A novel amidine-containing iNOS inhibitor with neuroprotective function and a preparation method and application thereof

Abstract

The application discloses a novel amidine-containing iNOS inhibitor with a neuroprotective function, and a preparation method and application thereof, and has a structural formula I, II or III. The compound takes the iNOS inhibitor 1400W, the neuroprotective agent NBP or the free radical scavenger Eda fragment as a skeleton, combines the advantages of the NBP and the Eda, and fuses the characteristics of the selective iNOS inhibitor 1400W, so that the compound can selectively inhibit iNOS and simultaneously has a neuroprotective capacity, plays a synergistic role, and better treats ischemic stroke.

Description

TECHNICAL FIELD

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

[0002] Stroke, also known as cerebral apoplexy (Mol. Basis. Dis., 2020, 1866(4): 165260.), is caused by the obstruction or rupture of cerebral blood vessels, resulting in insufficient or interrupted cerebral blood flow supply, thereby causing brain tissue damage (J. Clin. Neurosci., 2021, 93: 174-182.). The symptoms of stroke can vary from patient to patient, and common symptoms include sudden onset of limb weakness, numbness or weakness, sudden onset of speech difficulty, difficulty understanding, visual loss or blurring, and severe headache, dizziness, loss of balance ability, 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.). Among them, ischemic stroke refers to an acute cerebral vascular disease caused by multiple reasons of vascular damage, occlusion damage to the blood oxygen supply of the brain, ischemic and anoxic necrosis of the brain tissue, resulting in neurological impairment of the patient (Circulation, 2018, 137(12): e67-e492.). With the rapid increase in the aging population and the increasing incidence of cardiovascular disease, 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 disabilities 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] Current clinically used drugs, such as neuroprotective agent NBP and free radical scavenger Eda, show certain efficacy in anti-cerebral ischemia, but each has significant limitations. NBP has multi-target anti-cerebral ischemia activity, but its therapeutic effect is not outstanding (Neurosci. Lett., 2012, 516(2): 247-252); Eda needs to be administered by intravenous injection due to poor water solubility and low oral bioavailability, which increases the treatment cost and inconvenience. 1400W, as a highly selective iNOS inhibitor, shows good iNOS inhibitory activity in cell experiments, but its ability to improve cerebral blood flow is weak, and it fails to achieve ideal anti-cerebral ischemic effect (Neurochem. Res., 2011, 36: 476-486.).

[0005] SUMMARY

[0006] The first object of the present application is to provide a novel amidine-containing iNOS inhibitor with neuroprotective function which selectively inhibits iNOS and has neuroprotective ability; the second object of the present application is to provide a preparation method of the novel amidine-containing iNOS inhibitor with neuroprotective function; and the third object of the present application is the application of the novel amidine-containing iNOS inhibitor with neuroprotective function.

[0007] The novel amidine-containing iNOS inhibitor with neuroprotective function of the present application is a compound represented by formula I or II, or a pharmaceutically acceptable salt thereof.

[0008]

[0009] R 1 is selected from

[0010] R 2 is selected from -CH2- or R 3 is selected from R 4 is selected from

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

[0012] 3-(acetyliminomethyl)-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'-hydroxyacetamidino)methyl)benzamide

[0015]

[0016] 3-acetylimino-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'-hydroxyacetamidino)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 of 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 of Formula III is selected from the following compounds:

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

[0039]

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

[0041]

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

[0043] The dosage form of the pharmaceutical composition of the present application can be prepared by those skilled in the art according to the conventional method in the pharmaceutical field. For example, the active ingredient is mixed with one or more carriers (also known as excipients), and then it is made into the desired dosage form, including tablets, capsules, granules, aerosols; it can also be made into intravenous injection or intravenous injection lyophilized according to the conventional production method of injection.

[0044] The preparation method of the novel amidine-containing iNOS inhibitor with neuroprotective function of the present application, the compound of Formula I, when R 1 is R 2 is -CH2or The preparation method comprises the following steps:

[0045] (1) Compound 1 or Compound 2 is subjected to 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) subjecting intermediate 1-2 or 2-2 to nucleophilic substitution reaction with ethyl acetoimidate hydrochloride to obtain compound BN-1, BN-2, BN-3 or BN-4;

[0048] (4) subjecting compound BN-1, BN-2, BN-3 or BN-4 to 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 is R 2 is -CH2- or The preparation method comprises the following steps:

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

[0053] (2) subjecting intermediate 3-1 or 4-1 to 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, the preparation method of the compound shown in formula II, comprises the following steps:

[0056] (1) subjecting 6-NH2-NBP to nucleophilic addition reaction with acetonitrile under the action of concentrated hydrochloric acid to obtain compound BN-0;

[0057] (2) subjecting compound BN-0 to 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 amidine-containing iNOS inhibitor with neuroprotective function, the compound shown in formula III, when R 4 is The preparation method comprises the following steps:

[0060] (1) acetyl ethyl acetate and 4-cyanophenylhydrazine hydrochloride amide reaction to form ring to obtain intermediate 5-1;

[0061] (2) intermediate 5-1 reduction reaction cyanide is reduced to amino to obtain intermediate 5-2;

[0062] (3) intermediate 5-2 and ethyl acetyl imidazole hydrochloride nucleophilic substitution reaction to obtain compound CH-1; the synthetic route is as follows:

[0063]

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

[0065]

[0066] The application of the novel amidine-containing iNOS inhibitor with neuroprotective function in the preparation of a drug for preventing and / or treating cerebral apoplexy.

[0067] The application of the pharmaceutical composition in the preparation of a drug for preventing and / or treating cerebral apoplexy.

