Novel amidino-containing inos inhibitor having neuroprotective function, and preparation method therefor and use thereof

Abstract

Disclosed in the present invention are a novel amidino-containing iNOS inhibitor having a neuroprotective function and having a structural formula I, II or III, and a preparation method therefor and a use thereof. For the compound, an iNOS inhibitor 1400W, a neuroprotective agent NBP or a free radical scavenger Eda fragment is used as a scaffold; by combining the advantages of NBP and Eda and incorporating the characteristics of the selective iNOS inhibitor 1400W, the compound can selectively inhibit iNOS and has a neuroprotective capability; and the compound exerts a synergistic effect, thereby providing better treatment for ischemic stroke.

Description

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

[0001] The present application relates to a compound, in particular to a novel amidine-containing iNOS inhibitor with neuroprotective function and 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, 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 weakness, numbness or weakness of the limbs, sudden difficulty in speaking, difficulty in understanding, loss of vision or blurring, and 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.). Among them, ischemic stroke refers to an acute cerebral vascular disease caused by multiple causes of vascular damage, occlusion of the blood oxygen supply to the brain, ischemic and anoxic necrosis of the brain tissue, and resulting in neurological impairment in 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 butylphthalide (NBP) and free radical scavenger Edaravone (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 often 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, although it shows good iNOS inhibitory activity in cell experiments, 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 according to the present application is a compound represented by Formula I or II, or a pharmaceutically acceptable salt thereof.

[0008] wherein R 1 is selected from

[0009] R 2 is selected from -CH2- or R 3 is selected from R 4 is selected from

[0010] Preferably, the compound represented by Formula I is selected from the following compounds:

[0011] 3-(acetyliminomethyl)-N-(1-butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)benzamide

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

[0013] 3-acetylimino-N-(1 -butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)benzamide

[0014] N-(1 -butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-3-(N'-hydroxyacetylimino)benzamide

[0015] N-(3-(((1 -butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)amino)methyl)phenyl)acetamidine

[0016] N-(3-(((1 -butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)amino)methyl)phenyl)-N'- hydroxyacetamidine

[0017] N-(3-(((1 -butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)amino)methyl)benzyl)acetamidine

[0018] N-(3-(((1 -butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)amino)methyl)benzyl)-N'- hydroxyacetamidine

[0019] 4-(3-(((1 -butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)amino)methyl)benzyl)-3- methyl-1,2,4-oxadiazol-5(4H)-one

[0020] N-(1 -butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-3-((3-methyl-5-oxo-1,2,4-oxadiazol- 4(5H)-yl)methyl)benzamide

[0021] Preferably, the compound of formula II is selected from the group consisting of:

[0022] N-(1 -butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)acetamidine

[0023] N-(1 -butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-N'-hydroxyacetamidine

[0024] Preferably, the compound of formula III is selected from the group consisting of:

[0025] N-(4-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzyl)acetamidine

[0026] N-(3-(acetyliminomethyl)benzyl)-4-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzamide

[0027] A pharmaceutical composition comprising a therapeutically effective amount of a compound of the present application or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0028] 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.

[0029] The preparation method of the novel amidine-containing iNOS inhibitor with neuroprotective function of the present application, the compound represented by the formula I, when R 1 is R 2 is -CH2- or The preparation method comprises the following steps:

[0030] (1) Compound 1 or Compound 2 is subjected to amide condensation reaction with 6-NH2-NBP to obtain intermediate 1-1 or 2-1;

[0031] (2) The protecting group on the amino group of intermediate 1-1 or 2-1 is removed to obtain intermediate 1-2 or 2-2;

[0032] (3) Intermediate 1-2 or 2-2 is subjected to nucleophilic substitution reaction with ethyl acetylimidate hydrochloride to obtain compound BN-1, BN-2, BN-3 or BN-4;

[0033] (4) Compound BN-1, BN-2, BN-3 or BN-4 is subjected to nucleophilic addition-elimination reaction with hydroxylamine hydrochloride to obtain compound CN-1, CN-2, CN-3 or CN-4;

[0034] The synthesis route is as follows:

[0035] when R 1 is R 2 is -CH2- or The preparation method comprises the following steps:

