WO2017186138A1 - 环己烯类衍生物或其药学上可接受的盐及其用途 - Google Patents

环己烯类衍生物或其药学上可接受的盐及其用途 Download PDF

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WO2017186138A1
WO2017186138A1 PCT/CN2017/082155 CN2017082155W WO2017186138A1 WO 2017186138 A1 WO2017186138 A1 WO 2017186138A1 CN 2017082155 W CN2017082155 W CN 2017082155W WO 2017186138 A1 WO2017186138 A1 WO 2017186138A1
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injection
compound
hours
formula
pharmaceutically acceptable
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PCT/CN2017/082155
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English (en)
French (fr)
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张健存
李德耀
李宏
王坤
刘燕
吴妍
唐星
宋健波
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广州市恒诺康医药科技有限公司
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Priority to EP17788790.8A priority Critical patent/EP3450427A4/en
Priority to US16/096,778 priority patent/US20190167593A1/en
Publication of WO2017186138A1 publication Critical patent/WO2017186138A1/zh

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/56—Nitrogen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61J—CONTAINERS SPECIALLY ADAPTED FOR MEDICAL OR PHARMACEUTICAL PURPOSES; DEVICES OR METHODS SPECIALLY ADAPTED FOR BRINGING PHARMACEUTICAL PRODUCTS INTO PARTICULAR PHYSICAL OR ADMINISTERING FORMS; DEVICES FOR ADMINISTERING FOOD OR MEDICINES ORALLY; BABY COMFORTERS; DEVICES FOR RECEIVING SPITTLE
    • A61J3/00—Devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms
    • A61J3/02—Devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms into the form of powders
    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/451—Non condensed piperidines, e.g. piperocaine having a carbocyclic group directly attached to the heterocyclic ring, e.g. glutethimide, meperidine, loperamide, phencyclidine, piminodine
    • 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
    • A61K47/02—Inorganic compounds
    • 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
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses

Definitions

  • the present invention relates to the field of medical technology, and in particular to a cyclohexene derivative or a pharmaceutically acceptable salt thereof and use thereof.
  • Influenza is an acute viral respiratory infection caused by influenza viruses that is seriously harmful to human health.
  • the control and treatment of influenza in China is mainly the use of antipyretic, analgesic and cough medicine for symptomatic treatment, or inoculation with inactivated vaccine for prevention.
  • Some countries use amantadine and rimantadine to prevent influenza A, but both amantadine and rimantadine are ineffective against influenza B, and long-term use of amantadine has more adverse reactions.
  • Influenza virus neuraminidase (NA) inhibitors are a new class of antiviral drugs that have a strong inhibitory effect on influenza A and B.
  • Neuraminidase inhibitors selectively inhibit the activity of neuraminidase, preventing the replication and release of progeny virus particles in the host cell, thereby effectively preventing influenza and alleviating symptoms. Its principle of action is that the lipophilic group in the structure of the neuraminidase inhibitor binds to the hydrophobic portion of the enzyme, causing the virus to lose the ability to break down the glycosidic bond between the sialic acid residue and the glycoprotein, and block the virus after replication. The particles pass through the mucous layer of the respiratory tract; preventing the continued spread of the influenza virus to surrounding cells.
  • Tamiflu an influenza neuraminidase inhibitor
  • Tamiflu's generic name is Oseltamivir, a novel anti-influenza neuraminidase inhibitor developed by Gilead and Roche, with the molecular formula C 16 H 28 N 2 O 4 .H 3 PO 4 , the chemical name is (3R, 4R, 5S)-4-acetamido-5-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid ethyl ester phosphate ( Novel selective inhibitors of viral or bacterial neuraminidases, WO9626933).
  • Tamiflu is designed based on the three-dimensional structure of neuraminidase and selectively inhibits the activity of influenza virus neuraminidase. It acts on a relatively stable conserved site of the neuraminidase functional group and has a good effect on the virus and plays an important role in the prevention and treatment of influenza.
  • the virus is constantly emerging (Influenza Neuraminidase Inhibitors as Antiviral Agents. ANNUAL REPORTS IN MEDICINAL CHEMISTRY, 2006 (41): 287-297; In Vitro Generation of Neuraminidase Inhibitor Resistance in A (H5N1) Influenza Viruses. Antimicrob. Agents Chemother., 2009 (53): 4433-4440).
  • influenza epidemics (avian flu and swine flu) of different subtypes of different species indicate that the flu is also constantly mutating, indicating that the potential hazards of the flu are increasing.
  • Oxford University's research showed that the avian influenza virus began to develop resistance to Tamiflu, mainly due to mutations in the H274Y site of influenza neuraminidase.
  • Roche Pharmaceuticals then urged the addition of Duffy's dose to fight avian flu.
  • Duffy resistance caused by H274Y mutation reduced its activity by more than 300 times, and it is difficult to meet the clinical demand by increasing the dose.
  • the recently approved peramivir is also barely effective against the H274Y mutant influenza virus.
  • the cyclohexene derivative or a pharmaceutically acceptable salt thereof is a drug-resistant influenza A broad-spectrum anti-influenza drug that is effective against viruses.
  • the inventors discovered by accident that the above (3R,4R,5S)-4-acetamido-5-mercapto-3-((3S)-3-n-butylsulfonamidopiperidine-1) -
  • the compound of formula I ((3R,4R,5S)-4-acetamido-5-mercapto-3-( 3R)-3-n-butylsulfonamidopiperidine)-1-cyclohexene-1-carboxylic acid, the compound of formula I not only has a very good anti-influenza virus effect, especially a virus resistant to oseltamivir
  • the strain is still highly active and can be used as an effective and broad-spectrum anti-influenza neuraminidase inhibitor against Duffy.
  • the compound is a white solid at room temperature, is easy to be crystallized and purified, is industrially amplified, and is suitable for mass production.
  • the present invention also discloses an injection for an influenza virus which comprises the compound of the formula I or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the above injection may be an injection solution, a sterile powder for injection, or a concentrated solution for injection, and may be used for intramuscular injection, intravenous injection, intravenous drip, and selected according to specific clinical needs.
  • the concentrated solution for injection can be intravenously instilled before dilution.
  • the concentration of the active ingredient is from 1 mg/mL to 300 mg/mL based on the compound of formula I.
  • the injection can be made into a glass ampule, a glass bottle, a control bottle, a Kärner bottle, a vial, a prefilled syringe, a plastic bottle, an infusion bottle, a plastic bag, a prefilled syringe, and the like, and each injection or each bottle of injection
  • the amount in the range is between 0.5 and 500 ml, preferably 2 ml, 5 ml, 10 ml, 20 ml, 50 ml, 100 ml, 250 ml.
  • the pharmaceutically acceptable salt of the compound of Formula I is: lactate, hydrochloride, phosphate, acetate, malate, citrate, or aspartate Acid salt.
  • the compound of formula I is water-soluble and pH-dependent, and is not convenient for clinical use due to its unstable aqueous solution. Therefore, it is preferred that the compound of the formula I is salted with lactic acid, hydrochloric acid, phosphoric acid, acetic acid, malic acid, citric acid, or aspartic acid, and has good stability and solubility, and can satisfy clinical use. Need.
  • the pharmaceutically acceptable salt is lactate, wherein the molar ratio of the compound of formula I to lactic acid is from 1:1.5 to 2.5.
  • the present inventors have found through extensive experimental screening that the lactate of the compound of formula I is used, and the molar ratio of the two is limited to the above range, and has the best stability, solubility and body tolerance.
  • the injectable is a sterile powder for injection
  • the sterile powder for injection further comprises a pharmaceutically acceptable excipient.
  • the injection can be prepared as a sterile powder for injection, which can further improve the safety, effectiveness and stability of the preparation.
  • the sterile powder for injection can be obtained by adding a solvent and then adopting a solvent crystallization method, a spray drying method, or a freeze drying method or a low temperature drying method.
  • the pharmaceutically acceptable excipient includes at least one of an osmotic pressure adjusting agent, a pH adjusting agent, a solubilizing agent, an antioxidant, a bacteriostatic agent, and a filler.
  • the above excipients may be selected from conventional excipients in the art as desired.
  • the osmotic pressure adjusting agent includes, but not limited to, citric acid/citrate, acetate, mannitol, lactose, sorbitol, glucose, sodium chloride, potassium chloride, maltose, etc.;
  • the agent includes, but is not limited to, lactic acid, hydrochloric acid, phosphoric acid, acetic acid, malic acid, citric acid, aspartic acid, etc., and one or more of the above regulators may be added to a suitable pH value, and the pH is adjusted to pH 3 to pH 6. pH 3.5 - pH 5.0.
  • the solubilizing agents include, but are not limited to, Tween-40, Tween-60, Tween-80, lecithin, soybean phospholipid, cetyl alcohol, stearyl alcohol, poloxamer 188, polyoxyethylene castor oil One or a mixture of two or more of sodium deoxycholate.
  • the antioxidant includes, but is not limited to, sodium sulfite, sodium hydrogen sulfite, sodium metabisulfite, sodium thiosulfate, thiourea, dibutylhydroxytoluene, butylated hydroxyanisole, disodium edetate. One or a mixture of two or more.
  • the bacteriostatic agent includes, but is not limited to, one or a mixture of two or more of phenol, cresol, chlorocresol, chlorobutanol, benzyl alcohol, thimerosal.
  • the filler includes, but is not limited to, one or a mixture of two or more of sodium chloride, sorbitol, and mannitol.
  • the purified filling gas may also be added to the formulation package as needed, including but not limited to one or more of carbon dioxide and nitrogen.
