WO2021155651A1 - 4-氨基喹啉类化合物在治疗冠状病毒感染方面的应用 - Google Patents

4-氨基喹啉类化合物在治疗冠状病毒感染方面的应用 Download PDF

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WO2021155651A1
WO2021155651A1 PCT/CN2020/093989 CN2020093989W WO2021155651A1 WO 2021155651 A1 WO2021155651 A1 WO 2021155651A1 CN 2020093989 W CN2020093989 W CN 2020093989W WO 2021155651 A1 WO2021155651 A1 WO 2021155651A1
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formula
represented
chloroquine
pharmaceutically acceptable
hydroxychloroquine
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French (fr)
Inventor
钟武
肖庚富
胡志红
王曼丽
张磊砢
曹瑞源
李薇
樊士勇
周辛波
李松
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Academy of Military Medical Sciences AMMS of PLA
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Priority to JP2022546656A priority Critical patent/JP2023512093A/ja
Priority to US17/795,471 priority patent/US20230218606A1/en
Priority to EP20917330.1A priority patent/EP4101452A4/en
Publication of WO2021155651A1 publication Critical patent/WO2021155651A1/zh
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    • 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
    • A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This application relates to the following hydroxychloroquine represented by formula I or chloroquine represented by formula II, or their geometric isomers, pharmaceutically acceptable salts and/or both solvates and/or both hydrates, and containing the above-mentioned compounds
  • the pharmaceutical composition is used to treat SARS-CoV-2 infection.
  • Hydroxychloroquine (compound of formula I), chemical name is 2-[[4-[(7-chloro-4-quinolinyl)amino]pentyl]ethylamino]-ethanol; chloroquine (compound of formula II), chemical name N',N'-Diethyl-N-4-(7-chloro-4-quinolinyl)-1,4-pentanediamine.
  • the two types of compounds are 4-aminoquinoline antimalarial drugs, which have immunosuppressive, anti-inflammatory, reduce ultraviolet-induced reactions, sun protection, anti-thrombosis, affect porphyrin metabolism, anti-hyperlipidemia, anti-proliferation, and anti-pathogen The role of microorganisms.
  • hydroxychloroquine sulfate is clinically used for rheumatoid arthritis, juvenile chronic arthritis, discoid and systemic lupus erythematosus, and skin lesions caused or exacerbated by sunlight.
  • Chloroquine is clinically used to treat falciparum malaria, vivax malaria and vivax malaria that are sensitive to chloroquine. It can also be used for inhibitory prevention of malaria symptoms. It can also be used for the treatment of extraintestinal amebiasis, connective tissue disease, photosensitivity disease (such as sun erythema) and so on.
  • Hydroxychloroquine was artificially synthesized in 1944 and was initially used for antimalarial treatment. In 1955, it was used for the treatment of systemic lupus erythematosus (SLE). Compared with chloroquine phosphate, because hydroxychloroquine has a hydroxyl group, it retains the original efficacy of chloroquine, and its toxicity is half lower than that of chloroquine, which has better safety.
  • the IC 50 of hydroxychloroquine on DENV-2 infected cells is as follows: 10.1 ⁇ 1.6 ⁇ M (A549 cells); 12.9 ⁇ 4.2 ⁇ M (Hepa1-6 cells); 12.9 ⁇ 1.9 ⁇ M (WS-1 cells. cells).
  • 50-80 ⁇ M hydroxychloroquine can reduce the DENV-2 virus titer by 100 times.
  • Hydroxychloroquine has the effect of resisting ZIKV infection in pregnant mice, the dosage is 40mg/kg/day, the administration method is intraperitoneal administration, and the administration is started on the +1 day after ZIKV infection.
  • the results showed that the placenta of mice treated with hydroxychloroquine sustained a lower level of ZIKV infection compared to the control treated with PBS.
  • the 2019 Novel Coronavirus (2019-nCoV) is a new strain of coronavirus that has never been found in humans before.
  • ICTV International Commission for Classification of Viruses
  • SARS-CoV- 2 severe acute respiratory syndrome coronavirus 2
  • WHO World Health Organization
  • the symptoms of the new coronavirus infection in 2019 are mainly pneumonia, which can be divided into simple infections and infections according to the severity of the disease. Mild pneumonia, severe pneumonia, acute respiratory distress syndrome, sepsis, septic shock, etc.
