acetimidomethyl)benzyl)-4-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzamide

[0041]

[0042] A pharmaceutical composition comprises a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0043] The dosage form of the pharmaceutical composition of the present invention can be prepared by a person skilled in the art according to conventional methods in the pharmaceutical field. For example, the active ingredient is mixed with one or more carriers (also called excipients) and then made into a desired dosage form, including tablets, capsules, granules, and aerosols; it can also be made into an intravenous injection or an intravenous injection lyophilized agent according to conventional production methods for injections.

[0044] The preparation method of the novel iNOS inhibitor containing amidine group with neuroprotective function of the present invention comprises the compound shown in formula I, when R 1 for R 2 -CH2 or The preparation method comprises the following steps:

[0045] (1) Compound 1 or Compound 2 undergoes an amide condensation reaction with 6-NH2-NBP to obtain intermediate 1-1 or 2-1;

[0046] (2) removing the protecting group on the amino group of intermediate 1-1 or 2-1 to obtain intermediate 1-2 or 2-2;

[0047] (3) Intermediate 1-2 or 2-2 reacts with ethyl acetimidate hydrochloride to undergo nucleophilic substitution reaction to obtain compound BN-1, BN-2, BN-3 or BN-4;

[0048] (4) Compound BN-1, BN-2, BN-3 or BN-4 continues to undergo nucleophilic addition-elimination reaction with hydroxylamine hydrochloride to obtain compound CN-1, CN-2, CN-3 or CN-4;

[0049] The synthetic route is as follows:

[0050]

[0051] When R 1 for R 2 -CH2- or The preparation method comprises the following steps:

[0052] (1) Compound 3-(bromomethyl)benzoic acid or 3-(bromomethyl)benzaldehyde reacts with compound 3-methyl-5-oxo-1,2,4-oxadiazol-4-yl potassium to obtain intermediate 3-1 or 4-1;

[0053] (2) Intermediate 3-1 or 4-1 undergoes amide condensation reaction with 6-NH2-NBP to obtain PMN or PMK; the synthetic route is as follows:

[0054]

[0055] The preparation method of the novel amidine-containing iNOS inhibitor with neuroprotective function described in the present invention, the preparation method of the compound represented by formula II, comprises the following steps:

[0056] (1) 6-NH2-NBP undergoes a nucleophilic addition reaction with acetonitrile in the presence of concentrated hydrochloric acid to obtain compound BN-0;

[0057] (2) Compound BN-0 undergoes a nucleophilic addition-elimination reaction with hydroxylamine hydrochloride to obtain compound CN-0; the synthetic route is as follows:

[0058]

[0059] The preparation method of the novel iNOS inhibitor containing amidine group with neuroprotective function of the present invention comprises the compound represented by formula III, when R 4 for The preparation method comprises the following steps:

[0060] (1) Ethyl acetoacetate reacts with 4-cyanophenylhydrazine hydrochloride to form an amide ring to obtain intermediate 5-1;

[0061] (2) Intermediate 5-1 undergoes a reduction reaction, where the cyano group is reduced to an amino group to obtain intermediate 5-2;

[0062] (3) Intermediate 5-2 reacts with ethyl acetimidate hydrochloride to undergo nucleophilic substitution reaction to obtain compound CH-1; the synthetic route is as follows:

[0063]

[0064] When R 4 for The preparation method comprises the following steps: 1-(4-carboxyphenyl)-3-methyl-5-pyrazolone and 1400W undergo an amide condensation reaction to obtain compound CH-2. The synthesis route is as follows:

[0065]

[0066] The invention discloses an application of a novel amidine-containing iNOS inhibitor with neuroprotective function in preparing a medicine for preventing and / or treating cerebral stroke.

[0067] The invention relates to the use of the pharmaceutical composition in preparing a drug for preventing and / or treating stroke.

[0068] Mechanism of the invention: Multi-target drugs can act on multiple pathological mechanisms at the same time, providing a more comprehensive therapeutic effect. In the treatment of ischemic stroke, the advantages of multi-target drug design are particularly significant. The present invention designs and synthesizes compounds that can selectively inhibit iNOS and have neuroprotective capabilities at the same time, which can play a synergistic role and better treat ischemic stroke. These compounds combine the advantages of existing drugs NBP and Eda, and integrate the characteristics of the selective iNOS inhibitor 1400W, hoping to play a synergistic role in multiple pathological processes and achieve a better anti-cerebral ischemia effect.

[0069] Beneficial effects: Compared with the prior art, the present invention has the following advantages: (1) the compounds can effectively improve the survival rate of human neuroblastoma SH-SY5Y cells, mouse microglial cells BV-2 cells and human umbilical vein endothelial cell fusion cells EA.hy926 cells under OGD / R conditions; (2) compounds BN-1 and BN-4 have good iNOS inhibitory activity, and the molecular docking results show that compound BN-4 binds to iNOS in a binding mode similar to 1400W and has good selectivity; (3) compound BN-4 has a good inhibitory effect on tMCAO cerebral ischemia model. (4) Compound BN-4 has good pharmacokinetic properties. Its water solubility is more than one hundred times higher than that of NBP. Its blood-brain ratio is significantly improved compared with compound 1400W, and it has good drugability. (5) Compound BN-4 can reduce the ROS level of SH-SY5Y cells induced by OGD / R and alleviate cell necrosis and apoptosis. (6) The compound has high stability. (7) The preparation method is simple and the compound is easily available in synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 It is a schematic diagram of the design ideas and action mechanisms of a series of compounds;

[0071] Figure 2 is the effect of the compound at 1 μM and 10 μM on the viability of OGD / R-induced SH-SY5Y cells, Eahy-926 cells, and BV-2 cells;

[0072] Figure 3 is the effect of the compound at 1 μM on LPS-induced NO levels in Raw 264.7 cells;

[0073] Figure 4 The effects of a single administration of some compounds on the cerebral infarction area and Longa's score in tMCAO rats;

[0074] Figure 5 The effect of repeated administration of compound BN-4 on cerebral infarction area, mNSS score and corner test in tMCAO rats;

[0075] Figure 6 is the stability of compound BN-4 in rat plasma and liver microsomes;

[0076] Figure 7 It is the water solubility of compounds BN-4 and NBP. DETAILED DESCRIPTION

[0077] The present invention is further described below in conjunction with specific embodiments.

[0078] 1. Synthesis of intermediates

[0079] Example 1: The synthesis step of intermediate 1-1 is to place 6-NH2-NBP (205 mg, 1 mmol, 1 eq) and the corresponding benzoic acid derivative (1 mmol, 1 eq) in a 25 mL round-bottom flask, add 5 mL DMF as a solvent, and completely dissolve the reactants. Then add the condensation agent N, N, N', N'-tetramethylchloroformamidine hexafluorophosphate (420 mg, 1.5 mmol, 1.5 eq) and the acid binding agent N-methylimidazole (288 mg, 3.5 mmol, 3.5 eq). After the addition is completed, place it at 25 ° C and stir for 24 hours. During the reaction, the reaction solution gradually changes from turbid to clear, and the solid raw materials are completely dissolved. After the reaction is completed, it is concentrated under reduced pressure and the residue is diluted with water. Extract with EA, collect the supernatant, combine the organic phases, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent. The crude product is purified by column chromatography (PE: EA = 2: 1) to obtain a white solid, namely intermediate 1-1.