[0068] Invention mechanism: Multi-target drugs can act on multiple pathological mechanisms at the same time, providing more comprehensive therapeutic effect. In the treatment of ischemic stroke, the advantage of multi-target drug design is particularly significant. The compound designed and synthesized in the application can selectively inhibit iNOS and has neuroprotective ability, can play a synergistic effect, and better treat ischemic stroke. These compounds combine the advantages of existing drugs NBP and Eda, and fuse the characteristics of selective iNOS inhibitor 1400W, and are expected to play a synergistic effect 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 cells SH-SY5Y, mouse microglia BV-2 and human umbilical vein endothelial cell fusion cells EA.hy926 under OGD / R conditions; (2) Compounds BN-1 and BN-4 have good iNOS inhibitory activity. 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. (3) Compound BN-4 has a good effect on cerebral infarction and neurological function recovery in rats, and is superior to positive drug NBP, compound 1400W and the combination of the two; (4) Compound BN-4 has good pharmacokinetic properties, with water solubility more than 100 times higher than NBP, and blood-brain ratio significantly improved compared to compound 1400W, and has good drug-like properties; (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 readily available in synthesis. Attached Figure Description

[0070] Figure 1 This is a schematic diagram illustrating the design concept and mechanism of action of a series of compounds;

[0071] Figure 2 The effect of the compound at 1 μM and 10 μM on the survival rate of OGD / R-induced SH-SY5Y cells, Eahy-926 cells and BV-2 cells was investigated.

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

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

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

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

[0076] Figure 7 It is the water-soluble form of compounds BN-4 and NBP. Detailed Implementation

[0077] The present invention will be further described below with reference to specific embodiments.

[0078] I. Synthesis of intermediates

[0079] Example 1: Synthesis of intermediate 1-1 The procedure is to put 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 solvent, completely dissolve the reactants. Then add the condensing agent N,N,N',N'-tetramethylchloroformamidium hexafluorophosphate (420 mg, 1.5 mmol, 1.5 eq), the acid binding agent N-methylimidazole (288 mg, 3.5 mmol, 3.5 eq). After the addition is complete, place it in 25 °C and stir for 24 h. During the reaction, the reaction solution gradually changes from turbidity to clarity, and the solid raw material is completely dissolved. After the reaction is completed, the residue is diluted with water. Extract with EA, collect the upper clear liquid, combine the organic phases, dry over anhydrous sodium sulfate, concentrate under reduced pressure to remove the solvent, and purify the crude product by column chromatography (PE:EA = 2:1) to obtain a white solid, which is intermediate 1-1.

[0080] Example 2: Synthesis of intermediate 1-2 The procedure is to put intermediate 1-1 (1 mmol) in a 50 mL round bottom flask, add 8 mL of trifluoroacetic acid in dichloromethane solution (1:3) as solvent, place it in 25 °C, stir for 1 h, and the reaction is completed. Quench with water, extract with DCM three times, combine the organic phases, dry over anhydrous sodium sulfate, concentrate to dryness under reduced pressure, and obtain a white solid, which is directly used in the next step without further treatment.

[0081] Example 3: Synthesis of intermediate 2-1 The procedure is to put 6-NH2-NBP (205 mg, 1 mmol, 1 eq) and the corresponding benzaldehyde derivative (1 mmol, 1 eq) in a round bottom flask, dissolve the reactants in 10 mL of anhydrous ethanol, heat to 80 °C and reflux for 12 h, then slowly cool to 0 °C, add sodium borohydride (76 mg, 2 mmol, 2 eq) in small portions, and move to room temperature for 2 h. After the reaction is completed by TLC (developing agent PE:EA = 3:1), quench with water, extract with DCM, combine the organic phases, dry over anhydrous sodium sulfate, concentrate under reduced pressure to remove the solvent, and purify the crude product by column chromatography to obtain intermediate 2-1.

[0082] Example 4: Synthesis of intermediate 2-2 The procedure is to put intermediate 2-1 (1 mmol) in a 50 mL round bottom flask, add 8 mL of trifluoroacetic acid in dichloromethane solution (1:3) as solvent, place it in 25 °C, stir for 1 h, and the reaction is completed. Quench with water, extract with DCM three times, combine the organic phases, dry over anhydrous sodium sulfate, concentrate to dryness under reduced pressure, and obtain a white solid, which is directly used in the next step 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 of acetone to dissolve it, and then add 3-methyl-5-oxo-1,2,4-oxadiazol-4-yl potassium (138 mg, 1 mmol, 1 eq) to the flask, stir at 25°C for 18 h. After the reaction is completed, concentrate to dryness under reduced pressure, wash with anhydrous diethyl ether to obtain a white solid, which is directly used in the next step.

[0084] Example 6: The synthesis step of intermediate 4-1 is to place 3-(bromomethyl)benzaldehyde (198 mg, 1 mmol, 1 eq) in a 50 mL round-bottom flask, add 10 mL of acetone to dissolve it, and then add 3-methyl-5-oxo-1,2,4-oxadiazol-4-yl potassium (138 mg, 1 mmol, 1 eq) to the flask, stir at 25°C for 18 h. After the reaction is completed, concentrate to dryness under reduced pressure, wash with anhydrous diethyl 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 drop 5 mL of glacial acetic acid. The reaction is heated to 80°C to reflux for 12 h, and the system gradually changes from turbidity to clarity. After the reaction is completed, cool it to room temperature, extract with EA three times, combine the organic phase, concentrate to remove the solvent under reduced pressure, and purify the crude product by column chromatography (PE:EA = 1:1) to obtain intermediate 5-1.

[0086] Example 8: The synthesis step of intermediate 5-2 is to place intermediate 5-1 (199 mg, 1 mmol, 1 eq) in a 50 mL round-bottom flask, add 10 mL of MeOH to dissolve it, and then add 20 mg of palladium on carbon and 1 drop of glacial acetic acid, and react under hydrogen for 24 h. After the reaction is completed, filter the palladium on carbon, collect the filtrate, and concentrate to dryness under reduced pressure to obtain a yellow solid, which is directly used in the next step.

