WO2020007031A1 - 一种苯酞类衍生物及其制备方法和应用 - Google Patents

一种苯酞类衍生物及其制备方法和应用 Download PDF

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WO2020007031A1
WO2020007031A1 PCT/CN2019/072178 CN2019072178W WO2020007031A1 WO 2020007031 A1 WO2020007031 A1 WO 2020007031A1 CN 2019072178 W CN2019072178 W CN 2019072178W WO 2020007031 A1 WO2020007031 A1 WO 2020007031A1
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compound
butyl
hydroxy
formula
cyclopropanyl
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PCT/CN2019/072178
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French (fr)
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柳军玺
张亚明
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中国科学院兰州化学物理研究所
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Priority to US17/257,736 priority Critical patent/US11919857B2/en
Priority to EP19830370.3A priority patent/EP3819292B1/en
Publication of WO2020007031A1 publication Critical patent/WO2020007031A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/52—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring condensed with a ring other than six-membered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/44—Iso-indoles; Hydrogenated iso-indoles
    • C07D209/48—Iso-indoles; Hydrogenated iso-indoles with oxygen atoms in positions 1 and 3, e.g. phthalimide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers

Definitions

  • the invention belongs to the field of organic synthetic chemistry, and relates to a phthaloline derivative and a preparation method and application thereof.
  • the plants belonging to the genus Umbelliferae and the genus Astragalus contain phthalophthalic compounds, such as medicinal plants such as angelica and chuanxiong, which are the main components of plant volatile oils. It is rich in content, and has many types of compound structures. Such compounds are generally chemically unstable, and they are easily oxidized or degraded when exposed to light, heat, and enzymes in the environment. However, these compounds generally have good pharmacological activities, such as anti-oxidation, anti-platelet aggregation, promotion of microcirculation, analgesic and anti-inflammatory, central nervous system protection and anti-tumor activities.
  • Ligustilide is the main pharmacologically active ingredient in the volatile oils of umbelliferae plants such as Angelica sinensis and Ligusticum chuanxiong. Its content is as high as about 1%. It has significant cardio-cerebral vascular pharmacological activity. Pharmacological activity has been and is undergoing a lot of research work, and related research results on pharmacological activity have been published, such as ligustilide protects the brain nerves, improves microcirculation, relaxes blood vessels, inhibits vascular smooth muscle cell proliferation, antidepressant, anti-Alz Heimer's disease, analgesic and anti-inflammatory and other important pharmacological effects.
  • Ligustilide can significantly reduce cerebral infarction volume caused by cerebral ischemia-reperfusion, improve cerebral nerve function, and reduce damage to rat cortical neurons and hippocampal neurons.
  • HY Peng et al. Respectively studied transient and persistent cerebral ischemia-reperfusion models, and the results showed that ligustilide reduced the cerebral ischemia in a dose-dependent manner.
  • the content of malondialdehyde increases the activity of glutathione peroxidase and superoxide dismutase, enhances the expression of Bcl-2 and weakens the immune activities of Bax and Caspase-3.
  • the death mechanism plays a neuroprotective role in the injury caused by transient and persistent cerebral ischemia-reperfusion.
  • the study of cognitive impairment, biochemical changes and histopathological characteristics caused by chronic cerebral ischemia-reperfusion has also shown that the protective effect of ligustilide on cognitive deficits and brain damage is through antioxidant and increased Choline-like functional activity achieved (X Kuang, Y Yao, JRDu, YXLiu, CY Wang, ZM Qian, Neuroprotective role of Z-ligustilide against forebrain ischemic injury in ICR mice, Brain Research, 2006, 1102 (1): 145- 153; Haiyan Peng, Junrong Du, Guangyi Zhang, Xi Kuang, Yanxin Liu, Zhongming Qian, and Chenyuan Wang, Neuroprotective effect of Z-Ligustilide against permanent focal ischemic damage in rats, Biol Pharm Bull, 2007, 30 (2): 309 -312.).
  • Ligustilide by increasing cellular antioxidant capacity and inhibition of mitochondrial apoptosis pathway in PC12 cells under H 2 O 2 from damage neuroprotective effect (Yan Yu, Junrong Du, Chenyuan Wang, Zhongming Qian, Protection against hydrogen peroxide-induced injury by Z-ligustilide in PC12cells, Exp Brain Res, 2008, 184 (3): 307-312.).
  • HY Qi et al. Further investigated the biphasic toxicant excitatory regulation effect of ligustilide in hypoxic and hypoglycemic PC12 cells.
  • ligustilide can protect PC12 cells under low and high concentrations of hypoxia and hypoglycemic environment (Hongyi Qi, Yifan Han, Jianhui Rong, Potential roles of PI3K / Akt and Nrf2-Keap1pathways in regulating hormesis of Z-ligustilide in PC12cells against oxygen and glucose deprivation, Neuropharmacology, 2012, 62 (4): 1659-1670.).
  • Ligustilide significantly improved the neurological function and reduced the infarct volume of the ischemia-reperfusion rats, while significantly reducing the protein expression of the inflammatory factor NF- ⁇ B.
  • it can inhibit TNF- ⁇ , NO, IL-1 ⁇ , MCP-1 and other inflammatory mediators produced by LPS-induced glial cells, and it can mediate symptoms such as cognitive defects and neuropathology caused by ⁇ -amyloid peptides.
  • the improvement of the NF- ⁇ B pathway means that ligustilide can play an anti-inflammatory effect by inhibiting the NF- ⁇ B pathway.
  • ligustilide can also inhibit the inflammatory response of RAW 264.7 induced by LPS by inhibiting ROS production and down-regulating MAPK, NF- ⁇ B, and AP-1 signaling pathways.
  • ligustilide can also play a neuroprotective role through anti-inflammatory mechanisms, which is beneficial for the treatment of neuroinflammatory diseases (Yuwen Su, Wenfei Chiou, Shiouhuei Chao, Menghwan Lee, Chienchih Chen, Yingchieh Tsai, Ligustilide prevents LPS- induced iNOS expression in RAW 264.7 macrophages by preventing ROS production and down-regulating the MAPK, NF- ⁇ B and AP-1signaling pathways, International Immunopharmacology, 2011, 11 (9): 1166-1172.).
  • ligustilide inhibits ⁇ -amyloid-induced neurotoxicity through p38 and PI3K / Akt signaling pathways, and supports ligustilide to have Alzheimer's disease (AD) treatment effect.
  • AD Alzheimer's disease
  • Li Yang, Ji Li Protection against ⁇ -amyloid-induced neuratoxicity by naturally occurring Z-ligustilide through the concurrent regulation of p38 and PI3-K / Akt pathways, Neurochemistry, International, 2016, 100: 44-51.
  • Relevant documents at home and abroad show that ligustilide has significant pharmacological activity.
  • Phenylphthalein compounds are classified into benzene ring type such as n-butenylphthalide, dihydrobenzene ring type such as ligustilide, and tetrahydrobenzene ring type phthalide compound such as yangchuan lactone according to the degree of hydrogenation reduction of six-membered ring. H.
  • the side chain and the benzene ring have different substitution and oxidation degrees, resulting in a series of phthalophthalic compounds with various structures.
  • Various dimers and trimers can also be formed.
  • the ligustilide is a highly unsaturated conjugated system with three olefinic bonds and one carbonyl double bond, but it does not form a stable conjugated aromatic ring. It has more reactive sites, strong reactivity, and relatively free energy. Low, it is easy for Diels-Alder reaction of 1,2,1,4 and 1,6 to form complex dimer or trimer compounds.
  • Butylphthalide is China's first innovative drug with independent intellectual property rights. It was originally extracted and isolated from cress seed and then chemically synthesized. Butylphthalein has a unique dual mechanism of action, which can reconstruct the microcirculation and increase ischemic deperfusion, thereby protecting the integrity of the blood vessel structure and restoring the diameter of the blood vessel. Increase blood flow in the ischemic area and the number of surrounding microvessels, while protecting mitochondria and reducing cell death, thereby protecting the integrity of mitochondrial structure, increasing the activity of mitochondrial complex enzyme IV, increasing the activity of mitochondrial ATPase, and maintaining the stability of mitochondrial membrane, Double sniper to fight stroke. Studies have shown that compared with ligustilide's neuroprotective effects and toxic side effects, butylphthalide is inferior to ligustilide in pharmacological characteristics due to its significant chemical structural characteristics.
  • ligustilide has stronger pharmacological activity than butylphthalide.
  • butylphthalide has pharmaceutical characteristics (stable chemical structure). Approved for marketing, but due to its active and toxic side effects, clinicians have put forward a strong demand for butylphthalide to further optimize and improve its activity, which cannot meet the clinical requirements for the treatment of stroke. Therefore, it is necessary to artificially intervene in the structure of ligustilide's unique chemical structure, and optimize and screen high-efficiency and stable cardio-cerebral vascular therapeutic drugs based on these compounds.
  • the object of the present invention is to provide a phthaloline derivative, a preparation method and application thereof.
  • the remarkable feature of the phthalophthalic derivative is that its original lactone structure fragment is replaced by a lactam group, and the C-3 position is replaced by a hydroxyl group.
  • the present invention provides a phthalophthalic derivative of Formula I or Formula II, an optical isomer or a pharmaceutically acceptable salt thereof, or a mixture thereof (for example, a racemic mixture)
  • the phthalophthalic derivatives are N-cyclopropanyl-3-n-butyl-3-S-hydroxy-galactolactam, N-cyclopropanyl-3-n-butyl -3-R-Hydroxy-fluorenyllactam, N-cyclopropanyl-3-n-butyl-3-S-hydroxy-phthalolactam or N-cyclopropanyl-3-n-butyl-3-R -Hydroxy-phthalolactam.
  • the present invention provides a method for preparing a phthaloline derivative (for example, a phthaloline derivative of Formula I or Formula II), which comprises reacting a phthaloline compound with cyclopropylamine in an organic solvent, wherein benzene The original lactone structural fragment in the phthalate compound is replaced by a lactam group, and the C3 position is replaced by a hydroxyl group to obtain a phthalophthalic derivative of Formula I or Formula II.
  • a phthaloline derivative for example, a phthaloline derivative of Formula I or Formula II
  • the method of the invention comprises one, two, three or four of the following features:
  • the phthalophthalic compound is a n-butenyl-substituted benzene ring type, dihydrobenzene ring type or tetrahydrobenzene ring type phthalophthaline compound at the C3 position, preferably ligustilide or n-butene phthalide, wherein The ligustilide or n-butene phthalide is in the form of an isolated compound or in the form of a mixture of angelica or genus plant extracts, and more preferably the plant extract is angelica volatile oil extract or chuanxiong volatile oil extract Substances or mixtures thereof;
  • reaction is performed at -20 ° C to 60 ° C, preferably under stirring;
  • the organic solvent is a non-polar organic solvent, preferably selected from cyclohexane, petroleum ether, tetrahydrofuran, diethyl ether; and / or
  • the method further includes the step of chiral resolution of the enantiomers, preferably by chiral chromatography or chiral recrystallization.
