WO2020216349A1 - 一种肠道病毒抑制剂 - Google Patents

一种肠道病毒抑制剂 Download PDF

Info

Publication number
WO2020216349A1
WO2020216349A1 PCT/CN2020/086835 CN2020086835W WO2020216349A1 WO 2020216349 A1 WO2020216349 A1 WO 2020216349A1 CN 2020086835 W CN2020086835 W CN 2020086835W WO 2020216349 A1 WO2020216349 A1 WO 2020216349A1
Authority
WO
WIPO (PCT)
Prior art keywords
virus
pharmaceutically acceptable
compound
enterovirus
acceptable salt
Prior art date
Application number
PCT/CN2020/086835
Other languages
English (en)
French (fr)
Inventor
钟武
曹瑞源
程通
夏宁邵
李松
Original Assignee
中国人民解放军军事科学院军事医学研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国人民解放军军事科学院军事医学研究院 filed Critical 中国人民解放军军事科学院军事医学研究院
Priority to US17/606,243 priority Critical patent/US20220204501A1/en
Priority to EP20795446.2A priority patent/EP3960173A4/en
Priority to JP2021563068A priority patent/JP2022530070A/ja
Priority to CN202080030295.0A priority patent/CN113924093A/zh
Publication of WO2020216349A1 publication Critical patent/WO2020216349A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • 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
    • 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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • 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
    • 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
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses

Definitions

  • This application belongs to the field of medical technology, and specifically relates to 9-[3-(6-amino)-pyridyl]-1-[(3-trifluoromethyl)-phenyl]benzo[h][1,6]naphthalene Pyridin-2(1H)-one (formula I), its stereoisomers, its pharmaceutically acceptable salts and/or both solvates and/or hydrates, and pharmaceutical compositions containing the above compounds, for Broad-spectrum antiviral applications, especially in the treatment of enterovirus infectious diseases.
  • Enterovirus belongs to the Picornaviridae enterovirus genus. It is a non-enveloped single-stranded positive-stranded RNA virus, mainly including poliovirus, Coxsackie virus group A, and Coxsackie Virus group B, Echo virus and new enterovirus, among which Coxsackie virus and new enterovirus are the main pathogens of hand, foot and mouth disease. Enterovirus infection can cause serious neurological complications like polio, which is the main cause of severe cases and deaths of hand, foot and mouth disease. Hand, foot and mouth disease is mainly prevalent in the Asia-Pacific region, and many countries including Taiwan, Singapore and Vietnam have a large-scale epidemic.
  • influenza Newcastle disease virus
  • the highly pathogenic avian influenza that broke out at the end of 2003 not only caused the deaths of tens of millions of poultry and wild birds worldwide, but also caused more than 250 deaths, with a mortality rate as high as 60%.
  • a new type of influenza A which originated in North America in April 2009, quickly swept the world and caused more than 10,000 deaths.
  • humans have been aware of influenza for nearly a hundred years, the existing medical technology for influenza prevention and treatment (especially highly pathogenic influenza) is still very weak.
  • tamivir neuraminidase inhibitor oseltamivir
  • tamivir neuraminidase inhibitor oseltamivir
  • tamivir neuraminidase inhibitor
  • tamivir neuraminidase inhibitor
  • tamivir neuraminidase inhibitor
  • tamivir neuraminidase inhibitor
  • tamivir neuraminidase inhibitor
  • zanamivir and broad-spectrum The antiviral drug ribavirin is a three-class drug.
  • Amantadine has been used clinically for many years, and many subtypes of influenza strains have developed severe drug resistance to it. Tamiflu is expensive, and reports of virus resistance to it are increasing.
  • HRV Human rhinovirus
  • IFN interferon
  • Herpes virus family is a type of double-stranded DNA virus with an envelope structure.
  • Herpes simplex virus (HSV) belongs to the alpha subfamily. It exists widely in nature and can infect humans and many animals, especially human skin tissues. Strong addiction. The herpes virus mainly infects the host through skin, mucous membranes and nerve tissues and causes corresponding pathological changes. It is a common pathogen of human viral diseases. Herpes simplex virus can be divided into two serotypes, HSV-1 and HSV-2. HSV-1 infects the human body mainly to cause herpes labialis, pharyngitis, keratitis, and can also cause sporadic encephalitis and other serious diseases.
  • HSV-2 mainly causes genital herpes through damaged skin and mucous membrane infection.
  • epidemiological investigations have found that among the pathogens causing genital herpes viruses, HSV-1 and HSV-2 occupies the same important position, and both can be in the body for a long time. Lurking.
  • the latent infection process the structure and function of the herpes virus genome are not affected or destroyed, and a series of regulation related to viral gene transcription and expression are in a state of stagnation. This process does not have complete genome replication, but there is limited local gene transcription, and under certain conditions enter the stage of proliferative replication infection.
  • the incidence of genital herpes is increasing rapidly and it is easy to recur, which has caused great difficulties in the treatment and prevention of related diseases.
  • Vesicular stomatitis virus can cause vesicular stomatitis. It is a kind of pathogen of highly contact zoonotic diseases. The clinical symptoms after the onset are related to foot and mouth disease, swine vesicular disease and swine vesicular herpes Such diseases are very similar, and it is usually difficult to distinguish. The main clinical features are the appearance of small vesicles, ulcers and scabs on the lips, hoof crown and breasts. The pain caused by erosions and ulcers causes loss of appetite and secondary mastitis in animals, resulting in reduced productivity and even death of animals, causing serious economic losses to the breeding industry. At the same time, VSV can also infect humans, causing flu-like symptoms and severely leading to death.
  • Torin compound is an ATP analogue, which is a kind of serine/threonine protein kinase, also known as protein kinase B, is an important factor in the mTOR/AKT signaling pathway, and plays an important role in cell proliferation, differentiation, apoptosis and metabolism. It plays an important role in a series of physiological activities and is closely related to the occurrence of tumors. At present, the compound has undergone preclinical experiments.
  • the hand, foot and mouth disease (HFMD) mentioned above is a childhood infectious disease caused by a variety of human enteroviruses, and it is transmitted through fecal-oral or respiratory droplets, and can also be transmitted through contact with patients’ skin , Mucosal herpes fluid and infection.
  • the virus that causes hand, foot and mouth disease belongs to the genus of human enterovirus (HEV). According to the serotype, it can be divided into four subtypes: HEV-A, HEV-B, HEV-C and HEV-D.
  • Virus 71 Enterovirus71, EV71
  • Coxsackievirus A group 16 Cox Asckievirus 16, CA16
  • the antiviral drugs currently in preclinical research mainly include the following categories: 1) Antiviral drugs that target virus adsorption and enter the stage, such as Soluble scavenger receptor B2 (human scavenger receptor class B, member 2, SCARB2), P-selectin glycoprotein ligand-1 (PSGL-1), heparin or heparin analogs, and saliva Acid, etc.; 2) Antiviral drugs that target the virus uncoating stage, such as uncoating inhibitors WIN51711, BPROZ-194, BPROZ-112, etc.; 3) Antiviral drugs that target the viral RNA translation stage, such as Quinacrine , Amantadine, etc.; 4) Antiviral drugs that target the processing stage of viral proteins, such as 2A protease substrate analogue LVLQTM and 3C protein irreversible peptidase inhibitor AG7088, etc.; but most of them act on
  • the purpose of this application is to provide a new type of anti-enterovirus inhibitor in view of the scarcity of enterovirus drugs in clinical treatment.
  • It has the function of protecting cells infected by enterovirus, adenovirus, influenza virus, rhinovirus, herpes virus, vesicular stomatitis virus and herpes zoster virus, inhibiting virus replication, and is used in the treatment of enterovirus, adenovirus, Influenza virus, rhinovirus, herpes virus, vesicular stomatitis virus and herpes zoster virus disease have very good effects; in particular, it has significant anti-intestinal virus activity and can be developed as a new anti-intestinal virus drug , Has a wide range of application prospects.
  • the compound 9-[3-(6-amino)-pyridinyl]-1-[(3-trifluoromethyl)-phenyl]benzo[h][1,6]naphthyridine-2(1H) -Ketone can significantly inhibit the proliferation of enteroviruses EV-D68, Poliovirus I/II/III, CB3 and EV71 viruses in host cells.
  • the compound 9-[3-(6-amino)-pyridinyl]-1-[(3-trifluoromethyl)-phenyl]benzo[h][1,6]naphthyridine-2(1H) -Ketones can target the mTOR protein of host cells, and then regulate the PI3K/Akt/mTOR signaling pathway, thereby inhibiting the replication of enteroviruses, have obvious antiviral activity, and can be used to prepare anti-enteroviral drugs.
  • the first aspect of the application provides a compound represented by formula I, or a pharmaceutically acceptable salt, stereoisomer, hydrate or solvate thereof,
  • the second aspect of the present application provides a pharmaceutical composition, which comprises the compound of formula I described in the present application, its pharmaceutically acceptable salt, its stereoisomer, its hydrate, or its solvate.
  • the pharmaceutical composition described in the second aspect of the application further comprises a pharmaceutically acceptable carrier or adjuvant.
  • the pharmaceutical composition can be made into solid preparations, injections, external preparations, sprays, liquid preparations or compound preparations as required.
  • the compound of formula I can protect cells from the cytopathic effect (CPE) caused by virus infection, inhibit virus replication on cells, reduce the viral nucleic acid load in cell culture, and can also provide protection to virus-infected mice. Fully protected.
  • CPE cytopathic effect
  • the compound of formula I can reduce the infection of enterovirus, adenovirus, influenza virus, rhinovirus, herpes virus, vesicular stomatitis virus and herpes zoster virus at micromolar concentrations.
  • the compound of formula I can reduce the viral nucleic acid load level of cells infected by enterovirus, adenovirus, influenza virus, rhinovirus, herpes virus, vesicular stomatitis virus and herpes zoster virus at micromolar concentrations;
  • the compound of formula I can provide complete protection to mice infected with enterovirus, adenovirus, influenza virus, rhinovirus, herpes virus, vesicular stomatitis virus and herpes zoster virus at micromolar concentrations.
  • the third aspect of the application provides that the compound of formula I, its pharmaceutically acceptable salt, its stereoisomer, its hydrate or solvate or the pharmaceutical composition described in this application are prepared for the treatment of intestines.
  • Drugs for viral infectious diseases (such as hand, foot and mouth disease) caused by viruses such as tract virus, polio virus, adenovirus, influenza virus, rhinovirus, herpes simplex virus, vesicular stomatitis virus and/or herpes zoster virus the use of.
  • the fourth aspect of the application provides that the compound of formula I, its pharmaceutically acceptable salt, its stereoisomer, its hydrate or solvate or the pharmaceutical composition described in this application are used for intestinal inhibition.
  • the fifth aspect of the application provides a method of the application for treating and/or preventing diseases in a mammal in need, or inhibiting enterovirus, adenovirus, influenza virus, rhinovirus, herpes simplex in a mammal in need
  • a method for virus, vesicular stomatitis virus and/or herpes zoster virus replication which method comprises administering to a mammal in need thereof a therapeutically and/or prophylactically effective amount of the compound of formula I described in the present application, and its pharmaceutically acceptable
  • the sixth aspect of the application provides the compound of formula I, its pharmaceutically acceptable salt, pharmaceutically acceptable salt, its stereoisomer, its hydrate or solvate, or the pharmaceutical composition described in the application, which It is used to treat viral infectious diseases caused by viruses such as enterovirus, polio virus, adenovirus, influenza virus, rhinovirus, herpes simplex virus, vesicular stomatitis virus and/or herpes zoster virus (such as hand, foot and mouth disease) ).
  • viruses such as enterovirus, polio virus, adenovirus, influenza virus, rhinovirus, herpes simplex virus, vesicular stomatitis virus and/or herpes zoster virus (such as hand, foot and mouth disease) ).
  • the seventh aspect of the application provides the compound of formula I described in the application, its pharmaceutically acceptable salt, a pharmaceutically acceptable salt, its stereoisomer, its hydrate or solvate, or the pharmaceutical composition, which It is used to inhibit the replication of enterovirus, adenovirus, influenza virus, rhinovirus, herpes simplex virus, vesicular stomatitis virus and/or herpes zoster virus in host cells (such as mammalian cells).
  • the eighth aspect of this application provides the use of the compound represented by formula I, its stereoisomer, its hydrate or solvate in the preparation of a drug for inhibiting the replication of enterovirus in target cells,
  • the enterovirus described in this application is a virus of HEV-A, HEV-B, HEV-C, or HEV-D subtype.
  • the enterovirus described in this application is EV-D68, Poliovirus I/II/III, CA16, CA6, CA10, CB3 or EV71 virus.
  • the enterovirus described in this application is EV-D68, Poliovirus I/II/III, CB3 or EV71 virus.
  • the enterovirus described in this application is EV-D68 or CB3 virus.
  • the enterovirus described in this application is EV-D68.
  • the enterovirus described in the present application is Poliovirus I/II/III.
  • the enterovirus described in this application is a CA16 virus.
  • the enterovirus described in this application is a CA6 virus.
  • the enterovirus described in this application is a CA10 virus.
  • the enterovirus described in this application is a CB3 virus.
  • the enterovirus described in the present application is EV71 virus.
  • Poliovirus I/II/III refers to any type or a mixed strain of any two types or a mixed strain of three types among Poliovirus I, Poliovirus II, and Poliovirus III.
  • This application also relates to a pharmaceutical composition containing the compound of formula I and its pharmaceutically acceptable salt and/or its pharmaceutically acceptable solvate or its hydrate and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable salts of the compound of formula (I) described in this application include its inorganic or organic acid salts, and inorganic or organic base salts.
  • This application relates to all forms of the above-mentioned salts. Including but not limited to: sodium salt, potassium salt, calcium salt, lithium salt, meglumine salt, hydrochloride, hydrochloride, hydrocoholate, nitrate, sulfate, hydrogen sulfate, phosphate, Hydrogen phosphate, acetate, propionate, butyrate, oxalate, trimethyl acetate, adipate, alginate, lactate, citrate, tartrate, succinate , Maleate, fumarate, picrate, aspartate, gluconate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonic acid Salt and pamoate etc.
  • composition described in this application can be administered through various routes, such as oral tablets, capsules, powders, oral liquids, injections and transdermal preparations.
  • pharmaceutically acceptable carriers include diluents, fillers, disintegrants, wetting agents, lubricants, coloring agents, flavoring agents or other conventional additives.
  • Typical pharmaceutically acceptable carriers include, for example, microcrystalline cellulose, starch, crospovidone, povidone, polyvinylpyrrolidone, maltitol, citric acid, sodium lauryl sulfonate or magnesium stearate, etc. .