[0087] II. Synthesis of target compounds

[0088] Example 9: The synthetic route of the target compound BN-1 is to dissolve intermediate 1-2 (1 mmol, 1 eq) with anhydrous ethanol, cool to 0-5 °C, add ethyl acetimidate hydrochloride (128 mg, 0.9 mmol, 0.9 eq), anhydrous potassium carbonate (276 mg, 2 mmol, 2 eq), stir the reaction for 4 h. After the reaction is completed, the solvent is concentrated to dryness under reduced pressure, add water, extract with EA three times, combine the upper organic phase, dry with anhydrous sodium sulfate, concentrate the organic phase under reduced pressure to obtain a yellow sticky liquid. The product is subjected to flash column chromatography (DCM:MeOH = 15:1) to obtain BN-1 251 mg as a white solid with a yield of 66%. mp 220.2-220.8 °C; 1 H NMR (500 MHz, DMSO-d6) δ 10.77 (s, 1H), 8.40-8.32 (m, 1H), 8.15 (d, J = 7.8 Hz, 1H), 8.07 (s, 1H), 7.99 (d, J = 7.4 Hz, 1H), 7.67 (d, J = 8.3 Hz, 1H), 7.63-7.56 (m, 2H), 5.63 (dd, J = 7.6, 4.0 Hz, 1H), 4.58 (s, 2H), 2.24 (s, 3H), 2.05 (tt, J = 9.7, 5.2 Hz, 1H), 1.76-1.66 (m, 1H), 1.34-1.23 (m, 4H), 0.88 (t, J = 6.9 Hz, 3H); 13 C NMR (75 MHz, DMSO-d6) δ 170.38, 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] + calcd 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 target compound CN-1 is to put 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), anhydrous potassium carbonate (272 mg, 2 mmol, 2 eq). Heat to 70 °C and reflux for 5 h. Monitor using TLC method, PE:EA = 2:1, R f value is 0.5-0.6. The raw material is completely reacted, extracted with a small amount of EA for three times, the organic phase is combined, dried with anhydrous sodium sulfate, concentrated to dryness under reduced pressure, CN-1 crude product is obtained. Purify the crude product by column chromatography (PE:EA = 2:1) to obtain CN-1 300 mg as a white solid, yield 76%. mp 195.2-195.9 °C; MS (m / z): 418 [M+Na] + ; 1 H NMR (300 MHz, DMSO-d6) δ 10.58 (s, 1H), 8.98 (s, 1H), 8.30 (s, 1H), 8.07 (d, J = 8.2 Hz, 1H), 7.85 (s, 2H), 7.65 (d, J = 8.4 Hz, 1H), 7.51 (d, J = 5.3 Hz, 2H), 5.62 (d, J = 5.9 Hz, 1H), 4.26 (dd, J = 40.6, 6.1 Hz, 2H), 2.00 (d, J = 18.3 Hz, 1H), 1.66 (s, 1H), 1.33 - 1.22 (m, 4H), 0.85 (d, J = 7.1 Hz, 3H); 13 C NMR (75 MHz, DMSO-d6) δ 170.37, 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] + calcd 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 target compound BN-2 was to dissolve intermediate 1-2 (1 mmol, 1 eq) in anhydrous ethanol, cooled to about 5 °C, added ethyl acetyl imidate hydrochloride (128 mg, 0.9 mmol, 0.9 eq), anhydrous potassium carbonate (276 mg, 2 mmol, 2 eq), stirred for 4 h. After complete reaction, the treatment method was the same as BN-1, and a yellow viscous liquid was obtained. The product was obtained by flash column chromatography (DCM:MeOH = 15:1) to obtain BN-2 151 mg as a white solid, with a yield of 41%. mp 189.3-189.8 °C; 1 H NMR (400 MHz, Methanol-d4) δ 8.32 (d, J = 1.9 Hz, 1H), 8.03 - 7.96 (m, 2H), 7.89 (t, J = 2.0 Hz, 1H), 7.65 (t, J = 7.9 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.50 (dd, J = 7.9, 2.2 Hz, 1H), 5.56 (dd, J = 7.7, 4.0 Hz, 1H), 2.39 (s, 3H), 2.09 (dd, J = 9.6, 4.5 Hz, 1H), 1.77 - 1.69 (m, 1H), 1.40 - 1.30 (m, 4H), 0.91 (t, J = 7.0 Hz, 3H). 13 C NMR (126 MHz, Methanol-d4) δ 130.56, 127.13, 124.69, 122.51, 116.52, 81.98, 48.53, 48.19, 48.02, 47.85, 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] + calcd 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 target compound CN-2 was 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), anhydrous potassium carbonate (272 mg, 2 mmol, 2 eq). Heat to 70 °C and reflux for 5 h. Monitor by TLC method, PE:EA = 1:1, R fvalue of 0.5-0.6. After the reaction was completed, EA was added for extraction three times, the organic phase was combined, dried with anhydrous sodium sulfate, and the organic layer was concentrated to dryness under reduced pressure to obtain CN-2 crude product. The crude product was purified by column chromatography (PE:EA = 1:1) to obtain CN-2 300 mg in the form of a yellowish oil, with a yield of 76%. 1 H NMR (300 MHz, Methanol-d4) δ 8.25 (d, J = 2.0 Hz, 1H), 8.01 (dd, J = 8.3, 2.0 Hz, 1H), 7.71 - 7.64 (m, 2H), 7.54 (d, J = 4.9 Hz, 1H), 7.43 (s, 1H), 7.33 (d, J = 6.2 Hz, 1H), 5.56 - 5.51 (m, 1H), 2.05 (d, J = 6.5 Hz, 1H), 1.95 (d, J = 3.2 Hz, 3H), 1.77 - 1.68 (m, 1H), 1.35 - 1.29 (m, 4H), 0.90 (t, J = 3.9 Hz, 3H); 13 C NMR (75 MHz, Methanol-d4) δ 171.12, 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] + calcd for [C 21 H 24 N3O4] + 382.17613, found 382.17624, ppm error 1.18. HPLC. MeOH / H2O = 80:20.

[0092] Example 13: The synthetic route of the target compound BN-3 is to dissolve intermediate 2-2 (1 mmol, 1 eq) in anhydrous ethanol, cool to 0-5°C, and add a small amount of weighed ethyl acetimidate hydrochloride (128 mg, 0.9 mmol, 0.9 eq) and anhydrous potassium carbonate (276 mg, 2 mmol, 2 eq) in several portions, and stir at this temperature for 4 h. After the reaction is completed, the solvent is concentrated to dryness under reduced pressure, water is added, EA is extracted three times, the organic phase is combined, and the organic layer is concentrated to dryness under reduced pressure to obtain a yellow viscous liquid. The product is purified by flash column chromatography (DCM:MeOH = 15:1) to obtain BN-3 131 mg in the form of a yellowish solid, with a yield of 37%. mp 185.2-185.7°C; 1H NMR (300 MHz, Methanol-d4) δ 7.46 (d, J = 0.7 Hz, 2H), 7.35 (s, 1H), 7.25 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 2.0 Hz, 1H), 7.08 (d, J = 2.3 Hz, 1H), 6.80 (d, J = 2.2 Hz, 1H), 5.39 (dd, J = 7.5, 4.0 Hz, 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 = 3.6 Hz, 3H); 13 C NMR (75 MHz, Methanol-d4) δ 134.12, 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] + calcd for [C 21 H 26 N3O2] + 352.20195, found 352.20126, ppm error -1.54. HPLC. MeOH / H2O (with 0.1% Et3N) = 80:20.