  • the sterile powder for injection is a lyophilized powder for injection
  • the pharmaceutically acceptable excipient comprises a filler, which is mannitol, sorbitol, and sodium chloride. At least one, the filler and the compound of formula I or a pharmaceutically acceptable salt thereof are present in a weight ratio of from 0.19 to 1.9:1.
  • the inventors have found through extensive experimentation that the lyophilized powder for injection prepared by the above composition has the best stability, resolubility and body tolerance.
  • the present inventors have found in research that, especially when a filler is selected, the type and amount of the filler used have an influence on the physicochemical properties of the finally obtained lyophilized powder preparation, and the filler is selected as sodium chloride, and
  • the weight ratio of the compound of the formula I is preferably from 0.19 to 0.57:1, more preferably from 0.25 to 0.57:1, with better properties.
  • the invention also discloses a preparation method of the above-mentioned anti-influenza virus injection, which comprises the steps of: taking a compound of the formula I, adding an appropriate amount of water for injection, and then adding an organic acid or a mineral acid to the compound of the formula I and the organic acid. Or the inorganic acid is sufficiently reacted and dissolved, and then a predetermined amount of a pharmaceutically acceptable excipient is added, mixed, and then water for injection is added to a predetermined volume, and filtered.
  • the invention also discloses a preparation method of the above-mentioned anti-influenza virus injection, which comprises the following steps:
  • Solution preparation a compound of the formula I is added, an appropriate amount of water for injection is added, and then an organic acid or a mineral acid is added to fully react and dissolve the compound of the formula I with the organic or inorganic acid, followed by adding a predetermined amount of pharmaceutically acceptable Accept the excipients, mix, then add a predetermined amount of pharmaceutically acceptable excipients, mix and filter;
  • Lyophilization The drug solution obtained above is divided into parts and placed in a freeze-drying apparatus, and lyophilized to obtain.
  • the above-mentioned lyophilized powder for injection is obtained by a freeze-drying method, and can ensure that the active ingredient does not change during the preparation process, and has good stability and properties.
  • the lyophilized powder for injection is prepared according to the requirements of the preparation of the lyophilized powder for injection in the art, and the leak is measured by a reduced pressure method or other suitable method.
  • the pharmaceutically acceptable adjuvant is sodium chloride
  • the ratio by weight of the adjuvant to the compound of formula I is from 0.19 to 0.57 (preferably from 0.25 to 0.57): Adding an amount of 1 to the excipient;
  • lyophilization is carried out according to the following process conditions: setting the lamellar temperature to -30 to -50 ° C, and controlling the temperature to be maintained at -30 to -50 ° C for 3-5 hours;
  • the control temperature is gradually increased to 0 ° C in 0.5-1.5 hours, maintaining 1.5-2.5 hours;
  • the temperature is then gradually increased to 35-45 ° C in 0.5-1.5 hours for 4-10 hours.
  • the inventors fully considered the properties of each material, and carried out a large number of experimental explorations on the conditions of freezing speed, determination of the product temperature, and selection of the degree of vacuum, and obtained freeze-drying under the above-mentioned freeze-drying process conditions, and the appearance was full, A white, fine, loose porous solid, that is, the lyophilized powder has better properties.
  • the invention also discloses the use of the above cyclohexene derivative or a pharmaceutically acceptable salt thereof for preparing an anti-influenza virus medicament.
  • the present invention has the following beneficial effects:
  • the anti-influenza virus injection of the invention adopts the above-mentioned novel cyclohexene derivative, and has good inhibitory effect on influenza virus, especially the virus strain resistant to oseltamivir, and can still have high activity. It is a new broad-spectrum anti-influenza drug that can be used to treat influenza caused by influenza virus as an effective and broad-spectrum anti-influenza neuraminidase inhibitor against Duffy.
  • a method for increasing the water solubility thereof is provided, and the method is prepared as an injection, which effectively solves the clinical requirement that the influenza patient needs rapid and timely medication; and provides a freeze-dried powder injection. Convenient for long-term storage and long-distance transportation of drugs.
  • the preparation method of the anti-influenza virus injection of the invention can prepare the injection or the lyophilized powder for injection by a suitable method after fully considering the traits of the active ingredients and the auxiliary materials of the preparation, thereby ensuring the safety and effectiveness of the injection. It also improves the stability of the injection and can meet the needs of clinical applications.
  • Figure 1 is a photograph of a petri dish in the plaque assay virus titer experiment of Example 2;
  • A, B, C are infected with PR8 virus, 3 days after infection, photos were stained with crystal violet, D, E, F were infected with H274Y virus, and photos were immunostained 5 days after infection;
  • Figure 2-A is a graph showing the change in body weight of influenza virus PR8 mice in Example 2;
  • Figure 2-B is a graph showing the change in body weight of influenza virus H274Y-PR8 mice in Example 2;
  • Figure 3-A is a graph showing the survival rate of influenza virus PR8 mice in Example 2.
  • Figure 3-A is a graph showing the survival rate of influenza virus H274Y-PR8 mice in Example 2.
  • Figure 4 is a lyophilization curve 1 in Example 4.
  • Figure 5 is a lyophilization curve 2 in Example 4.
  • Figure 6 is a freeze-drying curve 3 in Example 4.
  • (3R,4R,5S)-4-acetamido-5-(2,3-di(tert-butoxycarbonyl)indolyl)-3-((3R)-3-n-butylsulfonamidopiperidine) 1-cyclohexene-1-carboxylic acid Place in a single-mouth bottle, add dilute hydrochloric acid solution at room temperature with stirring, continue to stir at room temperature until the reaction is completed, slowly add sodium hydroxide aqueous solution to precipitate a solid, filter, and dry the filter residue to obtain a white solid, which is (3R, 4R,5S)-4-Acetylamino-5-mercapto-3-((3R)-3-n-butylsulfonamidopiperidine)-1-cyclohexene-1-carboxylic acid.
  • NA enzyme neuroaminidase
  • Reference compound a compound of the following formula II, 1 mmol/L
  • stop solution 150 ⁇ l
  • the dynamic curve of the fluorescence value can be determined without adding a stop solution.
  • Oseltamivir, peramivir or a test compound is diluted 1:10, 1:100, 1:1000, 1:10000.
  • the compound of the formula I has a good inhibitory effect on the H274Y-resistant mutant neuraminidase, and its inhibitory effect is superior to that of the reference compound (the isomer of the compound of the formula I, derived from the patent CN201310130035. 2), better than oseltamivir phosphate and peramivir.
  • Test compound I 1 mmol/L; oseltamivir: 1 mmol/L; peramivir: 1 mmol/L.
  • the medium full of MDCK cells was aspirated, washed once with PBS, then 1 ml of Trypsin was added, digested at 37 ° C for 7 min, and then 5 ml of the medium was added to terminate the digestion, and the cells were gently blown to spread.
  • the cells were counted using a hemocytometer, and then the cells were diluted to 1 ⁇ 10 6 cells/ml, and 1 ml per well was plated into a 6-well cell culture plate, and cultured at 37 ° C, 5% CO 2 for 24 hours.
  • a DMEM virus dilution containing 0.3% BSA was prepared.
  • the packed small tube PR8 or H247Y virus stored in a -80 ° C refrigerator was taken out and thawed on ice.
  • the virus was diluted with the prepared virus dilution to the desired titer and volume, and the number of plaques in the virus control well was controlled at about 50.
  • the medium in the 6-well cell plate was aspirated, then washed twice with PBS, and PBS was discarded.
  • the virus infection solution was added, and the cells were incubated in a 37 ° C incubator for 2 h.
  • the 1.6% agarose gel was dissolved in a microwave oven and placed in a 40 ° C water bath for heat preservation; after the virus adsorption was completed, the virus infection solution was aspirated, washed once with PBS, and the prepared agarose medium was added, and the composition was (1 ⁇ MEM, 0.3% BSA, 1 x PS antibiotic, 1 ⁇ g/mL TPCK trypsin, corresponding final concentration of test compound, 0.8% agarose gel, 1 mL/well, were placed in a 37 ° C incubator. The morphology and number of plaques were observed after 3 to 5 days.
  • the cell plates were placed on ice for cooling, and after 15 minutes, the surface layer of agarose gel was picked. Different staining methods are selected according to the plaque characteristics of the virus.
  • the PR8 virus has a large spot size and is selected for crystal violet staining; the H274Y virus has a small spot size and is selected for immunostaining.
  • Crystal violet staining After picking up the gel, add 1ml/well of crystal violet staining solution (0.5% crystal violet formalin solution), and leave it at room temperature for 15min. After that, discard the coloring solution, wash it with water, observe plaques, and count.
  • the test compound and the control compound can effectively inhibit the formation of PR8 and H274Y plaques.
  • A, B, and C were infected with PR8 virus, and photos were stained with crystal violet 3 days after infection; D, E, and F were infected with H274Y virus, and photos were immunostained 5 days after infection.
  • A, D is the inhibition of viral plaque by the compound of formula I;
  • B, E is the inhibition of viral plaque by the control compound peramivir;
  • C F is the inhibition of viral plaque by the control compound oseltamivir.
  • the concentration of the compound is labeled above and below the cell well, and Virus represents the virus control without the compound.
  • Non-linear regression analysis was performed on the obtained data using GraphPad Prism 5 software to obtain IC 50 values as shown in the following table.
  • test compound inhibits plaque formation on the half H274Y concentration (IC 50) of 0.13uM, Oseltamivir lower than the IC 50 of about 115 times; Peramivir lower than the IC 50 of about 5 times.
  • IC 50 half maximal inhibitory concentration
  • Oseltamivir similar to the IC 50 (0.13uM) PR8 value of plaque formation.
  • the test compound has a significantly better inhibitory effect on the neuraminidase-resistant influenza virus than Oseltamivir, and also has a good inhibitory effect on the sensitive influenza virus.