[0080] Example 2: The synthesis steps of intermediate 1-2 are as follows: intermediate 1-1 (1 mmol) is placed in a 50 mL round-bottom flask, 8 mL of trifluoroacetic acid in dichloromethane solution (1:3) is added as a solvent, placed at 25°C, stirred for 1 h, the reaction is completed, quenched with water, extracted three times with DCM, the organic phases are combined, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain a white solid, which is directly used in the next reaction without further treatment.

[0081] Example 3: The synthesis steps of intermediate 2-1 are as follows: 6-NH2-NBP (205 mg, 1 mmol, 1 eq) and the corresponding benzaldehyde derivative (1 mmol, 1 eq) are placed in a round-bottom flask, 10 mL of anhydrous ethanol is added to dissolve the reactants, heated to 80°C for reflux reaction for 12 h, then slowly cooled to 0°C, sodium borohydride (76 mg, 2 mmol, 2 eq) is added in small amounts and multiple times, and the mixture is moved to room temperature for reaction for 2 h. After the reaction is completed by TLC (developing agent PE: EA = 3: 1), water is added to quench, DCM is extracted, the organic phases are combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure to remove the solvent, and the crude product is purified by column chromatography to obtain intermediate 2-1.

[0082] Example 4: The synthesis steps of intermediate 2-2 are as follows: intermediate 2-1 (1 mmol) is placed in a 50 mL round-bottom flask, 8 mL of trifluoroacetic acid in dichloromethane solution (1:3) is added as a solvent, the mixture is placed at 25°C, stirred for 1 h, the reaction is completed, water is added to quench, DCM is extracted three times, the organic phases are combined, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain a white solid, which is directly used in the next reaction without further treatment.

[0083] Example 5: The synthesis step of intermediate 3-1 is to place 3-(bromomethyl)benzoic acid (198 mg, 1 mmol, 1 eq) in a 50 mL round-bottom flask, add 10 mL acetone to dissolve it, then add 3-methyl-5-oxo-1,2,4-oxadiazol-4-yl potassium (138 mg, 1 mmol, 1 eq) to the flask, and stir at 25°C for 18 hours. After the reaction is completed, the mixture is concentrated to dryness under reduced pressure, washed with anhydrous ether to obtain a white solid, which is directly used in the next step.

[0084] Example 6: The synthesis steps of intermediate 4-1 are as follows: 3-(bromomethyl)benzaldehyde (198 mg, 1 mmol, 1 eq) is placed in a 50 mL round-bottom flask, 10 mL of acetone is added to dissolve it, and then 3-methyl-5-oxo-1,2,4-oxadiazol-4-yl potassium (138 mg, 1 mmol, 1 eq) is added to the flask, and stirred at 25° C. for 18 h. After the reaction is completed, the mixture is concentrated to dryness under reduced pressure, washed with anhydrous ether to obtain a white solid, which is directly used in the next step.

[0085] Example 7: The synthesis step of intermediate 5-1 is to place ethyl acetoacetate (130 mg, 1 mmol, 1 eq) and 4-cyanophenylhydrazine hydrochloride (170 mg, 1 mmol, 1 eq) in a 50 mL round-bottom flask, and slowly drip 5 mL of glacial acetic acid. The reaction is heated to 80 ° C and refluxed for 12 hours, and the system gradually changes from turbid to clear. After the reaction is completed, it is cooled to room temperature, extracted with EA three times, the organic phases are combined, and the solvent is removed by vacuum concentration. The crude product is purified by column chromatography (PE: EA = 1: 1) to obtain intermediate 5-1.

[0086] Example 8: The synthesis steps of intermediate 5-2 are as follows: intermediate 5-1 (199 mg, 1 mmol, 1 eq) is placed in a 50 mL round-bottom flask, 10 mL MeOH is added to dissolve, 20 mg of palladium carbon and 1 drop of glacial acetic acid are added, and the mixture is reacted under hydrogen for 24 hours. After the reaction is completed, the palladium carbon is filtered, the filtrate is collected, and it is concentrated to dryness under reduced pressure to obtain a yellow solid, which is directly used in the next step.

[0087] 2. Synthesis of target compounds

[0088] Example 9: The synthetic route of the target compound BN-1 is to dissolve the intermediate 1-2 (1mmol, 1eq) in anhydrous ethanol, cool to 0-5°C, add ethyl acetimidate hydrochloride (128mg, 0.9mmol, 0.9eq), anhydrous potassium carbonate (276mg, 2mmol, 2eq), and stir to react for 4h. After the reaction is completed, the solvent is concentrated to dryness under reduced pressure, water is added, and EA is extracted three times. The upper organic phase is combined, dried over anhydrous sodium sulfate, and the organic phase is concentrated under reduced pressure to obtain a yellow viscous liquid. The product is subjected to rapid column chromatography (DCM: MeOH = 15:1) to obtain BN-1 251mg, which is a white solid with a yield of 66%. mp 220.2~220.8℃; 1 H NMR(500MHz,DMSO-d6)δ10.77(s,1H),8.40–8.32(m,1H),8.15(d,J=7.8Hz, 1H),8.07(s,1H),7.99(d,J=7.4Hz,1H),7.67(d,J=8.3Hz,1H),7.63–7.56( m,2H),5.63(dd,J=7.6,4.0Hz,1H),4.58(s,2H),2.24(s,3H),2.05(tt,J=9 .7,5.2Hz,1H),1.76–1.66(m,1H),1.34–1.23(m,4H),0.88(t,J=6.9Hz,3H); 13 C NMR(75MHz,DMSO-d6)δ170.38,166.13,164.74,145.61,140.60,136.25,135.09,131.69,129.22,128.20,127 .67,126.99,126.24,123.29,115.85,81.54,45.71,34.04,26.85,22.37,19.16,14.31.HRMS-ESI(m / z):[M+H] + Calculate for [C 22 H 26 N3O3] + 380.19687, found 380.19596, ppm error-1.95. HPLC: MeOH / H2O (containing 0.1% Et3N) = 80:20.

[0089] Example 10: The synthetic route of the target compound CN-1 is to place BN-1 (379 mg, 1 mmol, 1 eq) in a 50 mL flask, add 15 mL of anhydrous THF to dissolve BN-1, then add hydroxylamine hydrochloride (100 mg, 1.5 mmol) and anhydrous potassium carbonate (272 mg, 2 mmol, 2 eq). Heat to 70 ° C and reflux for 5 h. Monitor by TLC method, PE: EA = 2: 1, R f The value is 0.5~0.6. The raw material reacted completely, extracted three times with a small amount of EA, combined the organic phases, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain crude CN-1. The crude product was purified by column chromatography (PE:EA=2:1) to obtain 300mg of CN-1 as a white solid with a yield of 76%. mp195.2~195.9℃;MS(m / z):418[M+Na] + ; 1 H NMR (300MHz, DMSO-d6) δ10.58(s,1H),8.98(s,1H),8.30(s,1H),8.07(d,J=8.2Hz,1H),7.85(s,2H),7.65(d,J=8.4Hz,1H),7.51(d,J=5.3H z,2H),5.62(d,J=5.9Hz,1H),4.26(dd,J=40.6,6.1Hz,2H),2.00(d,J=18.3Hz,1H),1.66(s,1H),1.33–1.22(m,4H),0.85(d,J=7.1Hz,3H); 13 C NMR(75MHz,DMSO-d6)δ170.37,166.40,145.58,141.40,140.55,135.16,130.66,129.05,126.96,126.82, 126.65,126.28,123.32,115.87,81.54,45.46,34.05,26.86,22.37,14.87,14.30.HRMS-ESI(m / z):[M+H] + Calculate for [C 22 H 26 N3O4] + 396.19178,found 396.19170,ppm error 0.39.HPLC.MeOH / H2O=80:20.