[0093] Example 14: The synthesis route of target compound CN-3 is to put BN-3 (351 mg, 1 mmol, 1 eq) in a flask, add 15 mL of anhydrous THF to completely dissolve, then add hydroxylamine hydrochloride (100 mg, 1.5 mmol, 1.5 eq), anhydrous potassium carbonate (272 mg, 2 mmol, 2 eq). Heat to 70°C and reflux for 5h. Monitor by TLC method, PE:EA = 1:2, R f value is 0.5-0.6. The raw material is completely reacted, extracted with EA for three times, the organic phase is combined, and the solvent is concentrated to dryness under reduced pressure to obtain CN-3 crude product. Purify the crude product by column chromatography (PE:EA = 1:2) to obtain CN-3 280 mg as a light yellow oil, with a yield of 76%. 1H NMR (300 MHz, Methanol-d4) δ 7.25 - 7.19 (m, 2H), 7.09 (d, J = 2.1 Hz, 2H), 6.99 (d, J = 2.3 Hz, 1H), 6.96 (dd, J = 2.4, 1.1 Hz, 1H), 6.83 (d, J = 2.2 Hz, 1H), 5.37 (dd, J = 7.5, 4.0 Hz, 1H), 4.34 (s, 2H), 1.98 - 1.94 (m, 1H), 1.77 (s, 3H), 1.60 (d, J = 4.6 Hz, 1H), 1.25 (s, 4H), 0.89 - 0.86 (m, 3H);13C NMR (75 MHz, Methanol-d4) δ 149.83, 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] + calcd for[C 21 H 26 N3O3] + 368.19687, found 368.19612, ppm error -1.61. HPLC. MeOH / H20 = 80:20.

[0094] Example 15: The synthetic route of target compound BN-4 is to dissolve intermediate 2-2 (1 mmol, 1 eq) in anhydrous ethanol, 5 °C, add ethyl acetyl imidate hydrochloride (128 mg, 0.9 mmol, 0.9 eq), anhydrous potassium carbonate (276 mg, 2 mmol, 2 eq), stir the reaction at this temperature for 4 h. The reaction is completed, the solvent is concentrated under reduced pressure, add water, extract with EA three times, combine the organic layer, dry with anhydrous sodium sulfate, concentrate the organic layer to dryness under reduced pressure, get light yellow solid. Purified by column chromatography (DCM:MeOH = 14:1) to get BN-4 310 mg as light yellow solid, yield 85%. mp 210.8-211.5 °C; 1H NMR (300 MHz, Methanol-d4) δ 7.35 (d, J = 1.3 Hz, 3H), 7.23 (d, J = 2.1 Hz, 2H), 7.02 (dd, J = 8.4, 2.2 Hz, 1H), 6.78 (d, J = 2.2 Hz, 1H), 5.36 (dd, J = 7.6, 4.0 Hz, 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 = 3.4 Hz, 4H), 0.86 (d, J = 2.3 Hz, 3H). 13 C NMR (75 MHz, Methanol-d4) δ 149.86, 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] + calcd for [C 22 H 28 N3O2] + 366.21760, found 366.21678, ppm error -1.87. HPLC MeOH / H2O (with 0.1% Et3N) = 80:20.

[0095] Example 16: The synthetic route of target compound CN-4 is to put BN-4 (365 mg, 1 mmol, 1 eq) in a flask, add 15 mL of anhydrous THF to completely dissolve it, then add hydroxylamine hydrochloride (100 mg, 1.5 mmol, 1.5 eq), anhydrous potassium carbonate (272 mg, 2 mmol, 2 eq). Heat to 70°C and reflux for 5h. Monitor by TLC method, PE:EA = 1:2, Rf value is 0.5-0.6. The raw material is completely reacted, and the EA extraction method is the same as CN-1 to obtain CN-4 crude product. Purify the crude product by column chromatography (PE:EA = 1:3) to obtain CN-4 295 mg as a white solid, with a yield of 77%. mp 199.5-199.8°C; 1H NMR (300 MHz, Methanol-d4) δ 7.32 (s, 1H), 7.27 (t, J = 1.8 Hz, 2H), 7.22 (d, J = 8.3 Hz, 1H), 7.17 (d, J = 2.5 Hz, 1H), 7.02 (dd, J = 8.3, 2.2 Hz, 1H), 6.85 (d, J = 2.2 Hz, 1H), 5.35 (s, 1H), 4.34 (d, J = 1.9 Hz, 4H), 1.95 (d, J = 1.5 Hz, 1H), 1.77 (s, 3H), 1.63 (d, J = 2.2 Hz, 1H), 1.33 (d, J = 5.5 Hz, 4H), 0.90 (d, J = 4.2 Hz, 3H). 13 C NMR (75 MHz, Methanol-d4) δ 140.12, 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] + calcd 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 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 of DMF as a solvent, and completely dissolve the reactants. Then add N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate (420 mg, 1.5 mmol, 1.5 eq), N-methylimidazole (288 mg, 3.5 mmol, 3.5 eq). After the addition is complete, place it in a 25°C water bath and stir the reaction for 24 h. Monitor the end of the reaction by TLC (developing agent PE:EA = 3:1), add water, extract with EA three times, and combine the organic phases and dry them over anhydrous sodium sulfate. Concentrate the organic layer to dryness under reduced pressure, and purify the crude product by column chromatography (PE:EA = 1:2) to obtain PMN 288 mg as a light yellow solid with a yield of 68%. mp 154.8-155.2 °C; 1H NMR (300 MHz, DMSO-d6) δ 10.80 - 10.51 (m, 1H), 7.73 (d, J = 98.3 Hz, 6H), 5.58 (s, 1H), 4.89 (d, J = 16.3 Hz, 1H), 2.25 - 2.14 (m, 2H), 1.25 (s, 4H), 0.85 (s, 3H). 13 C NMR (75 MHz, Methanol-d4) δ 160.45, 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] + calcd for [C 23 H 23 N3O5Na] + 444.15299, found 444.15299, ppm error 0.03. HPLC MeOH / H20 = 80:20.