[0036] (1) compound 3-(bromomethyl)benzoic acid or 3-(bromomethyl)benzaldehyde and compound 3-methyl-5-oxo-1,2,4-oxadiazol-4-yl potassium occur five generations of reaction to obtain intermediate 3-1 or 4-1;

[0037] (2) intermediate 3-1 or 4-1 and 6-NH2-NBP occur amide condensation reaction to obtain PMN or PMK; the synthetic route is as follows:

[0038] 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:

[0039] (1) 6-NH2-NBP is subjected to nucleophilic addition reaction with acetonitrile under the action of concentrated hydrochloric acid to obtain compound BN-0;

[0040] (2) compound BN-0 is subjected to nucleophilic addition-elimination reaction with hydroxylamine hydrochloride to obtain compound CN-0; the synthetic route is as follows:

[0041] 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:

[0042] (1) ethyl acetoacetate is subjected to amide reaction with 4-cyanophenylhydrazine hydrochloride to form a ring to obtain intermediate 5-1;

[0043] (2) intermediate 5-1 is subjected to reduction reaction, and the cyano group is reduced into an amino group to obtain intermediate 5-2;

[0044] (3) intermediate 5-2 is subjected to nucleophilic substitution reaction with ethyl acetoimidate hydrochloride to obtain compound CH-1; the synthetic route is as follows:

[0045] 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:

[0046] The novel amidine-containing iNOS inhibitor with neuroprotective function can be applied to the preparation of a drug for preventing and / or treating cerebral apoplexy.

[0047] The pharmaceutical composition can be applied to the preparation of a drug for preventing and / or treating cerebral apoplexy.

[0048] Inventive 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 present application designs and synthesizes compounds that can selectively inhibit iNOS and have neuroprotective ability at the same time, which can exert 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, which is expected to exert synergistic effect in multiple pathological processes and achieve better anti-cerebral ischemia effect.

[0049] Beneficial effects: Compared with the prior art, the present application has the following advantages: (1) The compounds can effectively improve the survival rate of human neuroblastoma cells 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, while having good selectivity; (3) Compound BN-4 has good effects on cerebral infarction and recovery of nerve function of tMCAO cerebral ischemia model rats, and is better than positive drugs NBP, compound 1400W and their combination; (4) Compound BN-4 has good pharmacokinetic properties, and its water solubility is more than one hundred times higher than that of NBP, and its blood-brain ratio is greatly improved compared with compound 1400W, and has good drug properties; (5) Compound BN-4 can reduce the ROS level of SH-SY5Y cells induced by OGD / R, and reduce cell necrosis and apoptosis; (6) The compound has high stability; (7) The preparation method is simple, and the compound is easy to obtain in synthesis. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 is a schematic diagram of the design idea and mechanism of action of the series of compounds;

[0051] Figure 2 is the effect of the compound on the survival rate of SH-SY5Y cells, Eahy-926 cells and BV-2 cells induced by OGD / R at 1 μM and 10 μM;

[0052] Figure 3 is the effect of the compound on the NO level of Raw 264.7 cells induced by LPS at 1 μM;

[0053] Figure 4 is the effect of single administration of some compounds on the cerebral infarction area and Longa's score of tMCAO rats;

[0054] Figure 5 is the effect of multiple administrations of compound BN-4 on the cerebral infarction area, mNSS score and corner test of tMCAO rats;

[0055] Figure 6 is the stability of compound BN-4 in rat plasma and liver microsomes;

[0056] Figure 7 is the water solubility of compound BN-4 and NBP. DETAILED DESCRIPTION

[0057] The present application will be further described in conjunction with specific examples.

[0058] I. Synthesis of intermediates

[0059] Example 1: Synthesis of intermediate 1-1 The synthesis steps are as follows: 6-NH2-NBP (205 mg, 1 mmol, 1 eq) and the corresponding benzoic acid derivative (1 mmol, 1 eq) are placed in a 25 mL round-bottom flask, 5 mL of DMF is added as a solvent, and the reactants are completely dissolved. Then, the condensing agent N,N,N',N'-tetramethylchloroformamidium hexafluorophosphate (420 mg, 1.5 mmol, 1.5 eq) and the acid-binding agent N-methylimidazole (288 mg, 3.5 mmol, 3.5 eq) are added. After the addition is complete, the mixture is placed in a 25 °C water bath and stirred for 24 h. During the reaction, the reaction solution gradually becomes clear from turbid, and the solid raw material is completely dissolved. After the reaction is complete, the solution is concentrated under reduced pressure, and the residue is diluted with water. The solution is extracted with EA, the upper clear liquid is collected, and the organic phases are combined. The mixture is dried over anhydrous sodium sulfate, and the solvent is removed under reduced pressure. The crude product is purified by column chromatography (PE:EA = 2:1) to obtain the intermediate 1-1 as a white solid.

