WO2017036392A1 - 一种线叶旋覆花内酯a衍生物 - Google Patents

一种线叶旋覆花内酯a衍生物 Download PDF

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WO2017036392A1
WO2017036392A1 PCT/CN2016/097481 CN2016097481W WO2017036392A1 WO 2017036392 A1 WO2017036392 A1 WO 2017036392A1 CN 2016097481 W CN2016097481 W CN 2016097481W WO 2017036392 A1 WO2017036392 A1 WO 2017036392A1
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acid
compound
dimethylamine
acetyl
formula
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French (fr)
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张卫东
孙青龑
李明花
杨保华
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Shanxi Zhendong Leading Biotechnology Co Ltd
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Shanxi Zhendong Leading Biotechnology Co Ltd
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Priority to US15/755,558 priority Critical patent/US10273220B2/en
Priority to CN201680028895.7A priority patent/CN107849002B/zh
Priority to EP16840821.9A priority patent/EP3345896A4/en
Publication of WO2017036392A1 publication Critical patent/WO2017036392A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D307/93—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems condensed with a ring other than six-membered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2013—Organic compounds, e.g. phospholipids, fats
    • A61K9/2018—Sugars, or sugar alcohols, e.g. lactose, mannitol; Derivatives thereof, e.g. polysorbates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A61K9/2059—Starch, including chemically or physically modified derivatives; Amylose; Amylopectin; Dextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • the present invention relates to medicinal chemistry, relating to dimethylamine 4-O-acetyl baseline inulinate A or a salt thereof and its use in pharmaceuticals, in particular, dimethylamine 4-O-acetyl baseline leaf rotation Intralactone A or a salt thereof and its preparation and use in the preparation of a medicament for treating multiple sclerosis.
  • MS Multiple sclerosis
  • the disease is a chronic, autoimmune disease that affects normal nerve transmission by destroying the myelin, a neurofibrous protective layer of the brain, spinal cord, and optic nerve, which causes physical disability.
  • the disease has become one of the most common and important neurological diseases due to its high recurrence rate and disability rate, chronic disease course and prone to young people.
  • the peak incidence of multiple sclerosis is around 30 years old. Therefore, it will seriously endanger the productivity and quality of life of the young and middle-aged population, and also impose a great burden on the state and the government, restricting social and economic development.
  • Glucocorticoids are mainly administered in the acute phase, and the main therapeutic drugs in the relapsing-remission phase include interferon-gamma, glatiramer acetate and the monoclonal antibody natalizumab. Severe patients can also consider autologous stem cell transplantation. Although Western medicine's hormone therapy can alleviate acute symptoms, it has obvious side effects, so it is not recommended for long-term use.
  • the immunomodulators interferon IFN- ⁇ , GA and natalizumab are not only expensive, but also long-term use for a single target. Drugs are prone to tolerance.
  • Inula lineariifolia Turcz. (syn. Inula linariaefolia) was extracted from the experimental autoimmune encephalomyelitis (EAE) model in mice.
  • EAE experimental autoimmune encephalomyelitis
  • This model is an ideal model for multi-sclerotherapy drug research, so the inventors have applied for the Chinese patent: 20120208157.4 "Application of the linear inulin a lactone A in the preparation of a medicament for treating multiple sclerosis".
  • the oral bioavailability of the linear inulinate A is low, and the inventors have structurally modified it to prepare a derivative of the linear inflorescence A: dimethylamine 4- O-acetyl baseline leaf inflorescence A.
  • the lactone ring will hydrolyze, and the carboxyl group formed after hydrolysis will form a lactone with the hydroxyl group at the 4-position, thereby forming the isomerization of the linear inflorescence A.
  • the body reduces the bioavailability of the linear inflorescence A. Therefore, the free hydroxyl group at the 4-position of the inflorescence A is first acetylated.
  • the ⁇ -methyl olefin- ⁇ -butyrolactone structural unit on the structure of the inflorescence A of the linear leaf is very unstable, and soon after entering the blood, it binds to cysteine, and the binding is stable. The bioavailability of the linear inflorescence A is decreased.
  • the test proves that the derivative has a good therapeutic effect in the experimental autoimmune encephalomyelitis (EAE) model in mice and has the effect of treating multiple sclerosis. It can be used for multiple sclerosis drugs; Compared with the inulinate A phase, the oral bioavailability is improved and further development is recommended.
  • EAE experimental autoimmune encephalomyelitis
  • the technical problem to be solved by the present invention is to overcome the above-mentioned deficiencies, and to study the design of the linear inflorescence A derivative and its application in pharmacy.
  • the present invention provides a dimethylamine 4-O-acetyl base leaf inflorescence A compound, which is represented by Formula I:
  • the present invention also provides a pharmaceutically acceptable salt of the dimethylamine 4-O-acetyl base leaf inflorescence A compound, as shown in Formula II:
  • the dimethylamine 4-O-acetyl base leaf inulinate A compound is pharmaceutically acceptable
  • the salt received was formed from dimethylamine 4-O-acetyl baseline leaf inflorescence A and HX.
  • HX is hydrochloric acid, sulfuric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, sulfurous acid, nitric acid, carbonic acid, boric acid, selenous acid, phosphomolybdic acid, phosphorous acid, citric acid, maleic acid, D- Malic acid, L-malic acid, DL-malic acid, D-lactic acid, L-lactic acid, DL-lactic acid, oxalic acid, sulfonic acid, benzenesulfonic acid, substituted benzenesulfonic acid, 1-naphthalenesulfonic acid, 2-naphthalenesulfonic acid , citric acid, tartaric acid, malonic acid, succinic acid, fumaric acid, citric acid, orotic acid, benzoic acid or substituted benzoic acid.
  • a pharmaceutically acceptable salt of the dimethylamine 4-O-acetyl base leaf inulinate A compound, preferably dimethylamine 4-O-acetyl base leaf inflorescence A hydrochloride salt III , dimethylamine 4-O-acetyl base leaf indola lactone A fumarate IV, nitrate V, hydrobromide VI, sulfate VII, orotate VIII, citrate IX, pair Tosylate X, succinate XI, most preferably hydrochloride III and fumarate IV.
  • Another object of the present invention is to provide a process for the preparation of the dimethylamine 4-O-acetyl base leaf inflorescence A compound and a pharmaceutically acceptable salt thereof.
  • the ratio of the amount of the alkali leaf and the acetic anhydride Ac 2 O is 1.0 eq: 1.0 to 2.0 eq: 1.0 to 2.0 eq.
  • the organic reagent refers to a reagent which is immiscible with water, such as one of ethyl acetate, dichloromethane, chloroform, carbon tetrachloride, diethyl ether, petroleum ether, n-butanol, benzene, toluene or tetrahydrofuran or Several mixed reagents, preferably dichloromethane.
  • the base is a lower trialkyl substituted amine such as trimethylamine, triethylamine, tripropylamine, tributylamine or pyridine or substituted pyridine, preferably p-lutidine.
  • the organic solvent is a lower aliphatic alcohol such as one or more combinations of methanol, ethanol, propanol, isopropanol, dichloromethane, chloroform, carbon tetrachloride, diethyl ether, petroleum ether or ethyl acetate.
  • ethanol preferably, ethanol.
  • the organic reagent refers to a reagent that is immiscible with water, such as one or more of ethyl acetate, dichloromethane, chloroform, carbon tetrachloride, diethyl ether, petroleum ether, n-butanol, benzene, toluene or tetrahydrofuran.
  • a mixed reagent preferably dichloromethane.
  • the base is a lower trialkyl substituted amine, and the alkyl group is a methyl group, an ethyl group, a propyl group, a butyl group.
  • pyridine substituted pyridine, preferably triethylamine.
  • the ratio of the amount of the dimethylamine 4-O-acetyl base leaf inflorescence A compound I to the acid HX is 1.0 eq: 1.0 to 1.5 eq.