[0090] Example 11: The synthetic route of the target compound BN-2 is to dissolve the intermediate 1-2 (1mmol, 1eq) in anhydrous ethanol, cool to about 5°C, add ethyl acetimidate hydrochloride (128mg, 0.9mmol, 0.9eq), anhydrous potassium carbonate (276mg, 2mmol, 2eq), and stir to react for 4h. The post-reaction treatment method is the same as BN-1 to obtain a yellow viscous liquid. The product is subjected to rapid column chromatography (DCM: MeOH = 15:1) to obtain BN-2 151mg, which is a white solid, with a yield of 41%. mp 189.3~189.8℃; 1 H NMR (400MHz, Methanol-d4) δ8.32(d,J=1.9Hz,1H),8.03–7.96(m,2H),7.89(t,J=2.0Hz,1H),7.65(t,J=7.9Hz,1H),7.57(d,J=8.3Hz,1H),7.50(dd,J =7.9,2.2Hz,1H),5.56(dd,J=7.7,4.0Hz,1H),2.39(s,3H),2.09(dd,J=9. 6,4.5Hz,1H),1.77–1.69(m,1H),1.40–1.30(m,4H),0.91(t,J=7.0Hz,3H). 13 C NMR(126MHz,Methanol-d4)δ130.56,127.13,124.69,122.51,116.52,81.98,48.53,48.19,48.02,4 7.85,47.68,47.51,47.34,47.17,33.99,26.63,22.12,20.75,18.13,12.87.HRMS-ESI(m / z):[M+H] + Calculate for [C 21 H 24 N3O3] + 366.18122, found 366.18073, ppm error-0.88.HPLC.MeOH / H2O (containing 0.1% Et3N) = 80:20.

[0091] Example 12: The synthetic route of the target compound CN-2 is to place BN-2 (365 mg, 1 mmol, 1 eq) in a 50 mL round-bottom flask, add 15 mL of anhydrous THF, then add hydroxylamine hydrochloride (100 mg, 1.5 mmol, 1.5 eq) and anhydrous potassium carbonate (272 mg, 2 mmol, 2 eq). Heat to 70 ° C and reflux for 5 h. Monitor by TLC, PE: EA = 1: 1, R fThe value is 0.5-0.6. After the reaction is completed, EA is added for extraction three times, the organic phases are combined, dried over anhydrous sodium sulfate, and the organic layer is concentrated to dryness under reduced pressure to obtain crude CN-2. The crude product is purified by column chromatography (PE:EA=1:1) to obtain 300 mg of CN-2 as a light yellow oil with a yield of 76%. 1 H NMR (300MHz, Methanol-d4) δ8.25(d,J=2.0Hz,1H),8.01(dd,J=8.3,2.0Hz,1H),7.71–7.64(m,2H),7.54(d,J=4.9Hz,1H),7.43(s,1H),7.33( d,J=6.2Hz,1H),5.56–5.51(m,1H),2.05(d,J=6.5Hz,1H),1.95(d,J=3.2Hz,3H),1.77–1.68(m,1H),1.35–1.29(m,4H),0.90(t,J=3.9Hz,3H); 13 C NMR(75MHz,Methanol-d4)δ171.12,150.01,146.05,135.47,129.09,126.94,126.73,126.26,122.89, 122.30,116.34,81.89,47.36,47.08,46.79,34.00,26.68,22.11,14.56,12.92.HRMS-ESI(m / z):[M+H] + Calculate for [C 21 H 24 N3O4] + 382.17613,found 382.17624,ppm error1.18.HPLC.MeOH / H2O=80:20.

[0092] Example 13: The synthetic route of the target compound BN-3 is to dissolve the intermediate 2-2 (1mmol, 1eq) in anhydrous ethanol, cool to 0-5°C, add ethyl acetimidate hydrochloride (128mg, 0.9mmol, 0.9eq) and anhydrous potassium carbonate (276mg, 2mmol, 2eq) in small amounts and multiple times, and stir the reaction at this temperature for 4h. After the reaction is completed, the solvent is concentrated to dryness under reduced pressure, water is added, and EA is extracted three times, the organic phases are combined, and the organic layer is concentrated to dryness under reduced pressure to obtain a yellow viscous liquid. The product is subjected to rapid column chromatography (DCM: MeOH = 15:1) to obtain BN-3 131mg, which is a light yellow solid with a yield of 37%. mp 185.2~185.7℃; 1H NMR (300MHz, Methanol-d4) δ7.46(d,J=0.7Hz,2H),7.35(s,1H),7.25(d,J=8.4Hz,1H),7.21(d,J=2.0Hz,1H),7.08(d,J=2.3Hz,1H),6.80( d,J=2.2Hz,1H),5.39(dd,J=7.5,4.0Hz,1H),4.44(s,2H),2.38(s,3H),1.99(s,1H),1.62(s,1H),1.32–1.28(m,4H),0.88(d,J=3.6Hz,3H); 13 C NMR (75MHz, Methanol-d4) δ134.12,130.22,127.38,123.57,123.51,122.26,120.85,81. 97,47.37,47.09,46.80,46.38,34.28,26.61,22.13,17.91,12.91.HRMS-ESI(m / z):[M+H] + Calculate for [C 21 H 26 N3O2] + 352.20195, found 352.20126, ppmerror-1.54.HPLC.MeOH / H2O (containing 0.1% Et3N) = 80:20.

[0093] Example 14: The synthetic route of the target compound CN-3 is to place BN-3 (351 mg, 1 mmol, 1 eq) in an eggplant-shaped bottle, add 15 mL of anhydrous THF to completely dissolve, then add hydroxylamine hydrochloride (100 mg, 1.5 mmol, 1.5 eq) and anhydrous potassium carbonate (272 mg, 2 mmol, 2 eq). Heat to 70°C and reflux for 5 h. Monitor by TLC, PE:EA=1:2, R f The value is 0.5-0.6. The raw material reacted completely, extracted three times with EA, combined the organic phases, and concentrated the solvent to dryness under reduced pressure to obtain crude CN-3. The crude product was purified by column chromatography (PE:EA=1:2) to obtain 280 mg of CN-3 as a light yellow oil with a yield of 76%. 1H NMR (300MHz, Methanol-d4) δ7.25–7.19(m,2H),7.09(d,J=2.1Hz,2H),6.99(d,J=2.3Hz,1H),6.96(dd,J=2.4,1.1 Hz,1H),6.83(d,J=2.2Hz,1H),5.37(dd,J=7.5,4.0Hz,1H),4.34(s,2H),1.98–1.94(m,1H),1.77(s,3H),1.60(d,J =4.6Hz,1H),1.25(s,4H),0.89–0.86(m,3H); 13CNMR(75MHz,Methanol-d4)δ149.83,122.90,122.24,122.08,120 .56,105.20,81.88,47.32,47.04,46.75,46.56,34.28,29.37,26.57,22.12,14.42,12.88.HRMS-ESI(m / z):[M+H] + Calculate for [C 21 H 26 N3O3] + 368.19687,found368.19612,ppm error-1.61.HPLC.MeOH / H2O=80:20.

