Butylphthalide impurity compound, preparation method and use thereof

Abstract

The present application relates to a butylphthalide impurity compound, a preparation method and use thereof. The impurity compound of the present application is similar to butylphthalide in structure, but has no significant effect on the secretion of TNF-alpha of LPS-induced mouse primary microglial cells; instead, the impurity compound has significant neurotoxicity on mouse brain neuroblastoma cell line (Neuro-2a), and can significantly inhibit the length of Neuro-2a nerve cell axon and the number of cells with axon. The content of peroxidized impurities has an effect on the treatment of butylphthalide composition on nerve function damage of tMCAO model rats, and the higher the content of peroxidized impurities, the greater the degree of nerve function damage. Limiting the impurity compound of the present application in the quality control of butylphthalide is beneficial to the quality control of butylphthalide drugs, and can ensure the effectiveness, safety and quality control of the drugs.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a butylphthalide impurity compound and a preparation method and application thereof, in particular to 3-peroxy-3-butyl isobenzofuranone and a preparation method and application thereof. BACKGROUND

[0002] Butylphthalide belongs to a C-3 substituted phthalide racemate compound, and is used for protecting and improving the brain nerve function of ischemic stroke. In recent years, new indications research and development have also achieved gratifying results, and are expected to be applied in chronic diseases such as senile dementia and depression. Butylphthalide is used very fast in the clinic, and the amount is large. Moreover, as the patent period ends, more manufacturers join the ranks of imitation, and therefore the attention to the quality control of the product is also higher. Phthalide compounds are unstable, and are easily affected by light, temperature, acid, alkali and other factors to change. The starting materials, intermediates, polymers, by-products and degradation products in the storage process, etc. brought in the production process all affect the content of related substances in the product. Therefore, the development of the detection method of the related substances is an important part in the quality research of the drug, and can improve the safety, effectiveness and quality controllability of the drug.

[0003] Butylphthalide sodium chloride injection acts on multiple pathological links of cerebral ischemia by improving the levels of NO and PGI2 of cerebral vascular endothelium, reducing the intracellular calcium ion concentration, inhibiting glutamate release, reducing arachidonic acid generation, eliminating oxygen free radicals, improving antioxidant enzyme activity, etc. Animal pharmacodynamic research shows that the product has a strong anti-cerebral ischemia effect, can obviously improve the microcirculation and blood flow of the cerebral ischemic area, increase the number of capillaries in the ischemic area, reduce brain edema, reduce the volume of rat cerebral infarction, improve brain energy metabolism, reduce neural cell apoptosis, and inhibit thrombus formation. Clinical research shows that the product has obvious therapeutic effect on ischemic cerebrovascular disease, and can promote the recovery of damaged nerve function of patients (Butylphthalide sodium chloride injection specification, State Drug Standard Code H20100041, Shenzhen Pharmaceutical Group Enbilu Pharmaceutical Co., Ltd.).

[0004] Most stroke patients will have residual neurological deficits. Therefore, it emphasizes the need to develop neurorestorative agents to reduce the extent of brain damage or restore brain function after stroke. Although the central nervous system has limited repair capacity, there is a certain degree of spontaneous repair after cerebral ischemia, and this repair process involves angiogenesis, neurogenesis, axon sprouting and synaptogenesis. This self-repairing ability is closely related to neural plasticity. Neural plasticity can be observed at multiple scales. From the perspective of microstructure, it refers to the branching of axons, dendrites, the morphology and density of dendritic spines, the size and number of synapses, receptor density, and the structure and number of neurons in certain brain regions. From the macrostructure, behavior, environmental stimuli, thoughts and emotions can also change through changes in neural activity of neural plasticity, which is of great significance to healthy development, learning, memory and recovery from brain injury. Studies have shown that healthy adult male C57BL / 6 mice were used to establish a dMCAO model by electric cauterization, and dl-3n-butyphthalide can promote the increase of dendritic branching in different parts of the brain after cerebral infarction in different degrees in the hyperacute phase, acute phase, subacute phase and recovery phase, suggesting that dl-3n-butyphthalide promotes the recovery of dendritic plasticity after cerebral infarction; Promote the increase of density and length of different types of dendritic spines and the growth of head width in the recovery period after cerebral infarction, suggesting that dl-3n-butyphthalide promotes the recovery of dendritic spine plasticity in the recovery period after cerebral infarction (Zhang Cong, dl-3n-butyphthalide promotes the recovery of dendritic plasticity in the recovery period after cerebral infarction. Doctoral thesis of Hebei Medical University, 2018).

[0005] Microglia belongs to mononuclear phagocyte family, and is widely considered as the main immune effector in the central nervous system (Giulian, 1987), which is involved in human nervous system disorder diseases such as HIV encephalopathy, Parkinson's disease, Alzheimer's disease (senile dementia), multiple sclerosis, etc. (Dickson, 1991; McGeer, 1993). Microglia is sensitive to central nervous system injury, can proliferate rapidly, increase or re-express MHC antigens, migrate and change into phagocyte-like morphology (amoeba-like), and at the same time, burst to secrete a large amount of cytokines and cytotoxic substances (Giulian, 1992), which is beneficial to the nutrition and repair of neurons in the later stage of inflammation caused by injury (Nagamoto-Combs k, 2007). The number of microglia is small, accounting for about 5% of all glial cells. This cell is a phagocyte that inhabits the brain, and its antigenicity increases, morphology stretches and function is active under inflammatory stimulation. Microglia is distributed in all parts of the brain, and the number in the gray matter is 5 times more than that in the white matter. The number of microglia in hippocampus, olfactory lobe and basal ganglia is more than that in thalamus and hypothalamus, while the number in brainstem and cerebellum is the least. Studies have shown that butylphthalide can inhibit the activation of microglia after trauma, reduce the release of inflammatory cytokines, reduce neuronal degeneration and improve neurological dysfunction (Liu Zhengwei, Neuroprotective Effect and Mechanism of Butylphthalide on Traumatic Brain Injury in Mice, Doctoral Dissertation of South Medical University, 2015). 118 patients with acute cerebral infarction (ACI) admitted from October 2016 to September 2017 were selected as the research object, and the influence of butylphthalide on serum inflammatory factors, oxidative stress and neurotrophic factors (NF) of ACI patients was investigated. Butylphthalide treatment of ACI patients can down-regulate inflammatory factors (such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and hypersensitive C-reactive protein (hs-CRP) expression), play an anti-inflammatory effect, increase antioxidant capacity to scavenge oxygen free radicals (OFR), improve neural cell metabolism, effectively play a neuroprotective and prognostic role (Li Ruilin et al., Influence of Butylphthalide on Serum Inflammatory Factors, Oxidative Stress and Neurotrophic Factors in Patients with ACI, Journal of Hainan Medical College, 2018, 24(01)).