  • Patients with simple infections may have non-specific symptoms, such as fever, cough, sore throat, nasal congestion, fatigue, headache, muscle pain or discomfort, the elderly and immunosuppressed people may have atypical symptoms.
  • Patients with mild pneumonia mainly cough, dyspnea + shortness of breath. Severe pneumonia can be seen in adolescents, adults or children. The main symptoms are increased respiratory rate, severe respiratory failure or dyspnea, central cyanosis, lethargy, unconsciousness or convulsions, gas pumping, etc.
  • the lung images of acute respiratory distress syndrome are bilateral ground-glass shadows, but they cannot be completely explained by effusion, lobar exudation, atelectasis, or pulmonary block shadows. Pulmonary edema is the main symptom. Patients with sepsis often have fatal organ dysfunction, and septic shock is the most critical patient with a higher probability of death.
  • the purpose of this application is to discover drugs with antiviral activity against coronaviruses, especially SARS-CoV-2, which can be used for related diseases caused by their infections such as simple infections such as fever, cough and sore throat, pneumonia, acute or severe acute respiratory infections , Hypoxic respiratory failure and acute respiratory distress syndrome, sepsis and septic shock.
  • the pharmaceutically acceptable salts of the compounds of the present application include their inorganic or organic acid salts, and inorganic or organic base salts.
  • the present 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, hydrogensulfate, 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.
  • the hydroxychloroquine represented by formula I or the chloroquine represented by formula II can inhibit virus replication on cells and reduce the viral nucleic acid load in cell culture.
  • the hydroxychloroquine represented by formula I or the chloroquine represented by formula II can reduce the viral nucleic acid load level of SARS-CoV-2 infected cells at a micromolar concentration.
  • compositions with acceptable carriers can be administered through various routes, such as oral tablets, capsules, powders, oral liquids, injections and transdermal preparations.
  • pharmaceutically acceptable carriers include diluents, fillers, disintegrating agents, 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. .
  • compositions which contains at least one pharmaceutically acceptable carrier of the compound of the application.
  • the pharmaceutical composition can be prepared into various forms according to different administration routes.
  • This application also relates to the preparation of hydroxychloroquine represented by formula I or chloroquine represented by formula II, or geometric isomers or pharmaceutically acceptable salts and/or solvates and/or hydrates thereof for the treatment of SARS- Diseases or infections caused by CoV-2 (such as respiratory diseases (including but not limited to simple infections such as fever, cough and sore throat, etc.), pneumonia, acute or severe acute respiratory infections, hypoxic respiratory failure, and acute respiratory distress syndrome , Sepsis and septic shock, etc.)) in the use of drugs,
  • SARS- Diseases or infections caused by CoV-2 such as respiratory diseases (including but not limited to simple infections such as fever, cough and sore throat, etc.), pneumonia, acute or severe acute respiratory infections, hypoxic respiratory failure, and acute respiratory distress syndrome , Sepsis and septic shock, etc.)
  • This application also relates to the preparation of hydroxychloroquine represented by formula I or chloroquine represented by formula II, or geometric isomers or pharmaceutically acceptable salts and/or solvates and/or hydrates thereof as SARS-CoV- 2
  • This application also relates to the use of the pharmaceutical composition in the preparation of drugs as SARS-CoV-2 inhibitors, or in the preparation of drugs for inhibiting the replication or reproduction of SARS-CoV-2 in cells (such as mammalian cells) use,
  • the pharmaceutical composition comprises hydroxychloroquine represented by formula I or chloroquine represented by formula II, or geometric isomers or pharmaceutically acceptable salts and/or solvates and/or hydrates thereof,
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or auxiliary material.
  • the pharmaceutical composition is a solid preparation, an injection, an external preparation, a spray, a liquid preparation, or a compound preparation.
