阅读说明：本技术 一种硫化氢的供体及其制备方法和用途 (Donor of hydrogen sulfide and preparation method and application thereof ) 是由 陈鲲 樊静 程魁 *** 胡滨雁 陈韵帆 胡佳钦 秦嘉裕 李海鹏 于 2019-07-11 设计创作，主要内容包括：本发明涉及一种硫化氢的供体及其制备方法和用途,属于医药技术领域。本发明提供的硫化氢的供体,其化学名称为S-(4-氟苄基)-N-(3,4,5-三甲氧基苯甲酰基)-L-半胱氨酸甲酯,化学结构式如式(Ⅰ)所示；本发明提供的硫化氢的供体(MTC),能有效治疗缺血性脑卒中的疾病,通过激活PI3K/AKT通路、抑制线粒体凋亡信号通路、降低内质网应激、激活ERK-MEK信号通路、增加神经细胞的存活率或保护神经元缺血性损伤的多种治疗靶点来实现有效治疗缺血性脑卒中的目的。<Image he="556" wi="603" file="DDA0002127037900000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention relates to a donor of hydrogen sulfide, a preparation method and application thereof, belonging to the technical field of medicines. The chemical name of the donor of hydrogen sulfide provided by the invention is S- (4-fluorobenzyl) -N- (3,4, 5-trimethoxybenzoyl) -L-cysteine methyl ester, and the chemical structural formula is shown as a formula (I); the hydrogen sulfide donor (MTC) provided by the invention can effectively treat ischemic stroke diseases by activating a PI3K/AKT pathway, inhibiting a mitochondrial apoptosis signal pathway, reducing endoplasmic reticulum stress, activating an ERK-MEK signal pathway, increasing the survival rate of nerve cells or protecting neuronal ischemic injuryThe aim of effectively treating the cerebral arterial thrombosis is fulfilled by a plurality of treatment targets.)

1. A donor of hydrogen sulfide, characterized in that the chemical name of the donor of hydrogen sulfide is S- (4-fluorobenzyl) -N- (3,4, 5-trimethoxybenzoyl) -L-cysteine methyl ester; the chemical structural formula of the hydrogen sulfide donor is shown as the formula (I):

2. the method of claim 1, wherein the method comprises the steps of:

(1) dissolving a compound shown as a formula (II) in thionyl chloride, carrying out reflux reaction at 70-80 ℃ until the reaction is complete, carrying out thin-layer chromatography tracking reaction until the reaction is finished, and carrying out reduced pressure spin drying on the solvent to obtain an intermediate product, namely a compound shown as a formula (III);

(2) dissolving the compound shown in the formula (III) in the step (1) in dichloromethane, adding Triethylamine (TEA) into the dichloromethane, stirring the mixture for reaction for 10-30 min under an ice bath condition, adding the compound shown in the formula (IV), continuously stirring the mixture until the reaction is finished, performing reduced pressure spin drying on the solvent, and performing chromatographic separation on the residue by using a silica gel column to obtain a donor of the target product hydrogen sulfide;

the structural formulas of the formula (II), the formula (III) and the formula (IV) are shown as follows:

3. the preparation method according to claim 2, wherein in the step (1), the compound represented by the formula (II) and thionyl chloride are added in a molar ratio of 1:40 to 45; in the step (2), the addition molar ratio of the compound shown in the formula (III), the dichloromethane, the triethylamine and the compound shown in the formula (IV) is 1: 45-50: 2-3: 1.

4. A pharmaceutical preparation characterized in that the active ingredient of the pharmaceutical preparation comprises a compound represented by the formula (i) as claimed in claim 1.

5. The pharmaceutical formulation of claim 4, wherein the compound of formula (I) is present in the pharmaceutical formulation in an amount of from 1% to 50%.

6. The pharmaceutical formulation of claim 4, further comprising pharmaceutically acceptable excipients comprising fillers, binders, disintegrants, lubricant glidants.

7. The pharmaceutical preparation of claim 6, wherein the content of the auxiliary materials is, in weight percent: 10 to 80 percent of filling agent, 1 to 45 percent of adhesive, 5 to 20 percent of disintegrating agent and 0.1 to 10 percent of lubricating flow aid.

8. The pharmaceutical preparation according to claims 4 to 7, wherein the pharmaceutical preparation is in the form of tablets, capsules or granules.