[0060] Example 2: Synthesis of intermediate 1-2 The synthesis steps are as follows: Intermediate 1-1 (1 mmol) is placed in a 50 mL round-bottom flask, 8 mL of a trifluoroacetic acid solution in dichloromethane (1:3) is added as a solvent, and the mixture is placed in a 25 °C water bath and stirred for 1 h. After the reaction is complete, the solution is quenched with water, extracted with DCM three times, and the organic phases are combined. The mixture is dried over anhydrous sodium sulfate, and the solvent is removed under reduced pressure to obtain a white solid. The product is used directly in the next reaction without further treatment.

[0061] Example 3: Synthesis of intermediate 2-1 The synthesis steps 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, and the mixture is heated to 80 °C and refluxed for 12 h. Then, the mixture is slowly cooled to 0 °C, and sodium borohydride (76 mg, 2 mmol, 2 eq) is added in small portions. The mixture is removed to room temperature and stirred for 2 h. After the reaction is complete, as determined by TLC (developing agent PE:EA = 3:1), the solution is quenched with water, extracted with DCM, and the organic phases are combined. The mixture is dried over anhydrous sodium sulfate, and the solvent is removed under reduced pressure. The crude product is purified by column chromatography to obtain the intermediate 2-1.

[0062] The synthesis step of Example 4: intermediate 2-2 is to place intermediate 2-1 (1 mmol) in a 50 mL round-bottom flask, add 8 mL of trifluoroacetic acid in dichloromethane (1:3) as a solvent, place it at 25°C, stir for 1 h, and after the reaction is complete, quench with water, extract with DCM three times, dry the combined organic phase with anhydrous sodium sulfate, and concentrate to dryness under reduced pressure to obtain a white solid, which is used directly in the next step without further treatment.

[0063] The synthesis step of Example 5: 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 complete, concentrate to dryness under reduced pressure, wash with anhydrous diethyl ether to obtain a white solid, which is used directly in the next step.

[0064] The synthesis step of Example 6: 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 complete, concentrate to dryness under reduced pressure, wash with anhydrous diethyl ether to obtain a white solid, which is used directly in the next step.

[0065] The synthesis step of Example 7: 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 into it. Heat the reaction to 80°C to reflux for 12 h, and the system gradually changes from turbidity to clarity. After the reaction is complete, cool it to room temperature, extract with EA three times, combine the organic phase, and concentrate to remove the solvent under reduced pressure. Purify the crude product by column chromatography (PE:EA = 1:1) to obtain intermediate 5-1.

[0066] The synthesis step of Example 8: 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 complete, filter the palladium on carbon, collect the filtrate, and concentrate to dryness under reduced pressure to obtain a yellow solid, which is used directly in the next step.

[0067] II. Synthesis of the target compound

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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 the weighed ethyl acetimidate hydrochloride (128 mg, 0.9 mmol, 0.9 eq) in small portions, anhydrous potassium carbonate (276 mg, 2 mmol, 2 eq), 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.

[0073] 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 / H2O = 80:20.

[0074] 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.

[0075] 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, use EA extraction method as CN-1, get CN-4 crude product. Purify the crude product by column chromatography (PE:EA = 1:3) to get CN-4 295 mg as a white solid, yield 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.

[0076] 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.

[0077] Example 18: The synthetic route of the target compound PMK is to place intermediate 4-1 (218 mg, 1 mmol, 1 eq) and 6-NH2-NBP (205 mg, 1 mmol, 1 eq) 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,found 430.17324,ppm error-0.83.HPLC MeOH / H2O=80:20.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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 yellowish 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.