  • the HX is hydrochloric acid, sulfuric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, sulfurous acid, nitric acid, carbonic acid, boric acid, selenous acid, phosphomolybdic acid, phosphorous acid, citric acid, maleic acid, D -malic acid, L-malic acid, DL-malic acid, D-lactic acid, L-lactic acid, DL-lactic acid, oxalic acid, sulfonic acid, benzenesulfonic acid, substituted benzenesulfonic acid, 1-naphthalenesulfonic acid, 2-naphthalenesulfonic acid Acid, citric acid, tartaric acid, malonic acid, succinic acid, fumaric acid, citric acid, orotic acid, benzoic acid or substituted benzoic acid, hydrochloric acid, fumaric acid, nitric acid, hydrobromic acid, sulfuric acid, whey Acid, citric
  • the solvent is a lower aliphatic alcohol such as one or more of methanol, ethanol, propanol, isopropanol, dichloromethane, chloroform, carbon tetrachloride, diethyl ether, petroleum ether or ethyl acetate. In combination, ethanol is preferred.
  • the preparation method is as follows:
  • the compound of the formula IV is beaten with methyl tert-butyl ether containing 0.2% of water for 2-5 hours, suction-filtered, vacuum-dried at 40-60 ° C, 0.03-0.1 MPa under reduced pressure for 4-24 h to obtain crystal form D.
  • the preparation method is as follows:
  • the compound of the formula IV is dissolved in 1-2 times methanol, stirring is continued, 2-5 times of diethyl ether is added to precipitate a large amount of crystals, and the mixture is dried under vacuum at 25-40 ° C, 0.03-0.1 MPa for 2-24 hours to obtain a compound of the F crystal form. IV.
  • the preparation method is as follows:
  • the compound of the formula I is dissolved in 2 times the amount of ethanol, and an equivalent amount of fumaric acid is added under stirring, stirring is continued for 0.5-2 h, 1-2 times the amount of methyl tert-butyl ether is added, crystals are precipitated, and suction filtration is carried out.
  • the tert-butyl ether was rinsed and dried under vacuum at 25-40 ° C to obtain a compound of formula G.
  • the inventors used the EAE animal model to carry out the EAE animal model efficacy test of dimethylamine 4-O-acetyl baseline leaf inulin a lactone A compound I. Because the EAE animal model is an ideal animal model for routine use in human multiple sclerosis (MS) in pharmacodynamic tests, it is commonly used in the study of mechanisms of immune activation and immunosuppression.
  • MS multiple sclerosis
  • the effect of Compound I on the experimental autoimmune encephalomyelitis (EAE) model in mice showed that the administration of Compound I and its fumarate IV, respectively, can significantly reduce the incidence of EAE (see figure 5. Figure 6).
  • the inflammatory cells in the brain tissue of EAE mice were infiltrated, the blood vessels and tissues were loosely arranged, and the edges were not clear.
  • the preparation of the medicament for treating multiple sclerosis according to the invention is dimethylamine 4-O-B
  • the acyl cord inulinate compound I or its salt II is used as a pharmaceutical composition prepared from a conventional pharmaceutical carrier as an active ingredient.
  • the pharmaceutical composition is a tablet, a dispersible tablet, a lozenge, an orally disintegrating tablet, a sustained release tablet, a capsule, a soft capsule, a dropping pill, a granule, an injection, a powder injection or an aerosol.
  • the dimethylamine 4-O-acetyl baseline inulinate A compound of the present invention has higher oral bioavailability than the linear inulinate A compound.
  • the invention provides a new medicine for the research and development of the treatment of multiple sclerosis, has a good clinical application prospect, and has greater social benefits.
  • Figure 1 is a powder X-ray diffraction pattern of Form A of the compound of Formula IV.
  • Figure 2 is a powder X-ray diffraction pattern of Form D of the compound of Formula IV.
  • Figure 3 is a powder X-ray diffraction pattern of Form F of the compound of Formula IV.
  • Figure 4 is a powder X-ray diffraction pattern of Form G of the compound of Formula IV.
  • Fig. 5 Changes in the incidence of EAE in mice after intragastric administration of Compound I (dose: 30 mg/kg), the ordinate is “EAE score”, and the abscissa is "day”.
  • Fig. 6 Changes in the incidence of EAE in mice after intragastric administration of Compound I (dose: 15 mg/kg), the ordinate is “EAE score”, and the abscissa is "day”.
  • FIG. 7 H&E staining of mouse brain tissue sections (1 blank group, 2EAE+solvent group, 3EAE+ Compound group I) and spinal cord LFB staining (4 blank group, 5EAE + solvent group, 6EAE + compound I group) (dose: 30 mg/kg).
  • FIG. 8 Mouse spinal cord tissue sections H&E staining (1 blank group, 2EAE + solvent group, 3EAE + compound IV group) and spinal cord LFB staining (4 blank group, 5EAE + solvent group, 6EAE + compound IV group) (dose: 15 mg/kg).
  • the obtained compound is firstly determined by mass spectrometry, molecular weight 366, molecular formula C 19 H 26 O 7 , and then subjected to nuclear magnetic resonance analysis to obtain carbon spectrum, hydrogen spectrum and two-dimensional spectrum data, and structural analysis is performed, and the known compound is in the line The data for ester A was consistently confirmed).
  • Example 2 The compound I obtained in Example 1 (10.0 g, 0.022 mol) was dissolved in 200 mL of anhydrous methanol, and passed through a standard HCl gas (500 mL), and stirred at room temperature (25 ° C) for half an hour, 40 ° C, 0.03 to 0.1 MPa. Concentration under pressure gave compound III (10.7 g, 0.022 mol).
  • Example 2 The compound I obtained in Example 1 (10.0 g, 0.022 mol) was dissolved in 200 mL of anhydrous methanol, and analytically purified fumaric acid (2.6 g, 0.022 mol) was added, and the reaction was stirred at room temperature (25 ° C) for half an hour, 40 ° C Methanol was concentrated under reduced pressure from 0.03 to 0.1 MPa to give Compound IV (12.6 g, 0.022 mol).
  • Example 2 The compound I (100 mg, 0.22 mmol) obtained in Example 1 was dissolved in 10 mL of ethyl acetate, and 1 mol/L hydrobromic acid solution (0.22 mL, 0.22 mmol) was added, and the reaction was stirred at room temperature (25 ° C) for half an hour. The ethyl acetate was concentrated under reduced pressure at 40 ° C, 0.03 to 0.1 MPa to give Compound VI (117 mg, 0.22 mmol).
  • Example 2 The compound I obtained in Example 1 (100 mg, 0.22 mmol) was dissolved in 10 mL of ethyl acetate, 1% sulfuric acid solution (0.6 mL, 0.11 mmol) was added, and the reaction was stirred at room temperature (25 ° C) for half an hour, 40 ° C, The ethyl acetate was concentrated under reduced pressure of 0.03 to 0.1 MPa to give Compound VII (110 mg, 0.22 mmol).
  • Example 2 The compound I (100 mg, 0.22 mmol) obtained in Example 1 was dissolved in 10 mL of ethyl acetate. citric acid (43 mg, 0.22 mmol) was added under stirring, and the reaction was stirred at room temperature (25 ° C) for half an hour, 40 ° C, 0.03 ° Ethyl acetate was concentrated under reduced pressure of 0.1 MPa to give Compound VII (143 mg, 0.22 mmol).
  • Example 5 The compound of the formula IV obtained in Example 5 was dissolved in 20 mL of ethyl acetate and continuously stirred. Next, 50 mL of n-hexane was added, a large amount of crystals were precipitated, and suction-filtered, and dried under reduced pressure at 40 ° C, 0.03 to 0.1 MPa to obtain crystal form A (8.5 g).
  • Example 5 The compound of the formula IV obtained in Example 5 was dissolved in 20 mL of methanol, and 50 mL of diethyl ether was added thereto with stirring, and a large amount of crystals were precipitated, and suction-filtered, and dried under reduced pressure at 40 ° C, 0.03 to 0.1 MPa for 4 hours to obtain crystal form A (8.9 g).
  • the compound of formula I (10.0 g) was dissolved in 2 mL of ethanol, and fumaric acid (2.6 g) was added with stirring. Stirring was continued for 0.5-2 h, 20 mL of methyl tert-butyl ether was added, crystals were precipitated, and the mixture was filtered. The tert-butyl ether was rinsed and dried under vacuum at 25-40 ° C for 2 h to obtain a crystal form of G (10.6 g).