  • A/Puerto Rico/8/34 H1N1 (PR8), H274Y virus, BALB/c mouse, phosphate buffer (PBS), isoflurane, test compound (#32) (ie, compound of formula I), Oseltamivir , Peramivir.
  • mice 2M LD 50 Respectively and H274Y-PR8 PR8 influenza virus infection of mice 2M LD 50.
  • the test was divided into the test compound (#32) group, the peramivir (Pera) group and the oseltamivir (Osel) drug group, and the infection control group and the normal control group were established at the same time.
  • 2M LD 50 /40 ⁇ l of influenza virus was instilled into the nasal cavity.
  • the test compound (#32) treatment group was administered intramuscularly 24 hours after infection (both leg muscles, 50 ⁇ l/side), 150 mg/kg dose, once daily; 75 mg/kg dose continuous Administration 5 times, once a day.
  • mice in the Pera drug-treated group were administered intramuscularly at 24 hours after infection, and administered once at a dose of 90 mg/kg; the dose of 45 mg/kg was administered 5 times a day, once a day.
  • the mice in the Osel drug treatment group were intragastrically administered with a dose of 10 mg/kg at a dose of 10 mg/kg 24 hours after infection (200 ⁇ l/mouse) for 5 times, once a day.
  • mice Morbidity and mortality of the mice were observed daily within 10 days after infection, and the body weight of the mice was weighed, and the changes in body weight (Fig. 2-A) and the survival rate curve (Fig. 3-A) were plotted.
  • mice infected with PR8 virus In the untreated group of mice infected with PR8 virus, symptoms of depression, dullness, and trembling of the back were observed on the third day after infection. As can be seen from Fig. 2-A, the body weight of the mice in this group began to decrease from the third day after infection, and all died on the 8th day after infection (Fig. 3-A).
  • mice in the Osel gavage treatment group also showed obvious disease symptoms. On the 8th day after infection, the body weight decreased by about 15% on average, and then began to recover. On the 10th day after infection, the body weight recovered to the pre-infection level.
  • the test compound (#32) was administered once at 150 mg/kg and 5 times at 75 mg/kg.
  • the mice in the treatment group showed no obvious disease characterization, and the body weight of the mice showed a slight decrease on the 7th day after infection, and then recovered. None of the mice in the treatment group died (Fig. 3-A).
  • mice were infected with 2MLD 50 NAI-resistant mutant H274Y-PR8 virus. Different groups of mice after 24 hours of infection were given intramuscular injection of test compound (#32), Pera treatment, and Osel treatment was given by intragastric administration.
  • mice were given 10 mg/kg Osel per day. After 5 consecutive days of administration, the mice still showed obvious disease symptoms, and 2 mice died on the 8th day after infection, and the mortality rate was 40% (Fig. 3-B).
  • test compound (#32) was administered at a dose of 75 mg/kg for 5 consecutive days, and the treated mice showed no symptoms of any disease, and the body weight was almost the same as that of the uninfected control mice; Pera was administered at a dose of 45 mg/kg for 5 consecutive days. The treated mice had mild disease symptoms and the average body weight decreased by about 6% on the 7th day after infection. ( Figure 2-B).
  • the test compound (#32) was administered at a dose of 150 mg/kg once, and the body weight was still significantly decreased after infection.
  • the compound disclosed by the present invention has a good inhibitory activity against influenza virus, especially a high inhibitory activity against the neuraminidase of the oseltamivir-resistant influenza virus strain, and small.
  • the weight of the mouse shows its application prospects in the preparation of anti-influenza drugs, and it is expected to be developed as a new drug.
  • solubility of the compound of the formula I with an acid such as lactic acid, hydrochloric acid, phosphoric acid, acetic acid, malic acid, citric acid or aspartic acid is greatly increased, and most of the pH is 3.8.
  • the solubility is greater than 100 mg/mL, wherein the molar ratio to the lactic acid is about 1:2, the solubility of the salt is 194 mg/mL, the pH of the solution is 3.8; and the molar ratio to malic acid is about 1:1, the solubility is 130 mg/mL;
  • the solution pH was 3.8; when the ratio of the acetic acid/aspartic acid substance was 1:2, the solubility was 35 mg/mL.
  • Preferred is lactic acid.
  • Vacuuming is carried out after drying according to the following freeze-drying process conditions.
  • the lyophilization conditions were: setting the lamellar temperature to -40 ° C, controlling the temperature below -40 ° C for 4 hours, and then evacuating. Then, the temperature was controlled to rise slowly to -30 ° C for 1 hour for 2 hours. Then, the control temperature rises slowly to within 1 hour. -20 ° C, hold for 3 hours. Then, the temperature was controlled to slowly rise to -10 ° C for 2 hours for 3 hours. Then, the control temperature was slowly raised to 0 ° C in 1 hour for 2 hours. Then, the temperature was controlled to rise slowly to 20 ° C in 1 hour for 2 hours. Then, the temperature was controlled to rise slowly to 40 ° C in 1 hour for 9 hours.
  • the experiment investigated the effects of three different freeze-drying processes on the finished product by designing different freeze-drying processes, as shown in Figures 4, 5 and 6.
  • the powder injection prepared by the freeze-drying process shown in Fig. 4 has a good appearance and resolubility.
  • the preparation method of the above injection solution is as follows:
  • the preparation method of the above injection solution is as follows:
  • the preparation method of the above injection solution is as follows:
  • a lyophilized powder for injection against influenza virus the prescription is as follows:
  • the lamellar temperature was set to -40 ° C, and the temperature of the control solution was below -40 ° C for 4 hours.
  • Vacuuming is carried out after drying according to the following freeze-drying process conditions.
  • the lyophilization conditions were: setting the lamellar temperature to -40 ° C, controlling the temperature below -40 ° C for 4 hours, and then evacuating. Then, the temperature was controlled to rise slowly to -30 ° C for 1 hour for 2 hours. Then, the temperature was controlled to rise slowly to -20 ° C for 1 hour for 3 hours. Then, the temperature was controlled to slowly rise to -10 ° C for 2 hours for 3 hours. Then, the control temperature was slowly raised to 0 ° C in 1 hour for 2 hours. Then, the temperature was controlled to rise slowly to 20 ° C in 1 hour for 2 hours. Then, the temperature was controlled to rise slowly to 40 ° C in 1 hour for 6 hours.
  • a lyophilized powder for injection against influenza virus the prescription is as follows:
  • a lyophilized powder for injection against influenza virus the prescription is as follows:
  • Vacuuming is carried out after drying according to the following freeze-drying process conditions.
  • a lyophilized powder for injection against influenza virus the prescription is as follows:
  • Preparation method measuring 50 ml of water for injection in a vial, adding 5.0 g of cyclohexene derivative, stirring, adding lactic acid, adjusting the pH to 4.0, completely dissolving the drug, adding 5 g of dextran, stirring uniformly, dissolving, filtering, Dispense to 20 bottles, spare.
  • test article (the sample prepared according to Example 9) was subjected to a long-term test, and the results are shown in the following table.