[0006] The national drug standard WS1-(X-124)-2005Z for butylphthalide discloses a method for detecting the content and related substances of butylphthalide. The method uses octadecylsilane-bonded silica gel as the filler, methanol: water (65:35) as the mobile phase, the detection wavelength is 280 nm, and the separation degree test solution is a methanol solution containing butylphthalide and propylphthalide. The standard can only separate a small number of impurities. With the increase of generic products, the requirements of pharmaceutical registration technology are increasing. The standard is no longer suitable for the quality control requirements of butylphthalide bulk drug and its preparations.

[0007] Chinese patent application CN201610395891.4 discloses a method for separating and determining butylphthalide and its related substances by high performance liquid chromatography. The method solves the problem of difficult separation of o-phthalic acid, intermediate I, intermediate II, propylphthalide, butenylphthalide, and dibutylphthalide impurities from the main component.

[0008] Chinese patent application CN201910466352.9 discloses a method for determining butylphthalide related substances by HPLC, which can effectively separate 2-(ɑ-hydroxy pentyl) potassium benzoate, phthalide, 2-(ɑ-carbonyl pentyl) benzoic acid, methyl phthalide, ethyl phthalide, 3-n-butyl-3-hydroxy-1(3H)-isobenzofuranone, propyl phthalide, pentyl phthalide, o-carboxybenzyl alcohol, o-carboxybenzaldehyde, isobutyl phthalide, sec-butyl phthalide, isobutyl phthalide, and 13 other impurities / related substances.

[0009] With the increase of generic products and the increase of newly developed dosage forms, the existing technology cannot meet the quality control requirements of butylphthalide, which has great hidden dangers for ensuring drug safety. SUMMARY

[0010] The inventors of the present application have found impurities that seriously affect the quality, efficacy, and safety of butylphthalide and its preparations through a large number of rigorous chemical research and biological tests. Specifically,

[0011] The inventors of the present application found a brand new butylphthalide impurity which was not identified in the prior art. The inventors isolated, identified and prepared the impurity, which was confirmed to be 3-peroxy-3-butylisobenzofuranone. Although the structure of 3-peroxy-3-butylisobenzofuranone is similar to that of butylphthalide, 3-peroxy-3-butylisobenzofuranone has no significant effect on the secretion of TNF-α of LPS-induced mouse primary microglial cells, but has a significant neurocytotoxic effect on mouse brain neuroblastoma cell line (Neuro-2a), and can significantly inhibit the length of Neuro-2a nerve cell axons and the number of axon cells. Therefore, 3-peroxy-3-butylisobenzofuranone has potential toxic side effects on the brain nerves. The inventors of the present application further used a rat middle cerebral artery transient ischemia-reperfusion (tMCAO) model to investigate the efficacy of butylphthalide compositions with different peroxide impurity contents on the model rats. The study found that both the butylphthalide composition group containing 3.0% peroxide impurity and the butylphthalide composition group containing 0.15% peroxide impurity can significantly reduce the cerebral infarction area of the tMCAO model rats, but the effect of the group containing 0.15% peroxide impurity is more significant. Relative to the model control group, the group containing 0.15% peroxide impurity has a significant effect on increasing the latency of the model animal rotarod test and reducing the behavior score, while the group containing 3.0% peroxide impurity has no statistical significance in increasing the latency of the model animal rotarod test and reducing the behavior score to a certain extent. The test shows that the peroxide impurity has an antagonistic effect on the protection of butylphthalide against nerve function damage in tMCAO rats, and the higher the peroxide content, the greater the nerve function damage in tMCAO rats.

[0012] 3-peroxy-3-butylisobenzofuranone is a new impurity of butylphthalide, which rapidly increases in the late production and storage period. Currently, there is no literature report on the discovery of this impurity in butylphthalide. In the existing separation technology, the retention time of this impurity is the same as or almost equal to that of some process impurities in the early stage of the process, making it difficult to be identified and separated by liquid chromatography and effectively controlled in quality, which greatly affects the quality of butylphthalide and its preparations, affects the efficacy, and even brings toxic side effects, which is not conducive to the rescue and rehabilitation of stroke patients. Therefore, the inventors of the present patent developed a new technology to solve the technical defects of the prior art.

[0013] Therefore, an object of the present application is to provide a butylphthalide impurity compound.

[0014] Another object of the present application is to provide a preparation method of the above-mentioned butylphthalide impurity compound (also referred to as peroxide impurity).

[0015] Still another object of the present application is to provide a pharmaceutical composition of butylphthalide.

[0016] Yet another object of the present application is to provide a preparation method of the pharmaceutical composition of butylphthalide.

[0017] Still another object of the present application is to provide an application of the butylphthalide impurity compound in butylphthalide quality control.

[0018] Still another object of the present application is to provide a method for preserving butylphthalide or a pharmaceutical composition of the butylphthalide.

[0019] A further object of the present application is to provide a liquid chromatography method for separating the above-mentioned butylphthalide impurity compound and other impurities.

[0020] The above-mentioned objects of the present application are achieved by adopting the following technical solutions.

[0021] In one aspect, the present application provides a butylphthalide impurity compound, which is 3-peroxyl-3-butylisobenzofuranone.

[0022] In the butylphthalide drug substance or its preparation, oxygen molecules are easy to attack the carbon atom at the benzyl position of butylphthalide at 20-40°C, thereby producing the above-mentioned impurity compound, which will also slowly increase during storage. This impurity is produced by oxidative degradation of butylphthalide, and it is necessary to control it during production and storage and to subscribe to quality standards. The impurity is identified as 3-peroxyl-3-butylisobenzofuranone, and its structure and molecular formula are as follows:

[0023] Structure formula:

[0024] Molecular formula: C 12 H 14 O4

[0025] Molecular weight: 222.09.

[0026] In another aspect, the present application provides a preparation method of the above-mentioned butylphthalide impurity compound, which comprises treating butylphthalide with an oxidizing agent. After treatment with the oxidizing agent, the target product can be obtained in pure form by column chromatography or liquid phase / preparation chromatography method. Specifically, butylphthalide can be oxidized to a peroxidized impurity under a corresponding oxidizing agent at a certain temperature, and the product after oxidation can be separated by column chromatography or liquid phase preparation to obtain 3-peroxyl-3-butylisobenzofuranone.

[0027] Preferably, the oxidizing agent is selected from oxygen, oxygen-containing air, hydrogen peroxide, meta-chloroperoxybenzoic acid (MCPBA) and peroxyacetic acid, and preferably is oxygen;

[0028] Preferably, the temperature for treating butylphthalide with the oxidizing agent is 20-110°C, and preferably is 45-75°C.

[0029] In still another aspect, the present application provides a pharmaceutical composition of butylphthalide, which comprises butylphthalide and 3-peroxyl-3-butylisobenzofuranone.

[0030] Preferably, the content of 3-peroxy-3-butylisobenzofuranone in the pharmaceutical composition is not more than 3 wt.%, preferably not more than 1 wt.%, more preferably not more than 0.3 wt.%, most preferably not more than 0.15 wt.%, further preferably not more than 0.1 wt.%.