[0094] Example 15: The synthetic route of the target compound BN-4 is to dissolve the intermediate 2-2 (1mmol, 1eq) in anhydrous ethanol, add ethyl acetimidate hydrochloride (128mg, 0.9mmol, 0.9eq) and anhydrous potassium carbonate (276mg, 2mmol, 2eq) at 5°C, and stir the reaction at this temperature for 4h. After the reaction is completed, the solvent is concentrated under reduced pressure, water is added, and EA is extracted three times. The organic layers are combined, dried over anhydrous sodium sulfate, and the organic layers are concentrated to dryness under reduced pressure to obtain a light yellow solid. Column chromatography purification (DCM: MeOH = 14: 1) gave BN-4 310mg, which is a light yellow solid, with a yield of 85%. mp210.8~211.5℃; 1H NMR (300MHz, Methanol-d4) δ7.35(d,J=1.3Hz,3H),7.23(d,J=2.1Hz,2H),7.02(dd,J=8.4,2.2Hz,1H),6.78(d,J=2.2Hz,1H),5.36(d d,J=7.6,4.0Hz,1H),4.41(s,2H),4.37(s,2H),2.21(s,3H),1.95(s,1H),1.60(s,1H),1.27(d,J=3.4Hz,4H),0.86(d,J=2.3Hz,3H). 13 C NMR(75MHz,Methanol-d4)δ149.86,140.51,138.74,134.67,128.93,126.86,126.35,126.33,122.12,120.76 ,104.85,81.91,47.35,47.07,46.78,46.69,45.81,34.30,26.63,22.13,17.51,12.91.HRMS-ESI(m / z):[M+H] + Calculate for [C 22 H 28 N3O2] + 366.21760, found 366.21678, ppmerror-1.87.HPLC MeOH / H2O (containing 0.1% Et3N) = 80:20.

[0095] Example 16: The synthetic route of the target compound CN-4 is to place BN-4 (365 mg, 1 mmol, 1 eq) in an eggplant-shaped bottle, add 15 mL of anhydrous THF to completely dissolve it, and then add hydroxylamine hydrochloride (100 mg, 1.5 mmol, 1.5 eq) and anhydrous potassium carbonate (272 mg, 2 mmol, 2 eq). Heat to 70°C and reflux for 5 hours. Monitor using TLC, PE:EA=1:2, Rf value is 0.5-0.6. The raw material reacts completely, and the EA extraction method is the same as CN-1 to obtain a crude CN-4. The crude product is purified by column chromatography (PE:EA=1:3) to obtain 295 mg of CN-4 as a white solid with a yield of 77%. mp 199.5-199.8°C; 1H NMR (300MHz, Methanol-d4) δ7.32(s,1H),7.27(t,J=1.8Hz,2H),7.22(d,J=8.3Hz,1H),7.17(d,J=2.5Hz,1H),7.02(dd,J=8.3,2.2Hz,1H),6.85(d,J=2 .2Hz,1H),5.35(s,1H),4.34(d,J=1.9Hz,4H),1.95(d,J=1.5Hz,1H),1.77( s,3H),1.63(d,J=2.2Hz,1H),1.33(d,J=5.5Hz,4H),0.90(d,J=4.2Hz,3H). 13 C NMR(75MHz,Methanol-d4)δ140.12,139.86,138.63,128.58,125.83,125.19,125.01,122.07,120.53,105 .15,81.87,47.36,47.07,46.92,46.79,45.27,34.31,26.65,22.14,13.11,12.93.HRMS-ESI(m / z):[M+H] + Calculate for [C 22 H 28 N3O3] + 382.21252,found 382.21177,ppm error-1.62.HPLC MeOH / H2O=80:20.

[0096] Example 17: The synthetic route of the target compound PMN is to place the intermediate 3-1 (208 mg, 1 mmol, 1 eq) and 6-NH2-NBP (205 mg, 1 mmol, 1 eq) in a 25 mL round-bottom flask, add 5 mL DMF as a solvent, and completely dissolve the reactants. Then add N, N, N', N'-tetramethylchloroformamidine hexafluorophosphate (420 mg, 1.5 mmol, 1.5 eq) and N-methylimidazole (288 mg, 3.5 mmol, 3.5 eq). After the addition is complete, place it at 25 ° C and stir to react for 24 hours. The reaction is monitored by TLC (developing agent PE: EA = 3: 1), water is added, EA is extracted three times, the organic phases are combined, and dried over anhydrous sodium sulfate. The organic layer is concentrated to dryness under reduced pressure, and the crude product is purified by column chromatography (PE: EA = 1: 2) to obtain PMN 288 mg, which is a light yellow solid with a yield of 68%. mp 154.8~155.2℃; 1H NMR(300MHz,DMSO-d6)δ10.80–10.51(m,1H),7.73(d,J=98.3Hz,6H),5.58( s,1H),4.89(d,J=16.3Hz,1H),2.25–2.14(m,2H),1.25(s,4H),0.85(s,3H). 13 C NMR (75MHz, Methanol-d4) δ160.45,156.68,129.26,122.55,122.35,116.37,115. 21,81.89,47.34,47.06,46.77,26.61,22.08,12.85,8.90.HRMS-ESI(m / z):[M+Na] + Calculate for [C 23 H 23 N3O5Na] + 444.15299, found 444.15299, ppm error 0.03. HPLC MeOH / H2O=80:20.

[0097] Example 18: The synthetic route of the target compound PMK is to place the intermediate 4-1 (218 mg, 1 mmol, 1 eq) and 6-NH2-NBP (205 mg, 1 mmol, 1 eq) in a flask, and add 10 mL of anhydrous ethanol to dissolve it. Heat to 80°C and reflux for 0.5 h to generate a light yellow solid. After the reaction is completed, let it cool to room temperature, filter and dry it under an infrared lamp to obtain an imine intermediate. Dissolve this compound in MeOH, cool to 5°C, slowly add sodium borohydride (76 mg, 2 mmol, 2 eq), monitor the completion of the reaction by TLC (developing agent PE: EA = 3: 1), add water to quench the reaction, extract with EA, and purify it in the same way as BN-1 245 mg to obtain a yellow solid with a yield of 60%. mp 134.8~135.2°C; 1 H NMR (500MHz, Methanol-d4) δ7.42(s,2H),7.34–7.28(m,2H),7.23(d,J=2.0Hz,1H),7.07(dd,J=8.3,2.2Hz,1H),6.85(d,J=2.2Hz,1H),5.45( dd,J=7.6,4.1Hz,1H),4.45(s,2H),2.10(s,3H),2.05(d,J=1.7Hz,1H),1.76–1.68(m,1H),1.40(dt,J=6.0,2.3Hz,4H),0.96(d,J=5.2Hz,3H). 13C NMR(75MHz,DMSO-d6)δ171.05,159.46,158.07,150.35,143.90,137.88,135.31,129.13,126.65,126.62,126.07,125.48,12 3.09,121.25,107.46,81.29,63.01,56.49,45.33,34.53,26.84,22.39,19.02,14.31,13.97,10.63.HRMS-ESI(m / z):[M+Na] + Calculate for [C 23 H 25 N3O4Na] + 430.17373, found430.17324, ppm error-0.83. HPLC MeOH / H2O=80:20.