  • This application also relates to the preparation of pharmaceutical compositions for the treatment of diseases or infections caused by SARS-CoV-2 (such as respiratory diseases (including but not limited to simple infections such as fever, cough and sore throat, etc.), pneumonia, acute respiratory infections or Severe acute respiratory infection, hypoxic respiratory failure and acute respiratory distress syndrome, sepsis and septic shock, etc.)) in a medicine, wherein the pharmaceutical composition comprises hydroxychloroquine shown in formula I or formula II Shows chloroquine, or its geometric isomers or pharmaceutically acceptable salts and/or solvates and/or hydrates thereof,
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or auxiliary material.
  • the pharmaceutical composition is a solid preparation, an injection, an external preparation, a spray, a liquid preparation, or a compound preparation.
  • This application also relates to a method for treating and/or preventing diseases or viral infections in a mammal in need, or a method for inhibiting SARS-CoV-2 replication or reproduction in a mammal in need, and the method includes Administering a therapeutically and/or prophylactically effective amount of hydroxychloroquine of formula I or chloroquine of formula II, or geometric isomers or pharmaceutically acceptable salts and/or solvates and/or hydrates thereof A substance or therapeutic and/or preventive effective amount comprising hydroxychloroquine represented by formula I or chloroquine represented by formula II, or geometric isomers or pharmaceutically acceptable salts and/or solvates and/or hydrates thereof Pharmaceutical composition,
  • the diseases described therein include diseases caused by SARS-CoV-2 (e.g., respiratory diseases (including but not limited to simple infections such as fever, cough and sore throat, etc., pneumonia, acute respiratory infections or severe acute respiratory infections (SARI), Hypoxic respiratory failure and acute respiratory distress syndrome, sepsis and septic shock, etc.)), the viral infections include infections caused by SARS-CoV-2.
  • respiratory diseases including but not limited to simple infections such as fever, cough and sore throat, etc., pneumonia, acute respiratory infections or severe acute respiratory infections (SARI), Hypoxic respiratory failure and acute respiratory distress syndrome, sepsis and septic shock, etc.
  • SARS-CoV-2 e.g., respiratory diseases (including but not limited to simple infections such as fever, cough and sore throat, etc., pneumonia, acute respiratory infections or severe acute respiratory infections (SARI), Hypoxic respiratory failure and acute respiratory distress syndrome, sepsis and septic shock, etc.)
  • the viral infections include infections caused by SARS-CoV-2.
  • This application also relates to hydroxychloroquine represented by formula I or chloroquine represented by formula II, or geometric isomers or pharmaceutically acceptable salts and/or solvates and/or hydrates thereof or containing the hydroxyl group represented by formula I
  • the pharmaceutical composition further contains a pharmaceutically acceptable carrier or excipient.
  • This application also relates to hydroxychloroquine represented by formula I or chloroquine represented by formula II, or geometric isomers or pharmaceutically acceptable salts and/or solvates and/or hydrates thereof or containing the hydroxyl group represented by formula I
  • the pharmaceutical composition further contains a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutically acceptable salt of hydroxychloroquine represented by formula I or chloroquine represented by formula II includes its inorganic or organic acid salts and inorganic or organic base salts,
  • the pharmaceutically acceptable salt includes, but is not limited to: the sodium salt, potassium salt, calcium salt, lithium salt, meglumine salt, hydrochloride, hydrochloride, hydrosidate, and nitrate of the compound , Sulfate, Bisulfate, Phosphate, Hydrogen Phosphate, Acetate, Propionate, Butyrate, Oxalate, Trimethylacetate, Adipate, Alginate, Lactate , Citrate, tartrate, succinate, maleate, fumarate, picrate, aspartate, gluconate, benzoate, methanesulfonate, ethanesulfonic acid Salt, benzenesulfonate, p-toluenesulfonate and pamoate, etc.
  • the pharmaceutically acceptable salt of chloroquine of formula II is chloroquine sulfate.
  • the pharmaceutically acceptable salt of chloroquine of formula II is chloroquine phosphate.
  • the disease caused by SARS-CoV-2 described in this application is COVID-19.
  • 2019-nCoV 2019-nCoV
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • 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. .
  • the mammals include bovines, equines, ovines, swines, canines, felines, rodents, primates, among which mammals are preferred Be human.
  • the pharmaceutical composition described in this application can be prepared into various forms according to different administration routes.