[0082] III. Effect verification

[0083] Example 22: In vitro study on OGD / R-induced SH-SY5Y cell, Eahy-926 cell and BV-2 cell survival rate

[0084] 1. Experimental method

[0085] 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, 100 μL of cell suspension was added to each well;

[0086] b. Place the 96-well cell culture plate in a 37°C, 5% CO2 incubator for 24 h;

[0087] c. Dilute the drug with the conditioned medium to the desired concentration, add 100 μL of the corresponding drug-containing medium to each well, and set up a negative control group;

[0088] d. Place the 96-well cell culture plate in a 37°C, 5% CO2 incubator for 24 h;

[0089] e. Replace the culture medium with DMEM medium without glucose and serum, and incubate the cells in a 5% CO2, 95% N2 environment for 2 h, then replace the culture medium with complete DMEM medium and incubate in a 5% CO2, 95% O2 environment for 24 h;

[0090] f. Perform CCK-8 staining of the 96-well plate at λ = 450 nm, and measure the OD value; A. Add 10 μL of CCK-8 to each well, and continue incubation in the incubator for 2 h; B. Gently mix on a shaker for 10 min; C. Read the OD value of each well at λ = 450 nm using an enzyme marker, and calculate the inhibition rate.

[0091] 2. Experimental results

[0092] As shown in FIG. 2, in SH-SY5Y cells, the survival rate of the OGD / R model group was significantly reduced, and the survival rate was significantly increased compared with the OGD / R group with the addition of NBP, 6-NH2-NBP and 1400W. At a concentration of 1 μM, the survival rate of CN-2 was higher than that of NBP or 6-NH2-NBP; at a concentration of 10 μM, the survival rate of BN-4, CN-1, CN-2, CN-4, CH-1 and PMK was also significantly higher than that of NBP or 6-NH2-NBP. In Eahy-926 cells and BV-2 cells, the survival rate of the OGD / R model group was significantly reduced, and the survival rate was significantly increased compared with the OGD / R group with the addition of NBP and 1400W. The survival rate of the test compound group was increased, among which BN-4 had the best effect and had similar activity to NBP, and could increase the survival rate at a concentration of 1 μM and 10 μM in both cells.

[0093] Example 23: Evaluation of iNOS inhibitory activity at the cellular and enzyme levels

[0094] 1. Experimental method

[0095] A. Evaluation of iNOS inhibitory activity at the cellular level

[0096] RAW 264.7 cells were seeded at 2 x 10 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.

[0097] B. Evaluation of iNOS inhibitory activity at enzyme levels (hemoglobin capture method)

[0098] 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.

[0099] 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.

[0100] c. Add 210 μL of the complete mixture to each well of the plate and keep it at 37°C.

[0101] d. Quickly add 20 μL of iNOS diluent to each well and mix the plate with the incubation track shaker for 15 s.

[0102] e. Place the plate into the reader (preheated to 37°C) and then begin reading the data.

[0103] 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.

[0104] 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)].

[0105] 2. Experimental Results

[0106] As shown in the cell experiment results of Figure 3, compounds BN-4, compounds CN-1 to CN-4, and compound 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 of Table 1 show that the IC50value of compound BN-4 for iNOS is 0.1707 μM, which is the best among the series of compounds and is basically the same as that of the positive control 1400W. 50 The IC50value of compound BN-4 for iNOS is 0.1707 μM, which is the best among the series of compounds and is basically the same as that of the positive control 1400W.

[0107] The IC50value of compound BN-4 for iNOS is 0.1707 μM, which is the best among the series of compounds and is basically the same as that of the positive control 1400W. 50

[0108] Example 24: Evaluation of single-dose administration on cerebral infarction area and recovery of neurological function of rats in tMCAO cerebral ischemia model