[0031] In yet another aspect, the present application provides a method for preparing a pharmaceutical composition of butylphthalide with low content of 3-peroxy-3-butylisobenzofuranone impurity, which comprises the following steps:

[0032] The crude butylphthalide is dissolved in an organic solvent, then washed with an alkali-alcohol solution formed by an alcohol and an aqueous alkali solution, the organic phase is separated and retained, filtered, concentrated, and the finished product is stored in a sealed container;

[0033] Preferably, the washing is carried out at a temperature of 20-70°C, preferably 40-50°C.

[0034] Preferably, the organic solvent is selected from tetrahydrofuran, methyl tert-butyl ether, dichloromethane, ethyl acetate, n-heptane, etc., and one or more mixed solvents thereof, preferably selected from tetrahydrofuran, methyl tert-butyl ether and dichloromethane, and most preferably methyl tert-butyl ether.

[0035] Preferably, the alcohol in the alkali-alcohol solution is selected from methanol, ethanol, isopropanol, and two or more mixed solvents thereof; preferably selected from methanol, ethanol;

[0036] Preferably, the alkali in the alkali-alcohol solution is selected from potassium hydroxide, sodium hydroxide, sodium carbonate, sodium bicarbonate; preferably selected from sodium hydroxide, sodium bicarbonate;

[0037] Preferably, the mass concentration of the alkali in the alkali-alcohol solution is 2%-20%, preferably 10%.

[0038] Preferably, the volume ratio between the alcohol and the aqueous alkali solution in the alkali-alcohol solution is 0.1:10-5:10, preferably 1:10-3:10.

[0039] The source of the crude butylphthalide can be found in various existing technologies, for example: butylphthalide can be obtained by using butenylphthalide as raw material and then hydrogenating with palladium on carbon, which can be specifically referred to in the literature: Synthesis of (±)-Apiole Li Shaobai, Zhang Shaoming, Li Yulin (Journal of Lanzhou University (Natural Science Edition) 1990, (01) 118-119).

[0040] The crude butylphthalide can also be obtained by reacting ortho-carboxybenzaldehyde with n-butyl halogen (magnesium bromide or chloride) Grignard reagent or n-butyllithium in an organic solution to obtain crude butylphthalide. For specific steps, refer to the literature: Synthesis and Anti-platelet Activity of Nitric Oxide Donor Type 3-Butylphthalide Derivatives by Zhang Yihua (Journal of China Pharmaceutical University, 2008, 39(5), 392-397).

[0041] The crude butylphthalide obtained by the above two methods can be preliminarily purified by distillation or without distillation.

[0042] The inventors have found that the finished butylphthalide should be stored in a sealed manner, preferably with an oxygen content of less than 1%, otherwise the impurities will rapidly increase with the extension of the storage time. In one embodiment, the effect of residual oxygen content on the content change of 3-peroxy-3-butylisobenzofuranone can be observed.

[0043] Thus, it can be seen that the present application actually provides a control method for the new related substance 3-peroxy-3-butylisobenzofuranone of butylphthalide.

[0044] In still another aspect, the present application provides the application of the impurity compound of butylphthalide in the quality control of butylphthalide. In the quality standard of butylphthalide bulk drug and its preparations, the limit control of 3-peroxy-3-butylisobenzofuranone is carried out to ensure that the content of the impurity compound of butylphthalide in the bulk drug, preparation, intermediate or crude butylphthalide does not exceed 3 wt.%, preferably does not exceed 1 wt.%, more preferably does not exceed 0.3 wt.%, most preferably does not exceed 0.15 wt.%, and further preferably does not exceed 0.1 wt.%.

[0045] To further improve the quality of butylphthalide products, 3-peroxy-3-butylisobenzofuranone is included in the quality standard of finished products, preparations and intermediates, which can improve the quality of products.

[0046] Therefore, in still another aspect, the present application provides the application of the impurity compound of butylphthalide as a standard or control in the quality control of butylphthalide.

[0047] Further, the present application provides a chromatographic separation method of the impurity compound of the above-mentioned butylphthalide and other impurities, wherein the chromatographic separation method has the following conditions: a phenyl-hexyl column or a phenyl column is used, the detection wavelength is 227 nm or 280 nm, the flow rate is 0.8-1.2 ml / min, the column temperature is 20-35℃, the injection volume is 10-100 μl, the mobile phase is composed of phase A and phase B, phase A is an aqueous solution with a pH value of 3-5, the volume ratio of acetonitrile to methanol in phase B is 1:0.8-1.2, the gradient elution program is as follows: at the beginning of the gradient elution, the volume percentage of phase B is 25%-35%, at the end of the gradient elution, the volume percentage of phase B is 50%-80%, the gradient elution time is 40-50 minutes, the change rate of phase B is 0-2.0% per minute at 0-10 minutes, and the change rate of phase B is 0.1-1.2% per minute at 10-50 minutes;

[0048] Preferably, the chromatographic separation method has the following conditions:

[0049] The chromatographic column is Agilent ZORBAX plus Pheny-Hexyl (4.6 mm x 250 mm, 5 um);

[0050] The mobile phase A is an aqueous solution of 0.07% acetic acid;

[0051] The mobile phase B is acetonitrile-methanol (1:1);

[0052] The detection wavelength is 227 nm; the flow rate is 1.0 ml / min, the column temperature is 30℃, and the injection volume is 20 μl;

[0053] The elution gradient is as follows:

[0054]

[0055]

[0056] or

[0057]

[0058] or

[0059]

[0060] Preferably, the other impurities include, but are not limited to, o-phthalic acid, 2-hydroxymethylbenzoic acid, o-carboxybenzaldehyde, phthalide, 2-pentanoylbenzoic acid, propylphthalide, sec-butylphthalide, isobutylphthalide, butenylphthalide, and pentylphthalide.

[0061] The application identifies, separates and identifies a new potential neurotoxicity impurity of butylphthalide: 3-peroxy-3-butylisobenzofuranone, develops a method for detecting the impurity, develops a process capable of reducing and removing the impurity, provides a new technology and quality control method for producing high-quality butylphthalide and its preparation, is beneficial to the quality control of butylphthalide and its preparation, and ensures the effectiveness, safety and controllability of the drug quality.