  • the molar ratio of the phthalide compound to cyclopropylamine is 1: 1 to 1.2.
  • the preparation method further includes the steps of recovering the organic solvent under reduced pressure and recrystallization to obtain the target product.
  • the solvent used for the recrystallization is one of petroleum ether, ethyl acetate, acetone, and ether. Or both.
  • the preparation method comprises dissolving a phthalophthalic compound in an organic solvent, controlling the temperature at -20 ° C to 60 ° C, adding a cyclopropylamine reaction solution in which the organic solvent is dissolved, and controlling the temperature at -20 ° C to The reaction was stirred at 60 ° C for 1-24 hours.
  • the organic solvent was recovered under reduced pressure, and the target product was obtained by recrystallization.
  • the present invention also provides a phthalophthalic derivative of Formula I or Formula II, an optical isomer or a pharmaceutically acceptable salt thereof, or a mixture thereof according to the present invention, which is prepared by the method described above.
  • the present invention also provides the use of a phthalophthalic derivative of formula I or formula II, an optical isomer or a pharmaceutically acceptable salt thereof, or a mixture thereof in the preparation of a medicament according to the present invention.
  • a phthalophthalic derivative of formula I or formula II an optical isomer or a pharmaceutically acceptable salt thereof, or a mixture thereof in the preparation of a medicament according to the present invention.
  • cardiovascular and cerebrovascular diseases depression, Alzheimer's disease, (neuro) inflammatory diseases, pain, neuronal cell damage, ischemia-reperfusion injury, cerebral infarction , Cognitive impairment or brain damage.
  • the medicament comprises a mixture of a compound of formula I and a compound of formula II as an active ingredient, wherein the mass percentage content of the compound of formula I in the mixture is 1-99%, preferably 90% or more.
  • the present invention provides a phthalide derivative, which is characterized in that the structure of the derivative is as follows:
  • the method for preparing a phthaloline derivative is as follows: the phthaloline compound is dissolved in an organic solvent, the temperature is controlled at -20 ° C-60 ° C, and the cyclopropylamine reaction solution dissolved in the organic solvent is added, and the temperature is controlled at- The reaction was stirred at 20 ° C-60 ° C for 1-24h, and the organic solvent was recovered under reduced pressure. The target product was obtained by recrystallization.
  • the molar ratio of the phthalide compound to cyclopropylamine is 1: 1 to 1.2.
  • the phthalophthalic compound is one or two or more of ligustilide, n-butenephthalide, and volatile oil extract of medicinal materials containing ligustilide or n-butenephthalide.
  • the medicinal material volatile oil extract is angelica volatile oil extract or chuanxiong volatile oil extract.
  • the organic solvent is a non-polar organic solvent, preferably cyclohexane, petroleum ether, tetrahydrofuran, and the like.
  • the solvent used for the recrystallization is one or two of petroleum ether, ethyl acetate, acetone, and ether.
  • the phthalophthalic derivative is a composition of a compound of formula I and a compound of formula II, and the mass percentage content of the compound of formula I in the composition is 1-100%.
  • the mass percentage of the compound of formula I is 90% or more (for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%).
  • the chemical structures of the phthaloline derivatives synthesized by the chemical structure derivation method are all through mass spectrometry (HR-ESI-MS), nuclear magnetic resonance (1D, 2D-NMR), single crystal diffraction (X-Ray), and liquid chromatography. (HPLC-DAD) and other detection techniques proved to be the target compound.
  • the phthalophthalic derivatives in the present invention have good pharmacological activity research in vitro and in vivo to prove that they have better antioxidant nerve cell protection and anti-platelet aggregation effects, and can be used as a chemical preventive and therapeutic drug for cardiovascular and cerebrovascular diseases.
  • the host compound of the present invention is an isoindolinone compound. Under the conditions of achiral synthesis, the compound of formula I undergoes a cyclization and nucleophilic substitution reaction within the molecule to generate a racemic stereostructure at the C-3 position. The enantiomers are then resolved by simple recrystallization to obtain the chiral compounds with single enantiomers in the S and R configuration at the C-3 hydroxyl group.
  • phthaloline derivatives constructed and prepared by chemical structure derivation are converted from the unstable oily substances of known phthalophthalic compounds into colorless crystalline solids.
  • Enhance the chemical structure stability of the compound effectively enhance the stability of natural or organic synthetic phthalide compounds, increase the medicinal properties of the compounds, and effectively mask the bad volatile odor of the compounds.
  • ligustilide is the main component of volatile oil. It is an oily substance, and its unstable drug-forming property is poor. Its instability is well-known in the industry. After derivatization of our structure, it becomes a crystalline solid, indicating that the stability is greatly enhanced.
  • the phthaloline derivatives prepared through chemical structure derivation form a unique stereochemical structure, which enhances the spatial binding degree of the compounds to the drug target and effectively enhances the pharmacological activity of the compounds.
  • cyclopropylamine first undergoes a nucleophilic substitution reaction with a carbonyl group in the ligustilide ring to form a key ring-opening intermediate of an amide and 1-pentanone.
  • the nitrogen atom in cyclopropane in the body has strong nucleophilic reaction performance, and continues to undergo nucleophilic addition reaction with 1-pentanone ketocarbonyl group, but at this time, the cyclopropanyl group in cyclopropylamine can be regarded as a highly co- The planar structure of the yoke, due to the effect of steric hindrance, the free rotation of the carbon-nitrogen bond formed by the carbonyl carbon and nitrogen in cyclopropylamine is inhibited, and the steric steric hindrance is reduced to promote the nucleophilic addition on the 1-pentanone group. The reaction takes place.
  • the three-dimensional structure and space volume of cyclopropane and hydroxyl group are relatively small, and at the same time, it is biased to the same side of the planar structure formed by lactam and hexadiene. Side, so phthalide compounds react with cyclopropylamine to form a unique stereochemical structure reaction product.
  • the phthalophthalic compound is obtained through the structural transformation of the present invention, which enriches the chemical structure library of the compound and provides a large number of lead compounds for drug screening of the compound.
  • Figure 1 shows the HPLC-DAD chromatographic test results of the compound N-cyclopropylalkyl-3-n-butyl-3-S-hydroxy-galactyllactam.
  • Detection instrument Agilent 1200 chromatographic system is equipped with DAD detector; chromatographic conditions: chromatographic column, XTerra MS C 18 (Waters); particle size of 5 ⁇ m, column length 4.6 ⁇ 250mm.
  • Fig. 2 UV spectrum of the DAD detection compound N-cyclopropanyl-3-n-butyl-3-S-hydroxy-galactamactam.
  • FIG. 3 is a 1 H-NMR chart of a compound N-cyclopropylalkyl-3-n-butyl-3-S-hydroxy-galactamactam.
  • FIG. 4 is a 13 C-NMR chart of a compound N-cyclopropylalkyl-3-n-butyl-3-S-hydroxy-galactamactam.
  • Figure 5 DEPT spectrum of the compound N-cyclopropylalkyl-3-n-butyl-3-S-hydroxy-galactamactam.
  • FIG. 6 is a 1 H- 1 HCOSY pattern of a compound N-cyclopropylalkyl-3-n-butyl-3-S-hydroxy-galactamactam.
  • FIG. 7 is an HSQC chart of a compound N-cyclopropylalkyl-3-n-butyl-3-S-hydroxy-galactamactam.
  • Figure 8 HMBC spectrum of the compound N-cyclopropanyl-3-n-butyl-3-S-hydroxy-galactamactam.
  • Figure 9 Single crystal diffraction (X-Ray) structure of compound N-cyclopropanyl-3-n-butyl-3-S-hydroxy-galactamactam, where a is a single crystal structure and b is a bicellular molecule
  • the single crystal structure shows a single enantiomer in which the C-3 stereo configuration is S configuration.
  • FIG. 10 HPLC-DAD hand of compound N-cyclopropanyl-3-n-butyl-3-hydroxy-phthalolactam and compound N-cyclopropyl-3-n-butyl-3-hydroxy-fluorenolactam Chromatogram.
  • Detection instrument Agilent 1200 chromatographic system is equipped with DAD detector; chromatographic conditions: chromatographic column, FMG-ACS-A01-NFC Chiral ND (2) (250mm ⁇ 4.5mm, 5 ⁇ m).
  • the detection wavelength is 283 nm, where S and R represent the corresponding enantiomers.
  • FIG. 11 is a circular dichroism (CD) spectrum chart of the enantiomers of the N-cyclopropanyl-3-n-butyl-3-S-hydroxy-fluorenolactam and S-forms.
  • Figure 12 Single crystal X-Ray derived structure diagram of compound N-cyclopropane-3-n-butyl-3-hydroxy-phthalolactam; where a is a single crystal structure, and b is a bi-molecular single crystal single in the unit cell The crystal structure shows that the stereo configuration at the C-3 position is a racemate in which both the R and S configurations exist.
  • FIG. 13 UV spectrum of the DAD detection compound N-cyclopropanyl-3-n-butyl-3-hydroxy-phthalolactam.
  • Figure 15 HPLC-DAD method for determining the target compounds N-cyclopropane-3-n-butyl-3-hydroxy-fluorenolactam and N-cyclopropyl-3-n-butyl-3- by area normalized content Content of hydroxy-phthalolactam.
  • Figure 16 Protective effect of phthaloline derivatives on hydrogen peroxide-induced oxidative damage to PC12 nerve cells.
  • ** means p ⁇ 0.01%, which has a significant difference
  • * means p ⁇ 0.05%, which has a significant difference.
  • FIG. 17 Therapeutic effect of phthaloline derivatives on acute cerebral ischemic infarction in Wistar rats with unilateral thread plug model.
  • Fig. 18 is a graph showing the time when phthaloline derivatives were administered orally to rats.
  • Fig. 19 is a graph showing the time when phthaloline derivatives were administered intravenously to rats.
  • the ligustilide (extracted from Angelica sinensis by the inventor's laboratory) (See Liu Rusi, Yue Meiying, Li Wenbing, Peng Cheng, Xiong Liang for the extraction method. Chemical composition of angelica oil and chuanxiong oil and its acute chemistry Study on the protective effects of traumatic brain injury, Chinese Pharmacology and Clinic, 2016, 32 (6): 105-108.), HPLC method using area normalized content detection was 98%) (1.900g, 0.010mol) dissolved in 50mL In tetrahydrofuran, cyclopropylamine (0.684 g, 0.012 mol) was dissolved in 20 mL of tetrahydrofuran, the temperature of the water bath was controlled at 25 ° C, and the tetrahydrofuran solution of cyclopropylamine was added dropwise to the tetrahydrofuran solution of ligustilide under mechanical stirring, and the reaction was stirred.