  • the mouse experimental autoimmune encephalomyelitis (EAE) model is an ideal animal model for human multiple sclerosis (MS) and is commonly used for mechanisms of immune activation and immunosuppression.
  • mice C57BL/6 mice, MOG35-55 300 ⁇ g/head, Mycobacterium tuberculosis H37Ra 400 ⁇ g/only, emulsified, 100 ⁇ l, subcutaneous injection; pertussis toxin PT 500 ng/only, 200 ⁇ l intraperitoneal injection on day 0 and day 2. Animals were grouped into groups of 6 each.
  • Formula compound I45.3g (prepared in Example 1) was suspended in 0.5% CMC-Na (carboxymethylcellulose sodium) to prepare a solution of 3 mg/ml, and was administered once a day from the 0th day of immunization. The dose was 30 mg/kg and was administered continuously for 30 days.
  • CMC-Na carboxymethylcellulose sodium
  • mice were administered by single gavage at 200 mg/kg, 600 mg/kg, 1800 mg/kg, and 5400 mg/kg, respectively, with 6 rats in each dose group, half male and half female.
  • the mice in the 200 mg/kg and 600 mg/kg groups had no obvious toxicity, one female mouse died in the 1800 mg/kg group, and 5 mice died in the 5400 mg/kg group.
  • the results show that Compound I is safer.
  • Compound I can be used to prepare a medicament for treating multiple sclerosis.
  • a sample of the compound A of the formula IV was dissolved in water for injection to prepare a solution of 1.5 mg/mL, which was administered once a day from the 0th day of immunization, at a dose of 15 mg/kg, for 30 days.
  • SD rats were intragastrically administered with compound of formula IV, 75, 150, 300 mg/kg. Each dose group consisted of 3 males and 3 females, once a day for 7 consecutive days. No toxic side effects were observed, indicating that the compound of formula IV is safe. .
  • Preparation method compound I, lactose and corn starch are mixed, uniformly moistened with water, the wetted mixture is sieved and dried, sieved, magnesium stearate is added, and then the mixture is tableted, each tablet weighs 300 mg, containing compound I25mg.
  • Preparation method Compound III and glucose were dissolved in 100 L of water for injection, and the resulting solution was filtered, and placed in an infusion bottle (100 ml per bottle) under aseptic conditions, and each bottle contained 5 mg of compound III.
  • Example 21 Preparation of lyophilized powder for injection
  • Preparation method Compound IV and mannitol were dissolved in 2 L of water for injection, and the resulting solution was filtered, and placed in a vial (10 ml vial, 2 ml per bottle) under aseptic conditions, and lyophilized, each containing 10 mg of compound IV.

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Abstract

一种线叶旋覆花内酯A衍生物，尤其是二甲基胺4-O-乙酰基线叶旋覆花内酯A或其盐及其制备与在制备治疗多发性硬化症药物中的应用。所述二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物，如式Ⅰ所示。

Description

一种线叶旋覆花内酯A衍生物 技术领域
本发明涉及药物化学，涉及二甲基胺4-O-乙酰基线叶旋覆花内酯A或其盐及其在制药中的应用，尤其涉及二甲基胺4-O-乙酰基线叶旋覆花内酯A或其盐及其制备与在制备治疗多发性硬化症药物中的应用。
背景技术
多发性硬化症(multiple sclerosis，MS)于1868年由法国医生Charcot首次提出，至今仍是整个医学界未解的难题之一。该病是一种慢性、自身免疫性疾病，通过破坏脑、脊髓以及视神经的神经纤维保护层髓磷脂，影响正常的神经传递而导致身体残疾。由于该病具有极高的复发率和致残率、呈慢性病程和倾向于年轻人罹患，已成为常见的、重要的神经系统疾病之一。另外，据统计，多发性硬化症的发病高峰在30岁左右，因此，会严重危害青壮年人口的生产力和生活质量，也给国家和政府造成极大负担，制约了社会和经济发展。
目前，临床上对多发性硬化症无特效治疗方法，缺乏令人满意的治疗药物。急性期主要给予糖皮质激素，复发-缓解期主要治疗药物包括:干扰素-γ，醋酸格拉替雷和单克隆抗体那他珠单抗。重度患者还可以考虑自体干细胞移植。西医的激素疗法虽然能够缓解急性期症状，但毒副作用明显，故不推荐长期使用；免疫调节剂干扰素IFN-β、GA和那他珠单抗等不仅费用昂贵，且长期使用针对单一靶点药物容易出现耐受。且上述药物均需经静脉、或注射给药，不能有效改善 MS反复复发和髓鞘脱失的根本问题，也即对于多发性硬化症无特效治疗方法，缺乏令人满意的治疗药物。因此，亟需要积极研发能有效控制多发性硬化症的复发、改善髓鞘脱失的口服药物，这具有十分重要的意义。而传统中医药具有多靶点、系统调节的作用特点，已在一些疑难杂症的治疗上显示了一定的优势。故而，针对多发性硬化症的复杂性、难治性疾病，探索有效的中药也是学者们研究的方向。
