CN107325058B - Diaryl methylene disulfide compound and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of medicine, in particular to a diarylmethylene disulfide compound and a preparation method and application thereof. The experimental results show that the diarylmethylene disulfide compounds provided by the present invention have the effect of inhibiting the neurotoxicity caused by excessive glutamate and the neurotoxicity caused by hydrogen peroxide in the cell model; The role of platelet aggregation in the mouse MCAO model can reduce the role of cerebral infarct size. These results indicate that the diarylmethylene disulfide compounds provided by the present invention can have preventive and/or therapeutic effects on stroke.

Description

Diarylmethylene disulfide compounds and preparation method and application thereof

technical field

The invention relates to the technical field of medicine, in particular to a diarylmethylene disulfide compound and a preparation method and application thereof.

Background technique

"Stroke" (cerebral stroke) is also known as "stroke", "cerebral vascular accident" (cerebralvascular accident, CVA). It is an acute cerebrovascular disease, which is a group of diseases that damage brain tissue due to the sudden rupture of blood vessels in the brain or the inability of blood to flow into the brain due to vascular blockage.

Worldwide, stroke is the third leading cause of death globally and the leading cause of disability in adults. According to statistics, there are 500,000 to 750,000 people in the United States each year. In China, the number of stroke cases reaches 2.5 million per year, and the number of deaths reaches 1.6 million per year. Stroke has surpassed myocardial infarction as the leading cause of death in China. Stroke not only has a high fatality rate, but surviving patients will also leave sequelae such as neurobehavioral impairment and cognitive impairment, resulting in a heavy social and family economic burden. It has the characteristics of high morbidity, high mortality and high disability rate. .

There are two main types of stroke. More than 70% are ischemic cerebral apoplexy, which refers to the blockage of cerebral blood vessels caused by various thrombus in different brain regions, thereby causing ischemic brain damage. The most common one is the blockage of the middle cerebral artery. Occlusion and stenosis of the internal carotid artery and vertebral artery can cause ischemic stroke, and most people are over 40 years old, more men than women, and severe cases can lead to death. In addition, less than 30% are cerebral hemorrhages caused by rupture of cerebral blood vessels, which eventually lead to cerebral insufficiency and ischemic brain damage. Cerebral ischemia is also one of the consequences of cardiac arrest.

Compared with the severity and harm of stroke, the current treatment drugs for stroke are still very limited. The current clinical drug treatment mainly includes thrombolytics, vasodilators, antiplatelet aggregation drugs and neuroprotective agents. For example, for ischemic stroke, the only internationally recognized drug therapy is acute phase thrombolysis of tissue plasminogen activator. However, due to the short therapeutic window of tissue plasminogen activator and significant side effects (such as causing cerebral hemorrhage and excitatory neurotoxicity), the total number of patients with cerebral ischemia that can be treated with tissue plasminogen activator is less than the total number of patients. 5%. Existing research also shows that stroke can cause a series of cellular and molecular response mechanisms in the brain, which are intertwined and eventually lead to brain damage. For example, the mechanisms of neuronal death or damage caused by ischemic stroke mainly include neurotoxicity caused by excessive reactive oxygen species, neuronal damage caused by excessive release of excitatory amino acids such as glutamate, and poly ADP ribose polymerase. Neuroinflammation injury mechanism caused by neuronal death caused by excessive activation and excessive inflammatory response in the brain caused by cerebral ischemia. These pathological damage mechanisms are of great significance to the research and development of cerebral apoplexy therapeutic drugs, and finding corresponding drugs for multiple pathological damage mechanisms at the same time has become an important strategy for the development of new stroke therapeutic drugs.

To sum up, in view of the great harm of stroke and the extremely limited current treatment methods, the development of new types of stroke prevention and/or treatment drugs has always been the focus of extensive attention by frontier scholars in this field, which is of great significance.

SUMMARY OF THE INVENTION

In view of this, the technical problem to be solved by the present invention is to provide a diarylmethylene disulfide compound and a preparation method and application thereof. The compounds provided by the invention not only have the functions of better inhibiting the neurotoxicity caused by excessive glutamate and scavenging excessive free radicals in the nerve cell model, have the effect of inhibiting platelet aggregation, but also have the effect of inhibiting the brain of mice with cerebral ischemia-reperfusion injury. It has a very good protective effect, so it has a preventive or therapeutic effect on stroke.