[0097] Example 18: The synthetic route of the target compound PMK is to put intermediate 4-1 (218 mg, 1 mmol, 1 eq) and 6-NH2-NBP (205 mg, 1 mmol, 1 eq) and in a flask, add 10 mL of anhydrous ethanol to dissolve it. Heat to 80 °C reflux reaction for 0.5 h, generate light yellow solid, the reaction is over, put it at room temperature, filter, dry under infrared lamp, get imine intermediate. Dissolve this compound in MeOH, cool to 5 °C, slowly add sodium borohydride (76 mg, 2 mmol, 2 eq), TLC (developing agent PE: EA = 3: 1) to monitor the end of the reaction, add water to quench the reaction, EA extraction, purification method is the same as BN-1 245 mg, get yellow solid, yield 60%. mp 134.8-135.2 °C; 1 H NMR (500 MHz, Methanol-d4) δ 7.42 (s, 2H), 7.34 - 7.28 (m, 2H), 7.23 (d, J = 2.0 Hz, 1H), 7.07 (dd, J = 8.3, 2.2 Hz, 1H), 6.85 (d, J = 2.2 Hz, 1H), 5.45 (dd, J = 7.6, 4.1 Hz, 1H), 4.45 (s, 2H), 2.10 (s, 3H), 2.05 (d, J = 1.7 Hz, 1H), 1.76 - 1.68 (m, 1H), 1.40 (dt, J = 6.0, 2.3 Hz, 4H), 0.96 (d, J = 5.2 Hz, 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] + calcd 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 for the target compound BN-0 was as follows: 6-NH2-NBP (205 mg, 1 mmol, 1 eq) was placed in a 50 mL round-bottom flask, and 20 mL of anhydrous acetonitrile was added to completely dissolve it. Then, 1 mL of concentrated hydrochloric acid was slowly added dropwise. The mixture was heated to 83 °C and refluxed for 5 h. The reaction changed from colorless to pale yellow, and was monitored by TLC. PE:EA = 1:1, R... f The value was 0.5–0.6. After the reaction was complete, the sample was extracted three times with ethyl acetate. The organic layers were combined, then washed separately with saturated sodium bicarbonate, twice with water, and once with brine. The mixture was dried over anhydrous sodium sulfate, and the organic layer was concentrated to dryness under reduced pressure to obtain crude BN-0. The crude product was subjected to rapid column chromatography (PE:EA = 1:1) to obtain 185 mg of BN-0 as a pale yellow solid, with a yield of 75%. mp 173.2–173.8℃; 1H NMR (300 MHz, DMSO-d6) δ 9.70 (s, 1H), 7.81 - 7.73 (m, 2H), 7.66 (dd, J = 8.1, 1.9 Hz, 1H), 5.67 (dd, J = 7.8, 3.9 Hz, 1H), 2.35 (s, 3H), 2.06 (dq, J = 14.2, 4.9 Hz, 1H), 1.72 (s, 1H), 1.31 (q, J = 6.3 Hz, 4H), 0.87 - 0.81 (m, 3H);13C NMR (75 MHz, DMSO-d6) δ 169.54, 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] + calcd for [C 14 H 19 N2O2] + 247.14465, found 247.14227, ppm error 8.61. HPLC MeOH / H2O (with 0.1% Et3N) = 80:20.

[0099] Example 20: The synthetic route of 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), anhydrous potassium carbonate (272 mg, 2 mmol, 2 eq). Place it in a room temperature for 5 h of stirring. Monitor using TLC method, PE:EA = 1:1, R f f = 0.5-0.6. The raw material is completely reacted, extracted with EA, the extraction method is the same as that of BN-0, concentrated under reduced pressure to obtain CN-0 crude product. Purify the crude product by column chromatography (PE:EA = 1:1) to obtain CN-0 150 mg as a white solid, with a yield of 57%. mp 188.6-189.3 °C; MS (m / z): 285 [M+Na] + ; 1 H NMR (300 MHz, Methanol-d4) δ 7.50 - 7.46 (m, 2H), 5.51 (dd, J = 7.7, 4.0 Hz, 1H), 2.04 (s, 1H), 1.93 (s, 3H), 1.68 (d, J = 3.6 Hz, 1H), 1.34 (dd, J = 4.8, 1.8 Hz, 4H), 0.88 (d, J = 2.7 Hz, 3H); 13C NMR (75 MHz, Methanol-d4) δ 171.00, 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] + calcd for [C 14 H 19 N2O3] + 263.13902, found 263.13801, ppm error -3.78. HPLC. MeOH / H2O = 80:20.

[0100] Example 21: The synthetic route of the target compound CH-1 is to dissolve intermediate 5-2 (203 mg, 1 mmol, 1 eq) in anhydrous ethanol, 5 °C, add ethyl acetyl imidate hydrochloride (128 mg, 0.9 mmol, 0.9 eq), anhydrous potassium carbonate (276 mg, 2 mmol, 2 eq), stir at this temperature for 4 h. The reaction is completed, methanol is concentrated to dryness under reduced pressure, add water, extract with ethyl acetate three times, combine the organic layer, dry with anhydrous sodium sulfate, concentrate the organic layer to dryness under reduced pressure, get light yellow solid. The product is purified by flash column chromatography (DCM:MeOH = 8:1) to get CH-1 110 mg as a light yellow solid, yield 45%. mp 167.2-167.8 °C; MS (m / z): 267 [M+Na] + ; 1 H NMR (300 MHz, Methanol-d4) δ 7.77 (d, J = 8.5 Hz, 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 (75 MHz, DMSO-d6) δ 127.19, 120.35, 47.69, 47.41, 47.13, 46.84, 46.56, 46.27, 45.99. HRMS-ESI (m / z): [M+H] + calcd for [C 13 H 17 N4O] + 245.13969, found 245.13961, 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 a flask, add 5 mL of DMF as the solvent, and completely dissolve the reactants. Then add N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate (420 mg, 1.5 mmol, 1.5 eq), N-methylimidazole (288 mg, 3.5 mmol, 3.5 eq). After the addition is complete, place it in a room temperature stir for 24 h. After the reaction is completed by TLC monitoring, add H2O, extract with EA three times, combine the organic layers, and dry over anhydrous sodium sulfate. The organic layer is concentrated to dryness under reduced pressure, and the product is obtained by flash column chromatography (PE:EA = 1:1) as a yellow oil, 151 mg, with a yield of 40%. MS (m / z): 400 [M+Na] + ; 1 H NMR (300 MHz, Methanol-d4) δ 8.23 (d, J = 1.9 Hz, 1H), 8.20 (d, J = 2.1 Hz, 1H), 8.06 (d, J = 4.7 Hz, 2H), 7.63 (d, J = 1.5 Hz, 3H), 7.52 (dd, J = 6.0, 2.3 Hz, 1H), 4.85 (d, J = 2.9 Hz, 2H), 4.71 (s, 2H), 3.24 (s, 2H), 2.50 (s, 3H), 2.50 (s, 3H). 13 C NMR (75 MHz, Methanol-d4) δ 168.77, 164.63, 139.00, 131.09, 129.43, 129.23, 128.49, 121.71, 121.14, 120.26, 118.92, 20.06, 18.25, 17.66, 14.09, 13.64, 8.32. HPLC. MeOH / H2O = 80:20.