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Abstract

本发明公开了一种环己烯类衍生物或其药学上可接受的盐及其用途，属于医药技术领域。该式（I）所示环己烯类衍生物或其药学上可接受的盐为一种新型广谱抗流感化合物，对流感病毒具有较好的抑制作用，特别是对奥司他韦耐药的病毒株仍然具有高活性，可作为对达菲耐药有效且广谱的抗流感神经氨酸酶抑制剂使用，可用于治疗由流感病毒引起的流感，为一种新型的广谱抗流感药物。

Description

环己烯类衍生物或其药学上可接受的盐及其用途 技术领域
本发明涉及医药技术领域，特别是涉及一种环己烯类衍生物或其药学上可接受的盐及其用途。
背景技术
流感是由流感病毒引起的一种严重危害人类健康的急性病毒性呼吸道传染病。目前国内流感的控制和治疗主要是应用解热、镇痛及止咳药进行对症治疗，或接种灭活疫苗进行预防。一些国家用金刚烷胺和金刚乙胺防治A型流感，但金刚烷胺和金刚乙胺都对B型流感无效，而且长期使用金刚烷胺不良反应较多。现在许多国家不建议再使用金刚烷胺治疗流感。又由于流感病毒抗原变异，常规疫苗尚不能有效预防流感爆发与流行，因此抗流感病毒药物研究在流感治疗中具有重要意义。
流感病毒神经氨酸酶(NA)抑制剂是一类新的抗病毒药物，对A，B型流感均有很强的抑制作用。神经氨酸酶抑制剂选择性地抑制神经氨酸酶的活性，阻止子代的病毒颗粒在宿主细胞的复制和释放，从而有效地预防流感和缓解症状。其作用原理为：神经氨酸酶抑制剂结构中的亲脂基团与酶的疏水部分相结合，使病毒失去分解唾液酸残基与糖蛋白之间糖苷键的能力，阻断复制后的病毒颗粒通过呼吸道黏液层；阻止流感病毒向周围细胞的继续扩散。
基于流感神经氨酸酶抑制剂的达菲(Tamiflu)已经成为了世界预防和治疗流感的首选药物。但随着临床的应用，它已经产生了耐药性。达菲通用名称为磷酸奥司他韦(Oseltamivir)，是由Gilead公司和Roche公司开发上市的新型抗流感病毒的神经氨酸酶抑制剂，分子式为C16H28N2O4.H3PO4，化学名为(3R,4R,5S)-4-乙酰胺基-5-氨基-3-(1-乙基丙氧基)-1-环己烯-1-羧酸乙酯磷酸盐(Novel selective inhibitors of viral or bacterial neuraminidases,WO9626933)。达菲是基于神经氨酸酶三维结构设计出来的，能选择性抑制流感病毒神经氨酸酶的活性。作用于神经氨酸酶功能团相对稳定的保守位点，对病毒具有很好的疗效，在防治流感中发挥了重要作用。但是，随着临床上应用的推广，病毒对其耐药性现象不断涌现(Influenza Neuraminidase Inhibitors as Antiviral Agents.ANNUAL REPORTS IN MEDICINAL CHEMISTRY,2006(41):287-297；In Vitro Generation of Neuraminidase Inhibitor Resistance in A(H5N1)Influenza Viruses.Antimicrob.Agents Chemother.,2009(53):4433-4440)。
近几年来，不同物种不同亚型的流感事件(禽流感以及猪流感)表明流感也在不断的变异，也说明了流感潜在的危害不断增大。与此同时，早在2005年牛津大学的研究结果显示禽流感病毒开始对达菲产生抗药性，主要是由于流感神经氨酸酶H274Y部位的突变引起。随后罗氏药厂敦促增加达菲的剂量对抗禽流感。但由于H274Y突变导致的达菲耐药使其活性降低了300倍以上，增加剂量很难满足临床需求。最近批准的帕拉米韦对于H274Y突变流感病毒也仅仅是勉强有效。
由于生态以及环境卫生条件的原因，我国是流感高发地区，再加上达菲耐药性的不断恶化，如何应对将来的高危害高致命流感病毒的到来，是整个社会应该思考和解决的问题。随着达菲耐药性也将日益严重，开发新型广谱包括对达菲耐药仍然有效的广谱抗流感药物有巨大的市场需求和社会意义。
发明内容
基于此，有必要针对上述问题，提供一种环己烯类衍生物或其药学上可接受的盐，该环己烯类衍生物或其药学上可接受的盐为一种对耐药型流感病毒有效的广谱抗流感药物。
式I所示的环己烯类衍生物或其药学上可接受的盐：
Figure PCTCN2017082155-appb-000001
本发明人在前期研究(专利申请CN201310130035.2)的基础上，对其中活性较好的部分化合物进行了进一步的深入研究，筛选出了活性较好的部分化合物进行稳定性、工艺放大等成药性考察，结果发现，部分化合物存在稳定性差无法满足成药要求的问题；部分化合物毒性较大；部分化合物存在理化性状较差，无法适应工业放大的要求，例如，(3R,4R,5S)-4-乙酰胺基-5-胍基-3-((S)-3-正丁磺酰胺基哌啶)-1-环己烯-1-羧酸，其具有很好的抑制流感病毒效果及较小的毒副作用，但是，该化合物室温下呈半固体，分离纯化困难，无法适应工业放大要求。
在上述研究基础上，本发明人尝试了多种不同的处理方式和手段，如结构改造或加入辅料等方法，力图在保持此类化合物活性的基础上，改善其稳定性和性状，提高其成药性。
最终，本发明人在实验中偶然发现，上述(3R,4R,5S)-4-乙酰胺基-5-胍基-3-((3S)-3-正丁磺酰胺基哌啶)-1-环己烯-1-羧酸化合物在改变特定手性中心的构型后，即得到式I化合物((3R,4R,5S)-4-乙酰胺基-5-胍基-3-((3R)-3-正丁磺酰胺基哌啶)-1-环己烯-1-羧酸，该式I化合物不但具有非常好的抑制流感病毒效果，特别是对奥司他韦耐药的病毒株仍然具有高活性，可作为对达菲耐药有效且广谱的抗流感神经氨酸酶抑制剂使用。而且，该化合物室温下为白色固体，易于结晶纯化，利于工业放大，适合大量制备。
本发明还公开了一种抗流感病毒的注射剂，该注射剂以式I所示化合物或其药学上可接受的盐作为活性成分。
可以理解的，上述注射剂可以是注射液、注射用无菌粉末、注射用浓溶液，可用于肌内注射、静脉注射、静脉滴注，根据具体临床需求选择。其中，注射用浓溶液可供临用前稀释后静脉滴注。最终注射用溶液中，以式I化合物计，活性成分的浓度为1mg/mL-300mg/mL。该注射剂可制成玻璃安瓿、玻璃瓶、管制瓶、卡氏瓶、西林瓶、预充注射器、塑料瓶、输液瓶、塑料袋、预灌封注射器等注射液包装形式，每支或每瓶注射剂中的量在0.5-500ml之间，优选的是2ml、5ml、10ml、20ml、50ml、100ml、250ml。
在其中一个实施例中，所述式I所示化合物药学上可接受的盐为：乳酸盐、盐酸盐、磷酸盐、醋酸盐、苹果酸盐、枸椽酸盐、或天冬氨酸盐。式I所示化合物水溶性具有pH依赖性，由于其水溶液不稳定，不便于临床应用。因此，优选的，将该式I化合物与乳酸、盐酸、磷酸、醋酸、苹果酸、枸椽酸、或天冬氨酸等成盐后，具有较好的稳定性和溶解性，能够满足临床使用的需要。
在其中一个实施例中，所述药学上可接受的盐为乳酸盐，其中，式I所示化合物与乳酸的摩尔比为1:1.5-2.5。本发明人通过大量实验筛选后发现，以式I化合物的乳酸盐使用，并将二者的摩尔比限定于上述范围内，具有最佳的稳定性、溶解度和机体耐受性。
在其中一个实施例中，该注射剂为注射用无菌粉末，所述注射用无菌粉末还包括药学上可接受的辅料。将该注射剂制备为注射用无菌粉末，能够进一步提高该制剂的安全性、有效性和稳定性。该注射用无菌粉末，可以添加辅料后，采取溶剂结晶法、喷雾干燥法或冷冻干燥或低温干燥法制得。可以理解的， 所述药学上可接受的辅料包括：渗透压调节剂、pH调节剂、增溶剂、抗氧剂、抑菌剂、填充剂中的至少一种。
可以理解的，上述辅料可根据需要从本领域常规辅料中选取。如：所述渗透压调节剂包括但不限于枸橼酸/枸橼酸盐、醋酸盐、甘露醇、乳糖、山梨醇、葡萄糖、氯化钠、氯化钾、麦芽糖等；所述pH调节剂包括但不限于乳酸、盐酸、磷酸、醋酸、苹果酸、枸椽酸、天冬氨酸等，可加入以上一种或多种调节剂至合适pH值，调节到pH3-pH6，最适为pH3.5-pH5.0。所述增溶剂包括但不限于吐温-40、吐温-60、吐温-80、卵磷脂、豆磷脂、十六烷醇、十八烷醇、泊洛沙姆188、聚氧乙烯蓖麻油、脱氧胆酸钠中的一种或两种以上混合物。所述抗氧剂包括但不限于亚硫酸钠、亚硫酸氢钠、焦亚硫酸氢钠、硫代硫酸钠、硫脲、二丁基羟基甲苯、丁基羟基茴香醚、乙二胺四乙酸二钠中的一种或两种以上混合物。所述抑菌剂包括但不限于苯酚、甲酚、氯甲酚、三氯叔丁醇、苯甲醇、硫柳汞中的一种或两种以上混合物。所述填充剂包括但不限于氯化钠、山梨醇、甘露醇中的一种或两种以上混合物。还可根据需要在制剂包装中加入净化后的填充气体，所述填充气体包括但不限于二氧化碳、氮气中的一种或两种以上。
在其中一个实施例中，所述注射用无菌粉末为注射用冻干粉，所述药学上可接受的辅料包括填充剂，所述填充剂为甘露醇、山梨醇、和氯化钠中的至少一种，所述填充剂与所述式I化合物或其药学上可接受的盐的重量份比为0.19-1.9：1。本发明人通过大量实验筛选后发现，以上述组方制备得到的注射用冻干粉，具有最佳的稳定性、复溶性和机体耐受性。本发明人在研究中发现，特别是选用填充剂时，所用填充剂的类型和量，对最终得到的冻干粉制剂的物化性质具有影响，而将填充剂选为氯化钠，且与所述式I化合物的重量份比优选0.19-0.57:1，更优选0.25-0.57:1，具有更好的性能。