  • the solvent was recovered under reduced pressure, and a single crystal was cultured using a petroleum ether / acetone solvent system, and the absolute stereo configuration of the compound was determined by X-Ray diffraction.
  • a target compound solution having a concentration of 0.1 mg / mL was prepared and passed through the circle.
  • Chromatography (CD) see Figure 11
  • optical rotation measurement assist in determining the absolute configuration of the compound.
  • Tr 7.623min is S-(+)-galactylcyclopropanelactam, accounting for 49.2%
  • N-cyclopropane-3-n-butyl-3-hydroxy-fluorenolactam in the S configuration in circular dichroism has a significant n-cotton effect at 254 nm
  • N-cyclopropan-3- N-butyl-3-hydroxy-galactamactam has a significant negative cotton effect at 254 nm
  • the two compounds have completely opposite CD spectral absorption characteristics.
  • n-butenylphthalide (1.880g, 0.010mol) (Alfa Aesar reagent company, purity 95%) in 50mL of tetrahydrofuran, and cyclopropylamine (0.684g, 0.012mol) in 20mL of tetrahydrofuran.
  • cyclopropylamine (0.684g, 0.012mol) in 20mL of tetrahydrofuran.
  • the tetrahydrofuran solution of cyclopropylamine was added dropwise to the tetrahydrofuran solution of ligustilide, and the reaction was stirred for 6 hours.
  • the tetrahydrofuran and the excess cyclopropylamine solution were recovered by rotary distillation under reduced pressure. 200 mL of petroleum ether was added to the above concentrate and mixed.
  • Example 3 Using angelica volatile oil for compound N-cyclopropanyl-3-n-butyl-3-hydroxy-galactamactam and compound N-cyclopropanyl-3-n-butyl-3-hydroxy-phthalide Synthesis of a mixture of both amides
  • the prepared angelica volatile oil was pre-separated by column chromatography using petroleum ether and ethyl acetate (3: 1, V / V) as eluents.
  • the angelica volatile oil (3.000 g) ) Dissolved in 60 mL of tetrahydrofuran, and cyclopropylamine (0.684 g, 0.012 mol) was dissolved in 30 mL of tetrahydrofuran. Under mechanical stirring, the tetrahydrofuran solution of cyclopropylamine was added dropwise to the tetrahydrofuran solution of the angelica volatile oil, and the reaction was stirred for 10 hours.
  • Tetrahydrofuran and excess cyclopropylamine solution were recovered by distillation under reduced pressure.
  • 200 mL of petroleum ether was added to the above concentrate, mixed well, and left to stand overnight for the crystals of the synthesized product to be washed out and filtered with suction to obtain 2.283 g of crude product.
  • the reaction yield was 76%.
  • the crude product was recrystallized from a mixed solvent of petroleum ether and acetone (10: 1, V / V) to obtain 1.872 g of a crystalline product with a crystallization yield of 82%.
  • HPLC-DAD method was used to determine the content of the target compound by area normalized content: the content of the compound N-cyclopropanyl-3-n-butyl-3-hydroxy-galactamactam was 90.0%; the compound N-cyclopropanyl- The content of 3-n-butyl-3-hydroxy-phthalolactam was 8.9% (see Fig. 14).
  • Example 4 Compound N-cyclopropanyl-3-n-butyl-3-hydroxy-fluorenolactam and compound N-cyclopropanyl-3-n-butyl-3-hydroxy-phthalide were prepared using chuanxiong volatile oil. Synthesis of a mixture of both amides
  • the prepared chuanxiong volatile oil was pre-separated by column chromatography using petroleum ether and ethyl acetate (3: 1, V / V) as eluents.
  • the chuanxiong volatile oil (3.500g) containing the higher part of ligustilide ,) Dissolved in 60mL of tetrahydrofuran, cyclopropylamine (0.684g, 0.012mol) was dissolved in 30mL of tetrahydrofuran, and under mechanical stirring, the tetrahydrofuran solution of cyclopropylamine was added dropwise to the tetrahydrofuran solution of the above chuanxiong volatile oil, and the reaction was stirred for 10 hours, Tetrahydrofuran and excess cyclopropylamine solution were recovered by rotary distillation under reduced pressure.
  • HPLC-DAD method was used to determine the content of the target compound by area normalized content: the content of the compound N-cyclopropane-3-n-butyl-3-hydroxy-galactam was 91.2%; the compound N-cyclopropanyl The content of 3-n-butyl-3-hydroxy-phthalolactam was 7.9%.
  • the prepared angelica volatile oil was pre-separated by column chromatography using petroleum ether and ethyl acetate (3: 1, V / V) as eluents.
  • the angelica volatile oil (3.000 g) ,) Dissolved in 60mL of tetrahydrofuran, cyclopropylamine (0.684g, 0.012mol) was dissolved in 30mL of tetrahydrofuran, and under mechanical stirring, the tetrahydrofuran solution of cyclopropylamine was added dropwise to the tetrahydrofuran solution of angelica volatile oil, and the water bath was heated to 45 The reaction was stirred at a constant temperature for 10 hours at °C, and the tetrahydrofuran and the excess cyclopropylamine solution were recovered by rotary evaporation under reduced pressure.
  • HPLC-DAD method was used to determine the content of the target compound by area normalized content: the content of the compound N-cyclopropanyl-3-n-butyl-3-hydroxy-galactam was 70.0%; the compound N-cyclopropanyl The content of 3-n-butyl-3-hydroxy-phthalolactam was 28.1%.
  • the prepared angelica volatile oil was pre-separated by column chromatography using petroleum ether and ethyl acetate (3: 1, V / V) as eluents.
  • the angelica volatile oil (3.000 g) ,) Dissolved in 60mL of tetrahydrofuran, cyclopropylamine (0.684g, 0.012mol) was dissolved in 30mL of tetrahydrofuran, and under mechanical stirring, the tetrahydrofuran solution of cyclopropylamine was added dropwise to the tetrahydrofuran solution of angelica volatile oil, and the reaction was stirred mechanically at room temperature.
  • the crude product was separated and purified by column chromatography using petroleum ether and ethyl acetate (3: 1, V / V) as eluents to obtain three components A, B, and C, respectively, to obtain component A (0.483 g).
  • component A Identified by thin-layer chromatography as N-cyclopropanyl-3-n-butyl-3-hydroxy-phthalolactam; obtained component B (0.420g) identified by thin-layer chromatography as compound N-cyclopropanyl-3 -A mixture of n-butyl-3-hydroxy-fluorenolactam and the compound N-cyclopropyl-3-n-butyl-3-hydroxy-phthalolactam; component C (1.128 g) is obtained.
  • Example 7 Compound N-Cyclopropylalkyl-3-n-butyl-3-hydroxy-liguslactam and compound N-cyclopropanyl-3-n-butyl-3-hydroxyl with volatile oil mixture of Angelica sinensis and Ligusticum chuanxiong -Synthesis of phthalolactam compounds
  • the prepared volatile oil mixture of Angelica sinensis and Ligusticum chuanxiong Hort was pre-separated by column chromatography with petroleum ether and ethyl acetate (3: 1, V / V) as eluents.
  • the volatile oil containing the higher part of ligustilide The mixture (3.000 g,) was dissolved in 60 mL of tetrahydrofuran, and cyclopropylamine (0.684 g, 0.012 mol) was dissolved in 30 mL of tetrahydrofuran. Under mechanical stirring, the tetrahydrofuran solution of cyclopropylamine was added dropwise to the tetrahydrofuran solution of the angelica volatile oil.
  • reaction was stirred at room temperature for 6 hours, and the tetrahydrofuran and the excess cyclopropylamine solution were recovered by rotary distillation under reduced pressure.
  • 200 mL of petroleum ether was added to the above concentrate and mixed uniformly. It was left overnight at -20 ° C until the synthesized product crystals were washed out, and filtered to obtain a crude product. 2.602 g, reaction yield 86.7%.
  • the crude product was recrystallized from a mixed solvent of petroleum ether and acetone (10: 1, V / V) to obtain 2.080 g of crystalline product with a crystal yield of 80%.
  • HPLC-DAD method was used to determine the content of the target compound by area normalized content: the content of the compound N-cyclopropane-3-n-butyl-3-hydroxy-galactam was 92.5%; the compound N-cyclopropanyl The content of 3-n-butyl-3-hydroxy-phthalolactam was 7.5% (as shown in Fig. 15).
  • Example 8 Study on the neuroprotective effect of phthaloline derivatives.
  • PC12 cells (Shanghai Institute of Cell Sciences, Chinese Academy of Sciences); phthaloline derivatives (recommended to be prepared in the above examples), high-sugar medium (DMEM, Nanjing Kaiji Biotechnology Development Co., Ltd.); dimethyl sulfoxide ( DMSO, Sigma); fetal bovine serum (Gibco, Thermo Fisher Scientific Co., Ltd.); H 2 O 2 (Nanjing Chemical Reagent Co., Ltd.); thiazole blue (MTT, Nanjing Shengxing Biotechnology Co., Ltd.), enzyme Standard instrument (American Bio-rad company).
  • DMEM high-sugar medium
  • DMSO dimethyl sulfoxide
  • fetal bovine serum Gibco
  • H 2 O 2 Najing Chemical Reagent Co., Ltd.
  • thiazole blue (MTT, Nanjing Shengxing Biotechnology Co., Ltd.), enzyme Standard instrument (American Bio-rad company).
  • PC12 cells were cultured. PC12 cells were inoculated into high-glucose DMEM medium. The medium contained 10% fetal bovine serum, penicillin 100U mL -1 , and streptomycin 100 ⁇ g mL -1 . The cells were cultured in a CO 2 cell culture incubator, changed every other day, and passaged with conventional trypsin digestion; the cells were used for experiments when they reached 70-80% confluence.
  • PC12 cells in logarithmic growth phase were seeded in a 96-well cell culture plate at a density of 1 ⁇ 10 4 cells / well. After incubation for 24 hours, 100 ⁇ l of phthalophthalate and The derivative solution was made to a final concentration of 1.25, 2.5, 5.0, 10, 20, 40, 80, and 160 ⁇ M, and the culture was continued for 24 hours. 100 ⁇ l of MTT (5 mg mL -1 ) was added. After 4 h of incubation, 150 ⁇ l of DMSO was added. After shaking for 10 min, the wavelength of 490 nm was selected and the absorbance (A) value of each well was detected.
  • MTT 5 mg mL -1
  • the experimental grouping was divided into normal blank control group, H 2 O 2 model group (250 ⁇ M), phthalophthalines and their derivatives. (2.5 ⁇ M, 5.0 ⁇ M, 10 ⁇ M, 25 ⁇ M, 50 ⁇ M) concentration group, positive control group (butylphthalide). 250 ⁇ M of H 2 O 2 was added to each group of phthalophthalines and their derivatives and the positive control group. In the experiment, phthalide and its derivative solution were respectively added to make the final concentration reach the corresponding value, H 2 O 2 was added after 4 hours, and MTT was added after 24 hours. The A value was measured to calculate the cell survival rate.