[0102] III. Effect verification

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

[0104] 1. Experimental method

[0105] a. Cell digestion, counting, and preparation of cell suspension with a concentration of 5 x 10 4 6 / mL in a 96-well cell culture plate, and adding 100 μL of cell suspension to each well;

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

[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. Incubate the 96-well cell culture plate at 37°C in a 5% CO2 incubator for 24 hours;

[0109] e. Replace the culture medium with DMEM medium without glucose and serum, and culture the cells in a 5% CO2, 95% N2 environment for 2 hours. Then replace the culture medium with complete DMEM medium and culture in a 5% CO2, 95% O2 environment for 24 hours.

[0110] f. Stain the 96-well plate with CCK-8 at λ = 450 nm and measure the OD value; A. Add 10 μL of CCK-8 to each well and continue incubation for 2 h; B. Gently mix by shaking for 10 min; C. At λ = 450 nm, read the OD value of each well with a microplate reader and calculate the inhibition rate.

[0111] 2. Experimental Results

[0112] like Figure 2 As shown, in SH-SY5Y cells, the cell viability of the OGD / R model group was significantly reduced. With the addition of NBP, 6-NH2-NBP, and 1400W, the cell viability significantly increased compared to the OGD / R group. At a concentration of 1 μM, CN-2 showed increased cell viability compared to the NBP or 6-NH2-NBP groups; at a concentration of 10 μM, BN-4, CN-1, CN-2, CN-4, CH-1, and PMK also showed significantly increased cell viability compared to the NBP or 6-NH2-NBP groups. In Eahy-926 and BV-2 cells, the cell viability of the OGD / R model group was significantly reduced. With the addition of NBP and 1400W, the cell viability significantly increased compared to the OGD / R group. All tested compounds improved cell viability, with BN-4 showing the best effect and similar activity to NBP, improving cell viability at both 1 μM and 10 μM concentrations in both cell types.

[0113] Example 23: Evaluation of iNOS inhibitory activity at 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 planted at a density of 2 × 10⁶ cells per well. 4Cells were seeded at a density of 100 μL of culture medium into black 96-well plates and incubated for 24 h. Cells were then pretreated with a compound for 1 h, followed by stimulation with LPS (1 μg / mL) for 6 h. The culture supernatant was removed, and 100 μL of NOS assay buffer was added to each well. Then, 100 μL of NOS analysis reaction solution (5% 0.1 mM NADPH, 0.2% DAF-FM DA, 39.8% MilliQ water, 50% NOS analysis buffer, 5% L-arginine solution) was added to each well and incubated at 37 °C for 2 h. Fluorescence was measured using a Bio-Tek fluorescent plate reader at excitation at 495 nm and emission at 515 nm.

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

[0118] a. Add 20 μL of the analyte 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. Add 10 μM L-arginine to HEPES buffer (100 mM, 10% glycerol, pH 7.4), which also contains 100 μM NADPH, 10 μM tetrahydrobiopterin and 4 μM human oxyhemoglobin.

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

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

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

[0123] f. The reaction was tracked by reading the absorbance at 401 nm using a dual-wavelength dynamic reader at 37°C. Dynamic readings were taken every 3-5 minutes.

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

[0125] 2. Experimental Results

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

[0127] Table 1 IC50 of compounds BN-1 to PMN on iNOS 50

[0128]

[0129]

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

[0131] 1. Experimental method

[0132] A. Preparation of rat tMACO model: In order to manufacture embolism model, nylon wire with diameter of 0.234 mm is selected, and the top end of the nylon wire is made thicker and is burned to a smooth ball with diameter of about 0.35 mm. A marker is made on the nylon wire at a distance of about 20 mm from the top end. The nylon wire is cleaned with 75% alcohol and is placed in 2500 U / mL heparin saline for use. The rats are anesthetized using a gas anesthesia machine (the anesthetic dose should not be too large to prevent the rats from dying due to excessive anesthesia), and the rats are fixed on the experimental table with their heads upward. An incision is made in the middle of the neck, and the skin is cut open using surgical scissors. The skin tissue is carefully torn open to find the blood vessels in the neck of the rat. First, the right common carotid artery (CCA) is found, and the right external carotid artery (ECA) and internal carotid artery are separated upward using a surgical thread. The two ends are ligated, and the thyroid superior artery and occipital artery, two branches of the external carotid artery, are cut. The ECA is doubly ligated near the CCA bifurcation about 6 mm away. A microartery clamp is used to clamp the blood vessel at the proximal end of the right common carotid artery, and a live knot is left at the proximal end of the right external carotid artery near the bifurcation. A V-shaped micro-incision with a diameter of about 0.2 mm is made between the proximal end of the ECA ligation and the common carotid artery bifurcation. The nylon wire head is gently inserted from the incision, and the knot is gently tightened. The internal carotid artery is cut between the two ligation lines to make it consistent with the direction of the internal carotid artery. The artery clamp is loosened, and the nylon wire is sent along the ECA and ICA into the cranium. The insertion depth is about 18 mm to 20 mm, and the resistance is stopped when the resistance is encountered. The nylon wire head is located at the beginning of the MCA, the blood flow of the MCA is blocked, the silk thread is tightened, the incision is sutured, and the tail of the nylon wire is left outside the body. After 2 h of ischemia, the modeling rats are anesthetized using a gas anesthesia machine. In order to perform reperfusion after ischemia, the top of the nylon wire needs to be returned to the micro-incision. The head end is gently pulled back into place using surgical forceps, and a slight resistance can be felt. At this time, the blood supply of the middle cerebral artery of the rat is restored, and the MCAO rat modeling is completed. At this time, the prepared drug is immediately injected into the tail vein for administration.