本发明还公开了上述的抗流感病毒的注射剂的制备方法，包括以下步骤：取式I所示化合物，加入适量注射用水，再加入有机酸或无机酸使所述式I化合物与所述有机酸或无机酸充分反应、溶解，随后加入预定量的药学上可接受的辅料，混匀，随后加入注射用水至预定体积，过滤，即得。
可以理解的，按照本领域对注射剂的制剂要求，配制得到药液后，还需无菌过滤或高温灭菌，按规定装量无菌法分装到灭菌后的洁净的包装中，熔封或严封，采用减压法或其它适宜的方法进行检漏。
本发明还公开了上述的抗流感病毒的注射剂的制备方法，包括以下步骤：
溶液配制：取式I所示化合物，加入适量注射用水，再加入有机酸或无机酸，使所述式I化合物与所述有机酸或无机酸充分反应、溶解，随后加入预定量的药学上可接受的辅料，混匀，随后加入预定量的药学上可接受的辅料，混匀，过滤；
冻干：将上述得到的药液分装后置于冷冻干燥设备中，冻干，即得。
上述注射用冻干粉，以冷冻干燥的方法制得，能够确保活性成分在制剂过程中不发生变化，并且具有较好的稳定性和性状。
同样，按照本领域对注射用冻干粉的制剂要求，制成注射用冻干粉后，采用减压法或其它适宜的方法进行检漏。
在其中一个实施例中，所述溶液配制步骤中，所述药学上可接受的辅料为氯化钠，按照辅料与所述式I化合物的重量份比为0.19-0.57(优选0.25-0.57)：1的量加入辅料；
所述冻干步骤中，按照下述工艺条件进行冻干：设定板层温度为-30～-50℃，控制温度在-30～-50℃保持3-5小时；
然后抽真空，控制温度在0.5-1.5小时内逐渐升高7-15℃，保持1.5-2.5小时；
然后控制温度在0.5-1.5小时内逐渐升高7-15℃，保持2-4小时；
然后控制温度在1.5-2.5小时内逐渐升高7-15℃，保持2-4小时；
控制温度在0.5-1.5小时内逐渐升高到0℃，保持1.5-2.5小时；
然后控制温度在0.5-1.5小时内逐渐升高到15-25℃，保持1.5-2.5小时；
然后控制温度在0.5-1.5小时内逐渐升高到35-45℃，保持4-10小时。
本发明人充分考虑了各物料性质，对冻结速度、品温的确定到真空度的选择等条件进行了大量的实验摸索后，得出以上述冻干工艺条件进行冷冻干燥，能够得到外观饱满、洁白、细腻、疏松多孔的固体，即该冻干粉的性状较好。
本发明还公开了一种上述的环己烯类衍生物或其药学上可接受的盐在制备抗流感病毒药物中的应用。
与现有技术相比，本发明具有以下有益效果：
本发明的一种新型的环己烯类衍生物或其药学上可接受的盐，为一种新型广谱抗流感化合物，该化合物特别对奥司他韦和帕拉米韦耐药的病毒株具有较高活性。
本发明的一种抗流感病毒的注射剂，采用上述新型的环己烯类衍生物，对流感病毒具有较好的抑制作用，特别是对奥司他韦耐药的病毒株仍然具有高活性，可作为对达菲耐药有效且广谱的抗流感神经氨酸酶抑制剂使用，可用于治疗由流感病毒引起的流感，为一种新型的广谱抗流感药物。
并且针对该化合物溶解度差的问题，提供了一种增加其水溶性的方法，并将其制成注射剂，有效解决了流感病人需要快速、及时用药的临床要求；提供了一种冻干粉针剂，方便药物长期存放和长途运输。
本发明的一种抗流感病毒的注射剂的制备方法，在充分考虑制剂有效成分和辅料的性状后，通过适宜的方法制备注射液或注射用冻干粉，既能确保该注射剂的安全有效性，又提高了该注射剂的稳定性，能够满足临床应用的需求。
附图说明
图1为实施例2中噬斑法测病毒滴度实验中的培养皿照片；
其中：A,B,C为感染PR8病毒，感染后3天用结晶紫染色照片，D,E,F为感染H274Y病毒，感染后5天用免疫法染色照片；
图2-A为实施例2中感染流感病毒PR8小鼠的体重变化曲线；
图2-B为实施例2中感染流感病毒H274Y-PR8小鼠的体重变化曲线；
图3-A为实施例2中感染流感病毒PR8小鼠的生存率曲线；
图3-A为实施例2中感染流感病毒H274Y-PR8小鼠的生存率曲线；
图4为实施例4中的冻干曲线1；
图5为实施例4中的冻干曲线2；
图6为实施例4中的冻干曲线3。
具体实施方式
为了便于理解本发明，下面将参照相关附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。
实施例1
(3R,4R,5S)-4-乙酰胺基-5-胍基-3-((3R)-3-正丁磺酰胺基哌啶)-1-环己烯-1-羧酸的制备。
a)(3R,4S,5S)-4-乙酰胺基-5-叠氮-3-((3R)-3-正丁基磺酰胺哌啶)-1-环己烯-1-羧酸乙酯的制备。
Figure PCTCN2017082155-appb-000002
(3R,4R,5S)-4-乙酰胺基-5-叠氮-3-乙酰氧基-1-环己烯-1-羧酸乙酯的制备：制法参见J.Am.Chem.Soc.,1997(119):681-690。
将20mmol的(3R,4R,5S)-4-乙酰氨基-5-叠氮-3-乙酰氧基-1-环己烯-1-羧酸乙酯和1mmol的四三苯基膦钯放置在干燥的双口瓶中，氮气置换两次体系内的空气后，用注射器加入40mL重蒸的DMF搅拌均匀，加入40mmol的DIPEA(N，N-二异丙基乙胺)并搅拌冷却到0℃，缓慢滴加(R)-3-正丁基磺酰胺基哌啶三氟乙酸盐的DMF(40mL)溶液,加完后继续0℃搅拌20分钟，移入油浴70℃反应1小时，TLC(DCM：MeOH＝10:1)显示反应完全，缓慢冷却到0℃，慢慢滴加等体积水，搅拌均匀后EA萃取，直接蒸干并用油泵抽走DMF得到褐色粘稠物。柱纯化(MeOH：DCM＝1:50)洗脱，得到黄白色粉末固体4.37g，即为产物(3R,4S,5S)-4-乙酰胺基-5-叠氮-3-((3R)-3-正丁基磺酰胺哌啶)-1-环己烯-1-羧酸乙酯，Y＝46.4％。
上述产物表征为：
1H-NMR(400MHz,CDCl3):δppm6.810(s,1H,NH),5.529-5.506(d,1H,2-CH),4.233-4.199(q,2H,COOCH 2CH3),4.067-3.991(m,1H,4-CH),3.655-3.589(m,2H,CH 2CH2CH2CH3),3.358-3.334(m,1H,3-CH),3.019-3.001(m,1H,NCH2CH),2.985-2.979(d,2H,NCH 2CH,NCH 2CH2CH2),2.947-2.904(m,2H,NCH 2CH,NCH 2CH2CH2),2.775-2.749(m,1H,5-CH),2.654-2.630(m,1H,6-CH),2.337-2.267(m,2H,6-CH,NCH2CH2CH 2),2.111(s,3H,COCH 3),1.819-1.744(m,2H,CH2CH 2CH2CH3),1.721-1.701(m,1H,NCH2CH2CH 2),1.689-1.664(m,1H,NCH2CH 2CH2),1.621-1.598(m,1H,NCH2CH 2CH2),1.484-1.427(m,2H,CH2CH2CH 2CH3),1.328-1.259(t,3H,COOCH2CH 3),0.970-0.934(t,3H,CH2CH2CH2CH 3).
ESI-MS m/z：471.6(M+H)+
b)(3R,4R,5S)-4-乙酰胺基-5-氨基-3-((3R)-3-正丁基磺酰胺基哌啶)-1-环己烯-1-羧酸乙酯的制备。
Figure PCTCN2017082155-appb-000003
(3R,4S,5S)-4-乙酰胺基-5-叠氮-3-((3R)-3-正丁基磺酰胺基哌啶)-1-环己烯-1-羧酸乙酯放置在单口瓶，加入催化量兰尼镍、40mL无水乙醇搅拌均匀后，用氢气置换体系空气，室温搅拌2小时，TLC(DCM：MeOH＝10:1)显示反应完成，用硅藻土滤去兰尼镍后蒸干溶剂，柱纯化(DCM：MeOH＝5:1)得到白色泡沫状固体，即为产物(3R,4R,5S)-4-乙酰胺基-5-氨基-3-((3R)-3-正丁基磺酰胺基哌啶)-1-环己烯-1-羧酸乙酯。
上述产物表征为：
1H-NMR(400MHz,CDCl3):δppm6.773(s,1H,NH),6.248-6.225(d,1H,2-CH),5.198-5.180(m,1H,4-CH),4.207-4.156(q,2H,COOCH 2CH3),3.894-3.821(m,1H,3-CH),3.708-3.656(t,2H,CH 2CH2CH2CH3),3.539-3.532(m,1H,5-CH),3.181-3.159(m,1H,NCH2CH),3.001-2.920(d,1H,NCH 2CH),2.834-2.799(m,2H,NCH 2CH2CH2),2.738-2.709(m,1H,NCH 2CH),2.608-2.588(m,1H,6-CH),2.281-2.236(m,1H,6-CH),2.078(s,3H,COCH 3),2.028-1.998(m,1H,NCH2CH2CH 2),1.798-1.724(m,2H,CH2CH 2CH2CH3),1.712-1.701(m,1H,NCH2CH2CH 2),1.692-1.598(m,2H,NCH2CH 2CH2),1.496-1.484(m,2H,CH2CH2CH 2CH3),1.296-1.212(t,3H,COOCH2CH 3),0.998-0.956(t,3H,CH2CH2CH2CH 3).