线叶旋覆花内酯A提取自线叶旋覆花[Inula lineariifolia Turcz.(syn.Inula linariaefolia)]，在小鼠实验性自身免疫性脑脊髓炎(EAE)模型有较好的治疗作用，该模型是多发硬化症药物研究的理想模型，因此本发明人已申请了中国专利：20120208157.4“线叶旋覆花内酯A在制备治疗多发硬化症药物中的应用”。然而进一步研究发现线叶旋覆花内酯A口服生物利用度较低，本发明人对其进行了结构修饰，制备了一种线叶旋覆花内酯A衍生物：二甲基胺4-O-乙酰基线叶旋覆花内酯A。线叶旋覆花内酯A在弱碱性环境下，内酯环会水解，水解后形成的羧基，会与4位的羟基形成内酯，从而形成线叶旋覆花内酯A的异构体，降低了线叶旋覆花内酯A的生物利用度，因此首先将线叶旋覆花内酯A结构上4位的游离羟基乙酰化。此外，线叶旋覆花内酯A结构上的α-甲基烯-γ-丁内酯结构单元很不稳定，入血后，很快与半胱氨酸结合，而且这种结合很稳定，降低了线叶旋覆花内酯A的生物利用度。通过将α-甲基烯-γ-丁内酯与二甲基胺反应，会使双键打开形成二甲基胺衍生物，该衍生物在中性和弱碱性条件下，会缓慢脱去二甲基胺，再形成α-甲基烯-γ-丁内酯，发挥药效。
试验证明：该衍生物在小鼠实验性自身免疫性脑脊髓炎(EAE)模型亦有较好的治疗作用具有治疗多发性硬化症的功效，可以用于多发性硬化症药物；并且与线叶旋覆花内酯A相比较，提高了口服生物利用度，宜作进一步研发。
发明内容
本发明所要解决的技术问题在于克服上述不足之处，研究设计线叶旋覆花内酯A衍生物及其在制药中的应用。
本发明提供一种二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物，以式Ⅰ所示：
Figure PCTCN2016097481-appb-000001
本发明还提供了所述二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物药学上可接受的盐，以式Ⅱ所示：
Figure PCTCN2016097481-appb-000002
所述二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物药学上可接 受的盐由二甲基胺4-O-乙酰基线叶旋覆花内酯A与HX生成。
其中HX为盐酸、硫酸、氢氟酸、氢溴酸、氢碘酸、磷酸、亚硫酸、硝酸、碳酸、硼酸、亚硒酸、磷钼酸、亚磷酸、柠檬酸、马来酸、D-苹果酸、L-苹果酸、DL-苹果酸、D-乳酸、L-乳酸、DL-乳酸、草酸、磺酸，苯磺酸、取代苯磺酸、1-萘磺酸、2-萘磺酸、酞酸、酒石酸、丙二酸、丁二酸、富马酸、枸橼酸、乳清酸、苯甲酸或取代苯甲酸。
所述二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物药学上可接受的盐，优选为二甲基胺4-O-乙酰基线叶旋覆花内酯A盐酸盐Ⅲ、二甲基胺4-O-乙酰基线叶旋覆花内酯A富马酸盐Ⅳ、硝酸盐V、氢溴酸盐VI、硫酸盐VII、乳清酸盐VIII、柠檬酸盐IX、对甲苯磺酸盐X、丁二酸盐XI，最优选为盐酸盐Ⅲ和富马酸盐Ⅳ。
Figure PCTCN2016097481-appb-000003
Figure PCTCN2016097481-appb-000004
Figure PCTCN2016097481-appb-000005
本发明的另一目的是提供所述二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物及其药学上可接受的盐的制备方法。
(一)制备二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物，该方法包括下列步骤：
(1)取线叶旋覆花内酯A，碱和乙酸酐Ac2O置于反应瓶中，加入有机溶剂溶解，0～50℃搅拌12～24h，TLC监测待原料反应完全后，加水淬灭，取有机层；
(2)有机层再加水萃取多次，直至有机层TLC检测反应产物4-O- 乙酰基线叶旋覆花内酯A中无其他杂质，弃去水层，有机层再用饱和食盐水洗涤，无水硫酸钠(Na2SO4)干燥，0.01～0.1MPa，20～80℃减压浓缩至原体积的1/10～1/20；
(3)浓缩液硅胶柱层析，以体积比为15:1～5:1的石油醚/乙酸乙酯系统梯度洗脱，TLC检测，得4-O-乙酰基线叶旋覆花内酯A；
以下列反应式表示：
Figure PCTCN2016097481-appb-000006
(4)取4-O-乙酰基线叶旋覆花内酯A和二甲胺盐酸盐(CH3NHCH3·HCl)置于反应瓶中，加入有机溶剂溶解，反应瓶置于-20～0℃冷却槽中，慢慢加入碱，然后将反应体系缓慢升至室温(25-30℃)，搅拌2～12h，TLC监测反应，待原料反应完全，0.01～0.1MPa20～80℃减压蒸除溶剂，得粗品，加有机试剂溶解，再加水萃取，弃去水层，重复此步骤，至有机层TLC监测反应产品二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物无其他杂质，有机层用饱和食盐水洗涤，无水硫酸钠(Na2SO4)干燥，硅胶柱层析，以体积比为15:1～1:1的石油醚/乙酸乙酯系统梯度洗脱，薄层层析检测，收集含化合物Ⅰ的洗脱液，0.01～0.1MPa，20～80℃减压蒸除溶剂，得化合物Ⅰ，
以下列反应式表示：
Figure PCTCN2016097481-appb-000007
本发明方法所述步骤(1)线叶旋覆花内酯A，碱和乙酸酐Ac2O的用量比为1.0eq：1.0～2.0eq：1.0～2.0eq。
所述有机试剂，指和水不互溶的试剂，如乙酸乙酯、二氯甲烷、三氯甲烷、四氯化碳、乙醚、石油醚、正丁醇、苯、甲苯或四氢呋喃中的一种或几种混合试剂，优选二氯甲烷。
所述碱为低级三烷基取代胺，如三甲基胺、三乙基胺、三丙基胺、三丁基胺或吡啶或取代吡啶，优选对二甲基吡啶。
所述步骤(4)4-O-乙酰基线叶旋覆花内酯A：二甲胺盐酸盐：碱的用量比为1.0eq：1.0～2.0eq：1.0～2.0eq。
所述有机溶剂为低级脂肪醇，如甲醇、乙醇、丙醇、异丙醇、二氯甲烷、三氯甲烷、四氯化碳、乙醚、石油醚或乙酸乙酯中的一种或多种组合，优选乙醇。
所述有机试剂指和水不互溶的试剂，如乙酸乙酯、二氯甲烷、三氯甲烷、四氯化碳、乙醚、石油醚、正丁醇、苯、甲苯或四氢呋喃中的一种或几种混合试剂，优选二氯甲烷。
所述碱为低级三烷基取代胺，烷基为甲基、乙基、丙基、丁基， 和吡啶、取代吡啶，优选三乙基胺。
(二)制备二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物的盐化合物Ⅰ(1.0eq)溶于溶剂中，与酸HX(1.0～1.5eq)，在0～50℃下搅拌反应2～24h，0.01～0.1MPa，20～80℃减压浓缩，得式Ⅱ的盐；
以下列反应式表示：
Figure PCTCN2016097481-appb-000008
上述方法中，所述二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物Ⅰ与酸HX的用量比为：1.0eq：1.0～1.5eq。
所述HX为盐酸、硫酸、氢氟酸、氢溴酸、氢碘酸、磷酸、亚硫酸、硝酸、碳酸、硼酸、亚硒酸、磷钼酸、亚磷酸、柠檬酸、马来酸、D-苹果酸、L-苹果酸、DL-苹果酸、D-乳酸、L-乳酸、DL-乳酸、草酸、磺酸，苯磺酸、取代苯磺酸、1-萘磺酸、2-萘磺酸、酞酸、酒石酸、丙二酸、丁二酸、富马酸、枸橼酸、乳清酸、苯甲酸或取代苯甲酸，盐酸、富马酸、硝酸、氢溴酸、硫酸、乳清酸、柠檬酸、对甲苯磺酸、丁二酸，最优选为盐酸和富马酸。
所述溶剂为低级脂肪醇，如甲醇、乙醇、丙醇、异丙醇、二氯甲烷、三氯甲烷、四氯化碳、乙醚、石油醚或乙酸乙酯中的一种或多种 组合，优选乙醇。
本发明的目的还在于提供二甲基胺4-O-乙酰基线叶旋覆花内酯A富马酸盐IV的A、D、F、G晶型及其制备方法。
所述式IV的A晶型具有以下特征：
其X-射线粉末衍射图2θ、d值和相对强度如下表所示：
Figure PCTCN2016097481-appb-000009
Figure PCTCN2016097481-appb-000010
其粉末X-射线衍射图见图1。
通过将式IV化合物溶于2-5倍量的乙酸乙酯，加入2-5倍量的正己烷，不断搅拌，至大量晶体析出，过滤，25-40℃减压真空干燥，获得A晶型的化合物IV。
所述式IV的D晶型具有以下特征：
其X-射线粉末衍射图2θ、d值和相对强度如下表所示：
Figure PCTCN2016097481-appb-000011
Figure PCTCN2016097481-appb-000012
其粉末X-射线衍射图见图2
制备方法如下：
将式IV化合物用含0.2％水分的甲基叔丁基醚打浆2-5h，抽滤，40-60℃，0.03-0.1MPa减压真空干燥4-24h，得晶型D。
所述式IV的F晶型具有以下特征：
其X-射线粉末衍射图2θ、d值和相对强度如下表所示：
Figure PCTCN2016097481-appb-000013
Figure PCTCN2016097481-appb-000014
其粉末X-射线衍射图见图3.