[0062] Compared with the prior art, the application has at least the following beneficial technical effects:

[0063] The application finds a new butylphthalide impurity compound, the impurity compound of the application has no significant effect on the secretion of TNF-alpha of LPS-induced mouse primary microglial cells, but has significant neurotoxicity on mouse brain neuroblastoma cell line (Neuro-2a), can significantly inhibit the length of Neuro-2a nerve cell axon and the number of axon cells. The content of peroxide impurity has an effect on the treatment of tMCAO model rats with neurologic impairment by butylphthalide composition, the higher the content of peroxide impurity, the greater the degree of neurologic impairment. Limiting the impurity compound of the application in the quality control of butylphthalide is beneficial to the quality control of butylphthalide, can ensure the effectiveness, safety and controllability of the drug quality. BRIEF DESCRIPTION OF DRAWINGS

[0064] Figure 1 Chromatogram of Comparative Example 1;

[0065] Figure 2 Chromatogram of change in peroxide content before and after treatment of Example 2, wherein Figure A is the chromatogram before treatment; and Figure B is the chromatogram after treatment;

[0066] Figure 3 Chromatogram of Example 6;

[0067] Figure 4 Chromatogram of Example 7;

[0068] Figure 5 Chromatogram of Example 8;

[0069] Figure 6 Neurotoxicity of 3-peroxy-3-butylisobenzofuranone at different concentrations on Neuro-2a, relative to the control group (CN group), *** P<0.001;

[0070] Figure 7 Effect of 3-peroxy-3-butylisobenzofuranone on low serum-induced Neuro-2a nerve cell axon regeneration, wherein, relative to the control group, ***P<0.001;

[0071] Figure 17 Morphology of in vitro Neuro-2a cells after treatment with A~E 3- peroxyl-3-butyl isobenzofuranone (0 μΜ, 5 μΜ, 10 μΜ, 20 μΜ and 50 μΜ);

[0072] Figure 19 Axonal changes in in vitro Neuro-2a cells after treatment with F 3- peroxyl-3-butyl isobenzofuranone;

[0073] Figure 20 Number of in vitro Neuro-2a cells with axons after treatment with G 3- peroxyl-3-butyl isobenzofuranone.

[0074] Figure 8 Figure 21 Effect of 3-peroxyl-3-butyl isobenzofuranone on LPS-induced inflammatory response in primary mouse microglial cells, relative to the control group (CN group), ***P < 0.001;

[0075] Figure 22 Toxicity of A 3-peroxyl-3-butyl isobenzofuranone in primary mouse microglial cells;

[0076] Figure 23 Effect of B 3-peroxyl-3-butyl isobenzofuranone on expression of TNF-α inflammatory factor in primary mouse microglial cells (ELISA method).

[0077] Figure 9 Figure 3 Chromatogram of an industrially prepared butylphthalide composition, where Figure A is a chromatogram of the crude product; and Figure B is a chromatogram of the finished product. DETAILED DESCRIPTION

[0078] The present application will be described in detail below with reference to the examples, which are merely used to illustrate the technical solutions of the present application and are not intended to limit the essence of the present application.

[0079] In the following examples, the crude butylphthalide was prepared according to the reference: Synthesis and Antiplatelet Activity of Nitric Oxide Donor Type 3-Butylphthalide Derivatives, Zhang Yihua (Journal of China Pharmaceutical University, 2008, 39(5), 392-397), unless otherwise specified.

[0080] Example 1 Preparation of 3-peroxyl-3-butyl isobenzofuranone

[0081] The crude butylphthalide 50 g was dissolved in methyl tert-butyl ether and subjected to continuous oxygen bubbling at 60°C. After stirring for 48 h, the reaction solution was subjected to silica gel column chromatography to obtain 20 g of 3-peroxyl-3-butyl isobenzofuranone impurities.

[0082] The structure determination data are as follows:

[0083] 1) UV-Vis absorption spectrum (UV-Vis)

[0084] The results determined by UV-visible absorption spectrometer are shown in Table 1 below.

[0085] Table 1: 3-Peroxyl-3-butyl isobenzofuranone UV-visible light analysis data table

[0086]

[0087]

[0088] 2) Infrared

[0089] The results determined by infrared spectrometer are shown in Table 2 below.

[0090] Table 2: 3-Peroxyl-3-butyl isobenzofuranone infrared spectrum analysis data table

[0091] Observed absorption peak cm -1 ]] Vibration type Group Intensity 3271.27 V O-H ]]> Peroxy alcohol Medium 2956.87,2856.58 V C-H ]]> Methyl Medium 2931.80 V C-H ]]> Methylene Medium 1772.58 V C＝O ]]> Lactone group Strong 1616.35,1606.70 Benzene ring skeleton vibration Benzene ring Medium

[0092] 3) High resolution mass spectrometry (ESI-MS)

[0093] Molecular formula C 12 H 14 O4, the theoretical monoisotopic molecular weight [M-H] - is 221.0819. The measured sample molecular ion peak ESI-MS (m / z): 221.0806 [M-H] - . At the same time, the positive ion detection mode mainly produces peroxyl positive ion fragments [M-OOH] + is 189.0924.

[0094] 4) Nuclear magnetic resonance spectrum (NMR)

[0095] Instrument model: Bruker 400MHZ

[0096] Solvent: deuterated dichloromethane (CDCl3)

[0097] Detection according to: Chinese Pharmacopoeia 2020 edition four general rules 0441 nuclear magnetic resonance spectroscopy

[0098] Test items: 1 H, 13 C, 1 H- 1 H COSY, HSQC and HMBC

[0099] Determination results and analysis

[0100]

[0101] Nuclear magnetic resonance 1 H-NMR, COSY spectrum data are shown in Table 3.

[0102] Table 3 NMR 1 H-NMR, COSY spectrum data list

[0103]

[0104] Example 2 Preparation method of butylphthalide with low content of 3-peroxy-3-butyl isobenzofuranone impurity of the present application (laboratory scale)

[0105] A solution of 1 kg of o-carboxybenzaldehyde in 10 L of tetrahydrofuran was added dropwise to a solution of 10 L of n-butylmagnesium bromide with a concentration of 2 M in tetrahydrofuran at 15 °C, and the reaction was allowed to proceed for 2 hours. Then, 30 L of a 20% aqueous solution of ammonium chloride was added dropwise, followed by the addition of 10% dilute salt solution until the pH reached 1. The organic phase was separated and concentrated to obtain 1.2 kg of butylphthalide crude product with a yield of 94% and a purity of 96.586%.

[0106] The butylphthalide crude product was dissolved in 10 L of methyl tert-butyl ether, and a previously prepared sodium hydroxide methanol aqueous solution (10 L of purified water, 1 L of methanol, and 1 kg of sodium hydroxide) was added. The mixture was heated to 50 °C and stirred for 0.5 h, and then the organic phase was separated and concentrated to obtain 0.96 kg of qualified butylphthalide product with a purity of 99.970% and a yield of 80%. The product was stored in a nitrogen-filled container, and the oxygen content was less than 1%.

[0107] Column: Agilent Phenyl-Hexyl 4.6 x 250 mm, 5 μm;

[0108] Mobile phase A: 0.07% acetic acid aqueous solution;

[0109] Mobile phase B: acetonitrile-methanol (1:1);

[0110] Detection wavelength: 227 nm; flow rate: 1.0 ml / min; column temperature: 30 °C; injection volume: 20 μl;

[0111] Dilution solvent: 30% acetonitrile;

[0112] Elution gradient:

[0113]

[0114] The impurities before and after the treatment of butylphthalide crude product by the above method of the present application are shown in Tables 4, 5, and Figure 2 .