[0098] Example 19: The synthetic route of the target compound BN-0 is to place 6-NH2-NBP (205 mg, 1 mmol, 1 eq) in a 50 mL round-bottom flask, add 20 mL of anhydrous acetonitrile to completely dissolve it, and then slowly drop 1 mL of concentrated hydrochloric acid. Heat to 83 ° C and reflux for 5 h. The reaction changes from colorless to light yellow. TLC is used for monitoring. PE:EA=1:1, R f The value is 0.5-0.6. The raw material reacts completely, the reaction is finished, and ethyl acetate is used for extraction three times. The organic layers are combined, and then washed with saturated sodium bicarbonate, washed with water twice, and washed with brine once, dried over anhydrous sodium sulfate, and the organic layer is concentrated to dryness under reduced pressure to obtain BN-0 crude product. The crude product is subjected to rapid column chromatography (PE: EA = 1: 1) to obtain BN-0 185 mg, which is a light yellow solid, with a yield of 75%. mp 173.2-173.8 ° C; 1H NMR(300MHz,DMSO-d6)δ9.70(s,1H),7.81–7.73(m,2H),7.66(dd,J=8.1,1.9Hz,1H),5.67(dd,J=7.8,3.9Hz ,1H),2.35(s,3H),2.06(dq,J=14.2,4.9Hz,1H),1.72(s,1H),1.31(q,J=6.3Hz,4H),0.87–0.81(m,3H); 13C NMR(75MHz,DMSO-d6)δ169.54,165.27,149.98,135.80,132.02,127.29,124.86, 122.37,81.77,45.75,33.87,26.90,22.35,19.41,14.28.HRMS-ESI(m / z):[M+H] + Calculate for [C 14 H 19 N2O2] + 247.14465, found 247.14227, ppmerror 8.61. HPLC MeOH / H2O (containing 0.1% Et3N) = 80:20.

[0099] Example 20: The synthetic route of the target compound CN-0 is to add BN-0 (246 mg, 1 mmol, 1 eq) into a 50 mL round-bottom flask, add 15 mL of anhydrous THF to completely dissolve it, then add hydroxylamine hydrochloride (100 mg, 1.5 mmol, 1 eq) and anhydrous potassium carbonate (272 mg, 2 mmol, 2 eq). Stir at room temperature for 5 h. Monitor using TLC method, PE:EA=1:1, R f The value is 0.5~0.6. The raw material reacts completely and is extracted with EA. The extraction method is the same as BN-0. The crude product is concentrated under reduced pressure to obtain CN-0 crude product. The crude product is purified by column chromatography (PE: EA = 1:1) to obtain CN-0 150mg as a white solid with a yield of 57%. mp 188.6~189.3℃; MS (m / z): 285[M+Na] + ; 1 H NMR(300MHz,Methanol-d4)δ7.50–7.46(m,2H),5.51(dd,J=7.7,4.0Hz,1H),2.04(s,1H) ,1.93(s,3H),1.68(d,J=3.6Hz,1H),1.34(dd,J=4.8,1.8Hz,4H),0.88(d,J=2.7Hz,3H); 13C NMR (75MHz, Methanol-d4) δ171.00,145.74,140.73,129.54,126.62,122.70,121.63,118. 10,81.91,47.41,47.13,46.84,34.03,26.73,22.12,14.62,12.97.HRMS-ESI(m / z):[M+H] + Calculate for [C 14 H 19 N2O3] + 263.13902,found 263.13801,ppmerror-3.78.HPLC.MeOH / H2O=80:20.

[0100] Example 21: The synthetic route of the target compound CH-1 is to dissolve the intermediate 5-2 (203 mg, 1 mmol, 1 eq) in anhydrous ethanol, add ethyl acetimidate hydrochloride (128 mg, 0.9 mmol, 0.9 eq) and anhydrous potassium carbonate (276 mg, 2 mmol, 2 eq) at 5°C, and stir at this temperature for 4 hours. After the reaction is completed, the methanol is concentrated to dryness under reduced pressure, water is added, and ethyl acetate is extracted three times, the organic layers are combined, dried over anhydrous sodium sulfate, and the organic layers are concentrated to dryness under reduced pressure to obtain a light yellow solid. The product is subjected to rapid column chromatography (DCM: MeOH = 8: 1) to obtain CH-1 110 mg, which is a light yellow solid, with a yield of 45%. mp 167.2~167.8°C; MS (m / z): 267[M+Na] + ; 1 H NMR(300MHz, Methanol-d4)δ7.77(d,J=8.5Hz,2H),7.34–7.29(m,2H),4.41(s,2H),3.33(s,2H),2.24(s,3H),2.10(s,3H).13C NMR(75MHz,DMSO-d6)δ127.19,120.35,47.69,47.41,47.13,46.84,46.56,46.27,45.99.HRMS-ESI(m / z):[M+H] + Calculate for [C 13 H 17 N4O] + 245.13969, found245.13961, ppm error-0.14. HPLC MeOH / H2O=80:20.

[0101] Example 22: The synthetic route of the target compound CH-2 is to place 1-(4-carboxyphenyl)-3-methyl-5-pyrazolone (218 mg, 1 mmol, 1 eq) and 1400W (177 mg, 1 mmol, 1 eq) in an eggplant-shaped bottle, add 5 mL of DMF as a solvent, and completely dissolve the reactants. Then add N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (420 mg, 1.5 mmol, 1.5 eq) and N-methylimidazole (288 mg, 3.5 mmol, 3.5 eq). After the addition is complete, place it at room temperature and stir for 24 hours. After the reaction is completed by TLC monitoring, add H2O, extract EA three times, combine the organic layers, and dry them over anhydrous sodium sulfate. The organic layer is concentrated to dryness under reduced pressure, and the product is subjected to rapid column chromatography (PE: EA = 1: 1) to obtain 151 mg of CH-2 as a light yellow oil with a yield of 40%. MS (m / z): 400 [M+Na] + ; 1 H NMR (300MHz, Methanol-d4) δ8.23(d,J=1.9Hz,1H),8.20(d,J=2.1Hz,1H),8.06(d,J=4.7Hz,2H),7.63(d,J=1.5 Hz,3H),7.52(dd,J=6.0,2.3Hz,1H),4.85(d,J=2.9Hz,2H),4.71(s,2H),3.24(s,2H),2.50(s,3H),2.50(s,3H). 13 C NMR(75MHz,Methanol-d4)δ168.77,164.63,139.00,131.09,129.43,129.23,128.49,121.7 1,121.14,120.26,118.92,20.06,18.25,17.66,14.09,13.64,8.32.HPLC.MeOH / H2O=80:20.

[0102] 3. Effect Verification

[0103] Example 22: In vitro study on the survival rate of SH-SY5Y cells, Eahy-926 cells and BV-2 cells induced by OGD / R

[0104] 1. Experimental methods

[0105] a. Digest and count cells and prepare to a concentration of 5×10 4 Cell suspension of 100 μL / mL was placed in a 96-well cell culture plate, and 100 μL of cell suspension was added to each well;

[0106] b. Place the 96-well cell culture plate in a 37°C, 5% CO2 incubator and culture for 24 hours;

[0107] c. Dilute the drug to the required concentration with conditioned medium, add 100 μL of the corresponding drug-containing medium to each well, and set up a negative control group at the same time;

[0108] d. Place the 96-well cell culture plate in a 37°C, 5% CO2 incubator for 24 hours;

[0109] e. The culture medium was replaced with DMEM medium without glucose and serum, and the cells were cultured in a 5% CO2, 95% N2 environment for 2 h, and then the culture medium was replaced with complete DMEM medium and cultured in a 5% CO2, 95% O2 environment for 24 h;

[0110] f. Stain the 96-well plate with CCK-8, λ=450nm, and measure the OD value; A. Add 10μL CCK-8 to each well and continue to culture in the incubator for 2h; B. Mix gently on a shaker for 10min; C. λ=450nm, read the OD value of each well with an enzyme-labeled instrument, and calculate the inhibition rate.