ESI-MS m/z：445.6(M+H)+
c)(3R,4R,5S)-4-乙酰胺基-5-(2,3-二(叔丁氧羰基)胍基)-3-((3R)-3-正丁基磺酰胺基哌啶)-1-环己烯-1-羧酸乙酯的制备。
Figure PCTCN2017082155-appb-000004
将0.5mmol的(3R,4R,5S)-4-乙酰胺基-5-氨基-3-((3R)-3-正丁基磺酰胺基哌啶)-1-环己烯-1-羧酸乙酯放置在单口瓶中，加入10mL乙腈室温搅拌均匀后，加入1.5mmolDIPEA和0.5mmol N,N'-二-BOC-1H-1-胍基吡唑，继续室温搅拌3h，TLC(PE：EA＝1:1)显示反应完成，加入20ml水，EA萃取，无水硫酸钠干燥后蒸干，柱纯化(PE：EA＝3:1)得到白色固体，即为产物(3R,4R,5S)-4-乙酰胺基-5-(2,3-二(叔丁氧羰基)胍基)-3-((3R)-3-正丁基磺酰胺基哌啶)-1-环己烯-1-羧酸乙酯。ESI-MS m/z：687.9(M+H)+
d)(3R,4R,5S)-4-乙酰胺基-5-(2,3-二(叔丁氧羰基)胍基)-3-((3R)-3-正丁基磺酰胺基哌啶)-1-环己烯-1-羧酸的制备。
Figure PCTCN2017082155-appb-000005
0.368mmol(3R,4R,5S)-4-乙酰胺基-5-(2,3-二(叔丁氧羰基)胍基)-3-((3R)-3-正丁基磺酰胺基哌啶)-1-环己烯-1-羧酸乙酯放置在单口瓶中，加入5.5ml 1,4-二氧六环和0.55ml水，0.55ml 1N KOH水溶液，室温搅拌过夜。TLC TLC(PE：EA＝1:1)显示反应完成，蒸干溶剂，油泵抽干后加入5ml甲醇溶解，酸性树脂调pH＝5，过滤蒸干柱纯化，DCM：MeOH＝10:1洗脱得到白色固体，即为产物(3R,4R,5S)-4-乙酰胺基-5-(2,3-二(叔丁氧羰基)胍基)-3-((3R)-3-正丁基磺酰胺基哌啶)-1-环己烯-1-羧酸。
上述产物表征为：
1H-NMR(400MHz,MeOD):δppm6.833(d,1H,2-CH),4.263-4.237(m,1H,4-CH),4.191-4.163(m,1H,3-CH),3.493-3.476(m,3H,5-CH,CH 2CH2CH2CH3),2.925-2.884(m,1H,NCH2CH),2.582-2.528(d,2H,NCH 2CH,NCH 2CH2CH2),2.388-2.342(m,1H,NCH 2CH2CH2),2.245-2.230(m,1H,NCH 2CH),2.201-1.190(m,1H,6-CH),2.154-2.143(m,1H,6-CH),1.929(s,3H,COCH 3),1.848-1.828(m,1H,NCH2CH2CH 2),1.772-1.717(m,3H,CH2CH 2CH2CH3,NCH2CH2CH 2),1.621-1.598(m,2H,NCH2CH 2CH2),1.518(s,9H,C(CH 3)3),1.458(s,9H,C(CH 3)3),1.347-1.281(m,2H,CH2CH2CH 2CH3),0.976-0.939(t,3H,CH2CH2CH2CH 3).
ESI-MS m/z：659.8(M+H)+
e)(3R,4R,5S)-4-乙酰胺基-5-胍基-3-((3R)-3-正丁基磺酰胺基哌啶)-1-环己烯-1-羧酸的制备。
Figure PCTCN2017082155-appb-000006
(3R,4R,5S)-4-乙酰胺基-5-(2,3-二(叔丁氧羰基)胍基)-3-((3R)-3-正丁基磺酰胺基哌啶)-1-环己烯-1-羧酸 放置在单口瓶中，室温搅拌下滴加稀盐酸溶液，滴完后继续室温搅拌至反应完成，慢慢滴加氢氧化钠水溶液至析出固体，过滤，干燥滤渣得到白色固体，即为(3R,4R,5S)-4-乙酰胺基-5-胍基-3-((3R)-3-正丁基磺酰胺基哌啶)-1-环己烯-1-羧酸。
式I化合物的纯化：将实施例中得到的粗品用水和异丙醇50℃加热打浆，然后自然冷却析晶，过滤，得到精制品。
上述产物表征为：
1H-NMR(400MHz,D2O):δppm6.814(d,1H,2-CH),4.473-4.447(m,1H,4-CH),4.435-4.424(m,1H,3-CH),3.989-3.922(m,1H,5-CH),3.670-3.644(m,2H,CH 2CH2CH2CH3),3.594-3.541(m,1H,NCH2CH),3.289-3.251(m,2H,NCH 2CH,NCH 2CH2CH2),2.388-2.3423.030-2.975(m,1H,NCH 2CH2CH2),2.511-2.439(m,1H,NCH 2CH),2.254-2.226(m,1H,6-CH),2.165-2.143(m,1H,6-CH),2.095(s,3H,COCH 3),1.998-1.898(m,1H,NCH2CH2CH 2),1.745-1.706(m,3H,CH2CH 2CH2CH3,NCH2CH2CH 2),1.484-1.429(m,2H,NCH2CH 2CH2),1.200-1.165(m,2H,CH2CH2CH 2CH3),0.945-0.908(t,3H,CH2CH2CH2CH 3).
ESI-MS m/z：459.6(M+H)+。
实施例2
一、流感病毒神经氨酸酶活性测定
实验材料：NA酶(神经氨酸酶)液：流感病毒感染鸡胚的尿囊液
酶促反应体系：330mmol/L的MES缓冲液(pH3.5)
200μmol/L的荧光底物MUNANA
4mmol/L的CaCl2溶液
终止液：14mmol/L的NaOH溶液(14mmol/L，83％的乙醇)
奥司他韦：1mmol/L
帕拉米韦：1mmol/L
参照化合物：下述式II化合物，1mmol/L
Figure PCTCN2017082155-appb-000007
酶活性测定：
Figure PCTCN2017082155-appb-000008
混匀孵育15min后,加入终止液：150μl
EX＝355nm XM＝460nm
注：为了验证酶反应系统是否正常，可以不加终止液，测定荧光值的动力曲线。
NA抑制剂筛选实验操作：
1、奥司他韦、帕拉米韦或受试化合物(式I，II化合物)按1：10，1：100，1：1000，1：10000稀释。
2、反应步骤：将NA酶液30μl与奥司他韦、帕拉米韦或受试化合物10μl混匀后孵育30min，加入 以下成分：MES(330mmol/L)：10μl；CaCl2(4mmol/L)：10μl；MUNANA(200μmol/L)：10μl；H2O：3μl。混匀后孵育15min，再加入终止液：150μl，最后混匀后测定荧光值：EX＝355nm XM＝460nm。
3、数据分析：GraphPad Prism Demo分析。
目标化合物活性测定结果如下表所示：
表1.化合物活性
化合物结构 野生型IC50(nM) 耐药突变型IC50(nM)
式I化合物 0.5002 1.695
参照化合物 0.8671 8.031
磷酸奥司他韦 0.7654 269.4
帕拉米韦 0.09845 26.93
从上述结果中可以看出，式I化合物对于H274Y耐药突变型神经氨酸酶具有很好的抑制作用，且其抑制效果优于参照化合物(式I化合物的异构体，来源于专利CN201310130035.2),更优于磷酸奥司他韦和帕拉米韦。
二、噬斑法测病毒滴度实验
1、实验材料
A/Puerto Rico/8/34(H1N1)(PR8)、H274Y病毒、Madin-Darby canine kidney(MDCK)cells、DMEM(Gibco，Cat#C11965500BT)、青霉素-链霉素双抗(Gibco，Cat#C11875)、Trypsin(含0.25％EDTA)(Gibco，Cat#25200-056)、BSA(Merck，Cat#126575)、Agrose(Biowest，Cat#111860)、Triton X-100(Amresco，Cat#9002931)、PR8小鼠高免血清，goat-anti mouse-IgG-HRP二抗(博士得，Cat#BA1050)、AEC显色试剂盒(Sigma，AEC101)、结晶紫(MBCHEM，Cat#548629)，DPBS(C14190500BT，life technology)、MOD EAGLE MED(MEM)(11935046，life technology)、TWEEN-20(P1379-6，SIGMA)
受试化合物I：1mmol/L；奥司他韦：1mmol/L；帕拉米韦：1mmol/L。
2、实验方法
将长满MDCK细胞的培养基吸弃，PBS洗一遍，然后加入1ml Trypsin，37℃消化7min，再加入5ml培养基终止消化，轻轻吹打使细胞散开。使用血球计数板对细胞进行计数，然后稀释细胞至1X 106个/ml，每孔1ml铺入6孔细胞培养板，37℃，5％CO2中培养24h。
配制含0.3％BSA的DMEM病毒稀释液。取出存放于-80℃冰箱中的分装好的小管PR8或H247Y病毒，于冰上解冻。用配制好的病毒稀释液稀释病毒至所需滴度和体积，使病毒对照孔的噬斑个数控制在50个左右。
将6孔细胞板中的培养基吸出，然后用PBS液洗两遍，吸弃PBS。加入病毒感染液，然后将细胞置于37℃培养箱中孵育2h。
1.6％琼脂糖凝胶在微波炉中溶化后放置于40℃水浴中保温；待病毒吸附完成后，吸弃病毒感染液，用PBS洗一遍，加入配置好的琼脂糖培养基，成分为(1×MEM，0.3％BSA，1×PS抗生素，1μg/mL TPCK胰酶，相应终浓度的受试化合物，0.8％琼脂糖凝胶)，1mL/孔，置于37℃培养箱培养。3～5日后观察噬斑形态与数量。