  • This application relates to a pharmaceutical composition, which contains the compound of formula I described in this application, its pharmaceutically acceptable salt, its stereoisomer, its hydrate, or its solvate, and at least one pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be prepared into various forms according to different administration routes.
  • the compound 9-[3-(6-amino)-pyridinyl]-1-[(3-trifluoromethyl)-phenyl]benzo[h][1,6]naphthyridine-2(1H) -Ketones can be made into a variety of pharmaceutically acceptable dosage forms, such as tablets, granules, powders, capsules, oral liquids, injections, etc., for anti-intestinal virus treatment.
  • the mammals include bovines, equines, ovines, swines, canines, felines, rodents, primates, among which mammals are preferred Be human.
  • the pharmaceutical composition can be administered in any of the following ways: oral administration, spray inhalation, rectal administration, nasal administration, buccal administration, vaginal administration, topical administration, parenteral administration such as subcutaneous, intravenous, intramuscular, Intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or medication with an explanted reservoir.
  • oral, intraperitoneal or intravenous administration is preferred.
  • the compound of formula I described in this application can be prepared into any orally acceptable preparation form, including but not limited to tablets, capsules, aqueous solutions or aqueous suspensions.
  • the carriers commonly used in tablets include lactose and corn starch, and lubricants such as magnesium stearate can also be added.
  • Diluents commonly used in capsule preparations include lactose and dried corn starch.
  • Aqueous suspension formulations usually mix the active ingredients with suitable emulsifiers and suspending agents. If necessary, some sweeteners, fragrances or coloring agents can be added to the above oral preparations.
  • the compound of formula I described in this application can generally be prepared in the form of a suppository, which is prepared by mixing the drug with a suitable non-irritating excipient.
  • the excipient presents a solid state at room temperature, but melts at the rectal temperature to release the drug.
  • excipients include cocoa butter, beeswax and polyethylene glycol.
  • topical medication especially for the treatment of affected surfaces or organs that are easily reached by topical application, such as eye, skin or lower intestinal neurological diseases
  • topical application such as eye, skin or lower intestinal neurological diseases
  • the compound of formula I described in this application can be made into different types according to different affected surfaces or organs.
  • Forms of topical preparations, the specific instructions are as follows:
  • the compound of formula I described in the present application can be formulated into a micronized suspension or solution, and the carrier used is isotonic sterile saline with a certain pH, with or without preservatives. Such as chlorinated benzyl alkoxide.
  • the compound can also be made into an ointment form such as petroleum jelly.
  • the compound of formula I described in this application can be prepared in the form of an appropriate ointment, lotion or cream preparation, wherein the active ingredient is suspended or dissolved in one or more carriers.
  • the carriers that can be used for the ointment here include, but are not limited to: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, emulsifying wax and water; the carriers that can be used for lotions or creams include but are not limited to: Mineral oil, sorbitan monostearate, Tween 60, cetyl ester wax, hexadecenyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • the compound of formula I described in the present application can be prepared as a rectal suppository preparation or a suitable enema preparation as described above, and a topical transdermal patch can also be used.
  • the compounds of formula I described in this application can also be administered in the form of sterile injection preparations, including sterile injection water or oil suspensions, or sterile injection solutions.
  • usable carriers and solvents include water, Ringer's solution and isotonic sodium chloride solution.
  • sterilized non-volatile oils can also be used as solvents or suspension media, such as monoglycerides or diglycerides.
  • therapeutically effective amount refers to an amount that is sufficient to treat or prevent the patient's disease but is low enough to avoid serious side effects (at a reasonable benefit/risk ratio) within the scope of reasonable medical judgment .
  • the therapeutically effective amount of the compound will depend on the specific compound selected (for example, considering the potency, effectiveness and half-life of the compound), the route of administration selected, the disease being treated, the severity of the disease being treated, and the patient's Factors such as age, size, weight and physical disease, medical history of the patient being treated, duration of treatment, nature of concurrent therapy, desired therapeutic effect and other factors have changed, but they can still be routinely determined by those skilled in the art.
  • the specific dosage and method of use of the compound of formula I described in this application for different patients are determined by many factors, including the patient’s age, weight, gender, natural health status, nutritional status, active strength of the compound, and time of administration, The metabolic rate, the severity of the disease, and the subjective judgment of the treating physician.
  • the preferred dosage here is between 0.01-100 mg/kg body weight/day.
  • Figure 1 is a fitting curve of compound concentration-mTOR kinase inhibition rate
  • Figure 2 shows the Western blot results of rapamycin and the compound of formula I.
  • the in vitro antiviral experiment of this application involves multiple subtypes of enterovirus, including EV-D68, Poliovirus I/II/III (type 3 mixed vaccine strain), CA16, CA6, CA10, CB3 (all the above strains are Provided by the Academy of Military Medical Sciences of the Chinese People’s Liberation Army) and EV71 virus (purchased from ATCC).
  • EV-D68 Poliovirus I/II/III (type 3 mixed vaccine strain)
  • CA16 CA6, CA10, CB3
  • EV71 virus purchased from ATCC.
  • EV-D68, Poliovirus I/II/III, CA16, CA6, CA10, and EV71 viruses were tested with RD cells (purchased from ATCC).
  • the experimental methods are as follows:
  • virus growth medium (DMEM+2% FBS, Gibco, article numbers are 11995-065, 1600-044, respectively) to dilute the virus seed to 100 TCID50, and add it to the above 96-well plate, 50 ⁇ l per well. Add an equal volume of virus growth solution to the cell control group.
  • the RD cells were cultured at 37°C for 4 days to test the plate.
  • the buffer of the chemiluminescent cell viability detection reagent (Promega) is mixed with the substrate in the dark to prepare a working solution.
  • inhibition rate (%) (experimental group-average value of virus group) / (average value of cell control group-average value of virus group) * 100
  • the inhibition rate-concentration curve was fitted to the S curve, and the IC 50 value of the test compound was calculated.
  • the final concentration of the drug is 0.5 times the pretreatment concentration.
  • the RD cells were cultured at 37°C for 4 days to test the plate.
  • the buffer of the chemiluminescent cell viability detection reagent is mixed with the substrate in the dark to prepare a working solution.
  • Inhibition rate (%) (the average value of the cell control group-the experimental group) / (the average value of the cell control group-the minimum value of the experimental group) * 100
  • the inhibition rate-concentration curve was fitted to the S curve, and the IC 50 value of the test compound was calculated.
  • the activity of CB3 virus was measured with Vero cells.
  • the experimental method is as follows:
  • Vero cells purchased from ATCC
  • 1Inoculate Vero cells (purchased from ATCC) into a 96-well plate with a white wall and transparent bottom at a concentration of 1*10 5 /ml 24 hours in advance, with 100 ⁇ l per well.
  • cell maintenance solution (DMEM+2% FBS, Gibco, article numbers are 11995-065, 1600-044, respectively) to dilute the test compound to a concentration of 800 ⁇ M, and then use a 3-fold dilution gradient to dilute the test compound. A total of 10 concentrations. Then add the diluted compound to the above 96-well plate, 50 ⁇ l per well. Both the cell control group and the virus control group were added with an equal volume of cell maintenance solution.
  • virus growth medium (DMEM+2% FBS, Gibco, article numbers are 11995-065, 1600-044, respectively) to dilute the virus seed to 100 TCID50, and add it to the above 96-well plate cells, 50 ⁇ l per well.
  • the cell control group was added with an equal volume of virus growth solution, the final volume per well was 200 ⁇ l, and the final drug concentration was 0.25 times the pretreatment concentration.
  • Vero cells were cultured at 37°C for 5 days to test the plate.
  • the buffer of the chemiluminescent cell viability detection reagent is mixed with the substrate in the dark to prepare a working solution.
  • the culture medium in the 96-well plate is discarded, and the plate is gently patted dry, 100 ⁇ l of detection reagent is added to each well, and the 96-well plate is shaken for 4 minutes with an orbital shaker to induce cell lysis. After the signal is stabilized in the dark for 15 minutes, the chemiluminescence unit is measured, and the plate reading program is the CellTiter-Glo preset program.
  • inhibition rate (%) (experimental group-average value of virus group) / (average value of cell control group-average value of virus group) * 100
  • the inhibition rate-concentration curve was fitted to the S curve, and the IC 50 value of the test compound was calculated.
  • Vero cells in a 96-well plate with a white wall and transparent bottom at a concentration of 1*105/ml 24 hours in advance, with 100 ⁇ l per well.
  • the buffer of the chemiluminescent cell viability detection reagent is mixed with the substrate in the dark to prepare a working solution.
  • Inhibition rate (%) (the average value of the cell control group-the experimental group) / (the average value of the cell control group-the minimum value of the experimental group) * 100
  • the inhibition rate-concentration curve was fitted to the S curve, and the IC 50 value of the test compound was calculated.
  • Basic buffer composition 50mM (mmol/L) HEPES (pH 7.5), 1mM EGTA, 0.01% Tween-20, 10mM MnCl2, 2mM DTT (diluted by 500mM when used).
  • 1Substrate buffer solution 1650 ⁇ L 2.5 ⁇ substrate buffer solution consists of 1559.6 ⁇ L 1 ⁇ basic buffer, 89.2 ⁇ L GFP-4E-BP1 (18.5 ⁇ M stock solution, purchased from Thermo Fisher, catalog number PV4759) and 1.2 ⁇ L ATP (10mM) Composition, the final concentration is 0.4 ⁇ M GFP-4E-BP1, 3 ⁇ M ATP.
  • 2mTOR kinase buffer solution 1650 ⁇ L 2.5 ⁇ mTOR kinase buffer solution consists of 1640.2 ⁇ L 1 ⁇ basic buffer, 9.8 ⁇ L mTOR (0.21mg/mL stock solution), and the final concentration is 0.5 ⁇ g/mL.
  • Detection buffer solution 3960 ⁇ L 2 ⁇ detection buffer solution consists of 3797.1 ⁇ L TR-FRET buffer diluent (purchased from Thermo Fisher, item number PV3574), 4.5 ⁇ L Tb-anti-p4E-BP1 antibody (3.49 ⁇ M stock solution, purchased from Thermo Fisher , The item number is PV4757), 158 ⁇ L EDTA (500mM stock solution), the final concentration is 2nM Tb-anti-p4E-BP1 antibody, 10mM EDTA.
  • the final concentration of mTOR reaction solution 0.5 ⁇ g/mL mTOR, 0.4 ⁇ M GFP-4E-BP1, 3 ⁇ M ATP.
  • Final concentration of test compound 50000, 16666, 5555, 1851, 617.3, 205.8, 68.58, 22.86, 7.62, 2.54 and 0.85 nM.
  • the final concentration of the DMSO solution is 1%.
  • Excitation wavelength is 340nm
  • emission wavelength 1 is 495nm
  • emission wavelength 2 is 520nm
  • the ratio of readings of 520nm/495nm is calculated as the TR-FRET value.
  • X Common logarithm of compound concentration
  • Y TR-FRET value (520nm/495nm).
  • mTORC1 and mTORC2 play a role by activating the phosphorylation of downstream substrates, by detecting the phosphorylation level of Thr389 of the downstream substrate of mTORC1 p70S6K1 and Ser473 of the downstream substrate of mTORC2, the phosphorylation level of the compound on mTORC1 and mTORC2 The inhibitory activity.
  • RD cells were cultured in DMEM medium containing 10% FBS and 1 ⁇ PS (penicillin and streptomycin concentrations were 100 IU and 100 ⁇ g/mL, respectively) at 37°C and 5% CO 2 concentration.
  • Preparation of insulin medium Dilute insulin in DMEM medium supplemented with 10% FBS and 1 ⁇ PS to make the final concentration of insulin reach 167 nM.
  • Pretreatment of test compound Dissolve the compound in DMSO so that the concentration of the test compound reaches 20 mM, and dilute the compound concentration to 20 ⁇ M in 167 nM insulin medium.
  • rapamycin solution dissolve rapamycin in DMSO to a concentration of 10mM, and dilute the rapamycin concentration to 20 ⁇ M in 167nM insulin medium.
  • 2Lysis Buffer Add 2 mL of 100 ⁇ protease inhibitor and 2 ⁇ phosphatase inhibitor Cocktails PhosSTOP (Beyotime, catalog number P1082) to 100 mL of cell extract, and gently stir until it is completely dissolved.
  • 10 ⁇ MOPS buffer solution add MOPS 52.33g, Tris base 30.29g, 0.5mol/L EDTA (pH8.5) 10mL, SDS 5g dissolved in 400mL double distilled water, stir to dissolve, adjust the pH to 7.5, then add heavy distilled water to 500mL;
  • 1 ⁇ MOPS 100mL 10 ⁇ MOPS is diluted to 1000mL with distilled water.
  • Transfer Buffer Dissolve 100mL 10 ⁇ transfer buffer (144g glucine, 30.3g trisbase, distilled water to 1L) and 400mL methanol in 1500mL double-distilled water, and add heavy-distilled water to 2000mL.
  • 510 ⁇ PBS Buffer (0.1M): Add 5 bags of PBS powder (Solarbio, item number P1010) to 800mL of distilled water, stir to dissolve, adjust the pH to 7.6, and then add distilled water to 1000mL.
  • 61 ⁇ PBS Buffer Dilute 100 mL 10 ⁇ PBS buffer to 1000 mL with re-distilled water.
  • Tween-20 Add 20mL Tween-20 to 180mL of distilled water, and stir well.
  • 81 ⁇ PBST Buffer Dilute 100 mL 10 ⁇ PBST buffer and 10 mL Tween-20 to 1000 mL with re-distilled water.
  • the RD cells were treated with a solution of rapamycin and a compound of formula I at a concentration of 20 ⁇ M for 2 hours, and the Western blot results are shown in Figure 2.
  • the compound can significantly down-regulate the phosphorylation level of Akt, while the positive control drug rapamycin cannot inhibit the phosphorylation of Akt, indicating that the compound 9-[3-(6-amino)-pyridyl]- 1-[(3-Trifluoromethyl)-phenyl]benzo[h][1,6]naphthyridin-2(1H)-one can effectively inhibit mTORC2 while inhibiting mTORC1, thereby better exerting mTOR Inhibition activity, thereby inhibiting virus replication and proliferation.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Virology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Pulmonology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)