[0111] 2. Experimental results

[0112] like Figure 2 As shown in the figure, in SH-SY5Y cells, the cell survival rate of the OGD / R model group was significantly reduced, and with the addition of NBP, 6-NH2-NBP and 1400W, the cell survival rate was significantly increased compared with the OGD / R group. At a concentration of 1μM, the cell survival rate of CN-2 was higher than that of the NBP or 6-NH2-NBP group; at a concentration of 10μM, the cell survival rates of BN-4, CN-1, CN-2, CN-4, CH-1 and PMK were also significantly higher than those of the NBP or 6-NH2-NBP group. In Eahy-926 cells and BV-2 cells, the cell survival rate of the OGD / R model group was significantly reduced, and with the addition of NBP and 1400W, the cell survival rate was significantly increased compared with the OGD / R group. All the tested compound groups could improve the cell survival rate, among which BN-4 had the best effect and had similar activity to NBP. In both cells, the cell survival rate could be improved at 1μM and 10μM concentrations.

[0113] Example 23: Evaluation of iNOS inhibitory activity at the cellular and enzyme levels

[0114] 1. Experimental methods

[0115] A. Evaluation of iNOS inhibitory activity at the cellular level

[0116] RAW 264.7 cells were plated at 2 × 10 per well. 4The cells were inoculated into a black 96-well culture plate at a density of 100 μL of culture medium and incubated for 24 hours. The cells were then pretreated with the compound for 1 hour and then stimulated with LPS (1 μg / mL) for 6 hours. The culture supernatant was removed and 100 μL of NOS assay buffer was added to each well. 100 μL of NOS assay reaction solution (5% 0.1 mM NADPH, 0.2% DAF-FM DA, 39.8% MilliQ water, 50% NOS assay buffer, 5% L-arginine solution) was then added to each well and incubated at 37°C for 2 hours. Fluorescence was measured with a fluorescence plate reader (Bio-Tek) at excitation of 495 nm and emission of 515 nm.

[0117] B. Evaluation of iNOS inhibitory activity at the enzyme level (hemoglobin capture method)

[0118] a. Add 20 μL of test buffer [compound (4 μM-50 nM) + HEPES buffer stock solution (100 mM)] to a 96-well plate and heat the plate in an incubator at 37°C for about 30 minutes.

[0119] b. 10 μM L-arginine was added to HEPES buffer (100 mM, 10% glycerol, pH 7.4) containing 100 μM NADPH, 10 μM tetrahydrobiopterin and 4 μM human oxyhemoglobin.

[0120] c. Add 210uL of the complete mixture to each well of the plate and keep it at 37°C.

[0121] d. Quickly add 20 μL of iNOS dilution to each well and mix the plate with an incubation orbital shaker for 15 s.

[0122] e. Place the plate into the reader (preheated to 37°C) and start reading.

[0123] f. Follow the reaction by reading absorbance at 401 nm using a dual wavelength kinetic plate reader at 37° C. Perform kinetic readings for 3-5 minutes.

[0124] g. Calculate IC using nonlinear regression using GraphPad Prism software 50 The standard error was calculated from LogIC50 and the Cheng-Prusoff equation [Ki = IC 50 / (1+[S] / Km)] to calculate the KI value.

[0125] 2. Experimental results

[0126] like Figure 3The results of cell experiments showed that compound BN-4, compounds CN-1 to CN-4, and compound CH-2 could significantly inhibit NO production in Raw 264.7 cells, indicating that they have certain iNOS inhibitory activity. Table 1 Enzyme level experimental results show that compound BN-4 has IC 50 The value of 0.1707 μM is the best among the series of compounds and is basically the same as the positive control 1400W.

[0127] Table 1 IC values ​​of compounds BN-1~PMN for iNOS 50

[0128]

[0129]

[0130] Example 24: Evaluation of single-dose administration on cerebral infarction area and neurological function recovery in rats with tMCAO cerebral ischemia model

[0131] 1. Experimental methods

[0132] A. Preparation of rat tMACO model: To make the embolism model, a nylon thread with a diameter of 0.234 mm should be selected, and the top of the nylon thread should be made thicker and burned into a smooth ball with a diameter of about 0.35 mm. A marker pen is used to mark the position about 20 mm from the top, cleaned with 75% alcohol, and placed in 2500U / mL heparin saline for use. The rat was anesthetized with a gas anesthesia machine (the anesthetic dose should not be too large to prevent the rat from excessive anesthesia and death), and the rat was fixed on the experimental table with its head up, and an incision was made in the middle of the neck. The skin was cut open with surgical scissors, and the skin tissue was carefully torn to find the blood vessels in the rat's neck. First, find the right common carotid artery (CCA), use surgical thread to separate the right external carotid artery (ECA) and internal carotid artery upwards, ligate both ends, cut the two external carotid artery branches of the superior thyroid artery and the occipital artery, double ligate the ECA about six millimeters near the bifurcation of the CCA, clamp the blood vessel with a micro-arterial clamp at the proximal end of the right common carotid artery, leave a slipknot (untightened slipknot) near the bifurcation of the right external carotid artery, make a V-shaped micro-incision with a diameter of about 0.2mm between the proximal ligation of the ECA and the bifurcation of the common carotid artery, gently insert the nylon thread head from the incision, gently tighten the knot, cut the internal carotid artery between the two ligatures, align it with the direction of the internal carotid artery, loosen the artery clamp, and send the nylon thread along the ECA through the ICA into the skull. The insertion depth is about 18mm to 20mm. Stop when you encounter slight resistance, so that the head of the nylon thread is located at the beginning of the MCA to block the MCA The blood flow is tightened by tightening the silk thread, suturing the incision, and leaving the tail end of the nylon thread outside the body. After 2 hours of ischemia, the model rats are anesthetized with a gas anesthesia machine. In order to perform reperfusion after ischemia, the top of the nylon thread needs to be returned to the micro-incision. Gently pull the nylon thread with surgical forceps to return the head end to its position, and a slight resistance can be felt. At this time, the blood flow supply to the middle cerebral artery of the rat is restored, and the MCAO rat model is completed. At this time, the prepared drugs are immediately administered by tail vein injection.

[0133] B.Testing indicators:

[0134] a. Determination of cerebral infarction volume (TTC staining method): After tMCAO modeling in rats, 24h or 72h later, the rats were anesthetized and killed, the heads were dissected to remove the brain tissue, and the tissue was quickly placed in the refrigerator fresh-keeping layer for 15min before being taken out, in order to harden the brain for slicing. After the brain was cut into 5 slices of uniform thickness, the brain slices were quickly placed in 5mL of PBS buffer solution containing 2% TTC prepared in advance, wrapped with tin foil and placed in an oven for incubation at 37°C for 15min. During this process, the slices were turned over every 2 minutes or so, in order to make the brain slices evenly stained. After incubation for 15min, the brain slices were taken out, and a black or blue background was selected to take pictures of the brain slices. After that, ophthalmic forceps were used to separate the pale area (infarction area) and the non-pale area (normal area), and the percentage of infarction was calculated by Image pro-plus 6.0 as follows:

[0135] Infarction percentage (%) = pale area / (pale area + non-pale area) × 100%

[0136] Infarct area inhibition rate (%) = model group infarction percentage (%) - drug group infarction percentage (%) / model group infarction percentage (%) × 100

[0137] b. Longa neurological function rating: Before modeling, 2h and 24h after reperfusion administration, the neurological deficits of animals were graded and scored according to Longa's method. The standards are as follows:

[0138] 0 points: normal neurological function;

[0139] 1 point: mild neurological deficit: when the tail is lifted, the animal's left forelimb flexes;

[0140] 2 points: moderate neurological deficit: the animal is placed on a smooth surface and walks, turning in circles to the left side while walking;

[0141] 3 points: moderate neurological deficit: in the resting state, the patient tilts to the left;

[0142] 4 points: decreased consciousness and no spontaneous limb movement;

[0143] 5 points: no response to stimulation or death.