  • the pharmaceutical composition can be administered in any of the following ways: oral, spray inhalation, rectal, nasal, buccal, vaginal, topical, parenteral, such as subcutaneous, intravenous, intramuscular, Intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or medication with the aid of an explanted reservoir.
  • oral, intraperitoneal or intravenous administration is preferred.
  • the chloroquine or hydroxychloroquine or its geometric isomers, pharmaceutically acceptable salts, solvates and/or hydrates thereof can be made into any oral acceptable preparation form, including but not limited to Tablets, capsules, aqueous solutions or 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 chloroquine or hydroxychloroquine or its geometrical isomers, pharmaceutically acceptable salts, solvates and/or hydrates thereof can generally be made into the form of suppositories, which can be prepared by combining the drug with a It is prepared by mixing suitable non-irritating excipients.
  • 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.
  • the chloroquine or hydroxychloroquine or its geometric isomers, or pharmaceutically acceptable Salts, their solvates and/or their hydrates can be made into different topical preparations according to different affected surfaces or organs.
  • the specific instructions are as follows:
  • the chloroquine or hydroxychloroquine or its geometric isomers, pharmaceutically acceptable salts, solvates and/or hydrates thereof can be formulated into a micronized suspension or solution.
  • the carrier used is isotonic sterile saline with a certain pH, which may or may not be added with preservatives such as benzyl alkanolate chloride.
  • the compound can also be made into an ointment form such as petrolatum ointment.
  • the chloroquine or hydroxychloroquine or its geometrical isomer, its pharmaceutically acceptable salt, its solvate and/or its hydrate can be made into an appropriate ointment, lotion or cream preparation form , 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 chloroquine or hydroxychloroquine or its geometrical isomers, pharmaceutically acceptable salts, solvates and/or hydrates thereof can be prepared into rectal suppository preparations as described above or suitable
  • topical transdermal patches can also be used.
  • the chloroquine or hydroxychloroquine or its geometric isomers, pharmaceutically acceptable salts, solvates and/or hydrates thereof can also be administered in the form of sterile injection preparations, including sterile injection water or oil suspensions, or Sterile injection solution.
  • 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 to be treated, the severity of the disease to be treated, and the patient's condition to be treated. Factors such as age, size, weight, and physical disease, the medical history of the patient being treated, the duration of treatment, the nature of concurrent therapy, the 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 chloroquine or hydroxychloroquine or its geometric isomers, pharmaceutically acceptable salts, solvates and/or hydrates thereof for different patients are determined by many factors, including The patient’s age, weight, gender, natural health status, nutritional status, active strength of the drug, taking time, metabolic rate, severity of the disease, and the subjective judgment of the physician.
  • the preferred dosage here is between 0.001-100 mg/kg body weight/day.
  • Figure 1 The effectiveness and safety test results of chloroquine phosphate on SARS-CoV-2 virus-infected vero-E6 cells, where (A) is the half effective concentration of chloroquine phosphate after 48 hours of SARS-CoV-2 virus infection ( ⁇ M) and the median lethal concentration ( ⁇ M) to cells; (B) is the immunofluorescence image of chloroquine phosphate treated with SARS-CoV-2 virus-infected cells.
  • Example 1 Chloroquine phosphate reduces the viral nucleic acid load of SARS-CoV-2 infected cells
  • Vero E6 cells purchased from ATCC, article number 1586
  • 2% cell maintenance solution formula: FBS (purchased from Gibco company, article number 16000044)
  • MEM purchased from Gibco, article number 10370021
  • SARS-CoV-2 2019-nCoV
  • SARS-CoV-2019BetaCoV/Wuhan/WIV04/2019 strain Stored by Wuhan Institute of Virology, Chinese Academy of Sciences
  • TCID 50 of virus Stored by Wuhan Institute of Virology, Chinese Academy of Sciences
  • RNA extraction kit was purchased from Qiagen Company, catalog number 74106.
  • the consumables spin column, 2ml collection tube without RNase, etc.
  • reagents RLT, RW1, RPE, RNase-free water, etc.
  • the following extraction steps are recommended steps in the kit instructions.
  • RNA reverse transcription kit (PrimeScript TM RT reagent Kit with gDNA Eraser, article number RR047Q) produced by TaKaRa was used for RNA reverse transcription. The steps are as follows.