[0109] 1. Experimental method

[0110] A. Preparation of rat tMACO model: In order to make the embolism model, nylon wire with a diameter of 0.234 mm is selected, and the top end of the nylon wire is made thicker and is burned and scalded into a smooth ball with a diameter of about 0.35 mm. A marker is made on the nylon wire at a position about 20 mm from the top end, and the nylon wire is cleaned with 75% alcohol and 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 from excessive anesthesia), and the rats are fixed on the experimental table with their heads facing upwards. An incision is made in the middle of the neck, and the skin is cut open with surgical scissors and carefully torn 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 upwards using a surgical thread, and both are ligated and cut. The superior thyroid artery and occipital artery, two branches of the external carotid artery, are cut, and the ECA is double-ligated near the CCA bifurcation about six millimeters away. A microartery clamp is used to clamp the blood vessel near the proximal end of the right common carotid artery, and a live knot is left at the proximal end of the ECA. A V-shaped micro-incision with a diameter of about 0.2 mm is made between the ligation site near the proximal end of the ECA and the bifurcation of the common carotid artery, and the head of the nylon wire is gently inserted from the incision. The knot is gently tightened, the internal carotid artery is cut between the two ligation lines, and the microartery clamp is loosened. The nylon wire is sent into the cranium along the ECA and ICA, and the insertion depth is about 18 mm to 20 mm. When resistance is encountered, the nylon wire is stopped, and the head end of the nylon wire is located at the beginning of the MCA. The blood flow of the MCA is blocked, 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 of the nylon wire 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.

[0111] B. Detection index:

[0112] a. Measurement of cerebral infarction volume (TTC staining method): After tMCAO modeling of rats, 24h or 72h later, the rats were sacrificed by anesthesia, the head was dissected to take out the brain tissue and quickly placed in the refrigerator preservation layer to cool for 15min, the purpose was to make the brain hard to facilitate sectioning. After cutting the brain into 5 pieces with uniform thickness, quickly place the brain slices in 5mL of pre-prepared PBS buffer solution containing 2% TTC, wrap with tin paper and put into the oven for 37℃ incubation for 15min, turn over every 2 minutes during this process, the purpose is to make the brain slices evenly dyed. After incubation for 15min, take out the brain slices, choose black or blue background to take pictures of the brain slices, then separate the pale area (infarction area) and non-pale area (normal area) with ophthalmic forceps, calculate the infarction percentage by Image pro-plus 6.0 as follows:

[0113] Infarction percentage (%) = pale area / (pale area + non-pale area) x 100%

[0114] Infarction area inhibition rate (%) = model group infarction percentage (%) - drug group infarction percentage (%) / model group infarction percentage (%) x 100

[0115] b. Longa neurological function rating: Before modeling, 2h and 24h after reperfusion, the animal's neurological function defects were graded according to Longa's method, the standard is as follows:

[0116] 0 points: normal neurological function;

[0117] 1 point: mild neurological dysfunction: when lifting the tail, the left forelimb of the animal is flexed;

[0118] 2 points: moderate neurological dysfunction: when walking on a smooth surface, the animal turns to the left side;

[0119] 3 points: moderate neurological dysfunction: in a stationary state, tilt to the left side;

[0120] 4 points: decreased consciousness, no spontaneous movement of limbs;

[0121] 5 points: no response to stimulation or death.

[0122] C. Inclusion and exclusion criteria of tMCAO model

[0123] Inclusion criteria: Longa score of 1-3 points;

[0124] Exclusion criteria: Longa score less than 1 and more than 3; concurrent subarachnoid hemorrhage at brain harvest; no ischemic lesion by TTC staining; death within 72 h of ischemia-reperfusion.

[0125] Statistical methods: The neurological deficit scores were expressed as median values, and the other data were expressed as Mean ± SD. The statistical differences between groups were analyzed by Kruskal-Wallis test and Mann-Whitney U test for neurological deficit scores, and by One-way ANOVA and Tukey’s test for the other data. The P value less than 0.05 was considered as significant.

[0126] 2. Experimental results

[0127] We selected some compounds with better performance at cellular and enzymatic levels for single-dose in vivo experiments. Each group of compounds was administered at an equimolar dose of NBP 5 mg / kg. As shown in Figure 4, compared with the model group, each group of compounds can significantly reduce the cerebral infarction volume induced by the 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%, and 25.51%, respectively, as shown in the figure. Among them, BN-4 had the best activity, with an inhibition rate of cerebral infarction volume of 51.19%, which was higher than that of the BN-1 group (45.04%), CN-1 group (41.36%), CN-4 group (42.15%), and CH-1 group (34.20%). Moreover, the BN-4 group was also better than the NBP (35.58%), 1400W (30.05%) single and combined administration groups (42.40%). At 2 h and 26 h after ischemia, the neurological behavior scores of each group were evaluated, and at 24 h after ischemia, the BN-1, CN-1, BN-4, CN-4, CH-1, and NBP groups, and the NBP + 1400W combined administration group can significantly improve the neurological function scores of rats. Among them, the BN-4 group had the most significant effect (P < 0.0001 vs MCAO group), which was better than the equimolar BN-1, CN-1, CN-4, CH-1 groups, and the NBP and 1400W single or combined administration groups.