The present invention provides the structural compound shown in formula I:

Wherein, Ar is 3,5,6-trimethylpyrazinyl or 4-COOCH ₃ -Ph.

The compound provided by the present invention is 1,2-bis[(3,5,6-trimethylpyrazin-2-yl)methyl]disulfide or 4,4'-disulfanediylbis(methylene base) methyl dibenzoate. The structures of the two compounds have not been disclosed before, but the present invention prepares the two compounds and verifies that they can achieve the preventive or therapeutic effect on stroke through various ways.

The application of the structural compound represented by the formula I of the present invention in the preparation of a preparation for protecting neuronal cells;

Wherein, Ar is 3,5,6-trimethylpyrazinyl, Ph, 4-COOCH ₃ -Ph, 2-BrPh, 3-BrPh, 4-BrPh, 3-FPh, 4-FPh, 2-NO ₂ Ph, 3-NO 2Ph, ₄ -NO2Ph, 4-ClPh, ₄ -CH3Ph or ₄ -CNPh.

The protective effect on neuronal cells includes the protective effect on neurotoxicity caused by glutamate or the protective effect on neurotoxicity caused by hydrogen peroxide. The neuron cells are mouse hippocampal neuron HT-22 cells. The protection specifically refers to: protecting HT22 cells from damage induced by glutamate, or protecting HT22 cells from damage induced by H ₂ O ₂ , indicating that the compound of formula I has the effect of reducing the toxicity of excitatory amino acids and scavenging free radicals generated by H ₂ O ₂ . Among them, 1,2-bis[(3,5,6-trimethylpyrazin-2-yl)methyl]disulfide had the best anti-glutamic acid toxicity effect. Compared with the glutamic acid group, Its cell viability was significantly increased.

The application of the structural compound shown in formula I in the preparation of a preparation for inhibiting platelet aggregation;

Wherein, Ar is 3,5,6-trimethylpyrazinyl, Ph, 4-COOCH ₃ -Ph, 2-BrPh, 3-BrPh, 4-BrPh, 3-FPh, 4-FPh, 2-NO ₂ Ph, 3-NO 2Ph, ₄ -NO2Ph, 4-ClPh, ₄ -CH3Ph or ₄ -CNPh.

The experiments of the present invention show that the compounds provided by the present invention can significantly reduce the maximum aggregation rate of platelets. (P<0.05, P<0.01), suggesting that 1,2-bis[(3,5,6-trimethylpyrazin-2-yl)methane base] disulfide has good anti-platelet aggregation activity.

The application of the structural compound represented by the formula I in the preparation of a preparation for preventing and treating brain injury after cerebral ischemia-reperfusion;

Wherein, Ar is 3,5,6-trimethylpyrazinyl, Ph, 4-COOCH ₃ -Ph, 2-BrPh, 3-BrPh, 4-BrPh, 3-FPh, 4-FPh, 2-NO ₂ Ph, 3-NO 2Ph, ₄ -NO2Ph, 4-ClPh, ₄ -CH3Ph or ₄ -CNPh.

The prevention and treatment of brain injury after cerebral ischemia-reperfusion specifically includes reducing infarct volumes of cortex, striatum and cerebral hemisphere. Experiments showed that 24 hours after middle cerebral artery occlusion, 1,2-bis[(3,5,6-trimethylpyrazin-2-yl)methan was reperfused for 3 hours compared with vehicle mice yl] disulfide can significantly reduce the infarct volume in the cortex, striatum and cerebral hemisphere, indicating that 1,2-bis[(3,5,6-trimethylpyrazin-2-yl)methyl]disulfide has a significant effect on Ischemic brain injury caused by middle cerebral artery occlusion has a significant protective effect.

Except for 1,2-bis[(3,5,6-trimethylpyrazin-2-yl)methyl]disulfide or 4,4'-disulfanediylbis(methylene)dibenzoic acid In addition to methyl ester, the present invention also found that other diarylmethylene disulfide compounds also have the effect of preventing and treating cerebral apoplexy. Its mechanism of action is to inhibit the neurotoxicity caused by excessive glutamate.

The application of the structural compound shown in formula I in the preparation of the preparation for preventing and treating cerebral apoplexy;

Wherein, Ar is 3,5,6-trimethylpyrazinyl, Ph, 4-COOCH ₃ -Ph, 2-BrPh, 3-BrPh, 4-BrPh, 3-FPh, 4-FPh, 2-NO ₂ Ph, 3-NO 2Ph, ₄ -NO2Ph, 4-ClPh, ₄ -CH3Ph or ₄ -CNPh.