制备方法如下：
将式IV化合物溶于1-2倍甲醇，不断搅拌，加入2-5倍量乙醚至大量晶体析出，25-40℃，0.03-0.1MPa真空减压干燥2-24h，获得F晶型的化合物IV。
所述式IV的G晶型具有以下特征：
其X-射线粉末衍射图2θ、d值和相对强度如下表所示：
Figure PCTCN2016097481-appb-000015
其粉末X-射线衍射图见图4
制备方法如下：
将式I化合物溶于2倍量的乙醇，搅拌下加入等当量的富马酸，继续搅拌0.5-2h，加入1-2倍量的甲基叔丁基醚，析出晶体，抽滤，用甲基叔丁基醚淋洗，25-40℃真空干燥，获得G晶型式IV化合物。
本发明的又一目的是提供所述二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物及其药学上可接受的盐在制备治疗多发性硬化症药物中的应用。
本发明人采用EAE动物模型，将二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物Ⅰ进行EAE动物模型药效试验。因为EAE动物模型是药效试验中常规使用于人类多发性硬化(MS)的理想动物模型，常用于免疫激活和免疫抑制方面的机制研究。通过化合物Ⅰ对小鼠实验性自身免疫性脑脊髓炎(EAE)模型的作用，试验结果表明：化合物Ⅰ和其富马酸盐IV分别灌胃给药，可明显降低EAE的发病程度(见图5、图6)。模型组EAE小鼠脑组织炎症细胞浸润，血管、组织排列松散，边缘不清晰；给药组炎症细胞减少，血管、组织基本恢复正常状态(见图7、图8)，化合物Ⅰ及其富马酸盐灌胃给药能较好的阻止EAE的发病过程,提示该化合物对多发性硬化症有较好的治疗作用，说明二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物Ⅰ或其盐Ⅱ可用于制备治疗多发性硬化症的药物。
本发明所述制备治疗多发性硬化症的药物为由二甲基胺4-O-乙 酰基线叶旋覆花内酯A化合物Ⅰ或其盐Ⅱ作为活性成分与常规药用载体制成的药物组合物。
所述药物组合物为片剂、分散片、含片、口崩片、缓释片、胶囊剂、软胶囊剂、滴丸、颗粒剂、注射剂、粉针剂或气雾剂等。
本发明的二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物比线叶旋覆花内酯A具有更高的口服生物利用度。本发明为治疗多发性硬化症研发提供了新的药物，有较好的临床应用前景，有较大的社会效益。
附图说明
图1式IV化合物晶型A的粉末X-射线衍射图。
图2式IV化合物晶型D的粉末X-射线衍射图。
图3式IV化合物晶型F的粉末X-射线衍射图。
图4式IV化合物晶型G的粉末X-射线衍射图。
图5灌胃给予化合物Ⅰ后小鼠EAE发病评分变化(剂量：30mg/kg)，纵坐标为“EAE评分”，横坐标为“天”。
(三条线从上到下依次为：EAE+溶剂组、EAE+化合物Ⅰ组、空白组)。
图6灌胃给予化合物Ⅰ后小鼠EAE发病评分变化(剂量：15mg/kg)，纵坐标为“EAE评分”，横坐标为“天”。
(三条线从上到下依次为：EAE+溶剂组、EAE+化合物ⅠV组、空白组)。
图7小鼠脑组织切片H&E染色(1空白组、2EAE+溶剂组、3EAE+ 化合物Ⅰ组)和脊髓LFB染色(4空白组、5EAE+溶剂组、6EAE+化合物Ⅰ组)(剂量：30mg/kg)。
图8小鼠脊髓组织切片H&E染色(1空白组、2EAE+溶剂组、3EAE+化合物ⅠV组)和脊髓LFB染色(4空白组、5EAE+溶剂组、6EAE+化合物ⅠV组)(剂量：15mg/kg)。
具体实施方式
以下实施例使用原料和试剂市售得到。
实施例1线叶旋覆花内酯A的制备
将线叶旋覆花干燥全草50Kg粉碎，以90％乙醇750L回流提取2次，每次2小时，合并提取液，提取液减压浓缩成流浸膏，流浸膏相当于1ml含线叶旋覆花1.0g，流浸膏加水50L稀释后，以石油醚每次50L萃取5次，得到石油醚部分。将石油醚部分应用硅胶柱层析，以体积比为100：0～1：1的石油醚/乙酸乙酯系统梯度洗脱，薄层层析检测，收集含线叶旋覆花内酯A的流分，再经C18反相柱层析，以重量比为50：100-70：100的甲醇/水进行洗脱，薄层层析检测，得线叶旋覆花内酯A纯品45.3g。
(得到的化合物先采用质谱测定分子量366，分子式C19H26O7，再进行核磁共振分析得到碳谱、氢谱以及二维谱数据，进行结构解析，与已知化合物线叶旋覆花内酯A的数据一致得到确证)。
实施例2 4-O-乙酰基线叶旋覆花内酯A的制备
取线叶旋覆花内酯A(10.0g,0.027mol，1.0eq)，对二甲氨基吡啶(DMAP)(4.0g,0.033mol,1.2eq)和乙酸酐(Ac2O)(3.34g,0.033 mol,1.2eq)置于500mL茄形瓶中，加入200mL无水二氯甲烷(CH2Cl2)，室温搅拌12h。薄层层析监测反应情况，待原料反应完全后，加水200mL淬灭，置分液漏斗中，弃去水层，有机层再加水100mL萃取，弃去水层，重复此步骤，直至有机层中检测不出DMAP(TLC法)，有机层再用饱和食盐水洗涤，无水硫酸钠(Na2SO4)干燥，40℃以下减压浓缩，硅胶柱层析，以体积比为15:1～5:1的石油醚/乙酸乙酯系统梯度洗脱，薄层层析检测，得4-O-乙酰基线叶旋覆花内酯A(10.5g,0.026mol)，产率94％。检测证实为该产品：
1H NMR(500MHz,Chloroform-d)δ6.14(dt,J＝3.8,1.1Hz,1H),5.51(dd,J＝3.2,1.6Hz,1H),5.26–5.19(m,1H),5.15(dt,J＝8.6,1.6Hz,1H),4.91(t,J＝7.8Hz,1H),4.49–4.42(m,1H),3.01–2.93(m,1H),2.43–2.36(m,1H),2.08–2.06(m,3H),2.03(q,J＝1.2Hz,3H),2.00–1.93(m,6H),1.89(dd,J＝12.0,5.9Hz,1H),1.48–1.38(m,1H),1.08(d,J＝1.2Hz,3H),0.97(dd,J＝6.4,1.4Hz,3H)；13C NMR(125MHz,Chloroform-d)δ170.7,170.3,169.7,168.9,137.9,121.2,76.1,75.0,73.7,73.6,52.4,50.3,50.3,43.9,34.2,29.8,21.2,21.1,20.9,20.3,17.3.
实施例3二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物Ⅰ的制备
取上述4-O-乙酰基线叶旋覆花内酯A(10.0g,0.024mol,1.0eq)，二甲胺盐酸盐(CH3NHCH3·HCl)(3.0g,0.037mol,1.5eq)置于500mL茄形瓶中，加入200mL乙醇溶解，反应瓶置于低温冷却槽中，0℃时，慢慢滴加三乙胺(NEt3)(5.1mL,0.037mol,1.5eq)，然后将反应体 系慢慢升至室温(25℃)，搅拌，薄层层析监测反应，4h后，原料反应完全，减压蒸除溶剂，得粗品，粗品加200mL二氯甲烷溶解，加水100mL萃取，弃去水层，重复此步骤，直至有机层检测不出二甲胺盐酸盐。有机层用饱和食盐水洗涤，无水硫酸钠(Na2SO4)干燥，硅胶柱层析，以体积比为15:1～1:1的石油醚/乙酸乙酯系统梯度洗脱，薄层层析检测，得到二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物Ⅰ(10.0g,0.022mol)，产率90％。
1H-NMR(500MHz,Chloroform-d)δ5.27(dd,J＝10.7,8.6Hz,1H),5.04(d,J＝9.5Hz,1H),4.92(ddd,J＝9.2,6.8,2.1Hz,1H),4.40(ddd,J＝11.7,10.1,2.8Hz,1H),2.69–2.61(m,2H),2.53(dd,J＝9.3,6.5Hz,1H),2.45(dd,J＝13.1,5.3Hz,1H),2.36(ddd,J＝13.2,4.5,2.8Hz,1H),2.18(s,6H),2.08–2.05(m,1H),2.03(s,4H),2.01(s,3H),1.97(s,3H),1.96–1.87(m,2H),1.40(dt,J＝13.1,11.9Hz,1H),1.12(s,3H),0.97(d,J＝6.5Hz,3H)；13C-NMR(125MHz,Chloroform-d)δ176.6,170.4,170.3,169.5,77.2,76.2,76.1,74.9,74.3,57.4,50.0,50.0,48.7,46.9,46.3,43.8,34.3,29.4,21.1,21.1,20.8,20.16,18.5。
实施例4化合物Ⅲ的制备
实施例1制得的化合物Ⅰ(10.0g，0.022mol)，溶于200mL无水甲醇中中，通入标准HCl气体500mL，室温(25℃)搅拌反应半小时，40℃，0.03～0.1MPa减压浓缩得化合物Ⅲ(10.7g，0.022mol)。
1H-NMR(500MHz,D2O)δ5.24–5.16(m,2H),5.02(td,J＝6.8,4.5Hz,1H),4.93–4.84(m,1H),3.68(dd,J＝13.4,11.5Hz,1H),3.45 (td,J＝11.4,4.0Hz,1H),3.37(s,1H),2.99(dd,J＝13.3,4.0Hz,7H),2.74(d,J＝11.5Hz,1H),2.44–2.36(m,1H),2.19(s,3H),2.16(d,J＝6.7Hz,1H),2.14(d,J＝1.0Hz,3H),2.13–2.07(m,2H),2.08(s,3H),1.53(q,J＝12.2Hz,1H),1.22(s,3H),0.99(d,J＝6.5Hz,3H)；13C NMR(125MHz,D2O)δ177.20,172.43,171.0(2×C),79.2,77.2(2×C),75.9,75.2,57.4,49.8,49.8(2×C),49.4,42.3,41.9,33.5,28.5,20.9,20.57,20.6,19.5,17.4。
实施例5化合物Ⅳ的制备
实施例1制得的化合物Ⅰ(10.0g，0.022mol)，溶于200mL无水甲醇中，加入分析纯富马酸(2.6g，0.022mol)，室温(25℃)搅拌反应半小时，40℃，0.03～0.1MPa减压浓缩除去甲醇得化合物Ⅳ(12.6g，0.022mol)。
1H-NMR(500MHz,D2O)δ6.72(d,J＝1.1Hz,2H),5.23–5.16(m,2H),5.00(td,J＝6.8,4.5Hz,1H),4.90–4.84(m,1H),3.66(dd,J＝13.4,11.5Hz,1H),3.44(td,J＝11.4,4.0Hz,1H),3.36(s,1H),2.98(dd,J＝13.3,4.0Hz,7H),2.72(d,J＝11.5Hz,1H),2.42–2.36(m,1H),2.17(s,3H),2.14(d,J＝6.7Hz,1H),2.12(d,J＝1.0Hz,3H),2.11–2.07(m,2H),2.06(s,3H),1.50(q,J＝12.2Hz,1H),1.20(s,3H),0.98(d,J＝6.5Hz,3H)；13C-NMR(125MHz,D2O)δ177.18,173.61,173.50,172.41,170.9(2×C),134.8(2×C),79.0,77.0(2×C),75.7,75.0,57.2,49.6,49.6(2×C),49.2,42.1,41.7,33.3,28.3,20.7,20.5,20.4,19.3,17.2.