[0128] Example 25: Evaluation of multiple-dose administration on cerebral infarction area and neurological function recovery in tMCAO cerebral ischemia model rats

[0129] 1. Experimental method

[0130] The same as example 24, three times of administration at 2h, 24h and 48h after ischemia.

[0131] Neurological score: A. mNSS B. Corner test

[0132] A. mNSS: The neurological status of tMCAO model at 24, 72h after ischemia was evaluated by mNSS. The score was 0-14 points, including motor, sensory, reflex and balance tests, normal state score was 0 points, the maximum defect score was 14 points. The cumulative score of 10-14 points, 5-9 points, 1-4 points was severe, moderate and mild injury, respectively.

[0133] B. Corner test: The purpose was to evaluate the sensory and motor function of rats. This test was performed after 24, 72 hours of ischemia in rats. Two pieces of wood (35 cm x 25 cm x 1.2 cm) were prepared in advance, one side of the wood was connected together, and the angle between them was acute angle not more than 35°. A small opening was left at the connection of the wood for light transmission to attract the rat to crawl into the corner. The rat was placed in front of the opening of the wood with its head facing the wood. The rat entered the corner, and the wood on both sides of the rat stimulated the rat to turn around to face the opening side. Each test was repeated 10 times, and the number of left turns of the rat was recorded. If it is a healthy non-acute cerebral ischemia rat, the probability of left and right turns 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%.

[0134] 2. Experimental results

[0135] 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-dose administration. As shown in Figure 5, the average cerebral infarction area of the model group was 35.64%, and that of 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 tMCAO, among which BN-4 had the best activity, with an inhibition rate of cerebral infarction volume of 53.68%, which was significantly higher than that of the NBP group (38.21%), indicating that compared with NBP, compound BN-4 had better inhibition effect on cerebral infarction. At 24h and 72h after ischemia-reperfusion, the mNSS and corner test scores of each group were evaluated. 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.

[0136] Example 26: In vitro stability experiment and water solubility

[0137] 1. Experimental method

[0138] A. Rat plasma and liver microsomes stability experiment

[0139] The test compound was prepared into a solution with a final concentration of 200 μM containing 1% DMSO / rat plasma, and incubated at 37°C on a shaker. Samples were taken at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 h, and analyzed by HPLC to observe the degradation of the original compound. The experiment was repeated three times. The test compound was prepared into a solution with a final concentration of 200 μM containing 1% DMSO / PBS, and added with rat liver microsomes in a corresponding proportion, with the organic phase proportion not exceeding 1%. The mixture was incubated at 37°C on a shaker. Samples were taken at 0, 10, 30, 60 and 120 min, and analyzed by HPLC to observe the degradation of the original compound. The experiment was repeated three times.

[0140] B. Water solubility experiment

[0141] 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.

[0142] 2. Experimental results

[0143] 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 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 exhibited excellent water solubility. The solubility test results 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.

[0144] Example 27: Pharmacokinetics of compound BN-4 in rats

[0145] 1. Experimental method

[0146] The selected compounds were studied in SD rats in vivo for pharmacokinetics. Rats (230-250 g, 3 animals per time point) were dosed intravenously with BN-4 (10 mg / kg) and 1400W (6.8 mg / kg). Blood samples (1.0 mL) were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 24 h after intravenous injection, and placed in heparinized tubes. Plasma was separated by centrifugation at 4 °C. Plasma samples were extracted by adding 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 and analyzed. The concentration of compounds in plasma was determined by high-performance liquid chromatography, and non-compartmental analysis was performed using WinNonlin software.