Abstract

本申请属于医药技术领域，具体涉及9-[3-(6-氨基)-吡啶基]-1-[(3-三氟甲基)-苯基]苯并[h][1,6]萘啶-2(1H)-酮(式I)、及其可药用的盐、其立体异构体、或其水合物或溶剂化物，及含有此化合物的药物组合物，用于广谱抗病毒，特别是用于治疗肠道病毒感染的用途。

Description

一种肠道病毒抑制剂
本申请是以CN申请号为201910346258.X，申请日为2019年4月26日的申请为基础，并主张其优先权，该CN申请的公开内容在此作为整体引入本申请中。
技术领域
本申请属于医药技术领域，具体涉及9-[3-(6-氨基)-吡啶基]-1-[(3-三氟甲基)-苯基]苯并[h][1,6]萘啶-2(1H)-酮(式I)、其立体异构体、其药物上可接受的盐和/或两者溶剂化物和/或水合物，及含上述化合物的药物组合物，用于广谱抗病毒方面的用途，特别是在治疗肠道病毒感染性疾病中的应用。
Figure PCTCN2020086835-appb-000001
背景技术
肠道病毒(Enterovirus)属于小RNA病毒科(Picornaviridae)肠道病毒属，是一种无包膜的单股正链RNA病毒，主要包括脊髓灰质炎病毒、柯萨奇病毒A组、柯萨奇病毒B组、埃可病毒及新肠道病毒等，其中柯萨奇病毒和新肠道病毒是手足口病的主要病原体。肠道病毒感染后可以导致脊髓灰质炎样的严重的神经系统并发症，是手足口病重症病例和死亡病例的主要原因。手足口病主要流行于亚太地区，包括台湾，新加坡和越南在内的许多国家都有较大规模的流行。最近几年，手足口病在我国大陆持续爆发流行，重症病例和死亡病例逐年增加，已经成为严重威胁我国儿童健康和社会稳定的公共卫生问题，受到社会各界的高度关注。一般来说，疫苗是对抗病毒性传染病的重要手段，但是手足口病一般是由多种肠道病毒感染所致，且一种疫苗只能对一种肠道病毒发挥作用对别的种类没有交叉保护效果。
近年来流感频繁爆发，严重危害人类和动物生命健康。2003年底爆发的高致病性禽流感，不仅造成全球数千万家禽、野禽死亡，而且导致250余人感染死亡，死亡率高达60％。2009年4月源于北美的新型甲流很快席卷全球，并导致万余人死亡。尽管人类对流感的认识已有近百年历史，但现有防治流感(特别是高致病性流感)的医药技术仍十分薄弱。可用于流感防治的药物品种很少，主要有M2离子通道抑制剂金刚烷胺和金刚乙胺、神经氨酸 酶抑制剂奥司米韦(俗称“达菲”)和扎那米韦及广谱抗病毒药利巴韦林三类药物。金刚烷胺临床上应用了多年，多种亚型流感毒株对其已产生严重的耐药性，而达菲价格昂贵，且病毒对其耐药性的报道也日渐增多。
人鼻病毒(HRV)属于小RNA病毒科，是一组单链小分子RNA病毒，至今已发现有100多种血清型。HRV是人类普通感冒的主要致病因素，急慢性支气管炎和其他呼吸道疾病也与之有关。尽管鼻病毒的感染是自限性的，但是病人会发生诸如哮喘、充血性心力衰竭、支气管扩张、包囊纤维化等并发症。特别是对于儿童和有基础疾病的人群，HRV感染会导致严重的后遗症。由于HRV有众多血清型，所以通过接种疫苗的方式来预防HRV感染，在理论上缺乏可行性。曾经认为干扰素(IFN)可作为控制HRV及其他核糖核酸病毒感染的主要物质，但是IFN治疗对已形成的HRV感染无效。
疱疹病毒家族是一类具有包膜结构的双链DNA病毒，单纯疱疹病毒(herpes simplex virus，HSV)属α亚科，广泛存在于自然界中，能感染人和许多动物，尤其对人体皮肤组织具有较强的嗜性。疱疹病毒主要通过皮肤、黏膜和神经组织感染宿主而引起相应的病变，是人类病毒性疾病的常见病原体。单纯疱疹病毒可分为HSV-1及HSV-2两种血清型，HSV-1感染人体后主要引起口唇疱疹、咽炎、角膜炎，也能引起散发性脑炎等严重疾病的发生。HSV-2主要通过破损皮肤及黏膜感染引起生殖器疱疹，近年来的流行病学调查发现，在引起生殖器疱疹病毒的病原中，HSV-1与HSV-2占有同样重要的地位，均可在体内长期潜伏。在潜伏感染过程中，疱疹病毒基因组的结构和功能均未受到任何影响和破坏，而病毒基因转录及表达相关的一系列调控都处于停滞状态。此过程没有完整的基因组复制，但存在有限的局部基因转录，并在特定条件下进入增殖性复制感染阶段。目前生殖器疱疹发病率迅速增加且易复发，这给相关疾病的治疗及预防造成了巨大困难。
水泡性口炎病毒(Vesicular stomatitis virus，VSV)可以引起水泡性口炎，是一类高度接触性的人畜共患传染病的病原体，发病后的临床症状与口蹄疫、猪水疱病及猪水疱疹病等疾病非常相似，通常情况下很难进行区分。以唇、蹄冠部和乳房等部位出现小囊泡、溃疡和结痂等为主要临床特征。由糜烂及溃疡引起的疼痛造成动物的食欲减退及继发性乳腺炎从而导致动物的生产力降低甚至死亡，给养殖业造成严重的经济损失。同时，VSV也可以感染人类，引发类似流感的症状，严重的导致死亡。
综上所述，研发一种具有特异性的小分子广谱抗病毒药物迫在眉睫。Torin类化合物是一种ATP类似物，该化合物是一种丝/苏氨酸蛋白激酶，又称蛋白激酶B，是mTOR/AKT信号通路的重要因子，在细胞的增生、分化、凋亡和代谢等一系列生理活动中起着重要作用，与肿瘤的发生息息相关。目前该化合物已经进行了临床前实验。
上文涉及的手足口病(hand,foot and mouth disease，HFMD)是一种由多种人类肠道病毒引起的儿童传染病，并经粪-口或呼吸道飞沫传播，亦可经接触患者皮肤、黏膜疱疹液 而发生感染。引发手足口病的病毒属于人类肠道病毒属(human enterovirus,HEV)，按血清型分类可分为HEV-A、HEV-B、HEV-C和HEV-D四种亚型，其中以肠道病毒71型(Enterovirus71，EV71)和柯萨奇病毒A组16型(Cox Asckievirus16，CA16)最为常见。近半个世纪以来，全球多地多次出现手足口病疫情，遍及各大洲，使其成为备受关注的世界性疾病。20世纪90年代，亚洲迎来了手足口病爆发的高峰，尤其在东南亚地区，以EV71感染为主的手足口病大规模流行，并引发中枢神经系统症状，导致死亡病例大量增加。
目前，尚无能够有效抑制肠道病毒的特效抗病毒药物上市，现阶段处于临床前研究的抗肠道病毒药物主要有以下几类：1)靶向病毒吸附和进入阶段的抗病毒药物，例如可溶性清道夫受体B2(human scavenger receptor class B，member 2，SCARB2)、P-选择素糖蛋白配体-1(P-selectin glycoprotein ligand-1，PSGL-1)、肝素或肝素类似物和唾液酸等；2)靶向病毒脱壳阶段的抗病毒药物，如脱壳抑制剂WIN51711、BPROZ-194、BPROZ-112等；3)靶向病毒RNA翻译阶段的抗病毒药物，如奎纳克林、金刚烷胺等；4)靶向病毒蛋白质加工阶段的抗病毒药物，如2A蛋白酶底物类似物LVLQTM和3C蛋白不可逆肽酶抑制剂AG7088等；但其大多作用于单个病毒亚型，不具有广谱学特性，且由于肠道病毒的高度变异性，以病毒自身为靶点的抗病毒药物极易产生耐药性。此外，一些抗肠道病毒的疫苗也在研发之中，如重组VP1蛋白疫苗和重组DNA疫苗，但由于应用时间较短、应用规模较低，目前尚缺乏充足的临床反馈。
发明内容
本申请的目的在于针对临床上治疗肠道病毒药物稀缺而提供一种新型的抗肠道病毒抑制剂。
本申请通过创造性的研究发现式I所示的9-[3-(6-氨基)-吡啶基]-1-[(3-三氟甲基)-苯基]苯并[h][1,6]萘啶-2(1H)-酮，
Figure PCTCN2020086835-appb-000002
其具有保护肠道病毒、腺病毒、流感病毒、鼻病毒、疱疹病毒、水泡性口炎病毒病毒及带状疱疹病毒感染的细胞，抑制病毒复制方面的功能，在治疗肠道病毒、腺病毒、流感病毒、鼻病毒、疱疹病毒、水泡性口炎病毒及带状疱疹病毒病方面具有很好的效果；特别 是具有显著的抗肠道病毒的活性，可以作为新的抗肠道病毒药物进行开发，具有广泛的应用前景。
所述化合物9-[3-(6-氨基)-吡啶基]-1-[(3-三氟甲基)-苯基]苯并[h][1,6]萘啶-2(1H)-酮可显著抑制肠道病毒EV-D68、Poliovirus Ⅰ/Ⅱ/Ⅲ、CB3和EV71病毒在宿主细胞中的增殖。
所述化合物9-[3-(6-氨基)-吡啶基]-1-[(3-三氟甲基)-苯基]苯并[h][1,6]萘啶-2(1H)-酮能够靶向宿主细胞的mTOR蛋白，进而调控PI3K/Akt/mTOR信号通路，从而抑制肠道病毒的复制，具有明显的抗病毒活性，可用于制备成抗肠道病毒药物。
本申请的第一方面提供式I所示化合物，或其药学上可接受的盐、其立体异构体、其水合物或溶剂合物，
Figure PCTCN2020086835-appb-000003
本申请的第二方面提供一种药物组合物，其包含本申请所述的式I化合物、其药学上可接受的盐、其立体异构体、其水合物、或其溶剂化物。
在一个优选的实施方案中，本申请第二方面所述的药物组合物，其还包含药学上可接受的载体或辅料。所述药物组合物可以根据需要制成固体制剂、注射剂、外用制剂、喷剂、液体制剂或复方制剂。
根据本申请，式I化合物可保护细胞因病毒感染所导致的细胞病变效应(CPE)，并在细胞上抑制病毒复制，减少细胞培养物中病毒核酸载量，还可以对病毒感染的小鼠提供完全保护。
本申请的发明人在经过长期研究后，发现了式I化合物在细胞内的一些新作用特点：
第一，式I化合物在体外抗病毒实验中，可以在微摩尔级浓度下降低肠道病毒、腺病毒、流感病毒、鼻病毒、疱疹病毒、水泡性口炎病毒病毒及带状疱疹病毒感染的细胞CPE水平；
第二，式I化合物可以在微摩尔级浓度下降低肠道病毒、腺病毒、流感病毒、鼻病毒、疱疹病毒、水泡性口炎病毒及带状疱疹病毒感染的细胞病毒核酸载量水平；
第三，式I化合物可以在微摩尔级浓度下对感染肠道病毒、腺病毒、流感病毒、鼻病毒、疱疹病毒、水泡性口炎病毒及带状疱疹病毒的小鼠提供完全保护。
本申请的第三方面提供本申请所述的式Ⅰ化合物、其药学上可接受的盐、其立体异构体、其水合物或溶剂合物或所述的药物组合物在制备用于治疗肠道病毒、脊髓灰 质炎病毒、腺病毒、流感病毒、鼻病毒、单纯疱疹病毒、水泡性口炎病毒和/或带状疱疹病毒等病毒引起的病毒感染性疾病(例如手足口病)的药物中的用途。
本申请的第四方面提供本申请所述的式Ⅰ化合物、其药学上可接受的盐、其立体异构体、其水合物或溶剂合物或所述的药物组合物在制备用于抑制肠道病毒、腺病毒、流感病毒、鼻病毒、单纯疱疹病毒、水泡性口炎病毒和/或带状疱疹病毒在宿主细胞(例如哺乳动物细胞)中复制的药物中的用途。
本申请的第五方面提供本申请一种在有需要的哺乳动物中治疗和/或预防疾病的方法或者在有需要的哺乳动物中抑制肠道病毒、腺病毒、流感病毒、鼻病毒、单纯疱疹病毒、水泡性口炎病毒和/或带状疱疹病毒复制的方法，该方法包括给有需要的哺乳动物施用治疗和/或预防有效量的本申请所述的式Ⅰ化合物、其药学上可接受的盐、其立体异构体、其水合物或溶剂合物或所述的药物组合物，其中所述的疾病包括肠道病毒、腺病毒、流感病毒、鼻病毒、单纯疱疹病毒、水泡性口炎病毒和/或带状疱疹病毒等病毒引起的病毒感染性疾病(例如手足口病)。
本申请的第六方面提供本申请所述的式Ⅰ化合物、其药学上可接受的盐可药用盐、其立体异构体、其水合物或溶剂合物或所述的药物组合物，其用于治疗肠道病毒、脊髓灰质炎病毒、腺病毒、流感病毒、鼻病毒、单纯疱疹病毒、水泡性口炎病毒和/或带状疱疹病毒等病毒引起的病毒感染性疾病(例如手足口病)。
本申请的第七方面提供本申请所述的式Ⅰ化合物、其药学上可接受的盐可药用盐、其立体异构体、其水合物或溶剂合物或所述的药物组合物，其用于抑制肠道病毒、腺病毒、流感病毒、鼻病毒、单纯疱疹病毒、水泡性口炎病毒和/或带状疱疹病毒在宿主细胞(例如哺乳动物细胞)中复制。
本申请的第八方面提供本申请所述式I所示化合物、其立体异构体、其水合物或溶剂合物在制备抑制肠道病毒在靶细胞中复制的药物中的应用，
在某些实施方案中，本申请所述肠道病毒为HEV-A、HEV-B、HEV-C或HEV-D亚型的病毒。
在某些实施方案中，本申请所述肠道病毒为EV-D68、Poliovirus Ⅰ/Ⅱ/Ⅲ、CA16、CA6、CA10、CB3或EV71病毒。
在某些实施方案中，本申请所述肠道病毒为EV-D68、Poliovirus Ⅰ/Ⅱ/Ⅲ、CB3或EV71病毒。