实施例6化合物V的制备方法
实施例1制得的化合物Ⅰ(100mg，0.22mmol)，溶于10mL乙酸乙酯中，加入1％的硝酸溶液(1mL，0.22mmol)，室温(25℃)搅拌反应半小时，40℃，0.03～0.1MPa减压浓缩除去乙酸乙酯得化合物V(113mg，0.22mmol)。
1H NMR(400MHz,(CD3)2SO)8.92(s,1H),5.03-5.10(m,2H),4.89-4.94(m,1H),4.65-4.71(m,1H),3.46-3.52(m,1H),3.36-3.39(m,1H),2.81-2.87(m,4H),2.73(s,3H),2.63(q,J＝10.0Hz,1H),2.18-2.21(m,1H),2.05(s,3H),2.02(s,3H),1.83-1.99(m,7H),1.41(q,J＝11.6Hz,1H),1.09(s,3H),0.89(d,J＝5.6Hz,3H).
实施例7化合物VI的制备方法
实施例1制得的化合物Ⅰ(100mg，0.22mmol)，溶于10mL乙酸乙酯中，加入1mol/L的氢溴酸溶液(0.22mL，0.22mmol)，室温(25℃)搅拌反应半小时，40℃，0.03～0.1MPa减压浓缩除去乙酸乙酯得化合物VI(117mg，0.22mmol)。
1H NMR(400MHz,(CD3)2SO)8.96(br,1H),5.01-5.09(m,2H),4.91-4.94(m,1H),4.66-4.72(m,1H),3.32-3.40(m,2H),2.59-2.85(m,8H),2.18-2.21(m,1H),2.05(s,3H),2.02(s,3H),1.83-1.99(m,7H),1.41(q,J＝12.0Hz,1H),1.10(s,3H),0.89(d,J＝5.6Hz,3H).
实施例8化合物VII的制备方法
实施例1制得的化合物Ⅰ(100mg，0.22mmol)，溶于10mL乙酸乙酯中，加入1％的硫酸溶液(0.6mL，0.11mmol)，室温(25℃)搅拌反应半小时，40℃，0.03～0.1MPa减压浓缩除去乙酸乙酯得化合物VII(110mg，0.22mmol)。
1H NMR(400MHz,(CD3)2SO)8.92(s,1H),5.01-5.09(m,2H),4.91-4.94(m,1H),4.66-4.72(m,1H),4.52(br,1H),3.47-3.53(m,1H),3.34-3.40(m,1H),2.79-2.88(m,4H),2.73(s,3H),2.63(q,J＝10.0Hz,1H),2.18-2.21(m,1H),2.05(s,3H),2.02(s,3H),1.83-1.99(m,7H),1.41(q,J＝11.6Hz,1H),1.10(s,3H),0.89(d,J＝5.6Hz,3H).
实施例9化合物VIII的制备方法
实施例1制得的化合物Ⅰ(100mg，0.22mmol)，溶于10mL乙酸乙酯中，搅拌下加入乳清酸(35mg，0.22mmol)，室温(25℃)搅拌反应半小时，40℃，0.03～0.1MPa减压浓缩除去乙酸乙酯得化合物VII(135mg，0.22mmol)。
1H NMR(400MHz,(CD3)2SO)11.05(s,1H),9.93(s,1H),5.83(s,1H),5.02-5.07(m,2H),4.90-4.93(m,1H),4.61-4.67(m,1H),3.68(br,1H),3.06-3.14(m,2H),2.79(d,J＝11.6Hz,1H),2.65(q,J＝10.0Hz,1H),2.56(s,6H),2.18-2.22(m,1H),2.02(s,3H),2.00(s,3H),1.82-1.98(m,7H),1.38(q,J＝12.0Hz,1H),1.09(s,3H),0.88(d,J＝6.0Hz,3H).
实施例10化合物IX的制备方法
实施例1制得的化合物Ⅰ(100mg，0.22mmol)，溶于10mL乙酸乙酯中，搅拌下加入柠檬酸(43mg，0.22mmol)，室温(25℃)搅拌反应半小时，40℃，0.03～0.1MPa减压浓缩除去乙酸乙酯得化合物VII(143mg，0.22mmol)。
1H NMR(400MHz,(CD3)2SO)11.44(br,1H),8.33(br,1H),5.97(d,J＝3.2Hz,1H),5.35(d,J＝2.8Hz,1H),5.01-5.10(m,2H),4.91-4.95 (m,1H),4.64-4.70(m,1H),3.22-3.29(m,2H),2.55(s,6H),2.54(dd,J＝31.2,15.2Hz,4H),2.17-2.22(m,1H),2.11(s,2H),2.03(s,3H),2.02(s,3H),1.91-2.01(m,7H),1.52(q,J＝12.0Hz,1H),1.07(s,3H),0.89(d,J＝6.4Hz,3H).
实施例11化合物X的制备方法
实施例1制得的化合物Ⅰ(100mg，0.22mmol)，溶于10mL乙酸乙酯中，搅拌下加入无水对甲苯磺酸(38mg，0.22mmol)，室温(25℃)搅拌反应半小时，40℃，0.03～0.1MPa减压浓缩除去乙酸乙酯得化合物VII(138mg，0.22mmol)。
1H NMR(400MHz,(CD3)2SO)8.92(s,1H),7.49(d,J＝8.0Hz,2H),7.12(d,J＝8.0Hz,2H),5.01-5.09(m,2H),4.91-4.94(m,1H),4.66-4.72(m,1H),3.47-3.53(m,1H),3.33-3.40(m,1H),2.81-2.88(m,4H),2.73(s,3H),2.63(q,J＝10.0Hz,1H),2.29(s,3H),2.17-2.20(m,1H),2.04(s,3H),2.02(s,3H),1.83-1.98(m,7H),1.41(q,J＝11.6Hz,1H),1.09(s,3H),0.88(d,J＝5.6Hz,3H)。
实施例12化合物XI的制备方法
实施例1制得的化合物Ⅰ(100mg，0.22mmol)，溶于10mL乙酸乙酯中，搅拌下加入丁二酸(26mg，0.22mmol)，室温(25℃)搅拌反应半小时，40℃，0.03～0.1MPa减压浓缩除去乙酸乙酯得化合物VII(126mg，0.22mmol)。
实施例13式IV化合物晶型A的制备
将实施例5制得的式IV化合物，溶于20mL乙酸乙酯中，不断搅拌 下，加入50mL正己烷，析出大量晶体，抽滤，40℃，0.03～0.1MPa减压干燥得晶型A(8.5g)。
实施例14式IV化合物晶型D的制备
向实施例5制得的式IV化合物中加入30mL甲基叔丁基醚，25℃打浆3h，45℃，0.03-0.1MPa减压真空4h干燥得晶型D(9.1g).