病毒对照孔形成明显噬斑后，将细胞板放置于冰上冷却，15分钟后挑去表层的琼脂糖胶。根据病毒的成斑特点选择不同的染色方式。PR8病毒成斑较大，选择结晶紫染色；H274Y病毒成斑较小，选择免疫染色法。
结晶紫染色：挑胶后，加入1ml/孔的结晶紫染色液(0.5％的结晶紫福尔马林溶液)，室温放置15min 后，弃掉显色液，用清水洗干净，观察噬斑、计数。
3.5.2免疫染色：挑胶后，每孔加入500μl 4％多聚甲醛固定15min，之后加入0.1％Triton-X100溶液破膜15min。吸走Triton-X100溶液，用500μL/孔的PBST(1×PBS加入0.05％Tween-20)溶液洗涤两次。加入500μL 1：1000稀释的小鼠抗PR8高免血清，室温轻摇1-2h，回收一抗，PBST洗涤三次，每次3min。加入500μL/孔1：2500稀释的HRP标记的山羊抗小鼠二抗，室温轻摇1-2h，回收二抗，PBST洗涤5次，每次3min。加入AEC染色缓冲液，300μL/孔，室温避光静置20min；噬斑观察、拍照，如图1所示，计数。
从图中可以看出，受试化合物及对照化合物能有效抑制PR8与H274Y噬斑形成。具体为：A,B,C为感染PR8病毒，感染后3天用结晶紫染色照片；D,E,F为感染H274Y病毒，感染后5天用免疫法染色照片。A,D为式I化合物对病毒噬斑的抑制；B,E为对照化合物帕拉米韦对病毒噬斑的抑制；C,F为对照化合物奥司他韦对病毒噬斑的抑制。化合物的浓度标记于细胞孔的上方和下方，Virus代表不加化合物的病毒对照。
使用GraphPad Prism 5软件对所得的数据进行非线性回归分析，得出IC50值，如下表所示。
表2.受试化合物、Peramivir和Oseltamivir对流感病毒复制的体外抑制作用
Figure PCTCN2017082155-appb-000009
结果表明受试化合物对H274Y噬斑形成的半数抑制浓度(IC50)为0.13uM,比Oseltamivir的IC50低约115倍；比Peramivir的IC50低约5倍。对PR8噬斑形成的半数抑制浓度(IC50)为0.25uM，与Oseltamivir的IC50(0.13uM)值相近。综上，受试化合物对神经氨酸酶耐药株流感病毒的抑制效果显著优于Oseltamivir，同时对敏感型流感病毒也有很好的抑制作用。
三、抗病毒活性，受试化合物对感染流感病毒PR8、H274Y小鼠的保护作用。
1、实验材料：
A/Puerto Rico/8/34(H1N1)(PR8)、H274Y病毒、BALB/c小鼠、磷酸缓冲液(PBS)、异氟烷、受试化合物(#32)(即式I化合物)、Oseltamivir、Peramivir。
2、实验方法：
分别用2M LD50的PR8及H274Y-PR8流感病毒感染小鼠。试验分为受试化合物(#32)组、帕拉米韦(Pera)组、奥司他韦(Osel)药物组，同时设立感染对照组及正常对照组。用异氟烷轻度麻醉小鼠后，经鼻腔滴入2M LD50/40μl的流感病毒。受试化合物(#32)治疗组小鼠在感染后24h，经腿部肌肉注射给药(两侧腿部肌肉，50μl/侧)，150mg/kg剂量，给药1次；75mg/kg剂量连续给药5次，每天1次。Pera药物治疗组小鼠在感染后24h，经腿部肌肉注射给药，90mg/kg剂量，给药1次；45mg/kg剂量连续给药5次，每天1次。Osel药物治疗组小鼠在感染后24h，按10mg/kg剂量，经灌胃针灌胃给药(200μl/只)，连续给药5次，每天1次。
感染后10天内每天观察小鼠发病与死亡情况，并称量小鼠体重，绘制体重变化(图2-A)和生存率曲线(图3-A)。
感染PR8病毒的未治疗组小鼠，在感染后第3天出现精神沉郁、被毛蓬乱、弓背发抖等疾病症状。从图2-A中可以看到，该组小鼠体重自感染后第3天开始出现持续下降，并在感染后第8天全部死亡(图3-A)。
Osel灌胃治疗组小鼠也出现明显的疾病症状，在感染后第8天，体重平均下降约15％，随后开始恢 复，至感染后第10天，体重基本恢复至感染前水平。
受试化合物(#32)150mg/kg给药1次与75mg/kg给药5次治疗组小鼠未出现明显的疾病表征，小鼠体重在感染后第7天出现微弱的下降，随后恢复。治疗组小鼠均没有死亡(图3-A)。
用2MLD50的NAI耐药突变株H274Y-PR8病毒感染小鼠，感染24h后的不同组别小鼠通过肌肉注射给予受试化合物(#32)、Pera治疗，灌胃给予Osel治疗。
感染H274Y-PR8病毒的未治疗组小鼠，在感染后第4天出现精神沉郁、倦态等症状，感染后第5天小鼠体重开始明显下降(图2-B)，并在感染后第9天全部死亡(图3-B)。
感染小鼠每天给予10mg/kg Osel，连续5天给药后，小鼠仍出现明显疾病症状，并在感染后第8天死亡2只小鼠，死亡率40％(图3-B)。
感染小鼠连续5天给予75mg/kg剂量的受试化合物(#32)后，治疗小鼠没有出现任何疾病症状，体重基本与未感染对照小鼠持平；连续5天给予45mg/kg剂量的Pera治疗小鼠有轻微的疾病症状，并在感染后第7天，平均体重下降约6％。(图2-B)。150mg/kg剂量的受试化合物(#32)1次给药治疗小鼠，感染后体重仍有明显下降，在感染后第8天死亡1只，死亡率20％；90mg/kg剂量的Pera药物1次给药治疗小鼠，疾病表型明显，体重明显下降(图2-B)，在感染后第8、9、10天各死亡1只小鼠，死亡率60％(图3-B)。
从以上测试结果可以看出，本发明公开的化合物对流感病毒具有较好的抑制活性，尤其是对奥司他韦耐药的流感病毒株的神经氨酸酶的表现较高的抑制活性，小鼠的体重显示出其在制备抗流感药物方面的应用前景，有望开发为新药。
实施例3
一、式I化合物不同盐的溶解度实验。
采用乳酸、盐酸、磷酸、醋酸、苹果酸、枸椽酸、天冬氨酸等不同的酸与式I化合物成盐反应，考察式I化合物不同盐的溶解度，结果如下表所示。
表3.式I化合物不同盐的溶解度
化合物类型 式I化合物与酸的摩尔比 溶解度 溶液pH值
式I化合物 / 2.5 7.8
式I化合物乳酸盐 1:2 194 3.8
式I化合物盐酸盐 1:1.5 108 3.8
式I化合物磷酸盐 1:1 160 3.5
式I化合物醋酸盐 1:2 35 3.9
式I化合物苹果酸盐 1:1 130 3.8
式I化合物枸椽酸盐 1:1 95 3.7
式I化合物天冬氨酸盐 1:2 35 4.5
从上述结果可以看出，式I化合物与乳酸、盐酸、磷酸、醋酸、苹果酸、枸椽酸、天冬氨酸等酸成盐后的溶解度均大幅提高，在pH＝3.8的情况下大部分溶解度大于100mg/mL，其中与乳酸摩尔比约为1:2时，成盐的溶解度达到194mg/mL，溶液pH为3.8；与苹果酸摩尔比约为1:1时，溶解度为130mg/mL；溶液pH为3.8；与醋酸/天冬氨酸物质的量比为1:2时，溶解度为35mg/mL。优选乳酸。
二、式I化合物乳酸盐的溶解度实验。
在上述研究基础上，进一步考察了加入不同量乳酸与式I化合物成盐的溶解度，结果如下表所示。
表4.式I化合物乳酸盐的溶解度
式I化合物与乳酸的摩尔比 pH值 溶解度
1:2 3.8 194mg/mL
1:1 4.0 92mg/mL
1:0.8 4.5 46mg/mL
1：0.75 5.0 20mg/mL
1:0.5 6.0 8.1mg/mL
无乳酸 7.8 2.5mg/mL
上述结果显示，加入不同比例乳酸时，溶液pH变化不大，但对溶解度有较在影响，乳酸加入量与溶解度成正比，加入越多溶解度越大。但进一步加大乳酸量，导致pH值过低，造成注射疼痛而不适合临床应用，故认为式I化合物与乳酸的摩尔比为1：2左右是最优的。
实施例4
一、注射用冻干粉的处方筛选。
实验通过设计不同处方，选择氯化钠、葡萄糖、乳糖、蔗糖、山梨醇、甘露醇、右旋糖酐等不同种类的辅料，具体如下表所示，考察对处方组成的影响，对冻干后的成品进行考察。
表5.处方设计
原料及辅料名称 处方1 处方2 处方3 处方4 处方5 处方6 处方7
环己烯类衍生物(mg) 265 265 265 265 265 265 265
乳酸(mg) 111 111 111 111 111 111 111
甘露醇(mg) 125 NA NA NA NA NA NA
山梨醇(mg) NA 125 NA NA NA NA NA
右旋糖酐(mg) NA NA 125 NA NA NA NA
蔗糖(mg) NA NA NA 125 NA NA NA
葡萄糖(mg) NA NA NA NA 125 NA NA
氯化钠(mg) NA NA NA NA NA 125 NA
乳糖(mg) NA NA NA NA NA NA 125
注射用水(ml) 2.5 2.5 2.5 2.5 2.5 2.5 2.5
注：NA表示未添加。
将上述处方按照以下制备方法制备：
一、溶液配制。
量取3ml注射用水于西林瓶中，加入式I化合物，搅拌，加乳酸，使药物完全溶解，加入不同填充剂，搅拌均匀，溶解后，过滤，分装，备用。
二、冻干。
1、预冻。
设定板层温度为-40℃，控制药品温度在-40℃以下，保持小时。
2、冻干。
抽真空，按照如下冻干工艺条件进行干燥后即得。
如图4所示，冻干条件为：设定板层温度为-40℃，控制温度在-40℃以下，保持4小时，然后抽真空。然后，控制温度在1小时内缓慢升高到-30℃，保持2小时。然后，控制温度在1小时内缓慢升高到 -20℃，保持3小时。然后，控制温度在2小时内缓慢升高到-10℃，保持3小时。然后，控制温度在1小时内缓慢升高到0℃，保持2小时。然后，控制温度在1小时内缓慢升高到20℃，保持2小时。然后，控制温度在1小时内缓慢升高到40℃，保持9小时。