  • ligustilide derivative When the concentration of ligustilide derivative was 10 ⁇ M, the cell viability was the highest, reaching 95%. Secondly, the activity of ligustilide is stronger than that of n-butenephthalide on the oxidative damage of nerve cells; ligustilide and the n-butenephthalide derivative (racemic mixture) prepared according to Example 2 are more effective against nerve The protection of cells from oxidative damage is almost the same.
  • Example 9 Study on the inhibitory effect of phthalide derivatives on cerebral infarction in acute ischemic stroke
  • mice Preparation of animal models: 75 rats will be purchased according to the standard, and they will be raised strictly in clean grade, with reference methods (Xiaomei Wu, Zhongming Qian, Li Zhu, Fang Du, Wingho Yung, Qi Gong and Ya Ya Ke, Neuroprotective effects of ligustilide against ischaemia-reperfusion, injury, via-regulation, erythropoietin, and down-regulation (RTP801, British J, Pharm.
  • MCAO model was established with a thread plug (rat middle cerebral artery occlusion), and 10% hydrated chlorine
  • a thread plug rat middle cerebral artery occlusion
  • 10% hydrated chlorine Intraperitoneal injection of aldehyde, anesthetized rats at a dose of 400 mg / kg, fixed in a supine position, exposed the neck, cut skin after shaving and cleaning the skin, gently manipulated and isolated the left common carotid artery, internal carotid artery, and external carotid artery Arteries and other main blood vessels, choose the external carotid artery to ligate and disconnect, to avoid common carotid artery bleeding, temporarily clamp the common carotid artery with an arterial clip, select the end of the external carotid artery, gently cut a small mouth and use it as a thread plug.
  • the prepared suture round head was placed with ophthalmic tweezers and gently inserted with a controlled depth of about 1.8-2.0 cm. Conventional incision was sutured. After 0.5 h of cerebral ischemia, the suture was pulled out to restore blood supply. At a certain time after the administration, the rats were sacrificed. The brain tissue was stained with 0.5% 2,3,5-triphenyltetrazolium chloride (TCC), and the area of cerebral infarction was calculated by scanning. The normal tissue was stained red and the infarcted tissue was stained. It was white after dyeing.
  • TCC 2,3,5-triphenyltetrazolium chloride
  • Animal grouping According to the model group, a pathological model of acute ischemia and hypoxia was given an equal dose of 8% Tween-80 saline, and the sham operation group (not administered, as a negative control, was given an equal dose of 8%). Tween-80 physiological saline), the prophylactic administration group (administration 3 days in advance) and the therapeutic administration group (administration 3 days after the blood supply was restored by embolization) were divided into groups.
  • ligustilide derivatives phthaloline derivatives: prepared according to Example 1, HPLC-DAD area normalization method for determination of N-cyclopropane-3-n-butyl-3-hydroxyl -The content of ligustilactam reaches 98%, dissolved with 8% Tween-80 in normal saline), dosage: 40mg / Kg.
  • Negative model control infarct area of 30.57%
  • B N-cyclopropanyl-3-n-butyl-3-hydroxy-galactamactam treatment group, (3 days after model administration, 40 mg / kg, abdominal cavity Injection), the left infarct area is 4%, the middle is completely recanalized (infarct area 0%), and the right side is calculated as a negative value according to the method of deedema
  • C N-cyclopropanyl-3-n-butyl-3-hydroxyl -Ligustilactam prophylaxis group (pre-dose for 3 days, modeling, and then for 3 days; 40 mg / kg, intraperitoneal injection), the infarct area is 22%.
  • the quantitative detection method of the compound was established.
  • the HPLC-DAD standard curve method was used to determine the detection limit (LOD), limit of quantification (LOQ), and linear range of the method.
  • N-Cyclopropylalkyl-3-n-butyl-3-hydroxy-galactam and N-Cyclopropylalkyl-3-n-butyl-3-hydroxy-phthalolactam standards were accurately weighed separately , Methanol was dissolved and diluted to obtain a standard stock solution of 2.0 mg / mL. Precisely measure and mix certain standard stock solutions and dilute with methanol to 0.1953, 0.395, 0.7813, 1.5625, 3.1250, 6.2500, 12.500, 25.0000, 50.0000, and 500.0 ⁇ g / mL.
  • Test sample preparation method The compound prepared in Example 7 (N-cyclopropylalkyl-3-n-butyl-3-hydroxy-fluorene) was prepared according to 10% ethanol, 20% Tween 80, and 70% physiological saline. The content of the present lactam is 92.5%; the content of the compound N-cyclopropanyl-3-n-butyl-3-hydroxy-phthalolactam is 7.5%) is prepared into an original stock solution with a concentration of 15mg / mL. Rats were administered by body weight, orally (intragastrically) at a dose of 90 mg / Kg, and intravenously at a dose of 40 mg / Kg.
  • Plasma sample pretreatment method Take 0.5mL of rat whole blood and place it in a 1.5mL centrifuge tube with 20uL sodium heparin, rotating speed 4 ⁇ 10 3 r. Centrifuge at high speed for 5 min at min -1 , take 200 ⁇ L of plasma, add 800 ⁇ L of dichloromethane, vortex and centrifuge for 10 min, and rotate at 1 ⁇ 10 5 r. min -1 , remove the lower layer solution and blow dry with nitrogen at room temperature, reconstitute with 300 ⁇ L methanol, elute and filter dry with nitrogen, add 30 ⁇ L methanol, centrifuge and take 20 ⁇ L of supernatant to determine the peak area by HPLC.
  • Twelve male Wistar rats were randomly divided into two groups, of which 6 were intravenously administered (i.v.), and 40 mg / kg of phthalide derivatives were administered.