In the present invention, a stroke is a stroke caused by cerebral vascular embolism and/or a stroke caused by cerebral hemorrhage.

Preferably, the stroke includes stroke caused by one or more of cerebral thrombosis, cerebral embolism, cerebral infarction, transient ischemic attack, cerebral ischemia caused by cardiac arrest, cerebral hemorrhage and cerebral arteriosclerosis.

In the present invention, the dosage of the compound of formula I ranges from 0.05 mg/kg to 90 mg/kg.

Preferably, the compound of formula I is 1,2-bis[(3,5,6-trimethylpyrazin-2-yl)methyl]disulfide.

A kind of medicine is characterized in that, comprises formula I compound and pharmaceutically acceptable adjuvant;

Wherein, Ar is 3,5,6-trimethylpyrazinyl, Ph, 4-COOCH ₃ -Ph, 2-BrPh, 3-BrPh, 4-BrPh, 3-FPh, 4-FPh, 2-NO ₂ Ph, 3-NO 2Ph, ₄ -NO2Ph, 4-ClPh, ₄ -CH3Ph or ₄ -CNPh.

Preferably, the dosage form of the preparation is an oral preparation, injection, suppository, inhalation or a dosage form that can be directly applied to cerebral ischemia.

Preferably, the formulations are in the form of capsules, microcapsules, tablets, granules, pills, dispersible powders, liquid preparations, decoctions, suspensions, syrups, gels, aerosols, patches, lipids body, oral, intravenous or intramuscular injection.

The preparation method of the structural compound shown in formula I of the present invention is as follows: reacting compound A with thiourea in the presence of manganese dioxide and PEG to obtain the compound of formula I;

Wherein, Ar is 3,5,6-trimethylpyrazinyl, Ph, 4-COOCH ₃ -Ph, 2-BrPh, 3-BrPh, 4-BrPh, 3-FPh, 4-FPh, 2-NO ₂ Ph, 3-NO ₂ Ph, 4-NO ₂ Ph, 4-ClPh, 4-CH ₃ Ph or 4-CNPh;

The compound A is 1-bromoligustrazine, benzyl bromide, 4-(bromomethyl) methyl benzoate, 2-bromobenzyl bromide, 3-bromobenzyl bromide, 4-bromobenzyl bromide, 3-fluorobenzyl bromide , 4-fluorobenzyl bromide, 2-nitrobenzyl bromide, 3-nitrobenzyl bromide, 4-nitrobenzyl bromide, 4-chlorobenzyl bromide, 4-methylbenzyl bromide, or 4-cyanobenzyl bromide.

The reaction is carried out under basic conditions. The present invention adopts sodium carbonate conditions to obtain an alkaline environment.

The molar ratio of compound A to thiourea was 0.97:1.45.

The molar ratio of the sodium carbonate to thiourea is 1:1.

The molar ratio of the manganese dioxide to compound A is 1:1.

The PEG is PEG200; preferably wet-PEG200; specifically, in the wet-PEG200, the volume ratio of PEG200 to H ₂ O is 10:1.

The reaction temperature was 40°C, and the reaction was stirred for 3 h.

After the compound of formula I is prepared, a purification step is also included. Specifically, the purification includes: extracting the reaction product with ethyl acetate, washing the extract with water, drying, suction filtration, and column chromatography (eluent [petroleum ether:ethyl acetate (v:v)=10:1]) ) to obtain the compound of formula I.

The present invention provides diarylmethylene disulfide compounds, and research shows that these compounds can prevent stroke through various ways. The experimental results show that the diarylmethylene disulfide compounds provided by the present invention have the effect of inhibiting the neurotoxicity caused by excessive glutamate in the cell model; also have the effect of inhibiting the neurotoxicity caused by hydrogen peroxide; The role of platelet aggregation; and the effect of reducing cerebral infarct size in the mouse MCAO model. These results indicate that the diarylmethylene disulfide compounds provided by the present invention can have preventive and/or therapeutic effects on various pathological mechanisms of stroke.