观察上述方法制得的注射用冻干粉成品的性状，进行复溶实验。高温40℃加速实验10天，观察成品性状和杂质含量，结果如下表所示。
表6.不同处方对成品的影响
Figure PCTCN2017082155-appb-000010
从上述结果中可以看出，以氯化钠、山梨醇、甘露醇为填充剂，制成的粉针剂稳定性和复溶性较好。
二、注射用冻干粉的工艺筛选。
实验通过设计不同的冻干工艺，如图4、5、6所示冻干曲线，考察三种不同冻干工艺对成品的影响。
具体如下：
表7.冻干工艺对比
Figure PCTCN2017082155-appb-000011
从上述结果中可以看出，以图4所示的冻干工艺制成的粉针剂外观和复溶性较好。
实施例5
一种抗流感病毒的注射液，处方如下：
式I化合物60g、枸橼酸25g、注射用水1200ml。
上述注射液的制备方法如下：
取式I化合物60g，加入500ml注射用水，搅拌，然后加入枸橼酸调pH值至3.5，使其溶解；用注射用水稀释至1200ml，粗滤；用0.22μm无菌滤膜过滤，超滤，检验，每瓶分装5ml，压塞，加铝塑复合盖扎盖，外包装，检验，即得产品。
实施例6
一种抗流感病毒的注射液，处方如下：
式I化合物25g、乳酸5.7g、注射用水1000ml。
上述注射液的制备方法如下：
取式I化合物25g，加入200ml注射用水，搅拌，然后加入乳酸调pH值至4.5，使其溶解；用注射 用水稀释至1000ml，粗滤；用0.22μm无菌滤膜过滤，超滤，检验，每瓶分装10ml，压塞，加铝塑复合盖扎盖，外包装，检验，即得产品。
实施例7
一种抗流感病毒的注射液，处方如下：
式I化合物2g、盐酸400mg、注射用水800ml。
上述注射液的制备方法如下：
取式I化合物2g，加入500ml注射用水，搅拌，然后加入盐酸调pH值至5.0，使其溶解；用注射用水稀释至800ml，粗滤；用0.22μm无菌滤膜过滤，超滤，检验，每瓶分装100ml，压塞，加铝塑复合盖扎盖，外包装，检验，即得产品。
实施例8
一种抗流感病毒的注射用冻干粉，处方如下：
式I化合物5.0g、乳酸2.1g、氯化钠1.8g、活性炭0.3g、注射用水60ml。
上述注射用冻干粉的制备方法如下：
一、溶液配制。
式I化合物原料药及辅料灭菌后，将5.0g式I化合物加入60ml注射用水中，慢慢加乳酸，调节pH至3.8，使药物完全溶解，加入1.8g氯化钠和0.3g活性炭，搅拌30分钟，过滤，无菌条件下分装为20瓶，备用。
二、冻干。
1、预冻。
设定板层温度为-40℃，控制药液温度在-40℃以下，保持4小时。
2、冻干。
抽真空，按照如下冻干工艺条件进行干燥后即得。
如图4所示，冻干条件为：设定板层温度为-40℃，控制温度在-40℃以下，保持4小时，然后抽真空。然后，控制温度在1小时内缓慢升高到-30℃，保持2小时。然后，控制温度在1小时内缓慢升高到-20℃，保持3小时。然后，控制温度在2小时内缓慢升高到-10℃，保持3小时。然后，控制温度在1小时内缓慢升高到0℃，保持2小时。然后，控制温度在1小时内缓慢升高到20℃，保持2小时。然后，控制温度在1小时内缓慢升高到40℃，保持6小时。
实施例9
一种抗流感病毒的注射用冻干粉，处方如下：
式I化合物2.0g、乳酸0.88g、氯化钠0.8g、活性炭100mg、注射用水加至20ml。
上述注射用冻干粉的制备方法如下：
一、溶液配制。
式I化合物原料药及辅料灭菌后，将2.0g式I化合物加入15ml注射用水中，慢慢加乳酸调节pH至3.8，使药物完全溶解，加入0.8g氯化钠和100mg活性炭，搅拌30分钟，过滤，无菌条件下分装为8瓶，准备冻干。
二、冻干。
1、预冻。
设定板层温度为-40℃，控制药品温度在-40℃以下，保持4小时。
2、冻干。
然后抽真空。按照实施例8的冻干工艺条件进行干燥后即得。
实施例10
一种抗流感病毒的注射用冻干粉，处方如下：
式I化合物500mg、乳酸200mg、甘露醇300mg、注射用水3ml。
上述注射用冻干粉的制备方法如下：
一、溶液配制。
量取3ml注射用水于西林瓶中，加入500mg式I化合物，搅拌，加乳酸，调节pH至4.0，使药物完全溶解，加入300mg甘露醇，搅拌均匀，溶解后，过滤，分装为2瓶，备用。
二、冻干。
1、预冻。
设定板层温度为-40℃，控制药品温度在-40℃以下，保持小时。
2、冻干。
抽真空，按照如下冻干工艺条件进行干燥后即得。
然后抽真空。按照实施例8的冻干工艺条件进行干燥后即得。
实施例11
一种抗流感病毒的注射用冻干粉，处方如下：
式I化合物5.0g、乳酸2.0g、右旋糖酐5g、注射用水50ml。
上述注射用冻干粉的制备方法如下：
一、溶液配制。
制备方法：量取50ml注射用水于西林瓶中，加入5.0g环己烯类衍生物，搅拌，加乳酸，调节pH至4.0，使药物完全溶解，加入5g右旋糖酐，搅拌均匀，溶解后，过滤，分装至20瓶，备用。
二、冻干。
1、预冻。
设定板层温度为-40℃，控制药品温度在-40℃以下，保持4小时。
2、冻干。
抽真空，按照实施例8的冻干工艺条件进行干燥后即得。
实施例12
根据药典对药品制剂的稳定性试验要求，对受试品(按实施例9所制备的样品)进行了长期试验的研究，结果见下表。
表8 长期试验结果(25℃)
Figure PCTCN2017082155-appb-000012
Figure PCTCN2017082155-appb-000013
从上述结果可以看出，以上长期留样样品在留样12个月和24个月时分别取样进行细菌内毒素和无菌检查，检查结果表明样品细菌内毒素和无菌均符合规定。说明本品在上市包装条件下，长期留样24个月后，样品外观色泽、pH值、澄明度、有关物质、含量和水分仍符合规定。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 式I所示的环己烯类衍生物或其药学上可接受的盐或其前药：
    Figure PCTCN2017082155-appb-100001
  2. 一种抗流感病毒的注射剂，其特征在于，该注射剂以式I所示化合物或其药学上可接受的盐或其前药作为活性成分。
  3. 根据权利要求1所述的抗流感病毒的注射剂，其特征在于，所述式I所示化合物药学上可接受的盐为：乳酸盐、盐酸盐、磷酸盐、醋酸盐、苹果酸盐、枸椽酸盐、或天冬氨酸盐。
  4. 根据权利要求3所述的抗流感病毒的注射剂，其特征在于，所述药学上可接受的盐为乳酸盐，其中，式I所示化合物与乳酸的摩尔比为1:1.5-2.5。
  5. 根据权利要求2所述的抗流感病毒的注射剂，其特征在于，该注射剂为注射用无菌粉末，所述注射用无菌粉末还包括药学上可接受的辅料。
  6. 根据权利要求5所述的抗流感病毒的注射剂，其特征在于，所述注射用无菌粉末为注射用冻干粉，所述药学上可接受的辅料包括填充剂，所述填充剂为甘露醇、山梨醇、和氯化钠中的至少一种，所述填充剂与所述式I化合物或其药学上可接受的盐的重量份比为0.19-1.9：1。
  7. 权利要求2所述的抗流感病毒的注射剂的制备方法，其特征在于，包括以下步骤：取式I所示化合物，加入适量注射用水，再加入有机酸或无机酸使所述式I化合物与所述有机酸或无机酸充分反应、溶解，随后加入预定量的药学上可接受的辅料，混匀，随后加入注射用水至预定体积，过滤，即得。
  8. 根据权利要求6所述的抗流感病毒的注射剂的制备方法，其特征在于，包括以下步骤：
    溶液配制：取式I所示化合物，加入适量注射用水，再加入有机酸或无机酸，使所述式I化合物与所述有机酸或无机酸充分反应、溶解，随后加入预定量的药学上可接受的辅料，混匀，随后加入预定量的药学上可接受的辅料，混匀，过滤；
    冻干：将上述得到的药液分装后置于冷冻干燥设备中，冻干，即得。
  9. 根据权利要求8所述的抗流感病毒的注射剂的制备方法，其特征在于，
    所述溶液配制步骤中，所述药学上可接受的辅料为氯化钠，按照辅料与所述式I化合物的重量份比为0.19-0.57：1的量加入辅料；
    所述冻干步骤中，按照下述工艺条件进行冻干：设定板层温度为-30～-50℃，控制温度在-30～-50℃保持3-5小时；
    然后抽真空，控制温度在0.5-1.5小时内逐渐升高7-15℃，保持1.5-2.5小时；
    然后控制温度在0.5-1.5小时内逐渐升高7-15℃，保持2-4小时；
    然后控制温度在1.5-2.5小时内逐渐升高7-15℃，保持2-4小时；
    控制温度在0.5-1.5小时内逐渐升高到0℃，保持1.5-2.5小时；
    然后控制温度在0.5-1.5小时内逐渐升高到15-25℃，保持1.5-2.5小时；
    然后控制温度在0.5-1.5小时内逐渐升高到35-45℃，保持4-10小时。
  10. 权利要求1所述的环己烯类衍生物或其药学上可接受的盐在制备抗流感病毒药物中的应用。
PCT/CN2017/082155 2016-04-28 2017-04-27 环己烯类衍生物或其药学上可接受的盐及其用途 WO2017186138A1 (zh)

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CN103224464A (zh) * 2013-04-15 2013-07-31 张健存 环己烯类衍生物或其药学上可接受的盐及其用途

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