9. Use of a donor of hydrogen sulfide as claimed in claim 1 for the preparation of a medicament for the treatment of ischemic stroke.

10. Use of a donor of hydrogen sulfide as claimed in claim 1 in the manufacture of a medicament for activating the PI3K/AKT pathway, inhibiting the mitochondrial apoptotic signaling pathway, or reducing endoplasmic reticulum stress.

11. Use of a donor of hydrogen sulfide as claimed in claim 1 for the manufacture of a medicament for activating the ERK-MEK signaling pathway, increasing the survival of nerve cells or protecting against neuronal ischemic injury.

Technical Field

The invention relates to a donor of hydrogen sulfide, a preparation method and application thereof, belonging to the technical field of medicines.

Background

The most effective method for clinically treating cerebral apoplexy at present is to rapidly dredge the cerebral ischemia, namely ischemia reperfusion, but reperfusion after cerebral ischemia can cause cerebral tissue injury and dysfunction aggravation under certain conditions, namely ischemia/reperfusion injury.

Hydrogen sulfide (hydrogen sulfide, H) ₂S) has been considered to be a third gas signal molecule following Nitric Oxide (NO) and carbon monoxide (CO). In recent years, the research finds that the brain tissue has physiological concentration of H ₂S is normally present, endogenous H ₂S is mainly derived from the desulfurization of cysteine (Cys). Found in the study that H ₂S exerts neuroprotective effects in the nervous system through a variety of mechanisms, protecting damaged neurons or glial cells, such as H ₂S can scavenge oxygen free radicals and also improve hypoxic neuron function by reducing expression of apoptotic proteins and activating anti-apoptotic proteins. In the central nervous system, at physiological concentrations of H ₂S modulates N-methyl-D-aspartate (NMDA) receptors in the hippocampus by promoting cAMP production, enhancing NMDA receptor-mediated neurological responses. Research results show that the anti-neuronal apoptosis effect of H2S is mainly due to the fact that the anti-neuronal apoptosis effect can protect the integrity of mitochondria, namely, the anti-neuronal apoptosis pathway of the mitochondria is inhibited. H ₂Propargyl cysteine (SPRC) and analogues thereof, including ethyl cysteine (SEC), allyl cysteine (SAC), allyl mercapto cysteine (SAMC), butyl cysteine (SBC) and amyl cysteine (SPEC) are all hydrogen sulfide donors, the compounds release endogenous hydrogen sulfide through cystathionine- β -synthetase (CBS) to protect ischemic stroke, but t1/2 in vivo is short and volatile, the administration amount cannot be controlled, and the compounds are easy to be oxidized and deteriorated in air due to the existence of amino in the structure of the compounds.

Disclosure of Invention

The gallic acid is widely present in rheum palmatum, eucalyptus robusta, dogwood and other plants, and is a natural nontoxic polyphenol compound, it is reported that gallic acid has antioxidant, anti-inflammatory, bacteriostatic, anti-free radical, anti-tumor and cardiovascular protective effects, and has neuroprotective effects in neurodegeneration, neurotoxicity and oxidative stress, 3 phenolic hydroxyl groups exist in its molecular structure, has strong reducibility, and is easily oxidized in the air, and its ester compound is widely used in pharmaceutical industry as an antioxidant.

In view of the biological activities of the gallic acid and the derivatives thereof as well as the propargyl cysteine and the analogues thereof, the inventor designs and synthesizes a series of conjugates of the propargyl cysteine analogue and the gallic acid ester compound according to the chemical hybridization principle, and obtains a compound MTC with better activity by screening, wherein the chemical name of the MTC is S- (4-fluorobenzyl) -N- (3,4, 5-trimethoxybenzoyl) -L-cysteine methyl ester.

The invention aims to overcome the defects of the prior art and provide a hydrogen sulfide donor (MTC) with a chemical name of S- (4-fluorobenzyl) -N- (3,4, 5-trimethoxybenzoyl) -L-cysteine methyl ester and a structural formula shown in a formula (I),

in addition, the present invention provides a method for preparing the above-mentioned hydrogen sulfide donor (MTC).

In addition, the present invention provides a pharmaceutical preparation, the active ingredient of which comprises the compound represented by the above formula (i).