  • 6 intravenously administered
  • 40 mg / kg of phthalide derivatives were administered.
  • blood was collected from the eyeballs by about 500 ⁇ L, and 200 ⁇ L of plasma was frozen after anticoagulation and centrifugation.
  • Six rats in the oral administration (po) group were given 90 mg / kg of phthalophthalic derivatives, 0, 0.5, 1, 2, 3, 4, 6, 8, 10, and
  • At 12h about 500 ⁇ L of blood was collected from the eye, and 200 ⁇ L of plasma was frozen after anticoagulation and centrifugation.

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Abstract

公开了一种苯酞类衍生物，该衍生物的结构如式(I)或式(II)所示。还公开了该衍生物的制备方法和应用。通过结构改造得到苯酞类化合物，增强了苯酞类化合物的化学稳定性和药理活性，有助于提高该类化合物的成药性。

Description

一种苯酞类衍生物及其制备方法和应用 技术领域
本发明属于有机合成化学领域，涉及一种苯酞类衍生物及其制备方法和应用。
背景技术
自然界中伞形科植物中当归属和藁本属的植物均含有苯酞类化合物，如当归和川芎等药用植物，该类化合物为植物挥发油的主要成分，大都具有浓烈的芳香气味，在植物中具有丰富的含量，且化合物结构类型较多，该类化合物普遍具有化学不稳定性，单独存在遇环境中的光、热以及酶等容易氧化或降解。但该类化合物普遍具有较好的药理活性，如抗氧化、抗血小板聚集、促进微循环、镇痛消炎、中枢神经保护以及抗肿瘤等显著的药理活性。
藁本内酯(ligustilide)是传统中药当归、川芎等伞形科植物挥发油中的主要药理活性成分，其含量高达1％左右，具有显著的心脑血管药理活性，国内外学者对藁本内酯的药理活性曾经和正在进行大量的研究工作，发表了相关的药理活性研究成果，如藁本内酯对脑神经保护，改善微循环，舒张血管，抑制血管平滑肌细胞增殖，抗抑郁，抗阿尔茨海默症，镇痛消炎等重要药理作用，同时研究人员对于藁本内酯的药理研究成果进行了大量的综述(A.Douglas Kinghorn,Heinz Falk,Simon Gibbons,and Jun’ichi Kobayashi edited,Progress in the Chemistry of Organic Natural Products,2017,P127,Springer International Published.；左爱华,王莉,肖红斌,藁本内酯药理学和药代动力学研究进展,中国中药杂志,2012,37(22):3350-3353.)。
藁本内酯能明显减少脑缺血再灌注引起的脑梗塞体积，改善脑神经功能，并且能减轻大鼠皮层神经元和海马神经元细胞的损伤。为进一步阐明其神经保护内在作用机制，X Kuang等和HY Peng等分别对短暂性和持久性脑缺血再灌注模型进行研究，结果表明藁本内酯呈剂量依赖性减少脑缺血脑组织中丙二醛含量，增加谷胱甘肽过氧化酶及超氧化物歧化酶活性，增强Bcl-2表达同时减弱Bax，Caspase-3酶免疫活性，揭示出藁本内酯是通过抗氧化及抗凋亡机制对短暂性和持久性脑缺血再灌注引起的损伤起神经保护作用。在此基础上，对慢性脑缺血再 灌注导致的认知缺损、生化变化及病理组织学特征进行研究，也说明藁本内酯对认知缺陷和脑损伤的保护作用是通过抗氧化及增加类胆碱功能活性实现的(X Kuang,Y Yao,J.R.Du,Y.X.Liu,CY Wang,ZM Qian,Neuroprotective role of Z-ligustilide against forebrain ischemic injury in ICR mice,Brain Research,2006,1102(1):145-153；Haiyan Peng,Junrong Du,Guangyi Zhang,Xi Kuang,Yanxin Liu,Zhongming Qian,and Chenyuan Wang,Neuroprotective effect of Z-Ligustilide against permanent focal ischemic damage in rats,Biol Pharm Bull,2007,30(2):309-312.)。藁本内酯通过提高细胞抗氧化能力和抑制线粒体凋亡等通路对H 2O 2损伤的PC12细胞起神经保护作用(Yan Yu,Junrong Du,Chenyuan Wang,Zhongming Qian,Protection against hydrogen peroxide-induced injury by Z-ligustilide in PC12cells,Exp Brain Res,2008,184(3):307-312.)。HY Qi等进一步研究了藁本内酯在缺氧缺糖PC12细胞中所起的双相毒物兴奋调节作用，它不仅能通过ROS的生成和谷胱甘肽的消耗引起氧化应激反应，而且能通过PI3K和Nrf2通路的交互作用激活促生存信号，使得藁本内酯对低、高浓度缺氧缺糖环境下的PC12细胞均能起到预先保护的作用(Hongyi Qi,Yifan Han,Jianhui Rong,Potential roles of PI3K/Akt and Nrf2-Keap1pathways in regulating hormesis of Z-ligustilide in PC12cells against oxygen and glucose deprivation,Neuropharmacology,2012,62(4):1659-1670.)。藁本内酯在显著改善缺血再灌注大鼠神经功能，减少梗塞体积的同时，使得炎症因子NF-κB的蛋白表达量明显减少。而且对LPS致神经胶质细胞产生的TNF-α，NO，IL-1α，MCP-1等炎症介质有抑制作用，对α-淀粉样多肽引起的认知缺陷，神经病理学等症状，能通过调解NF-κB通路方式使其得到改善，这些都说明藁本内酯能通过抑制NF-κB通路方式起到抗炎作用。另外，藁本内酯还能通过抑制ROS产生和下调MAPK，NF-κB，AP-1信号通路的方式对LPS致RAW 264.7的炎症反应起到抑制作用。总之藁本内酯也能通过抗炎机制起到神经保护的作用，这有利于神经炎症性疾病的治疗(Yuwen Su,Wenfei Chiou,Shiouhuei Chao,Menghwan Lee,Chienchih Chen,Yingchieh Tsai,Ligustilide prevents LPS-induced iNOS expression in RAW 264.7macrophages by preventing ROS production and down-regulating the MAPK,NF-κB and AP-1signaling pathways,International Immunopharmacology,2011,11(9):1166-1172.)。
藁本内酯在1-30μM浓度时，通过p38和PI3K/Akt信号通路，抑制β淀粉样蛋白诱导的神经毒作用，支持藁本内酯具有阿尔茨海默症(AD)治疗作用(Wei Xu,Li Yang,Ji Li,Protection againstβ-amyloid-induced neurotoxicity by naturally occurring Z-ligustilide through the concurrent regulation of p38and PI3-K/Akt pathways,Neurochemistry International,2016,100:44-51.)。国内外相关文献显示藁本内酯具有显著的药理活性。
苯酞类化合物根据六元环的氢化还原程度分为苯环型如正丁烯基苯酞，二氢苯环型如藁本内酯，四氢苯环型苯酞类化合物如洋川芎内酯H，同时侧链及苯环具有不同的取代情况和氧化程度，产生一系列结构多样的苯酞类化合物。同时也可以形成各种二聚和三聚体。
Figure PCTCN2019072178-appb-000001
藁本内酯由于具有3个烯键和1个羰基双键的高度不饱和共轭体系，但未形成芳香环的稳定共轭体系，反应位点较多，反应活性较强，相对自由能较低，故容易发生1,2、1,4和1,6的Diels-Alder反应，形成复杂的二聚或三聚化合物。
Frank R.Stermita曾用芳香胺通过对内酯环的亲核取代反应，试图得到酰胺类化合物，但这种酰胺通过分子内的环加成反应过渡态最终得到环合的反应产物(John J.Beck,Frank R.Stermitz,Addition of methyl thioglycolate and benzylamine to(Z)-ligustilide,a bioactive unsaturated lactone constituent of several herbal medicines.an improved synthesis of(Z)-ligustilide,Journal of Natural Products,1995,58(7):1047-1055.)，可见藁本内酯的每个双键或官能团均具有较高的反应活性，对于以藁本内酯为原料的化学反应必须进行严格的化学反应条件控制，才能达到理想的化学反应设计。
季晖等人曾通过对于丁苯酞的化学结构改造，试图通过引入硝酸甘油的药效团提高该类化合物对血小板聚集的抑制活性和通过NO抑制的抗炎活性，结果合成化合物活性比丁苯酞有了显著的提高(吴晶,凌菁菁,王旭亮,王晓丽,刘婧 超,季晖,张奕华,含单硝酸异山梨醇酯的丁苯酞衍生物的合成及抗血小板聚集活性研究,中国药物化学杂志,2012,22(6):483-489.)。
国内外文献显示对于苯酞类化合物进行结构改造可以显著提高该类化合物的药理活性，但对于高度不饱和的藁本内酯，其反应条件比较苛刻，反应产物难于控制，必须对反应条件及反应催化体系进行精心的设计和把控才能达到高效合成和目标合成。药理活性研究显示，显然对于藁本内酯通过Diels-Alder反应建立稳定体系的化合物，药理活性相对较差，故对于藁本内酯的结构衍生重点应该放在内酯环的取代和加成优化处理方面。
丁苯酞(Butylphthalide)是我国第一个拥有自主知识产权的创新药。最初是从水芹籽中提取分离得到，然后进行化学合成得到，丁苯酞具有独特的双重作用机制，既能重构微循环，增加缺血去灌注，从而保护血管结构完整、恢复血管管径、增加缺血区血流量及周围微血管数量，又能保护线粒体，减少细胞死亡，从而保护线粒体结构的完整、提高线粒体复合酶Ⅳ的活性、提高线粒体ATP酶的活性、维持线粒体膜的稳定性，双重狙击，对抗脑卒中。研究显示相较于藁本内酯的神经保护作用和化合物具有的毒副作用，就药理学特征丁苯酞比藁本内酯要差，原因在于显著的化学结构特征。
显而易见，由于苯酞类化合物普遍具有的化学不稳定性，虽然具有较好的药理活性，但大大限制了该类化合物在药物，保健品，食品和化妆品等领域相关产品的开发和广泛应用。因此要解决产品的开发和应用，必须首先解决该类化合物的稳定性。由于显而易见的化学结构特征，藁本内酯的药理活性要强于丁苯酞，但从药学的有效、安全和质量可控方面综合比较，丁苯酞具备药学特征(化学结构稳定)，故被SFDA批准上市，但目前临床反应由于其活性和毒副作用等原因，临床医生已经对丁苯酞提出进一步优化和提高活性的强烈需求，并不能满足临床对于脑卒中的治疗作用要求。因此有必要针对藁本内酯的独特化学结构进行结构的人为干预，优化和筛选高效和稳定的基于该类化合物的心脑血管治疗药物。
发明概述
本发明的目的在于提供一种苯酞类衍生物及其制备方法和应用。该苯酞类衍生物的显著特征在于其原有的内酯结构片段被内酰胺基团所取代，C-3位被羟基取代。
因此，本发明提供一种式I或式II的苯酞类衍生物，其旋光异构体或药用盐，或它们的混合物(例如，外消旋混合物)
Figure PCTCN2019072178-appb-000002
在一个优选实施方案中，所述的苯酞类衍生物为N-环丙烷基-3-正丁基-3-S-羟基-藁本内酰胺、N-环丙烷基-3-正丁基-3-R-羟基-藁本内酰胺、N-环丙烷基-3-正丁基-3-S-羟基-苯酞内酰胺或N-环丙烷基-3-正丁基-3-R-羟基-苯酞内酰胺。
在另一方面，本发明提供一种制备苯酞类衍生物(例如式I或式II的苯酞类衍生物)的方法，包括将苯酞类化合物与环丙胺在有机溶剂中反应，其中苯酞类化合物中原有的内酯结构片段被内酰胺基团所替换，C3位被羟基取代，以得到式I或式II的苯酞类衍生物。