实施例15式IV化合物晶型F的制备
将实施例5制得的式IV化合物，溶于20mL甲醇中，不断搅拌加入50mL乙醚，析出大量晶体，抽滤，40℃，0.03～0.1MPa减压干燥4h得晶型A(8.9g)。
实施例16式IV化合物晶型G的制备
将式I化合物(10.0g)溶于2mL的乙醇中，搅拌下加入富马酸(2.6g)，继续搅拌0.5-2h，加入20mL甲基叔丁基醚，析出晶体，抽滤，用甲基叔丁基醚淋洗，25-40℃真空干燥2h，获得G晶型(10.6g)。
实施例17化合物Ⅰ的动物药效试验
小鼠实验性自身免疫性脑脊髓炎(EAE)模型是人类多发性硬化(MS)的理想动物模型，常用于免疫激活和免疫抑制方面的机制研究。
17.1EAE造模方法
C57BL/6小鼠，MOG35-55 300μg/只，结核分支杆菌H37Ra 400μg/只，乳化，100μl，皮下注射；百日咳毒素PT 500ng/只，第0天和第2天腹腔注射200μl。动物分组，每组6只。
17.2给药方式
式化合物Ⅰ45.3g(实施例1制得)悬于0.5％的CMC-Na(羧甲基纤维素钠)配制成3mg/ml的溶液中，从免疫第0天开始每天给药1次，给药剂量30mg/kg，连续给药30天。
17.3实验结果
30mg/kg·d-1(30mg/kg每天1次)化合物Ⅰ灌胃给药，可明显延迟EAE的发病时间，降低EAE的发病程度(见图5)。模型组EAE小鼠脊髓组织炎症细胞浸润，血管、组织排列松散，边缘不清晰；给药组炎症细胞减少，血管、组织基本恢复正常状态；给药组髓鞘脱失情况明显减轻。(见图6)。
上述实验重复进行3次。效果同上所述。
17.4毒性
小鼠分别以200mg/kg、600mg/kg、1800mg/kg、5400mg/kg单次灌胃给药，每个剂量组6只，雌雄各半。200mg/kg、600mg/kg组小鼠无明显毒性反应，1800mg/kg组死亡一只雌性小鼠，5400mg/kg组死亡5只小鼠。结果显示化合物Ⅰ安全性较好。
17.5结论
化合物Ⅰ灌胃给药能较好的阻止EAE的发病过程。提示该化合物对多发性硬化症有较好的治疗作用。说明化合物Ⅰ可用于制备治疗多发性硬化症的药物。
实施例18式IV化合物的动物药效试验
18.1造模方式
同实施例17
18.2给药方式
将式IV化合物A晶型样品溶于注射用水中，制成1.5mg/mL的溶液，从免疫第0天开始每天给药1次，给药剂量15mg/kg，连续给药30天。
18.3实验结果
15mg/kg·d-1(15mg/kg每天1次)化合物ⅠV灌胃给药，可明显延迟EAE的发病时间，降低EAE的发病程度(见图7)。模型组EAE小鼠脊髓组织炎症细胞浸润，血管、组织排列松散，边缘不清晰；给药组炎症细胞减少，血管、组织基本恢复正常状态；给药组髓鞘脱失情况显著减轻(见图8)。
18.4毒性
SD大鼠灌胃给予式IV化合物，75、150、300mg/kg，每个剂量组雌雄各3只，每天给药一次，连续7天，未见任何毒副反应，说明式IV化合物安全性良好。
18.5结论
化合物IV灌胃给药能较好的阻止EAE的发病过程。提示该化合物对多发性硬化症有较好的治疗作用。说明化合物IV可用于制备治疗多发性硬化症的药物。
实施例19片剂制备
Figure PCTCN2016097481-appb-000016
Figure PCTCN2016097481-appb-000017
制备方法：化合物Ⅰ、乳糖和玉米淀粉混合，用水均匀湿润，把湿润后的混合物过筛并干燥，再过筛，加入硬脂酸镁，然后将混合物压片，每片重300mg，含式化合物Ⅰ25mg。
实施例20：注射液制备
实施例2制得的化合物Ⅲ     5g
葡萄糖                    50g
制备方法：将式化合物Ⅲ和葡萄糖溶解于100L注射用水中，过滤所得溶液，在无菌条件下装入输液瓶(每瓶100ml)中，每瓶含化合物Ⅲ5mg。
实施例21：注射用冻干粉针剂制备
实施例3制得的化合物Ⅳ    10g
甘露醇                   30g
制备方法：将化合物Ⅳ和甘露醇溶解于2L的注射用水中，过滤所得溶液，在无菌条件下装入西林瓶(10ml西林瓶，每瓶2ml)中，冻干，每支含化合物Ⅳ10mg。

Claims (19)

  1. 一种二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物，如式Ⅰ所示：
    Figure PCTCN2016097481-appb-100001
  2. 一种二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物药学上可接受的盐，如式Ⅱ所示：
    Figure PCTCN2016097481-appb-100002
    所述二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物药学上可接受的盐由二甲基胺4-O-乙酰基线叶旋覆花内酯A与HX生成。
  3. 根据权利要求2所述的一种二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物药学上可接受的盐，其特征在于，所述HX为盐酸、硫酸、氢氟酸、氢溴酸、氢碘酸、磷酸、亚硫酸、硝酸、碳酸、硼酸、亚硒酸、磷钼酸、亚磷酸、柠檬酸、马来酸、D-苹果酸、L-苹果酸、DL-苹果酸、D-乳酸、L-乳酸、DL-乳酸、草酸、磺酸，苯磺酸、取代苯磺酸、1-萘磺酸、2-萘磺酸、酞酸、酒石酸、丙二酸、丁二酸、富马酸、枸橼酸、乳清酸、苯甲酸或取代苯甲酸。
  4. 根据权利要求2所述的一种二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物药学上可接受的盐，其特征在于，优选为二甲基胺4-O-乙酰基线叶旋覆花内酯A盐酸盐Ⅲ、二甲基胺4-O-乙酰基线叶旋覆花内酯A富马酸盐Ⅳ、硝酸盐V、氢溴酸盐VI、硫酸盐VII、乳清酸盐VIII、柠檬酸盐IX、对甲苯磺酸盐X或丁二酸盐XI；结构式如下所示：
    Figure PCTCN2016097481-appb-100003
    Figure PCTCN2016097481-appb-100004
    Figure PCTCN2016097481-appb-100005
  5. 根据权利要求4所述的一种二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物药学上可接受的盐，其特征在于，更优选为盐酸盐Ⅲ或富马酸盐Ⅳ。
  6. 一种二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物及其药学上可接受的盐的制备方法，包括如下步骤：
    (一)制备二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物，该方法包括下列步骤：
    (1)取线叶旋覆花内酯A，碱和乙酸酐Ac2O置于反应瓶中，加入有机溶剂溶解，0～50℃搅拌12～24h，TLC监测待原料反应完全后，加水淬灭，取有机层；
    (2)有机层再加水萃取多次，直至有机层TLC检测反应产物4-O-乙酰基线叶旋覆花内酯A中无其他杂质，弃去水层，有机层再用饱和食盐水洗涤，无水硫酸钠(Na2SO4)干燥，0.01～0.1MPa，20～80℃ 减压浓缩至原体积的1/10～1/20；
    (3)浓缩液硅胶柱层析，以体积比为15:1～5:1的石油醚/乙酸乙酯系统梯度洗脱，TLC检测，得4-O-乙酰基线叶旋覆花内酯A；
    以下列反应式表示：
    Figure PCTCN2016097481-appb-100006