In addition, the invention provides application of the hydrogen sulfide donor (MTC) in preparation of a medicine for treating cerebral arterial thrombosis.

In addition, the invention provides application of the hydrogen sulfide donor (MTC) in preparing a medicine for activating a PI3K/AKT pathway, inhibiting a mitochondrial apoptosis signal pathway or reducing endoplasmic reticulum stress.

In addition, the invention provides the application of the hydrogen sulfide donor (MTC) in preparing medicines for activating an ERK-MEK signal pathway, increasing the survival rate of nerve cells or protecting neuron ischemic injury.

In order to achieve the purpose, the invention adopts the technical scheme that: a donor of hydrogen sulfide (MTC) having a chemical name of S- (4-fluorobenzyl) -N- (3,4, 5-trimethoxybenzoyl) -L-cysteine methyl ester; the chemical structural formula of the hydrogen sulfide donor is shown as the formula (I):

the invention provides a preparation method of a donor of hydrogen sulfide (MTC), which comprises the following steps:

(1) dissolving the compound shown in the formula (II) in thionyl chloride (SOCl) ₂) Carrying out reflux reaction (rf) at 70-80 ℃ until the reaction is complete, carrying out thin-layer chromatography tracking reaction until the reaction is finished, and carrying out decompression and spin-drying on the solvent to obtain a compound shown as an intermediate product formula (III);

(2) dissolving the compound shown in the formula (III) in the step (1) in Dichloromethane (DCM), adding Triethylamine (TEA) to react for 10-30 min under an ice bath condition by stirring, adding the compound shown in the formula (IV), continuously stirring until the reaction is finished, performing reduced pressure spin drying on the solvent, and performing chromatographic separation on the residue by using a silica gel column to obtain a donor (MTC) of the target product hydrogen sulfide;

the structural formulas of the formula (II), the formula (III) and the formula (IV) are shown as follows:

preferably, in the step (1), the addition molar ratio of the compound shown in the formula (II) to thionyl chloride is 1: 40-45; in the step (2), the addition molar ratio of the compound shown in the formula (III), the dichloromethane, the triethylamine and the compound shown in the formula (IV) is 1: 45-50: 2-3: 1.

The invention provides a pharmaceutical preparation, wherein the effective component of the pharmaceutical preparation comprises the compound shown in the formula (I) in the invention.

Preferably, the percentage of the compound shown in the formula (I) in the pharmaceutical preparation is 1-50%.

Preferably, the pharmaceutical preparation further comprises pharmaceutically acceptable auxiliary materials, wherein the auxiliary materials comprise a filling agent, a binding agent, a disintegrating agent and a lubricating glidant.

Preferably, the content of the auxiliary materials is as follows by weight percentage: 10 to 80 percent of filling agent, 1 to 45 percent of adhesive, 5 to 20 percent of disintegrating agent and 0.1 to 10 percent of lubricating flow aid.

Preferably, the dosage form of the pharmaceutical preparation is tablets, capsules or granules.

Preferably, the filler is selected from at least one of lactose, sucrose, starch, pregelatinized starch, mannitol, sorbitol, calcium hydrogen phosphate, calcium sulfate, calcium carbonate, microcrystalline cellulose.

Preferably, the binder is selected from at least one of dextrin, povidone, sodium carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, methylcellulose, polyethylene glycol, and pharmaceutically acceptable ethanol.

Preferably, the disintegrant is selected from at least one of crospovidone, croscarmellose sodium, low substituted carboxymethylcellulose sodium, croscarmellose sodium, and sodium starch glycolate.

Preferably, the lubricating glidant is selected from at least one of magnesium stearate, calcium stearate, stearic acid, sodium fumarate, sodium lauryl sulfate, glyceryl behenate, talc, silicon dioxide, polyethylene glycol and sodium stearyl fumarate.

The invention provides application of the hydrogen sulfide donor (MTC) in preparation of a medicine for treating ischemic stroke.

The invention provides application of the hydrogen sulfide donor (MTC) in preparation of medicines for activating a PI3K/AKT pathway, inhibiting a mitochondrial apoptosis signal pathway or reducing endoplasmic reticulum stress.

The invention provides application of the hydrogen sulfide donor (MTC) in preparation of medicines for activating an ERK-MEK signal pathway, increasing the survival rate of nerve cells or protecting neuron ischemic injury.