Description of drawings

Figure 1-a shows the effect of LYY-I ₁ on the survival rate of glutamate-induced injury mouse hippocampal neurons; Figure 1-b shows the effect of ligustrazine on the survival rate of glutamate-induced injury mouse hippocampal neurons Influence; in which ** shows a very significant difference compared with the glutamate group, P<0.01;

Figure 2-a shows the effect of LYY-I ₁ on the survival rate of mouse hippocampal neurons induced by hydrogen peroxide; Figure 2-b shows the effect of ligustrazine on the survival rate of mouse hippocampal neurons induced by hydrogen peroxide Influence; in which ** shows a very significant difference compared with the hydrogen peroxide group, P<0.01;

Figure 3 shows the effect of LYY-I ₁ on platelet aggregation rate; in which: * indicates a significant difference compared with the ADP group, P<0.05; ** indicates a very significant difference compared with the ADP group, P<0.01;

Figure 4 shows the cerebral infarction volume percentage of intraperitoneal administration of 0.22 mmol/kg LYY-I ₁ after 3 h of reperfusion in the mouse MCAO model, where * indicates a significant difference compared with the solvent group, P<0.05; ** indicates There was a very significant difference compared with the solvent group, P<0.01.

Detailed ways

The present invention provides a diarylmethylene disulfide compound, a preparation method and application thereof, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications herein without departing from the content, spirit and scope of the present invention, so as to realize and apply the present invention. Invention technology.

Below in conjunction with embodiment, the present invention is further elaborated:

Example 1 1,2-bis[(3,5,6-trimethylpyrazin-2-yl)methyl]disulfide (LYY- _{1 1} )

1-Bromoligustrazine (0.210g, 0.97mmol), thiourea (0.111g, 1.45mmol), sodium carbonate (0.154g, 1.45mmol), manganese dioxide (0.084g, 0.97mmol) were added to a 100mL reaction flask , and then add wet-PEG200 (20 mL) (PEG200:H ₂ O=10:1), heat to 40° C., and stir to react for 3 h. Cooled to room temperature, added water (20 mL), extracted three times with ethyl acetate (20 mL), washed three times with water (50 mL), dried over anhydrous magnesium sulfate, suction filtered, and subjected to column chromatography [eluent petroleum ether: ethyl acetate (v :v)=10:1], a yellow solid was obtained. Yield 45%. Mp: 73～75℃.

¹ H NMR (400 MHz, CDCl ₃ ), δ (ppm): 3.99 (s, 4H, CH ₂ ), 2.54 (s, 6H, CH ₃ ), 2.49 (s, 12H, CH ₃ ).

¹³ C-NMR (600MHz, CDCl ₃ ), δ (ppm): 150.17, 148.94, 148.64, 146.61, 42.67, 21.62, 21.40, 21.09.

HR-MS:[M+H] ⁺ ,Calcd:335.1364,Found:335.1360.

Example 2 1,2-dibenzyl disulfide (LYY- _{1 2} )

With reference to the synthetic method of compound LYY- ₁₁ , it is prepared from benzyl bromide and thiourea to obtain a white solid. Yield 57%. Mp: 64～68℃.

¹ H NMR (400 MHz, CDCl ₃ , δ (ppm): 7.35-7.32 (m, 2H, ArH), 7.32-7.28 (m, 4H, ArH), 7.24 (dd, J=5.8, 4.3 Hz, 4H, ArH) ), 3.60(s, 4H, CH ₂ ).

¹³ C-NMR (600MHz, CDCl ₃ ), δ (ppm): 137.60, 129.65, 128.72, 127.66, 43.03.

HR-MS:[M+H] ⁺ ,Calcd:247.0615,Found:247.0611.

Example 3 Methyl 4,4'-disulfanediylbis(methylene)dibenzoate (LYY- _{1 3} )

With reference to the synthetic method of compound LYY- ₁₁ , it is prepared from methyl 4-(bromomethyl)benzoate and thiourea to obtain a white solid. Yield 51%. Mp: 81～85℃.

1H NMR (400 MHz, CDCl ₃ ), δ (ppm): 8.01-7.97 (m, 4H, ArH), 7.28 (d, J=8.3 Hz, 4H, ArH), 3.92 (s, 6H, OCH ₃ ), 3.62 (s,4H,CH ₂ ).

13C-NMR (600MHz, CDCl ₃ ), δ (ppm): 166.80, 142.61, 129.86, 129.41, 129.35, 52.21, 42.91.

HR-MS:[M+H] ⁺ ,Calcd:363.0725,Found:363.0724.