在一个优选实施方案中，本发明的方法包括以下特征中的一个、两个、三个或四个：
1)所述苯酞类化合物为C3位正丁烯基取代的苯环型、二氢苯环型或四氢苯环型苯酞类化合物，优选藁本内酯或正丁烯苯酞，其中所述藁本内酯或正丁烯苯酞为单独分离的化合物形式或者处于当归属或藁本属植物提取物中的混合物形式，更优选所述植物提取物为当归挥发油提取物或川芎挥发油提取物或它们的混合物；
2)反应在-20℃至60℃下进行，优选在搅拌下进行；
3)有机溶剂为非极性有机溶剂，优选选自环己烷、石油醚、四氢呋喃、乙醚；和/或
4)所述方法还包括手性拆分对映异构体的步骤，优选通过手性色谱法或手性重结晶法。
在一个优选实施方案中，在所述的制备方法中，所述苯酞类化合物与环丙胺的摩尔比为1:1～1.2。
在一个优选实施方案中，所述的制备方法还包括减压回收有机溶剂和重结 晶得到目标产物的步骤，优选所述重结晶所用溶剂为石油醚、乙酸乙酯、丙酮和乙醚中的一种或两种。
在一个优选实施方案中，所述的制备方法包括将苯酞类化合物加入有机溶剂溶解，温度控制在-20℃至60℃，加入有机溶剂溶解的环丙胺反应液，控制温度在-20℃至60℃，搅拌反应1-24小时，减压回收有机溶剂，经重结晶得到目标产物。
本发明还提供通过以上所述方法制备的根据本发明的式I或式II的苯酞类衍生物，其旋光异构体或药用盐，或它们的混合物。
在另一方面，本发明还提供根据本发明的式I或式II的苯酞类衍生物，其旋光异构体或药用盐，或它们的混合物在制备药物中的应用，所述药物用作抗氧化剂和/或用于治疗或预防以下疾病：心脑血管疾病，抑郁症，阿尔茨海默症，(神经)炎性疾病，疼痛，神经元细胞损伤，缺血再灌注损伤，脑梗塞，认知缺陷或脑损伤。
在一个优选实施方案中，所述药物包含式I化合物和式II化合物的混合物作为有效成分，其中式I化合物在混合物中的质量百分含量为1-99％，优选90％以上。
发明详述
更具体地，本发明提供一种苯酞类衍生物，其特征在于该衍生物的结构如下：
Figure PCTCN2019072178-appb-000003
如上所述苯酞类衍生物的制备方法，具体步骤为：将苯酞类化合物加入有机溶剂溶解，温度控制在-20℃-60℃，加入有机溶剂溶解的环丙胺反应液，控制温度在-20℃-60℃，搅拌反应1-24h，减压回收有机溶剂，经重结晶得到目标产物。
所述苯酞类化合物与环丙胺的摩尔比为1:1～1.2。
所述苯酞类化合物为藁本内酯、正丁烯苯酞以及含藁本内酯或正丁烯苯酞的药材挥发油提取物中的一种或两种以上。
所述药材挥发油提取物为当归挥发油提取物或川芎挥发油提取物。
所述有机溶剂为非极性有机溶剂，优选环己烷、石油醚、四氢呋喃等。
所述重结晶所用溶剂为石油醚、乙酸乙酯、丙酮、乙醚中的一种或两种。
如上所述苯酞类衍生物在抗心脑血管疾病和抗氧化方面的应用。
所述苯酞类衍生物为式I化合物和式II化合物的组合物，组合物中式I化合物的质量百分含量为1-100％。
所述式I化合物的质量百分含量为90％以上(例如，91％，92％，93％，94％，95％，96％，97％，98％和99％)。
本发明中采用化学结构衍生方法合成的苯酞类衍生物的化学结构均通过质谱(HR-ESI-MS)，核磁(1D，2D-NMR)，单晶衍射(X-Ray)，液相色谱(HPLC-DAD)等多种检测技术证明为目标化合物。
本发明中苯酞类衍生物通过体内外药理活性研究证明具有较好的抗氧化神经细胞保护和抗血小板聚集作用，可以作为心脑血管疾病的化学预防和治疗药物的应用。
本发明的优点在于：
1、本发明的主体化合物属于异吲哚啉酮类化合物，式I化合物在非手性合成反应条件下，通过分子内的自身环化亲核取代反应，生成C-3位具有消旋的立体对映异构体，再通过重结晶简单拆分方法直接得到C-3位羟基为S和R构型的单一对映体的手性化合物。
2、借助环丙胺的独特化学结构和显著的药效基团，通过化学结构衍生构建和制备的苯酞类衍生物，由公知的苯酞类化合物不稳定的油状物转化为无色结晶型固体，增强了化合物的化学结构稳定性，有效增强了天然或有机全合成苯酞类化合物的稳定性，增加了该类化合物的成药性，并有效掩盖该类化合物的不良挥发性气味。例如，藁本内酯为挥发油主要成分，其本身为油状物，不稳定成药性差，其不稳定性为行业公知，我们结构衍生以后变成结晶性固体，说明稳定性大大增强。
3、通过化学结构衍生制备的苯酞类衍生物形成独特的立体化学结构，增强 了该类化合物与药物作用靶点的空间结合度，有效增强了该类化合物的药理活性。不受限于具体理论，据信在该类化合物反应中，首先环丙胺与藁本内酯环中的羰基进行亲核取代反应，形成酰胺和1-戊酮的开环关键中间体，该中间体中环丙烷中的氮原子具有较强的亲核反应性能，继续与1-戊酮基的酮羰基进行亲核加成反应，但此时环丙胺中的环丙烷基团可以看作是一个高度共轭的平面结构，由于空间位阻的影响，羰基碳与环丙胺中的氮形成的碳氮键进行自由旋转受到抑制，力求减小立体空间位阻，促使1-戊酮基上的亲核加成反应发生，结果环丙烷和羟基的立体结构和空间体积比较小，同时偏向于内酰胺和己二烯形成的平面结构的同一侧，而空间体积较大的4碳脂肪烷侧链趋向于另一侧，因此苯酞类化合物与环丙胺反应形成独特的立体化学结构反应产物。
4、通过本发明的结构改造得到苯酞类化合物，丰富了该类化合物的化学结构库，为该类化合物的药物筛选提供大量的先导化合物。
附图说明
图1化合物N-环丙烷基-3-正丁基-3-S-羟基-藁本内酰胺的HPLC-DAD色谱检测结果，图中T R＝14.863min处色谱峰为目标化合物。检测仪器：安捷伦1200色谱系统配备DAD检测器；色谱条件：色谱柱，XTerra MS C 18(Waters)；填料粒径5μm，柱长4.6×250mm。流动相：甲醇：水＝65:35(V/V)。手动进样器进样，每次20μL。检测波长280nm。
图2DAD检测化合物N-环丙烷基-3-正丁基-3-S-羟基-藁本内酰胺的UV光谱图。
图3化合物N-环丙烷基-3-正丁基-3-S-羟基-藁本内酰胺的 1H-NMR图谱。
图4化合物N-环丙烷基-3-正丁基-3-S-羟基-藁本内酰胺的 13C-NMR图谱。
图5化合物N-环丙烷基-3-正丁基-3-S-羟基-藁本内酰胺的DEPT图谱。
图6化合物N-环丙烷基-3-正丁基-3-S-羟基-藁本内酰胺的 1H- 1HCOSY图谱。
图7化合物N-环丙烷基-3-正丁基-3-S-羟基-藁本内酰胺的HSQC图谱。
图8化合物N-环丙烷基-3-正丁基-3-S-羟基-藁本内酰胺的HMBC图谱。
图9化合物N-环丙烷基-3-正丁基-3-S-羟基-藁本内酰胺的单晶衍射(X-Ray)结构，其中a为单晶结构，b为晶胞内双分子单晶结构，图示C-3位立体构型为 S构型的单一对映体。
图10化合物N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺和化合物N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺的HPLC-DAD手性拆分色谱图。检测仪器：安捷伦1200色谱系统配备DAD检测器；色谱条件：色谱柱，FMG-ACS-A01-NFC Chiral ND(2)(250mm×4.5mm,5μm)。流动相：正己烷：乙醇(V:V)＝90:10(v/v)。手动进样器进样，每次20μL。检测波长283nm，其中S和R分别表示相应的对映异构体。
图11R和S型N-环丙烷基-3-正丁基-3-S-羟基-藁本内酰胺对映异构体的圆二色谱(CD)谱图。
图12化合物N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺的单晶X-Ray衍生结构图；其中a为单晶结构，b为晶胞内双分子单晶单晶结构，图示C-3位立体构型为R和S构型同时存在的外消旋体。
图13DAD检测化合物N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺的UV光谱图。
图14化合物N-环丙烷基-3-正丁基-3-S-羟基-藁本内酰胺和化合物N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺的二者混合样品HPLC-DAD色谱检测结果，图中T R＝13.840min处色谱峰为N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺的色谱峰。T R＝15.348min处色谱峰为N-环丙烷基-3-正丁基-3-S-羟基-藁本内酰胺的色谱峰。色谱条件同图10中的色谱条件。
图15HPLC-DAD方法，通过面积归一化含量测定目标化合物N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺和N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺的含量。
图16苯酞衍生物对于过氧化氢诱导PC12神经细胞氧化损伤的保护作用。其中“ **”表示统计学上p<0.01％，具有较显著差异；“ *”表示统计学上p<0.05％，具有显著差异。
图17苯酞衍生物对于单侧线栓模型Wistar大鼠的急性脑缺血梗死的治疗作用。A、模型对照，梗死面积30.57％；B、藁本内酯衍生物治疗组完全再通，(造模后给药3天，40mg/kg，腹腔注射)；C、藁本内酯衍生物预防治疗组(预给药3天，造模，再给药3天；40mg/kg，腹腔注射)，梗死面积为22％。
图18大鼠灌胃给药苯酞类衍生物的药时曲线图。
图19大鼠静脉注射给药苯酞类衍生物的药时曲线图。
具体实施方式
实施例1
1)N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺的合成
将藁本内酯(本发明人实验室从当归中提取分离纯化所得(提取方法参见刘露丝,岳美颖,李文兵,彭成,熊亮.当归油与川芎油的化学成分及其对急性化学性脑损伤保护作用差异研究,中药药理与临床,2016,32(6):105-108.)，HPLC法采用面积归一化含量检测为98％)(1.900g，0.010mol)溶解在50mL的四氢呋喃中，环丙胺(0.684g，0.012mol)溶解在20mL的四氢呋喃中，水浴控制温度在25℃，机械搅拌下，将环丙胺的四氢呋喃溶液点滴加入到藁本内酯的四氢呋喃溶液中，搅拌反应4h，旋蒸减压回收四氢呋喃和过量的环丙胺溶液，将200mL石油醚加入到以上浓缩物中，混合均匀，放置过夜待合成产物结晶洗出，抽滤，得粗产物1.976克，反应产率80％，将粗产物用石油醚和丙酮(5:1，V/V)的混合溶剂重结晶，得到结晶产物0.97g，结晶产率49％。采用HPLC-DAD方法，面积归一化含量测定目标化合物的含量达到99.5％(见附图1、2)。目标化合物通过MS，NMR和X-Ray单晶衍射技术检测为目标化合物(见附图3-9)。
Figure PCTCN2019072178-appb-000004
N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺：无色针状或棱柱状结晶，熔点：116℃-118℃，HR-ESI-MS m/z 270.1465[M+H] +(计算值C 15H 21NNaO 2为270.1470), 1H-NMR(400MHz,Acetone-d 6):δ6.11(1H,dt,J＝12.0,4.0Hz,H-7),5.86(1H,dt,J＝8.0,4.0,Hz,H-6),2.49-2.43(1H,m,H-4),2.41-2.34(1H,m,H-4),2.39-2.36(2H,m,H-5),2.31-2.27(1H,m,H-12),2.17-2.11(1H,m,H-8),1.88-1.82(1H,m,H-8),1.35-1.31(2H,m.H-10),1.28-1.22(1H,m,1.27-1.25(1H,m,H-13),0.96-0.90(2H,m.H-9),0.88-0.85(3H,t,J＝8.0Hz,H-11),0.75-0.69(1H,m,H-14),0.64-0.58(1H,m,H-13),0.63-0.58(1H,m,H-14)； 13C-NMR(100MHz,Acetone-d 6):δ169.2(C-1), 152.6(C-3a),128.9(C-7a),128.3(C-6),117.4(C-7),92.4(C-3),33.9(C-8),25.9(C-9),22.6(C-10),21.1(C-12),13.7(C-11),5.1(C-14),2.7(C-13)。
晶胞参数：正交晶系，空间群P2 12 12 1，a＝7.8762(15)，b＝10.576(2)，c＝16.809(3)，α＝β＝γ＝90°，_晶胞体积V＝1400.1(5)，晶胞内分子数Z＝4，采用仪器：Bruker APEX II面检测仪，照射波长采用0.71073，放射源为MoKa，收集2876个独立衍射点，其中有效衍射点2037个，参加精修衍射点数目：2876；参加参数数目：165，对于全部衍射点的R 1值：0.0775；对于可观测衍射点的R 1值：0.0531；对于全部衍射点的wR 2值：0.1348；对于可观测衍射点的wR 2值：0.1203；对于可观测衍射点的S值：1.003；对于全部衍射点的S值：1.003；最后精修过程中的最大移动值：0.000。