[0115] Before the treatment, the peroxide impurity content was about 3%, as shown in Table 4 and Figure 2 A.

[0116] Table 4

[0117]

[0118]

[0119] After treatment, the peroxide impurity content is less than 0.1%, see Table 5 and Figure 2 B.

[0120] Table 5

[0121]

[0122] Example 3 Preparation of an industrial scale of butenafine composition

[0123] A solution of 20 kg of o-carboxybenzaldehyde in 200 L of tetrahydrofuran was added dropwise at 15°C to a solution of 200 L of n-butyl magnesium bromide in tetrahydrofuran at a concentration of 2 M, the reaction was carried out for 2 hours, 600 L of an aqueous solution of ammonium chloride at a concentration of 20% was added dropwise slowly, controlling the internal temperature to be less than 45°C, after the dropwise addition was completed, concentrated hydrochloric acid was used to adjust the pH to pH = 1, the organic phase was separated and concentrated to obtain 24.5 kg of butenafine crude product, the yield was 96.8%, the purity was 96.980%.

[0124] The butenafine crude product was dissolved in 200 L of methyl tert-butyl ether, a sodium hydroxide methanol aqueous solution (200 L of purified water, 20 L of methanol and 20 kg of sodium hydroxide) prepared in advance was added, the temperature was raised to 50 degrees and stirred for 0.5 h, then the organic phase was separated and concentrated to dryness to obtain 20.2 kg of qualified butenafine finished product, the purity was 99.970%, the yield was 82.5%, the finished product was stored in a sealed nitrogen atmosphere, the oxygen content was less than 1%. The detection method is the same as that of Example 2.

[0125] The impurities of the butenafine crude product are shown in Table 6 and Figure 9 A, the impurities of the butenafine finished product are shown in Table 7 and Figure 9 B.

[0126] Table 6

[0127]

[0128]

[0129] Table 7

[0130]

[0131] The method of the present application can be used to control the content of 3-peroxy-3-butyl isobenzofuranone to be less than 0.1%, long-term stability observation, under reasonable storage conditions, the impurity content increases slightly, but the content is not more than 0.15%.

[0132] Example 4 Long term observation test

[0133] The long term observation of the low level of 3-peroxy-3-butyl isobenzofuranone impurity in the butylphthalide obtained after the treatment of Example 3 was carried out. Observation conditions: residual oxygen content < 1% in a closed container, storage at 25 ± 2°C, RH 60 ± 5%, and the results are shown in Table 8.

[0134] Table 8

[0135]

[0136]

[0137] Example 5 Closed storage test

[0138] The butylphthalide product of the present application (prepared from Example 3) was stored in a closed, inert gas atmosphere, and the 3-peroxy-3-butyl isobenzofuranone impurity increased slowly, and was stable for a long time with a residual oxygen content of less than 1%, otherwise the impurity would increase rapidly with the extension of the storage time. This example studied the impurity increase under different storage conditions, and the results are shown in Table 9.

[0139] Table 9

[0140]

[0141] Example 6

[0142] Chromatographic column: Agilent ZORBAX plus Pheny-Hexyl (4.6 mm x 250 mm, 5 um);

[0143] Mobile phase A: 0.07% acetic acid in water;

[0144] Mobile phase B: acetonitrile-methanol (1:1);

[0145] Detection wavelength: 227 nm; flow rate: 1.0 ml / min, column temperature: 30°C; injection volume: 20 μl;

[0146] Elution gradient:

[0147]

[0148] Sample: Take butylphthalide, phthalic acid, 2-hydroxymethylbenzoic acid, o-carboxybenzaldehyde, phthalide, 2-pentanoylbenzoic acid, propylphthalide, 3- peroxy-3-butylisobenzofuranone, sec-butylphthalide, isobutylphthalide, butenylphthalide, pentylphthalide, and use 30% acetonitrile to prepare a mixed solution containing butylphthalide 0.7 mg / ml, phthalic acid, 2-hydroxymethylbenzoic acid, o- carboxybenzaldehyde, phthalide, 2-pentanoylbenzoic acid, propylphthalide, 3- peroxy-3-butylisobenzofuranone, sec-butylphthalide, isobutylphthalide, butenylphthalide, and pentylphthalide 7 μg / ml, as the sample.

[0149] Separation: The separation degree of each impurity peak is greater than 1.5.

[0150] Separation results are shown in Table 10 and Figure 3 .

[0151] Table 10

[0152]

[0153]

[0154] Example 7

[0155] Chromatographic column: Agilent phenyl-hexyl 4.6x250mm, 5μm

[0156] Mobile phase A: 0.07% acetic acid aqueous solution

[0157] Mobile phase B: acetonitrile-methanol (1:1)

[0158] Detection wavelength: 227nm; flow rate: 1.0ml / min; column temperature: 30℃; injection volume: 20μl

[0159] Dilution solvent: 30% acetonitrile

[0160] Elution gradient:

[0161]

[0162] Sample: Take butylphthalide, phthalic acid, 2-hydroxymethylbenzoic acid, o-carboxybenzaldehyde, phthalide, 2-pentanoylbenzoic acid, propylphthalide, 3- peroxy-3-butylisobenzofuranone, sec-butylphthalide, isobutylphthalide, butenylphthalide, pentylphthalide, and use 30% acetonitrile to prepare a mixed solution containing butylphthalide 0.7 mg / ml, phthalic acid, 2-hydroxymethylbenzoic acid, o- carboxybenzaldehyde, phthalide, 2-pentanoylbenzoic acid, propylphthalide, 3- peroxy-3-butylisobenzofuranone, sec-butylphthalide, isobutylphthalide, butenylphthalide, and pentylphthalide 7 μg / ml, as the sample.

[0163] Resolution: The resolution of each impurity peak is greater than 1.5.

[0164] The separation results are shown in Table 11 and Figure 4 .

[0165] Table 11

[0166]

[0167] Example 8

[0168] Column: Agilent phenyl-hexyl 4.6 × 250 mm, 5 μm

[0169] Mobile phase A: 0.07% aqueous acetic acid

[0170] Mobile phase B: Acetonitrile-methanol (1:1)

[0171] Detection wavelength: 227 nm; Flow rate: 1.0 ml / min; Column temperature: 30 °C; Injection volume: 20 μl

[0172] Diluent: 30% acetonitrile

[0173] Elution gradient:

[0174]

[0175] Sample: Weigh appropriate amounts of butylphthalide, phthalic acid, 2-hydroxymethylbenzoic acid, o-carboxybenzaldehyde, phthalide, 2-pentanoylbenzoic acid, propenylphthalide, 3-peroxy-3-butylisobenzofuranone, sec-butylphthalide, isobutylphthalide, butenylphthalide, and pentaphthalide. Prepare a mixed solution containing 0.7 mg / ml butylphthalide, 7 μg / ml each of phthalic acid, 2-hydroxymethylbenzoic acid, o-carboxybenzaldehyde, phthalide, 2-pentanoylbenzoic acid, propenylphthalide, 3-peroxy-3-butylisobenzofuranone, sec-butylphthalide, isobutylphthalide, butenylphthalide, and pentaphthalide using 30% acetonitrile.