Example 4 1,2-bis(2-bromobenzyl)disulfide (LYY- ₁₄ )

With reference to the synthetic method of compound LYY- ₁₁ , it is prepared from 2-bromobenzyl bromide and thiourea to obtain a white solid. Yield 36%. Mp: 93～96℃.

¹ H NMR (400 MHz, CDCl ₃ ), δ (ppm): 7.56 (d, J=7.8 Hz, 2H, ArH), 7.29-7.26 (m, 4H, ArH), 7.16-7.11 (m, 2H, ArH) ,3.80(s,4H,CH ₂ ).

¹³ C-NMR (600MHz, CDCl ₃ ), δ(ppm): 136.66, 133.06, 131.63, 129.11, 127.35, 124.56, 43.74.

HR-MS:[M+H] ⁺ ,Calcd:402.8825,Found:402.8819.

Example 5 1,2-bis(3-bromobenzyl)disulfide (LYY- ₁₅ )

With reference to the synthetic method of compound LYY- ₁₁ , it is prepared from 3-bromobenzyl bromide and thiourea to obtain a pale yellow solid. Yield 41%. Mp: 57～60℃.

¹ H NMR (400 MHz, CDCl ₃ ), δ (ppm): 7.44-7.39 (m, 2H, ArH), 7.38 (dd, J=7.7, 1.8 Hz, 2H, ArH), 7.23-7.17 (m, 2H, ArH), 7.17-7.13 (m, 2H, ArH), 3.55 (s, 4H, CH ₂ ).

¹³ C-NMR (600MHz, CDCl ₃ ), δ (ppm): 139.53, 132.32, 130.57, 130.07, 127.98, 122.43, 42.54.

HR-MS:[M+H] ⁺ ,Calcd:402.8825,Found:402.8811.

Example 6 1,2-bis (4-bromobenzyl) disulfide (LYY- _{1 6} )

With reference to the synthetic method of compound LYY- ₁₁ , it is prepared from 4-bromobenzyl bromide and thiourea to obtain a white solid. Yield 33%. Mp: 91～95℃.

¹ H NMR (600 MHz, CDCl ₃ ), δ (ppm): 7.45 (d, J=8.2 Hz, 4H, ArH), 7.09 (d, J=8.3 Hz, 4H, ArH), 3.56 (s, 4H, CH ₂ ).

¹³ C-NMR (600MHz, CDCl ₃ ), δ (ppm): 136.30, 131.62, 130.99, 121.47, 42.53.

HR-MS:[M+H] ⁺ ,Calcd:402.8825,Found:402.8817.

Example 7 1,2-bis(3-fluorobenzyl)disulfide (LYY- ₁₇ )

With reference to the synthetic method of compound LYY- ₁₁ , it is prepared from 3-fluorobenzyl bromide and thiourea to obtain a yellow liquid. Yield 37%.

¹ H NMR (400 MHz, CDCl ₃ ), δ (ppm): 7.32-7.26 (m, 2H, ArH), 7.01 (d, J=7.7Hz, 2H, ArH), 6.95 (ddd, J=9.2, 4.8, 2.1Hz, 4H, ArH), 3.59(s, 4H, CH ₂ ).

¹³ C-NMR (600MHz, CDCl ₃ ), δ (ppm): 163.53, 139.80, 129.99, 125.01, 116.13, 114.50, 42.72.

HR-MS:[M+H] ⁺ ,Calcd:283.0427,Found:283.0435.

Example 8 1,2-bis (4-fluorobenzyl) disulfide (LYY- _{1 8} )

Referring to the synthetic method of compound LYY- ₁₁ , it was prepared from 4-fluorobenzyl bromide and thiourea to obtain a white solid. Yield 28%. Mp: 67～71℃.

¹ H NMR (400 Hz, CDCl ₃ ), δ (ppm): 7.22-7.20 (m, 2H, ArH), 7.19 (d, J=5.5 Hz, 2H, ArH), 7.02 (d, J=8.2 Hz, 2H , ArH), 7.01(d, J=8.6Hz, 2H, ArH), 3.59(s, 4H, CH ₂ ).

¹³ C-NMR (600MHz, CDCl ₃ ), δ (ppm): 163.02, 133.09, 130.86, 115.30, 42.40.

HR-MS:[M+H] ⁺ ,Calcd:283.0427,Found:283.0432.