在某些实施方案中，本申请所述肠道病毒为EV-D68或CB3病毒。
在某些实施方案中，本申请所述肠道病毒为EV-D68。
在某些实施方案中，本申请所述肠道病毒为Poliovirus Ⅰ/Ⅱ/Ⅲ。
在某些实施方案中，本申请所述肠道病毒为CA16病毒。
在某些实施方案中，本申请所述肠道病毒为CA6病毒。
在某些实施方案中，本申请所述肠道病毒为CA10病毒。
在某些实施方案中，本申请所述肠道病毒为CB3病毒。
在某些实施方案中，本申请所述肠道病毒为EV71病毒。
本申请中，Poliovirus Ⅰ/Ⅱ/Ⅲ是指Poliovirus Ⅰ、Poliovirus Ⅱ、Poliovirus Ⅲ中任意一种型别或其中任意两种型别的混和毒株或三种型别的混合毒株。
本申请还涉及含有式I化合物及其药学上可接受的盐和/或其药物上可接受的溶剂化物或其水合物和药物上可接受的载体的药物组合物。
本申请所述式(I)化合物的药学上可接受的盐包括其无机或有机酸盐，以及无机或有机碱盐，本申请涉及上述盐的所有形式。其中包括但不限于:钠盐、钾盐、钙盐、锂盐、葡甲胺盐、盐酸盐，氢澳酸盐，氢腆酸盐，硝酸盐，硫酸盐，硫酸氢盐，磷酸盐，磷酸氢盐，乙酸盐，丙酸盐，丁酸盐，草酸盐，三甲基乙酸盐，己二酸盐，藻酸盐，乳酸盐，柠檬酸盐，酒石酸盐，琥珀酸盐，马来酸盐，富马酸盐，苦味酸盐，天冬氨酸盐，葡糖酸盐，苯甲酸盐，甲磺酸盐，乙磺酸盐，苯磺酸盐，对甲苯磺酸盐和双羟萘酸盐等。
本申请所述的药物组合物可以经多种途径施用，例如口服片剂，胶囊，粉剂，口服液，注射剂和透皮制剂。根据常规的药物上的惯例，药学上可接受的载体包括稀释剂、填充剂、崩解剂、润湿剂、润滑剂、着色剂、调味剂或其它常规添加剂。典型的药学上可接受的载体包括例如微晶纤维素、淀粉、交连聚维酮、聚维酮、聚乙烯吡咯烷酮、麦芽糖醇，柠檬酸，十二烷基磺酸钠或硬脂酸镁等。
本申请涉及药物组合物，其含有本申请所述式I化合物其药学上可接受的盐、其立体异构体、其水合物、或其溶剂化物，以及至少一种药学上可接受的载体。所述药物组合物可以根据不同给药途径而制备成各种形式。所述化合物9-[3-(6-氨基)-吡啶基]-1-[(3-三氟甲基)-苯基]苯并[h][1,6]萘啶-2(1H)-酮可制成多种药学上可接受的剂型，如片剂、颗粒剂、粉剂、胶囊、口服液、注射液等，用于抗肠道病毒的治疗。
在某些实施方案中，所述哺乳动物包括牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、啮齿类动物、灵长类动物，其中优选的哺乳动物为人。
根据申请，所述的药物组合物可以以下面的任意方式施用：口服、喷雾吸入、直肠用药、鼻腔用药、颊部用药、阴道用药、局部用药、非肠道用药如皮下、静脉、肌内、腹膜内、鞘内、心室内、胸骨内和颅内注射或输入、或借助一种外植储器用药。其中优选口服、腹膜内或静脉内用药方式。
当口服用药时，本申请所述式I化合物可制成任意口服可接受的制剂形式，包括但不限于片剂、胶囊、水溶液或水悬浮液。其中，片剂一般使用的载体包括乳糖和玉米淀粉，另外也可加入润滑剂如硬质酸镁。胶囊制剂一般使用的稀释剂包括乳糖和干燥玉米淀粉。 水悬浮液制剂则通常是将活性成分与适宜的乳化剂和悬浮剂混合使用。如果需要，以上口服制剂形式中还可加入一些甜味剂、芳香剂或着色剂。
当直肠用药时，本申请所述式I化合物一般可制成栓剂的形式，其通过将药物与一种适宜的非刺激性赋形剂混合而制得。该赋形剂在室温下呈现固体状态，而在直肠温度下熔化释出药物。该类赋形剂包括可可脂、蜂蜡和聚乙二醇。
当局部用药时，特别是治疗局部外敷容易达到的患面或器官，如眼睛、皮肤或下肠道神经性疾病时，本申请所述式I化合物可根据不同的患面或器官制成不同的局部用药制剂形式，具体说明如下：
当眼部局部施用时，本申请所述式I化合物可配制成一种微粉化悬浮液或溶液的制剂形式，所使用载体为等渗的一定pH的无菌盐水，其中可加入也可不加防腐剂如氯化苄基烷醇盐。此外对于眼用，也可将化合物制成膏剂形式如凡士林膏。
当皮肤局部施用时，本申请所述式I化合物可制成适当的软膏、洗剂或霜剂制剂形式，其中活性成分悬浮或溶解于一种或多种载体中。这里软膏即可使用的载体包括但不限于：矿物油、液体凡士林、白凡士林、丙二醇、聚氧化乙烯、聚氧化丙烯、乳化蜡和水；洗剂或霜剂可使用的载体包括但不限于：矿物油、脱水山梨糖醇单硬脂酸酯、吐温60、十六烷酯蜡、十六碳烯芳醇、2-辛基十二烷醇、苄醇和水。
当下肠道局部施用时，本申请所述式I化合物可制成如上所述的直肠栓剂制剂或适宜的灌肠制剂形式，另外也可使用局部透皮贴剂。
本申请所述式I化合物还可以无菌注射制剂形式用药，包括无菌注射水或油悬浮液，或无菌注射溶液。其中，可使用的载体和溶剂包括水，林格氏溶液和等渗氯化钠溶液。另外，灭菌的非挥发油也可用作溶剂或悬浮介质，如单甘油酯或二甘油酯。
如本文所述的，“治疗有效量”或“预防有效量”是指在合理的医学判断范围内，足以治疗或预防患者疾病但足够低地避免严重副作用(在合理的利益/风险比)的量。化合物的治疗有效量将根据所选择的具体化合物(例如考虑化合物的效力、有效性和半衰期)、所选择的给药途径、所治疗的疾病、所治疗的疾病的严重性、所治疗的患者的年龄、大小、体重和身体疾病、所治疗的患者的医疗史、治疗持续时间、并行疗法的性质、所需的治疗效果等因素发生变化，但仍可以由本领域技术人员常规确定。
另外需要指出，本申请所述式I化合物针对不同患者的特定使用剂量和使用方法决定于诸多因素，包括患者的年龄，体重，性别，自然健康状况，营养状况，化合物的活性强度，服用时间，代谢速率，病症的严重程度以及诊治医师的主观判断。这里优选使用剂量介于0.01-100mg/kg体重/天。
上述各种剂型的药物均可以按照药学领域的常规方法制备。
综上所述，本申请与现有技术相比具有以下优点和效果:
化合物9-[3-(6-氨基)-吡啶基]-1-[(3-三氟甲基)-苯基]苯并[h][1,6]萘啶-2(1H)-酮能够抑制肠道病毒EV-D68、Poliovirus Ⅰ/Ⅱ/Ⅲ、CA16、CA6、CA10、CB3和EV71病毒对细胞的感染，特别是EV-D68、Poliovirus Ⅰ/Ⅱ/Ⅲ、CB3和EV71病毒对细胞的感染，具有广谱的抗病毒活性。
化合物9-[3-(6-氨基)-吡啶基]-1-[(3-三氟甲基)-苯基]苯并[h][1,6]萘啶-2(1H)-酮作用于宿主细胞mTOR蛋白的ATP结合区域，能竞争性抑制ATP与mTOR蛋白结合位点的结合，阻断PI3K/Akt/mTOR信号通路，进而阻止病毒的转录复制。
附图说明
图1为化合物浓度-mTOR激酶抑制率拟合曲线；
图2为雷帕霉素和式I化合物Western blot结果。
具体实施方式
为了更好地理解本申请的内容，下面结合实验和结果对化合物9-[3-(6-氨基)-吡啶基]-1-[(3-三氟甲基)-苯基]苯并[h][1,6]萘啶-2(1H)-酮在抗肠道病毒中的应用作进一步说明。
实施例1
化合物9-[3-(6-氨基)-吡啶基]-1-[(3-三氟甲基)-苯基]苯并[h][1,6]萘啶-2(1H)-酮体外抗肠道病毒的活性及细胞毒性实验
本申请体外抗病毒实验中涉及多种亚型的肠道病毒，包括EV-D68、Poliovirus Ⅰ/Ⅱ/Ⅲ(3型混合疫苗毒株)、CA16、CA6、CA10、CB3(以上毒株均由中国人民解放军军史科学院军事医学研究院提供)和EV71病毒(购于ATCC)，具体方法如下:
其中，EV-D68、Poliovirus Ⅰ/Ⅱ/Ⅲ、CA16、CA6、CA10和EV71病毒用RD细胞(购于ATCC)测活性，实验方法如下：
(1)溶解化合物
①根据9-[3-(6-氨基)-吡啶基]-1-[(3-三氟甲基)-苯基]苯并[h][1,6]萘啶-2(1H)-酮的质量及分子量，将待测化合物用DMSO溶解至100mM。
(2)抗病毒活性筛选
①利用细胞维持液(DMEM+2％FBS，Gibco，货号分别为11995-065,1600-044)将待测化合物稀释至800μM浓度，之后以3倍为稀释梯度将待测化合物进行倍比稀释，共10个浓度，然后将稀释好的化合物加入白壁透明底的96孔板中，每孔50μl。细胞对照组及病毒对照组均加入等体积的细胞维持液。
②将所涉及病毒毒种从-80℃取出，平衡至室温。
③利用病毒生长液(DMEM+2％FBS，Gibco，货号分别为11995-065,1600-044)将毒种稀释至100 TCID50，加入上述96孔板中，每孔50μl。细胞对照组加入等体积的病毒生 长液。
④将RD细胞按照1*10 5/ml浓度接种至上述96孔板，每孔100μl，最终每孔体积为200μl，药物终浓度为预处理浓度的0.25倍。
⑤将RD细胞37℃培养4天测板。
⑥将
Figure PCTCN2020086835-appb-000004
化学发光细胞活力检测试剂(Promega)的Buffer和底物避光混合，配制为工作液。
⑦弃去96孔板中培养液，并将板子轻轻拍干后，每孔加入100μl检测试剂，利用轨道振荡器将96孔板震荡4min，以诱导细胞裂解。避光稳定信号15min后，使用MD5酶标仪(Molecular Devices)测定化学发光单位，读板程序为CellTiter-Glo预设程序。
药物对病毒的抑制率计算公式为：抑制率(％)＝(实验组-病毒组平均值)/(细胞对照组平均值-病毒组平均值)*100
利用origin8.0软件对抑制率-浓度进行S型曲线拟合，计算待测化合物的IC 50值。
(3)细胞毒性测定
①利用细胞维持液将待测化合物稀释至400μM浓度，之后以3倍为稀释梯度将待测化合物进行倍比稀释，共10个浓度。
②将稀释好的化合物加入白壁透明底的96孔板细胞，每孔100μl。细胞对照组加入等体积的细胞维持液。
③将RD细胞按照1*10 5/ml浓度接种至上述96孔板，每孔100μl，最终每孔体积为200μl，药物终浓度为预处理浓度的0.5倍。
④将RD细胞37℃培养培养4天测板。
⑤将
Figure PCTCN2020086835-appb-000005
化学发光细胞活力检测试剂的Buffer和底物避光混合，配制为工作液。
⑥弃去96孔板中培养液，并将板子轻轻拍干后，每孔加入100μl检测试剂，利用轨道振荡器将96孔板震荡4min，以诱导细胞裂解。避光稳定信号15min后，测定化学发光单位，读板程序为CellTiter-Glo预设程序。
按照以下公式计算药物各稀释度的抑制率：抑制率(％)＝(细胞对照组平均值-实验组)/(细胞对照组平均值-实验组的最小值)*100
(4)数据分析
利用origin8.0软件对抑制率-浓度进行S型曲线拟合，计算待测化合物的IC 50值。利用同样方法计算CC 50值，并根据IC 50及CC 50计算选择指数SI＝CC 50/IC 50。
CB3病毒用Vero细胞测活性，实验方法如下：
(1)溶解化合物
①根据9-[3-(6-氨基)-吡啶基]-1-[(3-三氟甲基)-苯基]苯并[h][1,6]萘啶-2(1H)-酮的质量 及分子量，将待测化合物用DMSO溶解至100mM。
(2)抗病毒活性筛选
①提前24小时将Vero细胞(购于ATCC)按照1*10 5/ml浓度接种至白壁透明底的96孔板，每孔100μl。
②用PBS洗板三次，每孔200μl，最后一次加入100μl/孔的细胞维持液。
③利用细胞维持液(DMEM+2％FBS，Gibco，货号分别为11995-065,1600-044)将待测化合物稀释至800μM浓度，之后以3倍为稀释梯度将待测化合物进行倍比稀释，共10个浓度。然后将稀释好的化合物加入至上述96孔板中，每孔50μl。细胞对照组及病毒对照组均加入等体积的细胞维持液。
④将CB3病毒毒种从-80℃取出，平衡至室温。
⑤利用病毒生长液(DMEM+2％FBS，Gibco，货号分别为11995-065,1600-044)将毒种稀释至100 TCID50，加入至上述96孔板细胞中，每孔50μl。细胞对照组加入等体积的病毒生长液，最终每孔体积为200μl，药物终浓度为预处理浓度的0.25倍。
⑥将Vero细胞37℃培养5天测板。
⑦将
Figure PCTCN2020086835-appb-000006
化学发光细胞活力检测试剂的Buffer和底物避光混合，配制为工作液。弃去96孔板中培养液，并将板子轻轻拍干后，每孔加入100μl检测试剂，利用轨道振荡器将96孔板震荡4min，以诱导细胞裂解。避光稳定信号15min后，测定化学发光单位，读板程序为CellTiter-Glo预设程序。