Compared with the prior art, the invention has the beneficial effects that:

(1) the hydrogen sulfide donor (MTC) with the chemical name of S- (4-fluorobenzyl) -N- (3,4, 5-trimethoxybenzoyl) -L-cysteine methyl ester can effectively treat ischemic cerebral apoplexy;

(2) the hydrogen sulfide donor (MTC) provided by the invention can realize the purpose of effectively treating ischemic stroke by activating a PI3K/AKT (mitochondrion activation/apoptosis) pathway, inhibiting a mitochondrial apoptosis signal pathway, reducing endoplasmic reticulum stress, activating an ERK-MEK (extracellular signal kinase-methyl-Ketone) signal pathway, increasing the survival rate of nerve cells or protecting various treatment targets of neuronal ischemic injury, and has no side effect of gastrointestinal hemorrhage;

(3) in the medicine for treating ischemic stroke, the invention firstly provides the function of the hydrogen sulfide donor (MTC) in the nervous system, and provides a new medicine selection for treating the ischemic stroke.

Drawings

FIG. 1 is a graph showing the effect of 7 compounds on the survival of ischemia reperfusion-induced damaged PC12 cells.

FIG. 2 is a schematic illustration of the effect of MTC on ischemic neuron survival; wherein, fig. 2A is a cell morphology of MTC injury to ischemia reperfusion induced PC 12; fig. 2B is a graph of the effect of MTC on the survival of cells damaged by ischemia reperfusion-induced PC 12.

Fig. 3 is a graph showing the effect of MTC on the karyotype of ischemia reperfusion-induced PC12 injury.

FIG. 4 is a graph showing the effect of MTC on ROS and SOD production; wherein, fig. 4A is the effect of MTC on ROS injury from ischemia reperfusion-induced PC 12; fig. 4B is a graph of the effect of MTC on damage to SOD by ischemia reperfusion-induced PC 12.

FIG. 5 is a graph showing the effect of MTC on the expression of PI3K, p-AKT and cleared caspase-3 protein levels following ischemia reperfusion-induced PC12 cell injury; wherein, FIG. 5A is a Western blot analysis showing that MTC increases the expression of PI3K protein; FIG. 5B shows Western blot analysis indicating that MTC activates p-AKT expression; FIG. 5C is a Western blot analysis showing that MTC inhibits clear caspase-3 expression.

FIG. 6 is a schematic representation of the effect of different concentrations of PD98059 pretreatment +1 μ M MTC on cell viability; wherein, fig. 6A shows the effect of different concentrations of PD98059 pretreatment for 2h and MTC treatment for 24h on the morphology of PC12 cells; FIG. 6B shows the effect of different concentrations of PD98059 pretreatment for 2h and MTC treatment for 24h on the survival rate of PC12 cells.

FIG. 7 is a schematic representation of the effect of different concentrations of PD98059 pretreatment +1 μ M MTC on cellular ROS and karyotype; wherein, FIG. 7A is the effect of different concentrations of PD98059 pretreatment + 1. mu.M MTC on karyotype; FIG. 7B is a graph of the effect of different concentrations of PD98059 pretreatment +1 μ M MTC on cellular ROS.

FIG. 8 is a schematic representation of the effects of MTC on ischemia-induced Endoplasmic Reticulum Stress (ERS) Bim of PC12 cells, capase-12, IP3, and the like; FIG. 8A is a Western blot analysis showing that MTC post-treatment inhibits the expression of Bim, caspase-12, IP3 proteins; FIG. 8B is a Western blot analysis showing that MTC post-treatment inhibits expression of GRP78, CHOP.

Fig. 9 is a graph showing the effect of MTC and SCGF on axon migration following ischemia reperfusion-induced PC12 cell injury.

FIG. 10 is a graph showing that MTC regenerates PC12 cell axons by elevating p-ERK levels; wherein, figure 10A is the effect of MTC treatment for 24h on axon growth after ischemia reperfusion-induced PC12 cell injury; FIG. 10B is a Western blot analysis showing that MTC treatment activates phosphorylation of ERK.

Figure 11 is a graph of the effect of MTC on hippocampal regions of mouse brain following ischemia reperfusion-induced PC12 cell injury.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.