Example 9 1,2-bis (2-nitrobenzyl) disulfide (LYY- _{1 9} )

With reference to the synthetic method of compound LYY- ₁₁ , it is prepared from 2-nitrobenzyl bromide and thiourea to obtain a white solid. Yield 21%. Mp: 108～111℃.

¹ H NMR (400 MHz, CDCl ₃ ), δ (ppm): 8.20-8.02 (m, 4H, ArH), 7.59-7.50 (m, 4H, ArH), 3.72 (s, 4H, CH ₂ ).

¹³ C-NMR (600MHz, CDCl ₃ ), δ (ppm): 148.25, 139.27, 135.25, 129.58, 124.08, 122.60, 42.08.

HR-MS:[M+H] ⁺ ,Calcd:337.0317,Found:337.0323.

Example 10 1,2-bis(3-nitrobenzyl)disulfide (LYY- ₁₁₀ )

With reference to the synthetic method of compound LYY- ₁₁ , it is prepared from 3-nitrobenzyl bromide and thiourea to obtain a white solid. Yield 47%. Mp: 97～101℃.

¹ H NMR (400 MHz, CDCl ₃ ), δ (ppm): 8.15 (dd, J=7.8, 1.4 Hz, 2H, ArH), 8.07 (s, 2H, ArH), 7.58 (d, J=7.6 Hz, 2H , ArH), 7.53(t, J=7.7Hz, 2H, ArH), 3.72(s, 4H, CH ₂ ).

¹³ C-NMR (600MHz, CDCl ₃ ), δ (ppm): 148.24, 139.27, 135.26, 129.58, 124.08, 122.60, 42.08.

HR-MS:[M+H] ⁺ ,Calcd:337.0317,Found:337.0312.

Example 11 1,2-bis(4-nitrobenzyl)disulfide (LYY- ₁₁₁ )

With reference to the synthetic method of compound LYY- ₁₁ , it is prepared from 4-nitrobenzyl bromide and thiourea to obtain a white solid. Yield 24%. Mp: 141～145℃.

¹ H NMR (600 MHz, CDCl ₃ ), δ (ppm): 8.19 (d, J=8.6 Hz, 4H, ArH), 7.43 (dd, J=12.9, 6.3 Hz, 4H, ArH), 3.68 (s, 4H) , CH ₂ ).

¹³ C-NMR (600MHz, CDCl ₃ ), δ (ppm): 129.71, 129.63, 123.85, 123.74, 35.18.

HR-MS:[M+H] ⁺ ,Calcd:337.0317,Found:337.0309.

Example ₁₂ 1,2-bis(4-chlorobenzyl)disulfide (LYY-112)

With reference to the synthetic method of compound LYY- ₁₁ , it is prepared from 4-chlorobenzyl bromide and thiourea to obtain a white solid. Yield 29%. Mp: 33～40℃.

¹ H NMR (600 MHz, CDCl ₃ ), δ (ppm): 7.27 (t, J=7.9 Hz, 4H, ArH), 7.19 (d, J=8.3 Hz, 4H, ArH), 3.54 (s, 4H, CH ₂ ).

¹³ C-NMR (600MHz, CDCl ₃ ), δ (ppm): 136.34, 130.63, 130.25, 128.62, 34.84.

HR-MS:[M+H] ⁺ ,Calcd:314.9836,Found:314.9828.

Example ₁₃ 1,2-bis(4-methylbenzyl)disulfide (LYY-113)

With reference to the synthetic method of compound LYY- ₁₁ , it is prepared from 4-methylbenzyl bromide and thiourea to obtain a white solid. Yield 34%. Mp: 52～55℃.

¹ H NMR (400 MHz, CDCl ₃ ), δ (ppm): 7.14-7.12 (m, 4H, ArH), 7.12-7.10 (m, 4H, ArH), 3.60 (s, 4H, CH ₂ ), 2.33 (s ,6H,CH ₃ ).

¹³ C-NMR (600MHz, CDCl ₃ ), δ (ppm): 137.11, 134.28, 129.28, 129.15, 43.04, 21.16.

HR-MS:[M+H] ⁺ ,Calcd:275.0928,Found:275.0924.

Example 14 1,2-bis(4-cyanobenzyl) disulfide (LYY-1 ₁₄ )

With reference to the synthetic method of compound LYY- ₁₁ , it is prepared from 4-cyanobenzyl bromide and thiourea to obtain a yellow solid. Yield 34%. Mp: 136～139℃.