药物对病毒的抑制率计算公式为：抑制率(％)＝(实验组-病毒组平均值)/(细胞对照组平均值-病毒组平均值)*100
利用origin8.0软件对抑制率-浓度进行S型曲线拟合，计算待测化合物的IC 50值。
(3)细胞毒性测定
①提前24小时将Vero细胞按照1*105/ml浓度接种白壁透明底的96孔板，每孔100μl。
②用PBS洗板三次，每孔200μl，最后一次加入100μl/孔的细胞维持液。
③利用细胞维持液将待测化合物稀释至400μM浓度，之后以3倍为稀释梯度将待测化合物进行倍比稀释，共10个浓度，将稀释好的化合物加入至上述96孔板，每孔100μl，最终每孔体积为200μl，药物终浓度为预处理浓度的0.5倍。细胞对照组加入等体积的细胞维持液。
④将Vero细胞37℃培养培养5天测板。
⑤将
Figure PCTCN2020086835-appb-000007
化学发光细胞活力检测试剂的Buffer和底物避光混合，配制为工作液。
⑥弃去96孔板中培养液，并将板子轻轻拍干后，每孔加入100μl检测试剂，利用轨道振荡器将96孔板震荡4min，以诱导细胞裂解。避光稳定信号15min后，使用MD5酶标 仪(Molecular Devices)测定化学发光单位，读板程序为CellTiter-Glo预设程序。
按照以下公式计算药物各稀释度的抑制率：抑制率(％)＝(细胞对照组平均值-实验组)/(细胞对照组平均值-实验组的最小值)*100
(4)数据分析
利用origin8.0软件对抑制率-浓度进行S型曲线拟合，计算待测化合物的IC 50值。利用同样方法计算CC 50值，并根据IC 50及CC 50计算选择指数SI＝CC 50/IC 50。
化合物9-[3-(6-氨基)-吡啶基]-1-[(3-三氟甲基)-苯基]苯并[h][1,6]萘啶-2(1H)-酮对病毒的抑制活性结果分别如下表1所示：
表1 化合物对肠道病毒的抑制活性及细胞毒性
肠道病毒亚型 IC 50(μM) CC 50(μM) SI(CC 50/IC 50)
EV71 0.01±0.00 0.04±0.01 4
EVD68 0.005±0.00 0.37±0.01 74
CA16 ﹥10.0±0.00 0.37±0.01 -
CB3 0.005±0.00 0.37±0.01 74
CA6 ﹥10.0±0.00 0.37±0.01 -
CA10 ﹥10.0±0.00 0.37±0.01 -
Polio Ⅰ/Ⅱ/Ⅲ 0.052±0.00 0.37±0.01 7.1
实验结果表明，化合物9-[3-(6-氨基)-吡啶基]-1-[(3-三氟甲基)-苯基]苯并[h][1,6]萘啶-2(1H)-酮对肠道病毒EV-D68、Poliovirus Ⅰ/Ⅱ/Ⅲ、CB3和EV71有明显的抑制作用，选择性安全指数高(表1)。说明化合物对EVD68、CB3这两种肠道病毒具有较强的抑制活性，同时具有一定的选择性。
实施例2
化合物9-[3-(6-氨基)-吡啶基]-1-[(3-三氟甲基)-苯基]苯并[h][1,6]萘啶-2(1H)-酮的mTOR激酶抑制活性实验
实验方法：
反应缓冲液配制：
基础缓冲液组成：50mM(mmol/L)HEPES(pH 7.5),1mM EGTA,0.01％Tween-20,10mM MnCl2,2mM DTT(用时由500mM稀释)。
①底物缓冲溶液：1650μL 2.5×底物缓冲液由1559.6μL 1×基础缓冲液、89.2μL GFP-4E-BP1(18.5μM原液，购于Thermo Fisher，货号为PV4759)和1.2μL ATP(10mM)组成，最终浓度为0.4μM GFP-4E-BP1、3μM ATP。
②mTOR激酶缓冲溶液：1650μL 2.5×mTOR激酶缓冲液由1640.2μL 1×基础缓冲液、9.8μL mTOR(0.21mg/mL原液)组成，最终浓度为0.5μg/mL.
③检测缓冲溶液：3960μL 2×检测缓冲液由3797.1μL TR-FRET缓冲稀释液(购于Thermo Fisher，货号为PV3574)、4.5μL Tb-anti-p4E-BP1抗体(3.49μM原液，购于Thermo  Fisher，货号为PV4757)、158μL EDTA(500mM原液)组成，最终浓度为2nM Tb-anti-p4E-BP1抗体、10mM EDTA。
实验步骤：
①将20μL含有5mM的测试化合物的100％DMSO溶液加入96孔板中。
②将化合物在DMSO中连续稀释3倍。
③取上一步骤中的化合物1μL用19μL mTOR激酶缓冲液稀释，转移至另一个96孔板。
④将4μL mTOR激酶溶液(购于Thermo Fisher，货号为PV4753)加入到384孔板中。
⑤取③中化合物2μL加入到具有mTOR激酶溶液的384孔板中，在室温下孵育15分钟。
⑥加入4μL底物溶液引发反应。
mTOR反应溶液终浓度：0.5μg/mL mTOR，0.4μM GFP-4E-BP1，3μM ATP。试验化合物终浓度：50000，16666，5555，1851，617.3，205.8，68.58，22.86，7.62，2.54和0.85nM。DMSO溶液的终浓度为1％。
⑦在室温下孵育60分钟。
⑧加入10μL检测缓冲液。最终浓度为2nM Tb-anti-p4E-BP1抗体和10mM EDTA。
⑨在室温下孵育30分钟。
⑩在MD5多模式读板仪(Molecular Devices)上读TR-FRET值。激发波长为340nm，发射波长1为495nm，发射波长2为520nm，计算出520nm/495nm读数比值，作为TR-FRET值。
数据处理：
由非线性回归方程拟合化合物IC 50：
Y＝Bottom+(Top-Bottom)/(1+10^((LogIC 50-X)*HillSlope))；
X：化合物浓度的常用对数值；Y：TR-FRET值(520nm/495nm)。
实验结果如表2和图1所示。
表2 化合物的mTOR激酶抑制活性
  Bottom Top HillSlope mTOR IC 50(nM)
Compound 3.74 103.60 1.14 3.01
参见表2和图1，实验结果表明，化合物9-[3-(6-氨基)-吡啶基]-1-[(3-三氟甲基)-苯基]苯并[h][1,6]萘啶-2(1H)-酮对mTOR激酶具有较强的抑制活性，其IC 50达到3.01nM，进一步验证了其通过抑制mTOR激酶活性，进而抑制病毒活性的机制。
实施例3
化合物9-[3-(6-氨基)-吡啶基]-1-[(3-三氟甲基)-苯基]苯并[h][1,6]萘啶-2(1H)-酮对mTORC1和mTORC2的抑制活性实验
为了测试所合成的化合物9-[3-(6-氨基)-吡啶基]-1-[(3-三氟甲基)-苯基]苯并[h][1,6]萘啶-2(1H)-酮对两种mTOR复合物的抑制活性，进行了mTORC1和mTORC2抑制活性实验。由于mTORC1和mTORC2通过激活下游底物磷酸化而发挥作用，因此通过检测mTORC1下游底物p70S6K1的Thr389位点和mTORC2下游底物Akt的Ser473位点的磷酸化水平，即可反映化合物对mTORC1和mTORC2的抑制活性。
实验方法：
化合物预处理：
①将RD细胞在含有10％FBS和1×PS(青霉素和链霉素浓度分别为100IU和100μg/mL)的DMEM培养基中于37℃、5％CO 2浓度下培养。
②将RD细胞(5×10 5细胞/2mL培养基)接种到6孔板中，并在37℃、5％CO 2浓度下孵育24小时。
③用PBS洗涤细胞一次，并将细胞在无血清培养基中无养分培养过夜。
④化合物的配制：
胰岛素培养基的制备：在添加10％FBS和1×PS的DMEM培养基中稀释胰岛素，使胰岛素最终浓度达到167nM。
待测化合物的预处理：将化合物溶解于DMSO中，使待测化合物浓度达到20mM，在167nM胰岛素培养基中将化合物浓度稀释至20μM。
雷帕霉素溶液的制备：将雷帕霉素溶解于DMSO中，使浓度达到10mM，在167nM胰岛素培养基中将雷帕霉素浓度稀释至20μM。
⑤除去每个孔中的无血清培养基。
⑥向每个孔中加入2mL含有DMSO的完全培养基作为对照载体。DMSO的最终浓度为0.2％。
⑦分别向指定的孔中加入2mL 20uM雷帕霉素溶液以及待测化合物20μM。DMSO的最终浓度为0.2％。
⑧在37℃、5％CO 2下孵育细胞2小时。
蛋白质提取和浓度测定：
①用冷藏PBS洗涤细胞一次，弃去PBS。
②将150μL细胞提取缓冲液(RIPA，APPLYGEN，货号C1053)移入各孔中，裂解细胞，然后在冰上孵育30分钟。
③在4℃下，14000rpm(13000×g)离心30分钟。
④将上清液转移到新的eppendorf管中，在检测前将细胞溶解液在-80℃下储存。
⑤采用BCA法测定蛋白质浓度。
缓冲溶液的配制：
①100×蛋白酶抑制剂(Beyotime，货号P1005)：将1mL重蒸水加入蛋白酶抑制剂中，轻轻搅拌，直到固体完全溶解。
②Lysis Buffer：在100mL细胞提取液中加入2mL 100×蛋白酶抑制剂和2×磷酸酶抑制剂Cocktails PhosSTOP(Beyotime，货号P1082)，轻轻搅拌至完全溶解。
③Electrophoresis Running Buffer：
10×MOPS缓冲液：加入MOPS 52.33g，Tris碱30.29g，0.5mol/L EDTA(pH8.5)10mL，SDS 5g溶于400mL重蒸水中，搅拌溶解，调节pH至7.5，再加重蒸水至500mL；
1×MOPS:100mL 10×MOPS用重蒸水稀释至1000mL。
④1×Transfer Buffer：将100mL 10×transfer buffer(144g glucine，30.3g trisbase，蒸馏水定容至1L)和400mL甲醇溶解于1500mL重蒸水中，再加重蒸水至2000mL。
⑤10×PBS Buffer(0.1M)：在800mL重蒸水中加入5袋PBS粉(Solarbio，货号P1010)，搅拌溶解，调节pH至7.6，再加入重蒸水至1000mL。
⑥1×PBS Buffer：用重蒸水将100mL 10×PBS缓冲液稀释至1000mL。
⑦10％吐温-20：在180mL重蒸水中加入20mL吐温-20，搅拌均匀。
⑧1×PBST Buffer：用重蒸水将100mL 10×PBST缓冲液和10mL吐温-20稀释至1000mL。
⑨初级抗体孵育：用0.1％吐温-20以1:1000的比例在封闭液(5％脱脂牛奶)中稀释一级抗体(Thermo Fisher，货号B2H9L2和PA5-85513)。
⑩二次抗体孵育：用0.1％吐温-20以1:500的比例在密闭缓冲液中稀释IRDye 800CW Goat anti-Rabbit IgG(Abcam，货号ab216773)。
Western Blot实验：
①在SDS-PAGE的样品孔中加入12μg总蛋白。在120V恒定电压下电泳，直到蓝色标记到达凝胶末端。
②在120V下，使用伯乐转膜仪把胶上的蛋白转移到pvdf膜40分钟。
③转膜后，用封闭缓冲液在室温下封闭2小时。
④将膜与相应的一级抗体溶液在恒温振荡器上置于4℃下孵育过夜。
⑤用1×PBST Buffer冲洗膜3×10min，再用二级抗体溶液于r.t.下孵育1小时。
⑥用1×PBST Buffer清洗膜3×10min，用Odyssey荧光扫描显影。
分别以浓度为20μM的雷帕霉素和式I化合物溶液处理RD细胞2小时，Western blot结果图2所示。
实验结果显示，发现在相同的检测条件下，雷帕霉素和化合物9-[3-(6-氨基)-吡啶基]-1-[(3-三氟甲基)-苯基]苯并[h][1,6]萘啶-2(1H)-酮处理后的细胞p70磷酸化表达水平均明显降低，说明两者均能抑制mTORC1。相反，在同样条件下，该化合物能够明显下调Akt磷酸化水平，而阳性对照药雷帕霉素则不能抑制Akt的磷酸化，说明化合物9-[3-(6-氨基)-吡啶基]-1-[(3-三氟甲基)-苯基]苯并[h][1,6]萘啶-2(1H)-酮在抑制mTORC1的同时能够有效抑制mTORC2，从而更好的发挥mTOR抑制活性，进而抑制病毒的复制与增殖。