[0144] C. Inclusion and exclusion criteria of tMCAO model

[0145] Inclusion criteria: Those with Longa scores of 1-3 points were included;

[0146] Exclusion criteria: Longa score lower than 1 or higher than 3; subarachnoid hemorrhage during brain extraction; no ischemic lesions were found in TTC staining; and death within 72 hours of ischemia-reperfusion.

[0147] Statistical methods: The neurological deficit grading score data were expressed as median, and the other data were expressed as Mean±SD. The statistical differences between the neurological deficit grading score data groups were analyzed using Kruskal-Wallis test and MannWhitney U test, and the statistical differences between the other data groups were analyzed using One-way ANOVA and Tukey's test. A P value less than 0.05 was considered to be significantly different.

[0148] 2. Experimental results

[0149] We selected some compounds that performed well at the cellular and enzyme levels for single-dose in vivo experiments, and each group of compounds was administered at an equimolar dose of NBP 5 mg / kg. Figure 4As shown, compared with the model group, each group of compounds can significantly reduce the volume of cerebral infarction induced by tMCAO model. The average cerebral infarction area of ​​the model group was 44.29%, and the BN-1 (9.98 mg / kg), CN-1 (10.41 mg / kg), BN-4 (9.61 mg / kg), CN-4 (10.03 mg / kg), CH-1 (6.42 mg / kg), NBP (5 mg / kg), 1400W (6.58 mg / kg) and NBP + 1400W (5 mg / kg + 6.58 mg / kg) groups were 24.34%, 25.97%, 20.96%, 25.62%, 29.14%, 28.54%, 30.98%, 25.51%, respectively, as shown in the figure. Among them, BN-4 had the best activity, with a cerebral infarction volume inhibition rate of 51.19%, which was higher than that of BN-1 group (45.04%), CN-1 group (41.36%), CN-4 group (42.15%) and CH-1 group (34.20%). And the BN-4 group was also better than the single and combined administration groups of NBP (35.58%) and 1400W (30.05%) (42.40%). Neurobehavioral scores were performed on each group 2h and 26h after ischemia. At 24h after ischemia, BN-1, CN-1, BN-4, CN-4, CH-1 and NBP groups, and the combined administration group of NBP+1400W could significantly improve the neurological function scores of rats. Among them, the effect of BN-4 group was the most significant (P<0.0001vs MCAO group), which was superior to the equimolar BN-1, CN-1, CN-4, CH-1 groups and the groups where NBP and 1400W were administered alone or in combination.

[0150] Example 25: Evaluation of the effect of multiple administrations on the cerebral infarction area and neurological function recovery in rats with tMCAO cerebral ischemia model

[0151] 1. Experimental methods

[0152] Basically the same as Example 24, the drug was administered three times at 2h, 24h and 48h after ischemia.

[0153] Neurological function score: A.mNSS B.Corner test

[0154] A. mNSS: mNSS was used to evaluate the neurological function of the tMCAO model 24 and 72 hours after ischemia. The score ranged from 0 to 14 points, including motor, sensory, reflex and balance tests. The normal state score was 0 points and the maximum defect score was 14 points. The cumulative scores of 10 to 14 points, 5 to 9 points, and 1 to 4 points were severe, moderate and mild injuries, respectively.

[0155] B. Corner test: The purpose is to evaluate the sensorimotor function of rats. This test is performed 24 and 72 hours after ischemia in rats. For this test, two wooden boards (35cm×25cm×1.2cm) are prepared in advance, and one side of the boards is connected together so that the acute angle between them does not exceed 35°. A small opening is left at the connection of the wooden boards to allow light to pass through and attract the rat to crawl to the corner. The rat is placed in front of the opening of the wooden board with its head facing the board. Let the rat enter the corner, and the wooden boards on both sides of the body will stimulate the rat to turn around and face the opening side. Each test is repeated 10 times, and the number of times the rat turns left is recorded. If it is a healthy rat without acute cerebral ischemia, the probability of turning left and right will be basically equal, but if it is an ischemic rat, the probability of turning to the healthy side will increase. Calculate the CT score R / 10%.

[0156] 2. Experimental results

[0157] Based on the excellent performance of BN-4 in the single-dose tMCAO model, we further evaluated the efficacy of BN-4 at the in vivo level by multiple administrations. Figure 5 As shown, the average cerebral infarction area in the model group was 35.64%, and that in the BN-4 (9.61 mg / kg) and NBP (5 mg / kg) groups was 16.62% and 23.30%, respectively. Compared with the model group, both BN-4 and NBP could significantly reduce the cerebral infarction induced by the tMCAO model, among which BN-4 had the best activity, and the cerebral infarction volume inhibition rate was 53.68%, which was significantly higher than that of the NBP group (38.21%), indicating that the compound BN-4 had a better inhibitory effect on cerebral infarction than NBP. At 24h and 72h after ischemia-reperfusion, the mNSS and corner test scores were performed on each group. At 24h after ischemia, the BN-4 administration group could significantly improve the mNSS behavior and corner test scores of rats, which was better than the compound NBP group. At 72h after ischemia, both the BN-4 group and the NBP group could significantly improve the mNSS behavior and corner test scores of rats, which was still better than the compound NBP.

[0158] Example 26: In vitro stability test and water solubility

[0159] 1. Experimental methods

[0160] A. Rat plasma and liver microsome stability experiments

[0161] The test compound was prepared to a final concentration of 200 μM, containing 1% DMSO / rat plasma solution, incubated at 37°C shaker, and samples were taken at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 hours and analyzed by HPLC to observe the degradation of the original compound. The experiment was repeated three times. The test compound was prepared to a final concentration of 200 μM, containing 1% DMSO / PBS solution, and rat liver microsomes in the corresponding proportion were added. The organic phase ratio should not exceed 1%, and the mixture was incubated at 37°C shaker. Samples were taken at 0, 10, 30, 60 and 120 minutes and analyzed by HPLC to observe the degradation of the original compound. The experiment was repeated three times.

[0162] B. Water Solubility Test

[0163] To determine the water solubility of the compound, it was dissolved in distilled water at different concentrations and a standard curve was established using HPLC. When complete solubility was not achieved, the maximum solubility of the compound in water was determined.

[0164] 2. Experimental results

[0165] Since BN-4 showed the best anti-cerebral infarction and improvement of neurological function scores in rats, it was selected as the preferred compound for further study. First, the stability of BN-4 in rat plasma and liver microsomes was tested. The results are as follows Figure 6 As shown in Figure 2, BN-4 is slowly degraded in rat plasma or liver microsomes, with a half-life of about 23 h in rat plasma and more than 120 min in liver microsomes, indicating that BN-4 is relatively stable in vitro. Figure 7 As shown, BN-4 exhibits excellent water solubility compared to NBP. The solubility test results performed by HPLC showed that the solubility of NBP in water was 0.25 mg / mL, while the solubility of BN-4 in water was 30 mg / mL, which was more than 100 times higher.