¹ H NMR (400 MHz, CDCl ₃ ), δ (ppm): 7.63 (d, J=8.2 Hz, 4H, ArH), 7.33 (d, J=8.2 Hz, 4H, ArH), 3.64 (s, 4H, CH ₂ ).

¹³ C-NMR (600MHz, CDCl ₃ ), δ (ppm): 142.65, 132.31, 129.96, 118.51, 111.45, 42.58.

HR-MS:[M+H] ⁺ ,Calcd:297.0520,Found:297.0513.

Example 15 2,2-[(3-hydroxy-4-hydroxymethyl-2-methyl-5-pyridyl)-methyl]disulfide (LYY-I ₁₅ )

Pyridinol hydrochloride (0.200 g, 0.5 mmol) was dissolved in water, and a saturated aqueous sodium carbonate solution was added dropwise to precipitate a white solid with a yield of 70%.

¹ H NMR (400MHz, CDCl ₃ ), δ (ppm): δ 8.35 (s, 2H, OH), 7.70 (s, 2H, ArH), 4.76 (s, 4H, OCH ₂ ), 3.83 (s, 4H , CH ₂ ), 2.34(s, 6H, CH ₃ ).

¹³ C-NMR (600MHz, CDCl ₃ ), δ(ppm): δ151.05, 147.12, 140.97, 132.05, 128.86, 56.98, 36.83, 19.84.

HR-MS:[M+H] ⁺ ,Calcd.:369.0938,Found:369.0940.

Example 16 Protective effect of LYY-I compounds on glutamate-induced injury of mouse hippocampal neuron HT-22 cell line

The test method refers to the literature [Cheng J, et al. Neurochemistry International, 2013, 62(8): 1072-1078.].

Samples were dissolved in DMSO. The experiment was divided into blank control group, glutamate group and sample group. The glutamate group was added with 5mM glutamate; the sample group was added with 5mM glutamate and LYY-I _1-15 or ligustrazine with a final concentration of 1, 5, 10, 50, 100 μM, respectively; the blank control group was added with the same volume of DMSO. 24 hours after dosing, the viable cells were detected by MTT method. Three parallel experiments were performed in each group, and the results are shown in Table 1.

Table 1 Effects of LYY-I compounds on the survival rate of glutamate-induced injury HT22 cells

** Shows significant difference compared with glutamate group, P<0.01

5 mM glutamate induced HT22 cell damage with <30% viability. It can be seen from the results in Table 1 that the compounds synthesized in Examples 1-15 with a final concentration of 1-100 μM can significantly improve the survival rate of HT22 cells induced by glutamate (P<0.05, P<0.01), and reduce nerve cell damage. Its effective protective concentration ranged from 10 to 100 μM, and the lowest effective protective concentration detected was 10 μM. Among them, LYY-I ₁ has the strongest protective effect on glutamate-induced injury HT22 cells, and the cell viability reaches 91.77-95.40% at 10-100 μM, which is significantly different from other compounds (P<0.01); but the same Ligustrazine has no anti-glutamic acid toxicity effect at the concentration, see Figure 1.

Example 17 The protective effect of LYY-I ₁ on the HT-22 cell line of mouse hippocampal neurons from hydrogen peroxide-induced injury

The test method refers to the literature [ChenZ, et al. Cancer Letters, 2013, 336(2): 281-289.].

The experiment was divided into blank control group, hydrogen peroxide group and sample group. The hydrogen peroxide group was treated with 100 μM hydrogen peroxide; the sample group was added with 100 μM hydrogen peroxide and LYY-I ₁ or ligustrazine with a final concentration of 1, 5, 10, 50, and 100 μM, respectively; the control group was added with the same volume of DMSO. 24 hours after dosing, the viable cells were detected by MTT method. Each group was repeated three times, and the experimental results are shown in Figure 2.

Figure 2 shows that in the H ₂ O ₂ group, the survival rate of HT-22 cells was 15.23%, indicating that 100 μM H ₂ O ₂ caused damage to HT-22 cells. After treatment with 50 and 100 μM LYY-I ₁ , the survival rate of HT-22 cells was significantly decreased (P<0.01), indicating that LYY-I ₁ has the effect of scavenging free radicals generated by H ₂ O ₂ and has a negative effect on H ₂ O ₂ damage. HT-22 cells have a good protective effect. However, at the same concentration, ligustrazine did not scavenge H ₂ O ₂ free radicals.