Claims (13)

  1. 式I所示化合物，或其药学上可接受的盐、其立体异构体、其水合物或溶剂合物，
    Figure PCTCN2020086835-appb-100001
  2. 一种药物组合物，其包含权利要求1所述的式Ⅰ化合物、其药学上可接受的盐、其立体异构体、其水合物或溶剂合物，
    优选地，所述的药物组合物还包含药学上可接受的载体或辅料，具体地，所述药物组合物为固体制剂、注射剂、外用制剂、喷剂、液体制剂、或复方制剂。
  3. 权利要求1所述的化合物，或其药学上可接受的盐、其立体异构体、其水合物或溶剂合物或权利要求2所述的药物组合物，其中所述药学上可接受的盐包括其无机或有机酸盐，以及无机或有机碱盐，例如:钠盐、钾盐、钙盐、锂盐、葡甲胺盐、盐酸盐，氢澳酸盐，氢腆酸盐，硝酸盐，硫酸盐，硫酸氢盐，磷酸盐，磷酸氢盐，乙酸盐，丙酸盐，丁酸盐，草酸盐，三甲基乙酸盐，己二酸盐，藻酸盐，乳酸盐，柠檬酸盐，酒石酸盐，琥珀酸盐，马来酸盐，富马酸盐，苦味酸盐，天冬氨酸盐，葡糖酸盐，苯甲酸盐，甲磺酸盐，乙磺酸盐，苯磺酸盐，对甲苯磺酸盐和双羟萘酸盐等。
  4. 权利要求1所述的式Ⅰ化合物、其药学上可接受的盐、其立体异构体、其水合物或溶剂合物或权利要求2所述的药物组合物在制备用于治疗肠道病毒、脊髓灰质炎病毒、腺病毒、流感病毒、鼻病毒、单纯疱疹病毒、水泡性口炎病毒和/或带状疱疹病毒等病毒引起的病毒感染性疾病(例如手足口病)的药物中的用途。
  5. 权利要求1所述的式Ⅰ化合物、其药学上可接受的盐、其立体异构体、其水合物或溶剂合物或权利要求2所述的药物组合物在制备用于抑制肠道病毒、腺病毒、流感病毒、鼻病毒、单纯疱疹病毒、水泡性口炎病毒和/或带状疱疹病毒在宿主细胞(例如哺乳动物细胞)中复制的药物中的用途。
  6. 一种在有需要的哺乳动物中治疗和/或预防疾病的方法或者在有需要的哺乳动物中抑制肠道病毒、腺病毒、流感病毒、鼻病毒、单纯疱疹病毒、水泡性口炎病毒和/或带状疱疹病毒复制的方法，该方法包括给有需要的哺乳动物施用治疗和/或预防有效 量的权利要求1所述的式Ⅰ化合物、其药学上可接受的盐、其立体异构体、其水合物或溶剂合物或权利要求2所述的药物组合物，其中所述的疾病包括肠道病毒、腺病毒、流感病毒、鼻病毒、单纯疱疹病毒、水泡性口炎病毒和/或带状疱疹病毒等病毒引起的病毒感染性疾病(例如手足口病)。
  7. 权利要求1所述的式Ⅰ化合物、其药学上可接受的盐可药用盐、其立体异构体、其水合物或溶剂合物或权利要求2所述的药物组合物，其用于治疗肠道病毒、脊髓灰质炎病毒、腺病毒、流感病毒、鼻病毒、单纯疱疹病毒、水泡性口炎病毒和/或带状疱疹病毒等病毒引起的病毒感染性疾病(例如手足口病)。
  8. 权利要求1所述的式Ⅰ化合物、其药学上可接受的盐可药用盐、其立体异构体、其水合物或溶剂合物或权利要求2所述的药物组合物，其用于抑制肠道病毒、腺病毒、流感病毒、鼻病毒、单纯疱疹病毒、水泡性口炎病毒和/或带状疱疹病毒在宿主细胞(例如哺乳动物细胞)中复制。
  9. 权利要求4至5中任一项所述的用途，权利要求6所述的方法或权利要求7至8中任一项所述的化合物、其药学上可接受的盐可药用盐、其立体异构体、其水合物或溶剂合物或药物组合物，其中所述肠道病毒为HEV-A、HEV-B、HEV-C或HEV-D亚型的病毒。
  10. 权利要求4至5中任一项所述的用途，权利要求6所述的方法或权利要求7至8中任一项所述的化合物、其药学上可接受的盐可药用盐、其立体异构体、其水合物或溶剂合物或药物组合物，其中所述肠道病毒为EV-D68、Poliovirus Ⅰ/Ⅱ/Ⅲ、CA16、CA6、CA10、CB3或EV71病毒。
  11. 权利要求10所述的用途、方法或化合物、其药学上可接受的盐可药用盐、其立体异构体、其水合物或溶剂合物或药物组合物，其中所述肠道病毒为EV-D68、Poliovirus Ⅰ/Ⅱ/Ⅲ、CB3或EV71病毒。
  12. 权利要求11所述的用途、方法或化合物、其药学上可接受的盐可药用盐、其立体异构体、其水合物或溶剂合物或药物组合物，其中所述肠道病毒为EV-D68或CB3病毒。
  13. 式I所示化合物、其立体异构体、其水合物或溶剂合物在制备抑制肠道病毒在靶细胞中复制的药物中的应用，
    优选地，所述肠道病毒为EV-D68、Poliovirus Ⅰ/Ⅱ/Ⅲ、CA16、CA6、CA10、CB3或EV71病毒，
    优选地，所述肠道病毒为EV-D68、Poliovirus Ⅰ/Ⅱ/Ⅲ、CB3或EV71病毒，
    优选地，所述肠道病毒为EV-D68或CB3病毒。
PCT/CN2020/086835 2019-04-26 2020-04-24 一种肠道病毒抑制剂 WO2020216349A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US17/606,243 US20220204501A1 (en) 2019-04-26 2020-04-24 Enterovirus inhibitor
EP20795446.2A EP3960173A4 (en) 2019-04-26 2020-04-24 ENTEROVIRUS INHIBITOR
JP2021563068A JP2022530070A (ja) 2019-04-26 2020-04-24 エンテロウイルス阻害薬
CN202080030295.0A CN113924093A (zh) 2019-04-26 2020-04-24 一种肠道病毒抑制剂