[0133] B. Detection index:

[0134] a. Measurement of cerebral infarction volume (TTC staining method): After the rat tMCAO model is established, the rats are sacrificed by anesthesia at 24 h or 72 h, and the head is dissected to remove the brain tissue. The brain tissue is quickly placed in the fresh layer of the refrigerator for 15 min to make the brain hard for slicing. The brain is cut into 5 pieces with uniform thickness, and then the brain slices are quickly placed in 5 mL of PBS buffer solution containing 2% TTC prepared in advance. The brain slices are wrapped with tin paper and placed in an oven for 37°C incubation for 15 min. During this process, the brain slices are turned over every 2 minutes to ensure uniform staining. After 15 min of incubation, the brain slices are removed, and the pale area (infarction area) and non-pale area (normal area) are separated using an ophthalmic forceps. The infarction percentage is calculated by Image pro-plus 6.0 as follows:

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

[0136] Infarction area inhibition rate (%) = model group infarction percentage (%) - administration group infarction percentage (%) / model group infarction percentage (%) x 100

[0137] b. Longa neurological function rating: Before modeling, 2h and 24h after reperfusion administration, the animal's neurological function defects were graded according to Longa's method, and the standard was as follows:

[0138] 0 points: normal neurological function;

[0139] 1 point: mild neurological dysfunction: when lifting the tail, the animal's left forelimb flexes;

[0140] 2 points: moderate neurological dysfunction: when walking on a smooth surface, the animal turns to the left side;

[0141] 3 points: moderate neurological dysfunction: in a stationary state, tilt to the left;

[0142] 4 points: decreased consciousness, no spontaneous movement of limbs;

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

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

[0145] Inclusion criteria: Longa score of 1-3 points;

[0146] Exclusion criteria: Longa score less than 1 point and score higher than 3 points; concurrent subarachnoid hemorrhage found when taking the brain; no ischemic lesion found by TTC staining; death within 72h of ischemia-reperfusion.

[0147] Statistical method: The neurological function defect grading score data is represented by median, and the rest of the data is represented by Mean ± SD; The statistical difference between groups of neurological function defect grading score data is analyzed by Kruskal-Wallis test and Mann Whitney U test, and the statistical difference between groups of the rest of the data is analyzed by One-way ANOVA and Tukey's test, P value less than 0.05 is considered to have significant difference.

[0148] 2. Experimental results

[0149] We selected some compounds that performed well at the cellular and enzyme levels for single-dose in vivo experiments. Each group of compounds was administered at an equal molar dose of NBP 5mg / kg. For example, Figure 4As shown, compared with the model group, each group of compounds can significantly reduce the cerebral infarction volume 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 has the best activity, and the cerebral infarction volume inhibition rate is 51.19%, which is 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 is also better than the NBP (35.58%), 1400W (30.05%) and the single and combined administration groups (42.40%). At 2h, 26h after ischemia, the neurological behavior scores of each group were evaluated, and at 24h after ischemia, BN-1, CN-1, BN-4, CN-4, CH-1 and NBP groups, and NBP+1400W combined administration group can significantly improve the neurological function score of rats. Among them, the BN-4 group has the most significant effect (P<0.0001 vs MCAO group), which is better than the equimolar BN-1, CN-1, CN-4, CH-1 group and NBP and 1400W single or combined administration group.

[0150] Example 25: Evaluation of cerebral infarction area and neurological function recovery of tMCAO cerebral ischemia model rats after multiple administrations

[0151] 1. Experimental method

[0152] Basically the same as in Example 24, three times of administration at 2h, 24h and 48h after ischemia.

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

[0154] A. mNSS: The neurological function status of tMCAO model at 24h and 72h after ischemia was evaluated by mNSS. The score was 0-14 points, including motor, sensory, reflex and balance tests. The normal state was scored as 0 points, and the maximum defect was scored as 14 points. The cumulative score of 10-14 points, 5-9 points and 1-4 points was severe, moderate and mild injury, respectively.

[0155] B. Corner Test: The purpose is to evaluate sensorimotor function in rats. This test is performed 24 and 72 hours after ischemia in rats. Two wooden boards (35cm × 25cm × 1.2cm) are prepared beforehand, joined together at one side, with the included angle not exceeding 35°. A small opening is left at the joint to allow light to pass through and attract the rat to crawl towards the corner. The rat is placed in front of the opening, head facing the board. As the rat enters the corner, the boards on either side stimulate it to turn to face the opening. Each test is repeated 10 times, recording the number of times the rat turns to the left. In healthy, non-acute cerebral ischemia rats, the probability of turning left and right is approximately equal; however, in ischemic rats, the probability of turning to the healthy side increases. The CT score R / 10% is calculated.

[0156] 2. Experimental Results

[0157] Based on the excellent performance of BN-4 in the single-dose tMCAO model, we further evaluated the in vivo efficacy of BN-4 through multiple dosing. Figure 5 As shown, the average infarct area in the model group was 35.64%, while 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 significantly reduced cerebral infarction induced by the tMCAO model. BN-4 showed the best activity, with an infarct volume inhibition rate of 53.68%, significantly higher than the NBP group (38.21%), indicating that compound BN-4 has a better inhibitory effect on cerebral infarction than NBP. At 24 h and 72 h after ischemia-reperfusion, the mNSS and corner test scores were assessed in each group. At 24 h after ischemia, the BN-4 group significantly improved rat mNSS behavior and corner test scores, superior to the NBP group. At 72 h after ischemia, both the BN-4 and NBP groups significantly improved rat mNSS behavior and corner test scores, still superior to NBP.