Example 18 The antagonistic effect of LYY-I ₁ on ADP-induced platelet aggregation in rats

The experimental method refers to the literature [Paul Jurasz, et al. PLoS One, 2013; 8(3): e59281.].

The experiment was divided into ADP (adenosine diphosphate) group, LYY-I ₁ group (adding ADP and 0.1, 0.5, 1 mM LYY-I ₁ ), and ligustrazine group (adding ADP and 0.1, 0.5, 1 mM ligustrazine). Set up 3 parallel tubes. Platelets were obtained from SD rats. The maximum aggregation rate (PAGm) of each group was determined on a platelet aggregometer.

As shown in Figure 3, the maximum platelet aggregation rate in the ADP group was 78.77%. When adding 0.1, 0.5, 1 mM LYY-I ₁ , the maximum platelet aggregation rate was significantly decreased (P<0.05, P<0.01), suggesting that LYY-I ₁ has a good anti-platelet aggregation activity. However, Ligustrazine had no anti-platelet aggregation effect at the same concentration.

Example 19 The brain protective effect of LYY-I ₁ in the middle cerebral artery occlusion model in mice

ICR mice, male, weighing 30±2.0 g, were purchased from Shanghai Experimental Animal Center, Chinese Academy of Sciences. 2,3,5-Triphenyltetrazolium chloride (TTC) (Sigma).

The experiment was carried out in the mouse middle cerebral artery occlusion (MCAO) model and divided into LYY-I ₁ group and solvent control group. 8 mice per group. The solvent group was injected with corn oil containing 25% DMSO. In the LYY-I ₁ group, 0.22 mmol/kg LYY-I ₁ prepared by the present invention was intraperitoneally administered 3 hours after ischemia-reperfusion. Mice were sacrificed 24 hours after ischemia-reperfusion, and the size of cerebral infarction was measured by TTC staining. Figure 4 shows the percentage of cerebral infarction volume in mice after 24h of reperfusion in MCAO. From the results in Figure 4, it can be seen that 24 hours after the middle cerebral artery occlusion, compared with the mice in the solvent group, injection of LYY-I ₁ after 3 hours of ischemia and reperfusion in mice significantly reduced the infarct volume of the cortex, striatum and cerebral hemisphere, indicating that LYY- I ₁ has a significant protective effect on ischemic brain injury caused by middle cerebral artery occlusion.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, some improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be regarded as It is the protection scope of the present invention.

Claims (10)

1. A compound of the structure of formula I:

wherein Ar is 3,5,6-trimethyl pyrazine-2-radical.

2. The application of the compound with the structure shown in the formula I in preparing a preparation for protecting neuron cells;

wherein Ar is 3,5,6-trimethyl pyrazine-2-radical.

3. The application of the compound with the structure shown in the formula I in the preparation of a preparation for inhibiting platelet aggregation;

wherein Ar is 3,5,6-trimethyl pyrazine-2-radical.

4. The application of the compound with the structure shown in the formula I in preparing a preparation for preventing and treating brain injury after cerebral ischemia reperfusion;

wherein Ar is 3,5,6-trimethyl pyrazine-2-radical.

5. The application of the compound with the structure shown in the formula I in preparing a preparation for preventing and treating cerebral apoplexy;

wherein Ar is 3,5,6-trimethyl pyrazine-2-radical.

6. Use according to claim 5, characterized in that the stroke is a stroke caused by cerebral embolism and/or a stroke caused by cerebral hemorrhage.

7. The use according to any one of claims 2 to 6, wherein the compound of formula I is administered in an amount of 0.05mg/kg to 90 mg/kg.

8. Use according to any one of claims 2 to 6, wherein Ar is 3,5, 6-trimethylpyrazin-2-yl.

9. A medicament comprising a compound of formula I and a pharmaceutically acceptable excipient;

wherein Ar is 3,5,6-trimethyl pyrazine-2-radical.

10. The preparation method of the compound with the structure shown in the formula I is characterized in that the compound A reacts with thiourea in the presence of manganese dioxide and PEG to prepare the compound shown in the formula I;

wherein Ar is 3,5, 6-trimethylpyrazin-2-yl; the compound A is 2-bromomethyl-3, 5,6-trimethyl pyrazine.

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