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910346258 2019-04-26
CN201910346258.X 2019-04-26

Publications (1)

Publication Number Publication Date
WO2020216349A1 true WO2020216349A1 (zh) 2020-10-29

Family

ID=72941355

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/086835 WO2020216349A1 (zh) 2019-04-26 2020-04-24 一种肠道病毒抑制剂

Country Status (5)

Country Link
US (1) US20220204501A1 (zh)
EP (1) EP3960173A4 (zh)
JP (1) JP2022530070A (zh)
CN (1) CN113924093A (zh)
WO (1) WO2020216349A1 (zh)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160339030A1 (en) * 2015-05-19 2016-11-24 University Of Maryland, Baltimore Treatment agents for inhibiting hiv and cancer in hiv infected patients
WO2018017426A1 (en) * 2016-07-16 2018-01-25 Florida State University Research Foundation, Inc. Compounds and methods for treatment and prevention of flavivirus infection

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2456869A4 (en) * 2009-07-23 2013-11-27 Trustees Of The University Of Princeton MTOR KINASE HEMMER AS ANTIVIRUS AGENT
US9879003B2 (en) * 2012-04-11 2018-01-30 Dana-Farber Cancer Institute, Inc. Host targeted inhibitors of dengue virus and other viruses
ITUB20153490A1 (it) * 2015-09-08 2017-03-08 Novamont Spa Miscele di esteri dell?acido pelargonico
WO2021207632A1 (en) * 2020-04-10 2021-10-14 The Regents Of The University Of California Small molecule therapeutics for the treatment of viral infections

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160339030A1 (en) * 2015-05-19 2016-11-24 University Of Maryland, Baltimore Treatment agents for inhibiting hiv and cancer in hiv infected patients
WO2018017426A1 (en) * 2016-07-16 2018-01-25 Florida State University Research Foundation, Inc. Compounds and methods for treatment and prevention of flavivirus infection

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3960173A4

Also Published As

Publication number Publication date
JP2022530070A (ja) 2022-06-27
EP3960173A4 (en) 2023-05-17
US20220204501A1 (en) 2022-06-30
CN113924093A (zh) 2022-01-11
EP3960173A1 (en) 2022-03-02

Similar Documents

Publication Publication Date Title
CN111557939B (zh) 法匹拉韦在制备用于治疗冠状病毒感染的药物中的应用
CN113289018B (zh) 金诺芬等老药及其组合物在抗单正链rna病毒中的应用
CN111743899B (zh) 硝唑尼特及其活性形式替唑尼特在制备用于治疗SARS-CoV-2感染的药物中的应用
CN113679726A (zh) 丹参提取物和醌类化合物在抗冠状病毒中的应用
JP2023512628A (ja) ＳＡＲＳ－ＣｏＶ－２感染症の治療における安息香酸塩化合物の利用
WO2021175224A1 (zh) 抗rna病毒药物及其应用
US11433080B2 (en) Antiviral treatment
WO2020216349A1 (zh) 一种肠道病毒抑制剂
US11957667B2 (en) Inhibitors of positive strand RNA viruses
CN116687932B (zh) ((3-氨甲酰-5-氟吡嗪-2-基)氧基)甲基异丁酸酯的医药用途
WO2021164672A1 (zh) 抗rna病毒药物及其应用
US20230225988A1 (en) Antiviral use of calixarenes
WO2023222018A1 (zh) Eidd-1931或其衍生物在治疗肠道病毒感染方面的应用
US20230071014A1 (en) Application of ritonavir in treating sars-cov-2 infection
US20240165096A1 (en) Anti Viral Therapy
JP2023525855A (ja) コロナウイルスによる疾患の治療及び／又は予防のための化合物及びその使用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20795446

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021563068

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020795446

Country of ref document: EP

Effective date: 20211126