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

[0159] 1. Experimental Methods

[0160] A. Rat plasma and liver microsomal stability test

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

[0162] B. Water solubility experiment

[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 high-performance liquid chromatography. When complete dissolution was not possible, the maximum solubility of the compound in water was determined.

[0164] 2. Experimental results

[0165] Since BN-4 exhibited the best anti-cerebral infarction and improved neurological score 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, and the results are shown in Figure 6 BN-4 was slowly degraded in rat plasma or liver microsomes, with a half-life of about 23 h in rat plasma and a half-life of more than 120 min in liver microsomes, indicating that BN-4 was relatively stable in vitro. In addition, as shown in Figure 7 compared with NBP, BN-4 showed excellent water solubility. The solubility test 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 in vivo

[0167] 1. Experimental method

[0168] The selected compounds were pharmacokinetic studied in SD rats. Rats (230-250g, 3 animals per time point) were administered BN-4 (10 mg / kg) and 1400W (6.8 mg / kg) intravenously. 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 and placed in centrifuge tubes containing heparin sodium. Plasma was separated by centrifugation at 4°C. Plasma samples were extracted with acetonitrile containing a universal internal standard (carbamazepine). Samples were centrifuged at 12,000 rpm for 20 min at 4°C, and the supernatant was collected for analysis. The concentrations of compounds in plasma were determined by high-performance liquid chromatography (HPLC), 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. The experimental results showed that the C60 of BN-4... max AUC 0-∞ The values ​​were 11.21 ± 2.85 nmol·mL, respectively. -1 and 16.47±1.27h·nmol·mL -1 All were above 1400 W (9.81 ± 2.85 nmol·mL). -1 and 5.07±0.38h·nmol·mL -1 This indicates that BN-4 has a higher in vivo distribution compared to intravenous administration of 1400W, potentially leading to better therapeutic effects. Furthermore, BN-4's T... 1 / 2 MRT 0-t and MRT 0-∞ The values ​​were 5.21 ± 0.46 h and 1.62 ± 0.36 mL·h, respectively. -1 ·kg -1 and 1.91±0.49 mL·h -1 ·kg -1 All are higher than 1400W T 1 / 2 (2.79±0.13h, 0.96±0.19mL·h -1 ·kg -1 1.11±0.22 mL·h -1 ·kg -1 This indicates that BN-4 has a longer blood retention time compared to 1400W. Finally, we investigated 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 cross the blood-brain barrier (Table 2).

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

[0172]

Claims

1. A novel amidine-containing iNOS inhibitor having a neuroprotective function, characterized by, A compound of Formula I, II or a pharmaceutically acceptable salt thereof; or ; wherein R 1 is selected from , , , , or ; R 2 is selected from -CH2- or ; R 3 is selected from or .

2. The novel interamidinium-containing iNOS inhibitor with neuroprotective function according to claim 1, characterized in that, The compound of Formula I is selected from the following compounds: 3-(acetylimidomethyl)- N - (1 -butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)benzamide 、 N - ( 1 -butyl-3-oxo- 1,3-dihydroisobenzofuran-5-yl)-3- ( 1 -hydroxyethyl)benzamide N' - ( 1 -butyl-3-oxo- 1,3-dihydroisobenzofuran-5-yl)-3- ( 1 -hydroxyethyl)benzamide 、 3-acetylimino- N - (1 -butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)benzamide 、 N - ( 1 -butyl-3-oxo- 1,3-dihydroisobenzofuran-5-yl)-3- (hydroxyacetylamino)benzamide N' - ( 1 -butyl-3-oxo- 1,3-dihydroisobenzofuran-5-yl)-3- (hydroxyacetylamino)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) ethanimidamide 、 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 or 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 interamine-containing iNOS inhibitor with neuroprotective function according to claim 1, characterized in that, The compound of Formula II is selected from the following compounds: N - (1 -butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)acetamidine or N - (1 -butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)- N' - hydroxyacetamidine 。 4. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises a therapeutically effective amount of the compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

5. A method for preparing the novel amidine-containing iNOS inhibitor having a neuroprotective function according to claim 1 or 2, characterized by, said compound of formula I, when R 1 is , , or , R 2 is -CH2 or , is prepared by the following steps: (1) Amide condensation reaction of Compound 1 or Compound 2 with 6-NH2-NBP to obtain intermediate 1-1 or 2-1; (2) Removal of the protecting group on the amino group of intermediate 1-1 or 2-1 to obtain intermediate 1-2 or 2-2; (3) Nucleophilic substitution reaction of intermediate 1-2 or 2-2 with ethyl acetoimidate hydrochloride to obtain Compound BN-1, BN-2, BN-3 or BN-4; (4) Nucleophilic addition-elimination reaction of Compound BN-1, BN-2, BN-3 or BN-4 with hydroxylamine hydrochloride to obtain Compound CN-1, CN-2, CN-3 or CN-4; The synthesis route is as follows: ， ； when R 1 is or , R 2 is -CH2- or ; the method of preparation comprising the steps of: (1) Pentad reaction of Compound 3-(bromomethyl)benzoic acid or 3-(bromomethyl)benzaldehyde with Compound 3-methyl-5-oxo-1,2,4-oxadiazol-4-yl potassium to obtain intermediate 3-1 or 4-1; (2) Amide condensation reaction of intermediate 3-1 or 4-1 with 6-NH2-NBP to obtain PMN or PMK; the synthesis route is as follows: ， 。 6. A method for preparing the amidine-containing novel iNOS inhibitor having a neuroprotective function according to claim 1 or 3, characterized by, The preparation method of the compound of Formula II comprises the following steps: (1) Nucleophilic addition reaction of 6-NH2-NBP with acetonitrile under the action of concentrated hydrochloric acid to obtain Compound BN-0; (2) Nucleophilic addition-elimination reaction of Compound BN-0 with hydroxylamine hydrochloride to obtain Compound CN-0; the synthesis route is as follows: 。 7. Use of the amidine-containing novel iNOS inhibitor with neuroprotective function of any one of claims 1-3 in the preparation of a medicament for preventing and / or treating cerebral stroke.

8. Use of the pharmaceutical composition of claim 4 in the preparation of a medicament for preventing and / or treating cerebral stroke.

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