[0147] 2. Experimental results

[0148] 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 1400W. The results showed that the Cmax, AUC0-t, and AUC0-∞ of BN-4 were 11.21 ± 2.85 nmol·mL-1, 16.47 ± 1.27 h·nmol·mL-1, and 16.47 ± 1.27 h·nmol·mL-1, respectively, all of which were higher than those of 1400W (9.81 ± 2.85 nmol·mL-1, 5.07 ± 0.38 h·nmol·mL-1, and 5.07 ± 0.38 h·nmol·mL-1, respectively). This indicates that BN-4 has a higher in vivo distribution volume than 1400W, which may lead to a better therapeutic effect. In addition, the T1 / 2, MRT, and MRT of BN-4 were 5.21 ± 0.46 h, 1.62 ± 0.36 mL·h-1·kg-1, and 1.91 ± 0.49 mL·h-1·kg-1, respectively, all of which were higher than those of 1400W (2.79 ± 0.13 h, 0.96 ± 0.19 mL·h-1·kg-1, and 1.11 ± 0.22 mL·h-1·kg-1, respectively), 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). max 0-∞ -1 -1 -1 -1 1 / 2 0-t 0-∞ -1 -1 -1 -1 1 / 2 -1 -1 -1 -1 BN-4 27.36% 5.21 ± 0.46 h 1.62 ± 0.36 mL·h-1·kg-1 1.91 ± 0.49 mL·h-1·kg-1​​​​​​​​​​​​​​​​​

[0149] Table 2 Pharmacokinetic parameters of 1400W and BN-4 in whole blood after i.v. administration in SD rats (n=3)

Claims

1. A novel amidine-containing iNOS inhibitor having a neuroprotective function, characterized by, Compounds of the formulae I, II or pharmaceutically acceptable salts thereof; wherein R 1 selected from R 2 selected from -CH2- or R 3 selected from R 4 selected from 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-(acetyliminomethyl)-N-(1 -butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)benzamide N-(1 -butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-3-((N'-hydroxyacetamidinyl)methyl)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-(N'-hydroxyacetamidino)benzamide N-(3-(((1 -butyl-3-oxo- 1,3-dihydroisobenzofuran-5-yl)amino)methyl)phenyl)formamidine N-(3-(((1 -butyl-3-oxo- 1,3-dihydroisobenzofuran-5-yl)amino)methyl)phenyl)-N'- hydroxyethaneimidamide N-(3-(((1 -butyl-3-oxo- 1,3-dihydroisobenzofuran-5-yl)amino)methyl)benzyl)ethane- 1,2-diaminium N-(3-(((1 -butyl-3-oxo- 1,3-dihydroisobenzofuran-5-yl)amino)methyl)benzyl)-N'- hydroxyethaneimidamide 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 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 N-(1 -butyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-N'-hydroxyethaneimidamide 4. The novel interamidinium-containing iNOS inhibitor with neuroprotective function according to claim 1, characterized in that, The compound of formula III is selected from the following compounds: N-(4-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzyl)ethanimidamide N-(3-(acetyliminomethyl)benzyl)-4-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzamide 5. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises a therapeutically effective amount of the compound of claims 1-4 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

6. 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 R 2 is -CH2or The preparation method comprises 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 synthetic route is as follows: when R 1 is R 2 is -CH2- or The preparation method comprises the following steps: (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 of intermediate 3-1 or 4-1 with 6-NH2-NBP to give PMN or PMK; the synthetic route is as follows:

7. 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 synthetic route is as follows:

8. A method for preparing the amidine-containing novel iNOS inhibitor having a neuroprotective function according to claim 1 or 4, characterized by, said compound of formula III, when R 4 is The method of preparation comprises the steps of: (1) Amide reaction of ethyl acetoacetate with 4-cyanophenylhydrazine hydrochloride to form a ring to obtain intermediate 5-1; (2) Reduction reaction of intermediate 5-1 to reduce the cyano group to an amino group to obtain intermediate 5-2; (3) nucleophilic substitution reaction of intermediate 5-2 with ethylacetoimidate hydrochloride to obtain compound CH-1; the synthetic route is as follows: when R 4 is The preparation method comprises the following steps: subjecting 1-(4-carboxyphenyl)-3-methyl-5-pyrazolone to an amide condensation reaction with 1400W to obtain compound CH-2, and the synthesis route is as follows:

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

10. Use of the pharmaceutical composition of claim 5 in the preparation of a medicament for preventing and / or treating cerebral stroke.

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