[0176] Resolution: The resolution of each impurity peak is greater than 1.5.

[0177] The separation results are shown in Table 12 and Figure 5 .

[0178] Table 12

[0179]

[0180] Comparative Example 1 Existing quality control methods for the impurity 3-peroxy-3-butylisobenzofuranone in butylphthalide

[0181] Referring to the national drug standard for butylphthalide WS1-(X-124)-2005Z, HPLC was used to separate and detect butylphthalide, 3-peroxy-3-butylisobenzofuranone, and other related substances.

[0182] Column: C18, 4.6 mm × 250 mm, 5 μm;

[0183] Mobile phase: methanol-water (65:35);

[0184] Detection wavelength: 280 nm, flow rate: 1.0 ml / min, column temperature: 30 ℃, injection volume: 20 μl.

[0185] Diluent: Methanol.

[0186] Sample: Weigh appropriate amounts of butylphthalide, phthalic acid, 2-hydroxymethylbenzoic acid, phthalide, 2-butyrylbenzoic acid, 3-peroxy-3-butylisobenzofuranone, propenphthalide, sec-butylphthalide, and pentaphthalide, dissolve and dilute with methanol to prepare a mixed solution containing approximately 0.1 mg / ml of butylphthalide and approximately 0.05 mg / ml of phthalic acid, 2-hydroxymethylbenzoic acid, phthalide, 2-butyrylbenzoic acid, 3-peroxy-3-butylisobenzofuranone, propenphthalide, sec-butylphthalide, and pentaphthalide as the sample.

[0187] The separation results are shown in Table 13 below. Figure 1 As shown.

[0188] Table 13

[0189]

[0190] The results showed that the peaks of 3-peroxy-3-butylisobenzofuranone and propylphthalide were poorly separated, indicating that the existing national drug standard WS1-(X-124)-2005Z method cannot effectively separate the impurity 3-peroxy-3-butylisobenzofuranone in propylphthalide from other substances.

[0191] Test Example 1 Activity assay of 3-peroxy-3-butylisobenzofuranone in neurons and microglia

[0192] 1. Experimental Methods

[0193] 1.1 Nerve Cell Culture

[0194] (1) Cell culture of neural cell lines:

[0195] Mouse brain neuroblastoma cell line (Neuro-2a) was cultured in MEM medium containing 10% FBS and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator, and the medium was changed every day. When the cells grew to 70%-80% of the bottom area, the cells were gently blown off to make a cell suspension, and the cells were subcultured at 1:2.

[0196] (2) Extraction and culture of primary mouse microglial cells

[0197] 1) Coat T75 culture flask with PLL 12h in advance, place at room temperature overnight, wash T75 culture flask with PBS, discard PBS, and ultraviolet irradiate in a clean bench for 1h;

[0198] 2) Put ice box and sterilized surgical instruments (microfine forceps, surgical scissors, surgical forceps) into the clean bench, and ultraviolet irradiate for 0.5h;

[0199] 3) Alcohol disinfect 1-2d old suckling mice, decapitate, open the skull of the suckling mouse, take out the brain tissue, and put it into pre-cooled HBSS buffer. Remove the striatum, globus pallidus, and corpus callosum with fine forceps, and leave the cortical tissue;

[0200] 4) Transfer the brain cortex tissue to a 15mL centrifuge tube with a pipette, add 0.25% trypsin for digestion, add serum-containing DMEM / F12 medium to terminate digestion, and blow evenly with a 10mL pipette;

[0201] 5) Centrifuge the completely digested brain tissue at 1500r / s for 10min, discard the supernatant, resuspend with 4mL serum-containing DMEM / F12 medium, blow evenly with a 10mL pipette, pass the cell suspension through a cell sieve with a pore size of 70μm to remove undigested tissue fragments, and transfer the cell suspension to a PLL-coated T75 culture flask;

[0202] 6) Replace the fresh serum-containing DMEM / F12 medium every two days to remove unattached cells;

[0203] 7) After two medium changes, culture in a cell culture incubator for 6 days, and observe that the microglial cells adhere to the bottom of the astrocytes;

[0204] 8) After the microglial cells mature, transfer them to a centrifuge tube, centrifuge at 1500r / s for 10min, discard the supernatant, resuspend with serum-containing DMEM / F12 medium, and plate.

[0205] 1.2 CCK-8 assay of cell viability

[0206] The detection method for evaluating cell viability in this experiment is CCK-8 (Cell Counting Kit-8) method, which is a rapid and sensitive reagent widely used in cell proliferation and cytotoxicity based on WST-8 (Chinese name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazole monosodium salt). The detection principle of CCK-8 is that WST-8 can be reduced to orange-yellow water-soluble tetrazole in the presence of electron coupling reagent by dehydrogenase in cell mitochondria. The greater the cytotoxicity, the lighter the color, and within a certain range, the amount of water-soluble tetrazole generated, i.e. the color depth, is linearly related to the number of living cells.

[0207] When the density of Neuro-2a cells inoculated in the 24-well plate reached 70%, the Neuro-2a cells were treated with different concentrations of 3-peroxy-3-butyl isobenzofuranone for 48 h, and the cell viability of each group was determined by CCK-8 method. The specific method is as follows: the supernatant is aspirated, then 100 μL of MEM medium (without serum) containing 10 μL of CCK-8 reagent is added to each cell, and it is placed in a 37°C, 5% CO2 cell incubator for about 1 h, 90 μL of supernatant is aspirated and placed in a 96-well cell culture plate, and the absorbance value (OD 450 ) at 450 nm wavelength is measured by a multifunctional enzyme label instrument. The absorbance value of all sample wells is subtracted from the absorbance value of the blank well, which is the corrected absorbance value, then the average value of the corrected absorbance value of the Control group cells is calculated, and the cell viability of the sample well is calculated according to the formula: corrected absorbance value of sample well / corrected average absorbance value of Control group cells x 100%.

[0208] 1.3 Establishment of lipopolysaccharide (LPS) induced primary mouse microglial cell inflammation model

[0209] After the dotting, the original culture medium was aspirated, and serum-free medium containing 1 μM, 5 μM and 10 μM of the compound was added to pretreat the cells for 3 h, and the control group was given the same volume of DMSO solution. Subsequently, LPS (final concentration of 100 ng / mL) was added to treat the cells for 2 h. The secretion of TNF-α in the cell supernatant was detected by ELISA kit, and the degree of cell damage was detected by CCK-8.