[0166] Example 27: Pharmacokinetics of Compound BN-4 in Rats

[0167] 1. Experimental methods

[0168] The pharmacokinetic study of the selected compounds was conducted in SD rats. BN-4 (10 mg / kg) and 1400W (6.8 mg / kg) were administered intravenously to rats (230-250 g, 3 animals at each time point). Blood samples (1.0 mL) were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after intravenous injection of the dose, placed in a centrifuge tube containing sodium heparin, and centrifuged at 4°C to separate plasma. Plasma samples were extracted by adding acetonitrile containing a universal internal standard (carbamazepine). Samples were centrifuged at 12,000 rpm for 20 minutes at 4°C, and the supernatant was collected and analyzed. The concentration of the compounds in plasma was determined by high performance liquid chromatography, and non-compartmental analysis was performed using WinNonlin software.

[0169] 2. Experimental results

[0170] To further investigate the pharmacokinetics of BN-4 in vivo, we studied the pharmacokinetic parameters of BN-4 in whole blood after intravenous injection and compared them with those of 1400W. max , AUC 0-∞ 11.21±2.85nmol·mL -1 and 16.47±1.27h·nmol·mL -1 , all higher than 1400W (9.81±2.85nmol·mL -1 and 5.07±0.38h·nmol·mL -1 ). This suggests that BN-4 has a higher body distribution volume than intravenous injection of 1400W, which may lead to better therapeutic effects. In addition, the T 1 / 2 , MRT 0-t and MRT 0-∞ 5.21±0.46h, 1.62±0.36mL·h -1 kg -1 and 1.91±0.49mL·h -1 kg -1 , both higher than 1400W T 1 / 2 (2.79±0.13h, 0.96±0.19mL·h -1 kg -1 、1.11±0.22mL·h -1 kg -1 ), indicating that BN-4 has a longer blood retention time than 1400W. Finally, we studied the blood-brain distribution of BN-4 and 1400W. At the same molar concentration, the blood-brain ratios of BN-4 and 1400W were 27.36% and 10.96%, respectively, indicating that BN-4 has a better ability to penetrate the blood-brain barrier (Table 2).

[0171] Table 2 Pharmacokinetic parameters of 1400W and BN-4 in whole blood of SD rats after intravenous administration (n=3)

[0172]

Claims

1. A novel iNOS inhibitor containing an amidine group having a neuroprotective function, characterized in that: A compound represented by formula I or II or a pharmaceutically acceptable salt thereof; Among them, R 1 Selected from R 2 Selected from -CH2- or R 3 Selected from R 4 Selected from 2. The novel iNOS inhibitor containing an amidine group having a neuroprotective function according to claim 1, characterized in that: The compound represented by formula I is selected from the following compounds: 3-(Acetimidomethyl)-N-(1-butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)benzamide N-(1-Butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-3-((N'-hydroxyacetimidyl)methyl)benzamide 3-Acetimido-N-(1-butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)benzamide N-(1-Butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-3-(N'-hydroxyacetimidyl)benzamide N-(3-(((1-butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)amino)methyl)phenyl)acetamidine N-(3-(((1-butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)amino)methyl)phenyl)-N'-hydroxyacetamidine N-(3-(((1-butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)amino)methyl)benzyl)acetamidine N-(3-(((1-butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)amino)methyl)benzyl)-N'-hydroxyacetamidine 4-(3-(((1-butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)amino)methyl)benzyl)-3-methyl-1,2,4-oxadiazol-5(4H)-one N-(1-Butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-3-((3-methyl-5-oxo-1,2,4-oxadiazol-4(5H)-yl)methyl)benzamide 3. The novel iNOS inhibitor containing an amidine group having a neuroprotective function according to claim 1, characterized in that: The compound represented by formula II is selected from the following compounds: N-(1-Butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)acetamidine N-(1-Butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-N'-hydroxyacetamidine 4. The novel iNOS inhibitor containing an amidine group having a neuroprotective function according to claim 1, characterized in that: The compound represented by formula III is selected from the following compounds: N-(4-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzyl)acetamidine N-(3-(acetimidomethyl)benzyl)-4-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzamide 5. A pharmaceutical composition, characterized in that The invention comprises a therapeutically effective amount of the compound according to claim 1 to 4 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or auxiliary material.

6. A method for preparing the novel amidine-containing iNOS inhibitor with neuroprotective function according to claim 1 or 2, characterized in that: The compound represented by formula I, when R 1 for R 2 -CH2 or The preparation method comprises the following steps: (1) Compound 1 or Compound 2 undergoes an amide condensation reaction with 6-NH2-NBP to obtain intermediate 1-1 or 2-1; (2) removing the protecting group on the amino group of intermediate 1-1 or 2-1 to obtain intermediate 1-2 or 2-2; (3) Intermediate 1-2 or 2-2 reacts with ethyl acetimidate hydrochloride to undergo nucleophilic substitution reaction to obtain compound BN-1, BN-2, BN-3 or BN-4; (4) Compound BN-1, BN-2, BN-3 or BN-4 continues to undergo nucleophilic addition-elimination reaction with hydroxylamine hydrochloride to obtain compound CN-1, CN-2, CN-3 or CN-4; The synthetic route is as follows: When R 1 for R 2 -CH2- or The preparation method comprises the following steps: (1) Compound 3-(bromomethyl)benzoic acid or 3-(bromomethyl)benzaldehyde reacts with compound 3-methyl-5-oxo-1,2,4-oxadiazol-4-yl potassium to obtain intermediate 3-1 or 4-1; (2) Intermediate 3-1 or 4-1 undergoes amide condensation reaction with 6-NH2-NBP to obtain PMN or PMK; the synthetic route is as follows:

7. A method for preparing the novel amidine-containing iNOS inhibitor with neuroprotective function according to claim 1 or 3, characterized in that: The preparation method of the compound represented by formula II comprises the following steps: (1) 6-NH2-NBP undergoes a nucleophilic addition reaction with acetonitrile under the action of concentrated hydrochloric acid to obtain compound BN-0; (2) Compound BN-0 undergoes a nucleophilic addition-elimination reaction with hydroxylamine hydrochloride to obtain compound CN-0; the synthetic route is as follows:

8. A method for preparing the novel amidine-containing iNOS inhibitor with neuroprotective function according to claim 1 or 4, characterized in that: The compound represented by formula III, when R 4 for The preparation method comprises the following steps: (1) Ethyl acetoacetate reacts with 4-cyanophenylhydrazine hydrochloride to form an amide ring to obtain intermediate 5-1; (2) Intermediate 5-1 undergoes a reduction reaction, where the cyano group is reduced to an amino group to obtain intermediate 5-2; (3) Intermediate 5-2 reacts with ethyl acetimidate hydrochloride to undergo nucleophilic substitution reaction to obtain compound CH-1; the synthetic route is as follows: When R 4 for The preparation method comprises the following steps: 1-(4-carboxyphenyl)-3-methyl-5-pyrazolone and 1400W undergo an amide condensation reaction to obtain compound CH-2. The synthesis route is as follows:

9. Use of the novel amidine-containing iNOS inhibitor with neuroprotective function as claimed in any one of claims 1 to 4 in the preparation of a drug for preventing and / or treating stroke.

10. Use of the pharmaceutical composition according to claim 5 in preparing a drug for preventing and / or treating stroke.

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