2)N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺对映异构体的手性拆分及构型确定
将以上1)中合成的化合物500mg溶解在2mL正己烷和乙醇混合溶剂(V:V，90:10)中，利用三取代3,5-二甲基苯异氰酸酯衍生的纤维素手性涂覆型键合固定相为色谱柱，采用正相色谱系统(正己烷：乙醇，V:V，90:10)为流动相，连续液相色谱进行化合物手性拆分制备，收集7.623min和10.544min流份(见附图10)，减压回收溶剂，采用石油醚/丙酮溶剂系统培养单晶，进行X-Ray衍射确定化合物绝对立体构型，配制浓度为0.1mg/mL的目标化合物溶液进行通过圆二色谱(CD)(见附图11)和旋光测定，辅助进行化合物绝对构型确定。
色谱条件为：FMG-ACS-A01-NFC Chiral ND(2)(250mm×4.5mm,5μm)，流动相：正己烷：乙醇(V:V)＝90:10，检测波长：254nm。Tr＝7.623min为S-(+)-藁本环丙内酰胺占比49.2％，Tr＝10.544min为R-(-)-藁本环丙内酰胺，占比50.8％。通过手性色谱的R和S型化合物的面积归一化比例，可以确定藁本内酯与环丙烷的合成反应生成的手性产物为等量的对映异构体。
圆二色谱中S构型的N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺在254nm有明显的正cotton效应，而R构型的N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺在254nm有明显的负的cotton效应，两个化合物具有完全相反的CD谱特征吸收。R和S构型的N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺的旋光数据测定：(S)-N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺(N-环丙烷基-3-正丁基-3-S-羟基-藁本内 酰胺)的旋光值
Figure PCTCN2019072178-appb-000005
(R)-N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺(N-环丙烷基-3-正丁基-3-R-羟基-藁本内酰胺)的旋光值
Figure PCTCN2019072178-appb-000006
Figure PCTCN2019072178-appb-000007
实施例2：N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺的合成。
将正丁烯基苯酞(1.880g，0.010mol)(Alfa Aesar试剂公司产品，纯度95％)溶解在50mL的四氢呋喃中，环丙胺(0.684g，0.012mol)溶解在20mL的四氢呋喃中，常温机械搅拌下，将环丙胺的四氢呋喃溶液点滴加入到藁本内酯的四氢呋喃溶液中，搅拌反应6h，旋蒸减压回收四氢呋喃和过量的环丙胺溶液，将200mL石油醚加入到以上浓缩物中，混合均匀，放置过夜待合成产物结晶洗出，抽滤，得粗产物2.083克，反应产率85％，将粗产物用石油醚和丙酮(10:1，V/V)的混合溶剂重结晶，得到结晶产物1.666g，结晶产率80％。采用HPLC-DAD方法，面积归一化含量测定目标化合物的含量达到99.6％。目标化合物通过MS，NMR和X-Ray单晶衍射技术检测为目标化合物(见附图12，13)。按照实施例1的手性拆分方法，通过高效液相色谱的手性拆分确定该化合物为一对手性对映异构体(见图10)。
Figure PCTCN2019072178-appb-000008
N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺：无色针状或棱柱状结晶，HR-ESI-MS m/z 246.1492[M+H] +(计算值C 15H 20NO 2为246.1489), 1H-NMR(400MHz,Acetone-d 6):δ7.63(1H,d,J＝8.0Hz,H-7),7.60(1H,t,J＝8.0,4.0Hz,H-5),7.55(1H,d,J＝8.0Hz,H-4),7.49(1H,t,J＝8.0,4.0Hz,H-4),2.45-2.50(1H,td,J＝8.0,4.0Hz,H-8),2.38-2.38(1H,td,J＝12.0,4.0Hz,H-8),2.25-2.16(1H,td,J＝16.0,4.0Hz,H-9),2.05-2.07(1H,m,H-12),1.49-1.42(1H,m,H-9),1.30-1.18(2H,m,H-10),0.89-1.00(1H,m,H-13),0.82-0.87(1H,m,H-13),0.79(3H,t,J＝16.0,8.0Hz H-11),0.58-0.76(2H,m,H-14)； 13C-NMR(100MHz,Acetone-d 6):δ166.2,145.8,130.4,130.0,127.3,120.6,120.2,90.2,34.4,24.0,20.5,19.7,11.6,3.20,0.90。
实施例3：采用当归挥发油进行化合物N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺和化合物N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺二者混合物的合成
将制备得到的当归挥发油采用柱色谱法，以石油醚和乙酸乙酯(3:1，V/V)为洗脱剂进行预分离，将含藁本内酯较高部分的当归挥发油(3.000g)溶解在60mL的四氢呋喃中，环丙胺(0.684g，0.012mol)溶解在30mL的四氢呋喃中，机械搅拌下，将环丙胺的四氢呋喃溶液点滴加入到以上当归挥发油的四氢呋喃溶液中，搅拌反应10h，旋蒸减压回收四氢呋喃和过量的环丙胺溶液，将200mL石油醚加入到以上浓缩物中，混合均匀，放置过夜待合成产物结晶洗出，抽滤，得粗产物2.283克，反应产率76％，将粗产物用石油醚和丙酮(10:1，V/V)的混合溶剂重结晶，得到结晶产物1.872g，结晶产率82％。采用HPLC-DAD方法，面积归一化含量测定目标化合物的含量：化合物N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺的含量90.0％；化合物N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺的含量为8.9％(见图14)。
实施例4：采用川芎挥发油进行化合物N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺和化合物N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺二者混合物的合成
将制备得到的川芎挥发油采用柱色谱法，以石油醚和乙酸乙酯(3:1，V/V)为洗脱剂进行预分离，将含藁本内酯较高部分的川芎挥发油(3.500g，)溶解在60mL的四氢呋喃中，环丙胺(0.684g，0.012mol)溶解在30mL的四氢呋喃中，机械搅拌下，将环丙胺的四氢呋喃溶液点滴加入到以上川芎挥发油的四氢呋喃溶液中，搅拌反应10h，旋蒸减压回收四氢呋喃和过量的环丙胺溶液，将200mL石油醚加入到以上浓缩物中，混合均匀，放置过夜待合成产物结晶洗出，抽滤，得粗产物1.982克，反应产率56.7％，将粗产物用石油醚和丙酮(10:1，V/V)的混合溶剂重结晶，得到结晶产物1.685g，结晶产率85％。采用HPLC-DAD方法，面积归一化含量测定目标化合物的含量：化合物N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺的含量为91.2％；化合物N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺的含量为7.9％。
实施例5：采用当归挥发油进行N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺和化合物N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺混合物的合成制备
本实施例为考察温度对于反应产率和两个化合物相对含量的影响，因为藁本内酯不稳定，所以加热处理容易发生芳构化反应，减低N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺的生成量，增加N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺的生成量。
将制备得到的当归挥发油采用柱色谱法，以石油醚和乙酸乙酯(3:1，V/V)为洗脱剂进行预分离，将含藁本内酯较高部分的当归挥发油(3.000g，)溶解在60mL的四氢呋喃中，环丙胺(0.684g，0.012mol)溶解在30mL的四氢呋喃中，机械搅拌下，将环丙胺的四氢呋喃溶液点滴加入到以上当归挥发油的四氢呋喃溶液中，水浴加热至45℃，恒温搅拌反应10h，旋蒸减压回收四氢呋喃和过量的环丙胺溶液，将200mL石油醚加入到以上浓缩物中，混合均匀，放置过夜待合成产物结晶洗出，抽滤，得粗产物2.562克，反应产率85.4％，将粗产物用石油醚和丙酮(10:1，V/V)的混合溶剂重结晶，得到结晶产物2.050g，结晶产率80％。采用HPLC-DAD方法，面积归一化含量测定目标化合物的含量：化合物N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺的含量为70.0％；化合物N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺的含量为28.1％。
实施例6：采用当归挥发油进行N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺和化合物N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺单体化合物的合成制备
将制备得到的当归挥发油采用柱色谱法，以石油醚和乙酸乙酯(3:1，V/V)为洗脱剂进行预分离，将含藁本内酯较高部分的当归挥发油(3.000g，)溶解在60mL的四氢呋喃中，环丙胺(0.684g，0.012mol)溶解在30mL的四氢呋喃中，机械搅拌下，将环丙胺的四氢呋喃溶液点滴加入到以上当归挥发油的四氢呋喃溶液中，常温机械搅拌反应10h，旋蒸减压回收四氢呋喃和过量的环丙胺溶液，将200mL石油醚加入到以上浓缩物中，混合均匀，放置过夜待合成产物结晶洗出，抽滤，得粗产物2.302克，反应产率76.7％。
将粗产物用石油醚和乙酸乙酯(3:1，V/V)为洗脱液，进行柱色谱分离纯化，分别得到A、B和C三个组分，得到组份A(0.483g)，经薄层色谱鉴定为N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺；得到组份B(0.420g)经薄层色谱鉴定为化合物N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺和化合物N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺的混合物；得到组份C(1.128g)。经薄层色谱鉴定 为化合物N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺；采用HPLC-DAD方法，面积归一化进行目标化合物的含量测定：组份A为99.4％；组份B为：化合物N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺的含量为66.9％，化合物N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺的含量为36.5％；组份C为99.6％。
实施例7：采用当归和川芎的挥发油混合物进行化合物N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺和化合物N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺化合物的合成
将制备得到的当归和川芎的挥发油混合物采用柱色谱法，以石油醚和乙酸乙酯(3:1，V/V)为洗脱剂进行预分离，将含藁本内酯较高部分的挥发油混合物(3.000g，)溶解在60mL的四氢呋喃中，环丙胺(0.684g，0.012mol)溶解在30mL的四氢呋喃中，机械搅拌下，将环丙胺的四氢呋喃溶液点滴加入到以上当归挥发油的四氢呋喃溶液中，常温搅拌反应6h，旋蒸减压回收四氢呋喃和过量的环丙胺溶液，将200mL石油醚加入到以上浓缩物中，混合均匀，-20℃放置过夜待合成产物结晶洗出，抽滤，得粗产物2.602克，反应产率86.7％，将粗产物用石油醚和丙酮(10:1，V/V)的混合溶剂重结晶，得到结晶产物2.080g，结晶产率80％。采用HPLC-DAD方法，面积归一化含量测定目标化合物的含量：化合物N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺的含量为92.5％；化合物N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺的含量为7.5％(如图15所示)。