[0210] 1.4 Data analysis

[0211] All data in this study are expressed as Mean ± SD, and statistical analysis was performed using GraphPad Prism 7.0. One-way ANOVA was used for fitting, and Bonferroni test was used for comparison. P < 0.05 indicates a statistically significant difference between groups, P < 0.01 indicates a highly statistically significant difference between groups, and P < 0.001 indicates a very highly statistically significant difference between groups.

[0212] 2. Experimental results

[0213] 2.1 Toxicity of 3-peroxy-3-butylisobenzofuranone on Neuro-2a cells

[0214] We used different concentrations of 3-peroxy-3-butylisobenzofuranone to treat Neuro-2a cells for 48 h, and CCK-8 was used to determine cell viability to investigate the effect of 3-peroxy-3-butylisobenzofuranone on Neuro-2a cell viability. The results showed that, compared with the Control group, 3-peroxy-3-butylisobenzofuranone (10 μM, 20 μM and 50 μM) had a significant neurotoxic effect, as shown in Figure 6 .

[0215] 2.2 Effect of 3-peroxy-3-butylisobenzofuranone on the axonal regeneration model of Neuro-2a cells in vitro

[0216] Neuro-2a cells were seeded in culture dishes and induced to regenerate axons in MEM medium containing 0.1% FBS; then 5 μM, 10 μM, 20 μM and 50 μM of 3-peroxy-3-butylisobenzofuranone were added to the Neuro-2a cells for 48 h, and the effect of 3-peroxy-3-butylisobenzofuranone on axonal regeneration of the nerve cells was investigated by measuring the length of the axons and the number of cells with axons. The results are shown in Figure 2 , compared with the solvent control group, 3-peroxy-3-butylisobenzofuranone (5 μM, 10 μM, 20 μM and 50 μM) significantly inhibited the length of the axons and the number of cells with axons, as shown in Figure 7 .

[0217] 2.3 Effect of 3-peroxy-3-butylisobenzofuranone on inflammation mediated by mouse primary microglial cells

[0218] To investigate the effect of 3-peroxy-3-butyl isobenzofuranone on the inflammatory response of LPS-induced mouse primary microglial cells, we first examined the cytotoxicity of 3-peroxy-3-butyl isobenzofuranone on mouse primary microglial cells by CCK-8 assay. The experimental results showed that, compared with the control group, 3-peroxy-3-butyl isobenzofuranone (1 μM, 5 μM and 10 μM) incubation had no cytotoxic effect on mouse primary microglial cells (P > 0.05) (Figure 1), indicating that 1-10 μM of 3-peroxy-3-butyl isobenzofuranone had no cytotoxic effect on mouse primary microglial cells. Figure 8

[0219] To evaluate the effect of 3-peroxy-3-butyl isobenzofuranone on the inflammatory response of LPS-induced microglial cells, we incubated microglial cells with 3-peroxy-3-butyl isobenzofuranone (1 μM, 5 μM and 10 μM) for 3 h, then induced the inflammatory response of microglial cells with LPS, and detected the expression of TNF-α inflammatory factor by ELISA. The ELISA experimental results showed that, compared with the CN group, the secretion of TNF-α inflammatory factor in the supernatant of microglial cells in the LPS modeling group was significantly increased, and compared with the LPS modeling group, 3-peroxy-3-butyl isobenzofuranone (1 μM, 5 μM and 10 μM) had no significant effect on the secretion of TNF-α in the supernatant (P > 0.05) (Figure 2). Figure 8

[0220] Test Example 2 Therapeutic effect of butylphthalide composition on rat transient middle cerebral artery occlusion (tMCAO) model

[0221] In this study, the therapeutic effect of butylphthalide composition with different peroxide impurity contents on the model rats was investigated by using the rat transient middle cerebral artery occlusion (tMCAO) model.

[0222] Methods:

[0223] The rat tMCAO model was based on the intraluminal line method of Longa et al. The main operation steps are as follows:

[0224] The rats were placed in an anesthesia induction box and given 3%-4% isoflurane for induction of anesthesia. After the rats were anesthetized, they were fixed in a supine position on the operation table. The rats were given a breathing mask and 1.5%-2.5% isoflurane to maintain an anesthetized state.

[0225] The rat's neck skin was wiped with a 75% alcohol cotton ball, and the skin was incised in the middle of the neck. The muscles and soft tissues were separated with a curved forceps, the right common carotid artery was exposed, and a nylon thread was placed for use.

[0226] ​​The soft tissue was bluntly separated from the common carotid artery towards the rat head side, and the external carotid artery and the internal carotid artery were exposed. It should be noted that the vagus nerve should be avoided from being damaged. The common carotid artery and the internal carotid artery were clamped with an artery clamp, and the external carotid artery was ligated at the distal and proximal ends, respectively. The blood vessel was cut with an ophthalmic scissors between the two ligation points.

[0227] A small oblique incision was made on the free end of the external carotid artery, and a silicone thread plug was inserted into the external carotid artery. Then, the external carotid artery end was pulled into a straight line with the internal carotid artery, and the thread plug was smoothly pushed into the internal carotid artery. The internal carotid artery clamp was loosened, and the thread plug was pushed further into the intracranial internal carotid artery. The length of the thread plug was adjusted, and the right brain blood flow was detected by a laser speckle blood flow imaging system. When the right brain blood flow decreased to 25% or less of that before modeling, the modeling was considered successful.

[0228] The internal carotid artery was ligated to fix the thread plug and prevent bleeding. The muscle and skin were sutured, and the rat was placed on a 37°C warming pad to maintain body temperature.

[0229] After 2 hours of ischemia, the rat was anesthetized with isoflurane, and the thread plug was slowly pulled out. The external carotid artery end was ligated to achieve reperfusion of the right brain blood flow. When the right brain blood flow increased to 75% or more of that before modeling, the reperfusion was considered successful.

[0230] The rats in the sham control group were operated on in the same way as the modeling groups, but the thread plug was pulled out immediately after being inserted. The test groups were divided as follows:

[0231] The animals were randomly divided into a model control group, a butylphthalide composition containing peroxide impurities (peroxide impurities were 3.0% and 0.15%, respectively), and a sham control group (normal control group) in which the thread plug was pulled out immediately after being inserted. After 4 hours, 24 hours, and 48 hours of ischemia, the tail vein of each group was given the corresponding test substance. The dose of the drug group was butylphthalide 5 mg / kg, and the sham control group and the model control group were given the same volume of solvent.

[0232] Table 14 Dose Design Table

[0233]

[0234]

[0235] *Note: NBP of Example 2 was added with corresponding amount of peroxide impurities, so that the impurity content reached 3.0% and 0.15%, respectively.

[0236] a: The number of animals is 10, which is the number of animals taken from each group for brain tissue. The number of animals that died during modeling and drug administration is not included.