    (4)取4-O-乙酰基线叶旋覆花内酯A和二甲胺盐酸盐(CH3NHCH3·HCl)置于反应瓶中，加入有机溶剂溶解，反应瓶置于-20～0℃冷却槽中，慢慢加入碱，然后将反应体系缓慢升至室温(25-30℃)，搅拌2～12h，TLC监测反应，待原料反应完全，0.01～0.1MPa20～80℃减压蒸除溶剂，得粗品，加有机试剂溶解，再加水萃取，弃去水层，重复此步骤，至有机层TLC监测反应产品二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物无其他杂质，有机层用饱和食盐水洗涤，无水硫酸钠(Na2SO4)干燥，硅胶柱层析，以体积比为15:1～1:1的石油醚/乙酸乙酯系统梯度洗脱，薄层层析检测，收集含化合物Ⅰ的洗脱液，0.01～0.1MPa，20～80℃减压蒸除溶剂，得化合物Ⅰ，
    以下列反应式表示：
    Figure PCTCN2016097481-appb-100007
    (二)制备二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物的盐；化合物Ⅰ(1.0eq)溶于溶剂中，与酸HX(1.0～1.5eq)，在0～50℃下搅拌反应2～24h，0.01～0.1MPa，20～80℃减压浓缩，得式Ⅱ的盐；以下列反应式表示：
    Figure PCTCN2016097481-appb-100008
  7. 根据权利要求6所述的一种二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物及其药学上可接受的盐的制备方法，其特征在于：
    所述(一)制备二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物中，所述步骤(1)线叶旋覆花内酯A，碱和乙酸酐Ac2O的用量比为1.0eq：1.0～2.0eq：1.0～2.0eq；
    所述有机试剂，指和水不互溶的试剂，如乙酸乙酯、二氯甲烷、三氯甲烷、四氯化碳、乙醚、石油醚、正丁醇、苯、甲苯或四氢呋喃 中的一种或几种混合试剂，优选二氯甲烷；
    所述碱为低级三烷基取代胺，如三甲基胺、三乙基胺、三丙基胺、三丁基胺或吡啶或取代吡啶，优选对二甲基吡啶；
    所述步骤(4)4-O-乙酰基线叶旋覆花内酯A：二甲胺盐酸盐：碱的用量比为1.0eq：1.0～2.0eq：1.0～2.0eq；
    所述有机溶剂为低级脂肪醇，如甲醇、乙醇、丙醇、异丙醇、二氯甲烷、三氯甲烷、四氯化碳、乙醚、石油醚或乙酸乙酯中的一种或多种组合，优选乙醇；
    所述有机试剂指和水不互溶的试剂，如乙酸乙酯、二氯甲烷、三氯甲烷、四氯化碳、乙醚、石油醚、正丁醇、苯、甲苯或四氢呋喃中的一种或几种混合试剂，优选二氯甲烷；
    所述碱为低级三烷基取代胺，烷基为甲基、乙基、丙基、丁基，和吡啶、取代吡啶，优选三乙基胺。
  8. 根据权利要求6所述的一种二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物及其药学上可接受的盐的制备方法，其特征在于：所述(二)制备二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物的盐的步骤中，所述二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物Ⅰ与酸HX的用量比为：1.0eq：1.0～1.5eq；
    所述HX为盐酸、硫酸、氢氟酸、氢溴酸、氢碘酸、磷酸、亚硫酸、硝酸、碳酸、硼酸、亚硒酸、磷钼酸、亚磷酸、柠檬酸、马来酸、D-苹果酸、L-苹果酸、DL-苹果酸、D-乳酸、L-乳酸、DL-乳酸、草酸、磺酸，苯磺酸、取代苯磺酸、1-萘磺酸、2-萘磺酸、酞酸、酒 石酸、丙二酸、丁二酸、富马酸、枸橼酸、乳清酸、苯甲酸或取代苯甲酸，盐酸、富马酸、硝酸、氢溴酸、硫酸、乳清酸、柠檬酸、对甲苯磺酸或丁二酸，优选为盐酸或富马酸；
    所述溶剂为低级脂肪醇，如甲醇、乙醇、丙醇、异丙醇、二氯甲烷、三氯甲烷、四氯化碳、乙醚、石油醚或乙酸乙酯中的一种或多种组合，优选乙醇。
  9. 一种二甲基胺4-O-乙酰基线叶旋覆花内酯A富马酸盐IV的A晶型，所述式IV的A晶型具有以下特征：
    其X-射线粉末衍射图2θ、d值和相对强度如下表所示：
    Figure PCTCN2016097481-appb-100009
    Figure PCTCN2016097481-appb-100010
    其粉末X-射线衍射图见图1。
  10. 一种二甲基胺4-O-乙酰基线叶旋覆花内酯A富马酸盐IV的A晶型的制备方法，其特征在于，通过将式IV化合物溶于2-5倍量的乙酸乙酯，加入2-5倍量的正己烷，不断搅拌，至晶体析出，过滤，25-40℃减压真空干燥，获得A晶型的化合物IV。
  11. 一种二甲基胺4-O-乙酰基线叶旋覆花内酯A富马酸盐IV的D晶型，所述式IV的D晶型具有以下特征：
    其X-射线粉末衍射图2θ、d值和相对强度如下表所示：
    Figure PCTCN2016097481-appb-100011
    Figure PCTCN2016097481-appb-100012
    其粉末X-射线衍射图见图2。
  12. 一种二甲基胺4-O-乙酰基线叶旋覆花内酯A富马酸盐IV的D晶型的制备方法，其特征在于，将式IV化合物用含0.2％水分的甲基叔丁基醚打浆2-5h，抽滤，40-60℃，0.03-0.1MPa减压真空干燥4-24h，得晶型D。
  13. 一种二甲基胺4-O-乙酰基线叶旋覆花内酯A富马酸盐IV的F晶型，所述式IV的F晶型具有以下特征：
    其X-射线粉末衍射图2θ、d值和相对强度如下表所示：
    Figure PCTCN2016097481-appb-100013
    Figure PCTCN2016097481-appb-100014
    其粉末X-射线衍射图见图3。
  14. 一种二甲基胺4-O-乙酰基线叶旋覆花内酯A富马酸盐IV的F晶型的制备方法，其特征在于，所述式IV的F晶型制备方法如下：
    将式IV化合物溶于1-2倍甲醇，不断搅拌，加入2-5倍量乙醚至大量晶体析出，25-40℃，0.03-0.1MPa真空减压干燥2-24h，获得F晶型的化合物IV。
  15. 一种二甲基胺4-O-乙酰基线叶旋覆花内酯A富马酸盐IV的G晶型，所述式IV的G晶型具有以下特征：
    其X-射线粉末衍射图2θ、d值和相对强度如下表所示：
    Figure PCTCN2016097481-appb-100015
    其粉末X-射线衍射图见图4。
  16. 一种二甲基胺4-O-乙酰基线叶旋覆花内酯A富马酸盐IV的G晶型的制备方法，其特征在于，所述式IV的G晶型制备方法如下：
    将式I化合物溶于2倍量的乙醇，搅拌下加入等当量的富马酸，继续搅拌0.5-2h，加入1-2倍量的甲基叔丁基醚，析出晶体，抽滤，用甲基叔丁基醚淋洗，25-40℃真空干燥，获得G晶型式IV化合物。
  17. 一种二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物及其药学上可接受的盐在制备治疗多发性硬化症药物中的应用。
  18. 一种二甲基胺4-O-乙酰基线叶旋覆花内酯A化合物Ⅰ或其盐Ⅱ作为活性成分与常规药用载体制成的药物组合物在制备治疗多发性硬化症药物中的应用。
  19. 根据权利要求18所述的应用，所述药物组合物为片剂、分散片、含片、口崩片、缓释片、胶囊剂、软胶囊剂、滴丸、颗粒剂、注射剂、粉针剂或气雾剂。
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