实施例8：苯酞类衍生物神经保护作用研究。
1、材料：PC12细胞(中科院上海细胞所)；苯酞衍生物(建议改为上述实施例制备)，高糖培养基(DMEM，南京凯基生物科技发展有限公司)；二甲基亚砜(DMSO，Sigma公司)；胎牛血清(Gibco，赛默飞世尔科技有限公司)；H 2O 2(南京化学试剂股份有限公司)；噻唑蓝(MTT，南京生兴生物技术有限公司)，酶标仪(美国Bio-rad公司)。
2、方法：PC12细胞培养，取PC12细胞接种于高糖DMEM培养基中，培养基中含有10％胎牛血清、青霉素100U mL -1，链霉素100μg mL -1，置于37℃、5％的CO 2细胞培养箱中培养，隔日换液，常规胰蛋白酶消化传代；待细胞长至70-80％汇合时用于实验。
3、苯酞及其衍生物对PC12细胞增殖的影响：取对数生长期的PC12细胞以1×10 4细胞/孔密度接种于96孔细胞培养板中，孵育24h后，加入100μl苯酞及其衍生物溶液，使其终浓度分别为1.25、2.5、5.0、10、20、40、80、160μM，继续培养24h，加入100μl的MTT(5mg mL -1)，孵育4h后，加入150μl的DMSO，振荡10min后，选择490nm波长，检测各孔吸光度(A)值。
细胞存活率(％)＝(A 实验组–A 空白组)/(A 对照组-A 空白组)×100％。
结果显示200μM以上的藁本内酯，藁本内酯衍生物，正丁烯苯酞、正丁烯苯酞衍生物才会对PC12细胞的生长受到抑制。
4、氧化损伤模型建立：取对数生长期的PC12细胞以5×10 3细胞/孔密度接种于96孔细胞培养板中，加入不同浓度的H 2O 2，培养4h，按以上MTT测定法项下操作加入MTT，测定各孔A值，最终选择降低细胞存活率50％的H 2O 2浓度为造模浓度。
结果显示250μM的H 2O 2可以使50％的PC12细胞生长受到抑制。故选择250μM作为神经细胞氧化受损的诱导浓度。
5、分组及药物处理：实验分组为正常空白对照组、H 2O 2模型组(250μM)、苯酞类及其衍生物。(2.5μM，5.0μM，10μM，25μM，50μM)浓度组、阳性对照组(丁苯酞)。苯酞类及其衍生物各组和阳性对照组分别加入H 2O 2 250μM。实验首先分别加入苯酞类及其衍生物溶液，使其终浓度达到相应值，4h后加入H 2O 2，24h后加入MTT，检测A值，计算细胞存活率。
研究结果见附图16，图中“ **”表示该结果与阳性对照比较有统计学较显著性差异P<0.01％，图中“ *”表示该结果与阳性对照比较有统计学显著性差异P<0.05％。图中可以看出，根据实施例1制备的藁本内酯衍生物(外消旋混合物)的对于PC12神经细胞的过氧化氢保护作用最强，在最低实验浓度2.5μM时，可以使细胞活度提高80％，相较于模型组的52％和阳性药物对照组的68％，该化合物可以显著保护过氧化氢对于PC12的氧化损伤。藁本内酯衍生物的浓度在10μM时，细胞活度最高，达到95％。其次藁本内酯的活性较正丁烯苯酞对于神经细胞的氧化损伤保护作用较强；藁本内酯与根据实施例2制备的正丁烯苯酞衍生物(外消旋混合物)对于神经细胞的氧化损伤保护作用几乎相同。
实施例9：苯酞类衍生物对急性缺血性脑卒中的脑梗塞抑制作用研究
1、实验动物：四周龄Wistar雄性大鼠(230-250g)由中国农业科学院兰州兽医研究所提供。
2、动物模型制备：将按标准采购的75只大鼠，严格清洁级饲养，参考文献方法(Xiaomei Wu,Zhongming Qian,Li Zhu,Fang Du,Wingho Yung,Qi Gong and Ya Ke,Neuroprotective effect of ligustilide against ischaemia-reperfusion injury via up-regulation of erythropoietin and down-regulation of RTP801,British J Pharm.2011,164,332-343.)，以线栓建立MCAO模型(大鼠大脑中动脉阻塞)，用10％水合氯醛腹腔注射，按剂量400mg/kg麻醉大鼠，用仰卧位方法固定，将颈部暴露，皮肤剃毛清洁后开始切皮，轻柔操作并分离出左侧颈总动脉、颈内动脉、颈外动脉等主要血管，选择颈外动脉结扎后离断，避免颈总动脉出血，以动脉夹暂时夹闭颈总动脉，选择颈外动脉末端处，轻轻剪一小口并作为线栓置入处，准备好的线栓圆头用眼科镊置，轻柔置入，控制深度约1.8-2.0cm，常规切口缝合，脑缺血0.5h后，拔出线栓恢复血供。给药后一定时间，处死大鼠，取脑组织采用0.5％的2,3,5-三苯基氯化四氮唑(TCC)染色，扫描计算脑梗死面积，正常组织染色为红色，梗塞组织染色后为白色。
3、动物分组：按模型组为急性缺血缺氧病理模型，给与等剂量8％的吐温-80的生理盐水，假手术组(不给药，作为阴性对照，给与等剂量8％的吐温-80的生理盐水)，预防给药组(提前3天给药)和治疗给药组(栓塞恢复血供后给药3天)分组。
4、给药方法：腹腔注射藁本内酯衍生物(苯酞衍生物：根据实施例1制备，HPLC-DAD面积归一化法测定N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺的含量达到98％，采用含8％的吐温-80的生理盐水溶解)，剂量：40mg/Kg。
5、结果：从附图17可以看出，大鼠在急性脑缺血模型建模以后给与N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺，相较于模型组(A组)可以有效抵抗脑梗死的发生，具有很好的治疗效果(B组)。但预防性给药(C组)由于该类化合物的扩张血管活性使得大鼠的劲总动脉血管发生扩张，使得造模动物不可平行，故容易造成造模动物死亡，使得该组生存动物的抗栓塞效果相对较弱。A、阴性模型对照，梗死面积30.57％；B、N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺治疗组，(造模后给药3天，40mg/kg，腹腔注射)，左侧梗死面积4％，中间完全再通(梗死面积0％)，右侧按照去水肿的方法计算为负值；C、N-环丙烷基-3-正丁 基-3-羟基-藁本内酰胺预防治疗组，(预给药3天，造模，再给药3天；40mg/kg，腹腔注射)，梗死面积为22％。
实施例10：苯酞衍生物大鼠体内药代动力学研究
1.实验方法：
1.1化合物的定量检测方法学建立，采用HPLC-DAD标准曲线法，确定方法的检测限(LOD)、定量限(LOQ)和线性范围。
分别精确称量一定量的N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺和N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺标准品，甲醇溶解稀释得2.0mg/mL的标准储备溶液。分别精密量取一定的标准储备溶液混合后用甲醇稀释至0.1953、0.395、0.7813、1.5625、3.1250、6.2500、12.500、25.0000、50.0000和500.0μg/mL。各个样品取20uL进HPLC系统分析，依次测定N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺和N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺的峰面积(A)并与浓度C(μg/mL)进行线性回归，得回归方程及线性范围、检测限(LOD)、定量限(LOQ)。
表1 HPLC-DAD检测方法的检测线、定量限和线性范围
Figure PCTCN2019072178-appb-000009
1.2供试样品制备方法：按10％的乙醇，20％的吐温80，70％的生理盐水将实施例7制备的化合物(N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺的含量为92.5％；化合物N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺的含量为7.5％)制备成浓度为15mg/mL的原始储备溶液，根据大鼠体重给药，口服给药(灌胃)剂量90mg/Kg，静脉注射给药剂量按40mg/Kg。
1.3血浆样品前处理方法：取大鼠全血0.5mL置于有20uL肝素钠的1.5mL离心管中，转速4×10 3r﹒min -1下高速离心5min，取血浆200μL，加入800μL的二氯甲烷，涡旋混匀后离心10min，转速为1×10 5r﹒min -1，取下层溶液并在室温下氮气吹干，用300μL甲醇复溶，洗脱过滤用氮气吹干后，加入30μL的甲醇，离心后取20μL上清液进HPLC测定峰面积。
1.4苯酞类衍生物大鼠体内药代动力学参数测定
取12只雄性Wistar大鼠随机均分为两组，其中6只为静脉注射给药组(i.v.)，给予40mg/kg的苯酞类衍生物。在给药后0(给药前)、0.167、0.333、0.5、1、2、3、4和6h时分别眼眦取血约500μL，抗凝并离心后取200μL血浆冻存。灌胃给药(po)组6只大鼠给予90mg/kg的苯酞类衍生物，在给药后0(给药前)、0.5、1、2、3、4、6、8、10和12h时分别眼眦取血约500μL，抗凝并离心后取200μL血浆冻存。将血浆样品恢复至室温后进行前处理，取20μL样品溶液进液相色谱分析，根据标准曲线计算各时间点的血药浓度，利用DAS2.0软件拟合出药时曲线并计算药动学参数。图18和19分别显示了灌胃给药药时曲线和静脉注射给药药时曲线。
表2 灌胃和静脉注射N-环丙烷基-3-正丁基-3-羟基-藁本内酰胺(藁本环丙内酰胺)和N-环丙烷基-3-正丁基-3-羟基-苯酞内酰胺(正丁环丙内酰胺)时化合物药代动力学参数
Figure PCTCN2019072178-appb-000010
Figure PCTCN2019072178-appb-000011
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (10)

  1. 式I或式II的苯酞类衍生物，其旋光异构体或药用盐，或它们的混合物
    Figure PCTCN2019072178-appb-100001
  2. 如权利要求1所述的苯酞类衍生物，其为N-环丙烷基-3-正丁基-3-S-羟基-藁本内酰胺、N-环丙烷基-3-正丁基-3-R-羟基-藁本内酰胺、N-环丙烷基-3-正丁基-3-S-羟基-苯酞内酰胺或N-环丙烷基-3-正丁基-3-R-羟基-苯酞内酰胺。
  3. 一种制备苯酞类衍生物的方法，包括将苯酞类化合物与环丙胺在有机溶剂中反应，其中苯酞类化合物中原有的内酯结构片段被内酰胺基团所替换，C3位被羟基取代。
  4. 如权利要求3所述的方法，其中
    1)所述苯酞类化合物为C3位正丁烯基取代的苯环型、二氢苯环型或四氢苯环型苯酞类化合物，优选藁本内酯或正丁烯苯酞，其中所述藁本内酯或正丁烯苯酞为单独分离的化合物形式或者处于当归属或藁本属植物提取物中的混合物形式，更优选所述植物提取物为当归挥发油提取物或川芎挥发油提取物或它们的混合物；
    2)反应在-20℃至60℃下进行，优选在搅拌下进行；
    3)有机溶剂为非极性有机溶剂，优选选自环己烷、石油醚、四氢呋喃、乙醚；和/或
    4)所述方法还包括手性拆分对映异构体的步骤，优选通过手性色谱法或手性重结晶法。
  5. 如权利要求3所述的制备方法，其中所述苯酞类化合物与环丙胺的摩尔比为1:1～1.2。
  6. 如权利要求3所述的制备方法，所述方法还包括减压回收有机溶剂和重结晶得到目标产物的步骤，优选所述重结晶所用溶剂为石油醚、乙酸乙酯、丙酮和乙醚中的一种或两种。
  7. 如权利要求3所述的方法，包括将苯酞类化合物加入有机溶剂溶解，温度控制在-20℃至60℃，加入有机溶剂溶解的环丙胺反应液，控制温度在-20℃至60℃，搅拌反应1-24小时，减压回收有机溶剂，经重结晶得到目标产物。
  8. 通过权利要求3至7中任一项所述的方法制备的如权利要求1或2所述的式I或式II的苯酞类衍生物，其旋光异构体或药用盐，或它们的混合物。
  9. 如权利要求1或2所述的式I或式II的苯酞类衍生物、其旋光异构体或药用盐或它们的混合物在制备药物中的应用，所述药物用作抗氧化剂和/或用于治疗或预防以下疾病：心脑血管疾病，抑郁症，阿尔茨海默症，(神经)炎性疾病，疼痛，神经元细胞损伤，缺血再灌注损伤，脑梗塞，认知缺陷或脑损伤。
  10. 如权利要求9所述的应用，其中所述药物包含式I化合物和式II化合物的混合物作为有效成分，其中式I化合物在混合物中的质量百分含量为1-100％，优选90％以上。
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