[0237] Detection: (1) TTC staining infarct area percentage in brain after 72 hours post-operation, (2) Rotarod test latency before operation, 24 hours, 48 hours, 72 hours post-operation, (3) Longa behavior score 24 hours, 48 hours, 72 hours post-operation.

[0238] Results:

[0239] (1) Effect on infarct area percentage in brain

[0240] The infarct area percentage in brain after TTC staining in rats is shown in Table 15.

[0241] As shown in Table 15, compared with the sham control group, the infarct area in the model control group increased, with significant statistical significance (p<0.01); compared with the model control group, the infarct area in the 3.0% and 0.15% impurity groups decreased, with significant statistical significance (p<0.05, p<0.01); the 0.15% impurity group was superior to the 3.0% impurity group. There was no statistical significance between the two groups (p>0.05).

[0242] Table 15 Effect of the Butylphthalide Composition on Infarct Area Percentage in Brain n=10

[0243]

[0244] vs normal control group: ##p<0.01; vs model control group: *p<0.05, **p<0.01.

[0245] a: variance is not uniform, Dunnett T3 test.

[0246] (2) Effect on Rotarod Test Latency

[0247] The latency of each animal in the Rotarod test is shown in Table 16.

[0248] The longer the latency, the smaller the damage to the rat's neural function. As shown in Table 16, before operation, there was no difference in latency among the animals in each group (p>0.05); compared with the sham control group, the latency of the model control group decreased at 24h, 48h, 72h post-operation, with significant statistical significance (p<0.05, p<0.01); compared with the model control group, the latency of the 0.15% impurity group increased at 24h, 48h, 72h post-operation, with significant statistical significance (p<0.05), and the latency of the 3.0% group increased to a certain extent at each time point, but there was no statistical significance (p>0.05). There was no statistical significance between the 3.0% and 0.15% groups (p>0.05).

[0249] Table 16 Effect of the Butylphthalide Composition on Rotarod Test Latency n=10

[0250]

[0251] vs normal control group: #p<0.05, ##p<0.01; vs model control group: *p<0.05, **p<0.01.

[0252] a: homogeneity of variance, LSD test; b: heterogeneity of variance, independent t test.

[0253] (3) Effect on Longa behavioral score

[0254] The results of Longa behavioral score are shown in Table 17.

[0255] The higher the Longa score, the more severe the neurological impairment of the rats. As shown in Table 17, compared with the sham control group, the behavioral scores of the model control group were increased at 24 h, 48 h and 72 h after surgery, with significant statistical significance (p<0.01); compared with the model control group, the behavioral scores of the 0.15% group were decreased at 48 h and 72 h after surgery, with significant statistical significance (p<0.05), and the behavioral scores of the 0.15% group at 24 h, the 3.0% group at 24 h, 48 h and 72 h were decreased to a certain extent, but without statistical significance (p>0.05). There was no statistical significance between the 3% and 0.15% groups (p>0.05).

[0256] Table 17 Effect of the butylphthalide composition on behavioral score n=10

[0257]

[0258]

[0259] vs normal control group: ##p<0.01; vs model control group: *p<0.05, **p<0.01.

[0260] a: homogeneity of variance, LSD test; b: heterogeneity of variance, Dunnett T3 test; c: constant, single-sample t test.

[0261] Conclusion: In summary, both the 3.0% impurity group and the 0.15% impurity group can significantly reduce the cerebral infarction area of the tMCAO model rats, but the effect of the 0.15% impurity group is more significant. Compared with the model control group, the 0.15% impurity group has a significant effect on increasing the latency of the rotarod test of the model animals and reducing the behavioral score, while the 3.0% impurity group increases the latency of the rotarod test of the model animals and reduces the behavioral score to a certain extent, but without statistical significance.

[0262] The above discloses the basic technology of the present application, if based on the technology of the present application, the proportion of the butylphthalide composition is changed, additional gradient elution or isocratic elution is increased, and there is no substantial benefit change, still belongs to the scope of the claims of the present application.

Claims

1. A method for preparing the butyrphthalide impurity compound shown in the following formula, the method comprising treating butyrphthalide with an oxidizing agent; in, The oxidant is selected from oxygen, hydrogen peroxide, m-chloroperbenzoic acid, and peracetic acid; the temperature for treating butylphthalide with the oxidant is 45–75°C.

2. A method for preparing a butylphthalide pharmaceutical composition, the method comprising the following steps: The crude butylphthalide was dissolved in an organic solvent, then washed with an alkali-alcohol solution formed by an aqueous solution of alcohol and alkali. The organic phase was separated, filtered, concentrated, and the finished product was stored in a sealed container. The washing was carried out at a temperature of 20–70°C. in, The pharmaceutical composition comprises butylphthalide and butylphthalide impurity compound prepared by the preparation method of claim 1, wherein the content of the butylphthalide impurity compound in the pharmaceutical composition does not exceed 0.1 wt.%. The organic solvent is selected from tetrahydrofuran and methyl tert-butyl ether; The alcohol in the alkali-alcohol solution is selected from methanol, ethanol, isopropanol, and two or more mixed solvents thereof; The alkali in the alkali-alcohol solution is selected from potassium hydroxide, sodium hydroxide, sodium carbonate, and sodium bicarbonate; The mass concentration of alkali in the alkali-alcohol solution is 2% to 20%. The volume ratio between the alcohol and the aqueous solution of the alkali in the alkali-alcohol solution is 0.1:10 to 5:

10.

3. The preparation method according to claim 2, wherein, In the pharmaceutical composition, the content of the butylphthalide impurity compound does not exceed 0.02 wt.%.

4. The preparation method according to claim 2, wherein, The washing is carried out at a temperature of 40–50°C.

5. The preparation method according to claim 2, wherein, The organic solvent is methyl tert-butyl ether.

6. The preparation method according to claim 2, wherein, The alcohol in the alkaline-alcohol solution is selected from methanol and ethanol; The alkali in the alkali-alcohol solution is selected from potassium hydroxide and sodium bicarbonate; The alkali-alcohol solution has a alkali concentration of 10% by mass. The volume ratio between the alcohol and the aqueous solution of the alkali in the alkali-alcohol solution is 1:10 to 3:

10.

7. A chromatographic separation method for the butylphthalide impurity compound prepared according to claim 1 and other impurities, wherein, The conditions for the chromatographic separation method are as follows: Column: Agilent ZORBAX plus Pheny-Hexyl, 4.6mm × 250mm, 5µm; Mobile phase A: 0.07 v / v % aqueous acetic acid; Mobile phase B: Acetonitrile-methanol in a volume ratio of 1:1; The detection wavelength was 227 nm; the flow rate was 1.0 ml / min; the column temperature was 30 °C; and the injection volume was 20 μl. The elution gradient is: or or The other impurities include phthalic acid, 2-hydroxymethylbenzoic acid, o-carboxybenzaldehyde, phthalide, 2-pentanoylbenzoic acid, propenylphthalide, sec-butylphthalide, isobutylphthalide, butenylphthalide, and pentophthalide.

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