  • 1gDNA removal Collect RNA samples of each experimental group, and take 1 ⁇ g for reverse transcription. First, add 2 ⁇ l 5 ⁇ gDNA Eraser Buffer to the RNA of each experimental group, fill up the reaction system to 10 ⁇ l with RNase Free water, mix well, and remove the g DNA that may exist in the sample in a water bath at 42°C for 2 minutes;
  • Fluorescence quantitative PCR was used to detect the number of copies per milliliter of the original virus solution.
  • RBD-qR CTCAAGTGTCTGTGGATCACG
  • Cycle parameters 95°C for 15 seconds, 54°C for 15 seconds, 72°C for 30 seconds, a total of 40 cycles.
  • MEM purchased from Gibco, product number 10370021
  • FBS Gibco company, product number 10370021
  • Cell viability (%) (A (drug treatment group) -A (blank control) )/(A (negative control) -A (blank control) ) ⁇ 100%
  • the results of the virus proliferation inhibition experiment showed that the test compound chloroquine phosphate at concentrations of 50 ⁇ M, 16.67 ⁇ M, and 5.56 ⁇ M can effectively inhibit the replication of the SARS-CoV-2 virus genome in the infection supernatant.
  • the half-maximal effective concentration (EC 50 ) of chloroquine phosphate is 1.13 ⁇ M
  • the half-cytotoxic concentration (CC 50 ) for cells is greater than 100 ⁇ M
  • the selectivity index (SI) is greater than 88.5.
  • Example 2 Chloroquine phosphate and hydroxychloroquine under 4 different infection titers (MOI) to reduce the viral nucleic acid load of SARS-CoV-2 infected cells
  • Vero E6 cells Inoculate Vero E6 cells into a 24-well plate, culture for 24 hours, and then carry out virus infection.
  • Set four groups of different infection doses respectively 0.01MOI, 0.02MOI, 0.2MOI and 0.8MOI.
  • fluorescence quantitative PCR was used to detect the number of copies per milliliter of the original virus solution.
  • cytotoxicity showed that when the test concentration was 100 ⁇ M, the treatment of the test compound did not change the cell viability, that is, the test compound had no toxic effect on the cells at this concentration.
  • the CC 50 of hydroxychloroquine is greater than 249.50 ⁇ M, and the CC 50 of chloroquine phosphate is greater than 273.20 ⁇ M.
  • the EC 50 of the test compound is as follows:
  • Example 3 The mechanism experiment of inhibiting SARS-CoV-2 virus invasion by chloroquine phosphate and hydroxychloroquine

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Abstract

一种羟基氯喹或氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物，及含上述化合物的药物组合物，用于治疗SARS-CoV-2引起的疾病或感染的用途。

Description

4-氨基喹啉类化合物在治疗冠状病毒感染方面的应用
本申请是以CN申请号为202010078808.7，申请日为2020年2月3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
本申请涉及下面式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或它们的几何异构体、药物上可接受的盐和/或两者溶剂化物和/或两者水合物，及含上述化合物的药物组合物，用于治疗SARS-CoV-2感染方面的用途。
Figure PCTCN2020093989-appb-000001
背景技术
羟基氯喹(式Ⅰ化合物)，化学名为2-[[4-[(7-氯-4-喹啉基)氨基]戊基]乙氨基]-乙醇；氯喹(式Ⅱ化合物)，化学名为N’,N’-二乙基-N-4-(7-氯-4-喹啉基)-1,4-戊二胺。
二类化合物均为4-氨基喹啉类抗疟药，具有免疫抑制、抗炎、减少紫外线诱发反应及日光保护、抗血栓、影响卟啉代谢、抗高脂血症、抗增生、抗病原微生物等作用。目前硫酸羟基氯喹临床用于类风湿关节炎，青少年慢性关节炎，盘状和系统性红斑狼疮，以及由阳光引发或加剧的皮肤病变。氯喹临床用于治疗对氯喹敏感的恶性疟、间日疟及三日疟。并可用于疟疾症状的抑制性预防。也可用于治疗肠外阿米巴病、结缔组织病、光敏感性疾病(如日晒红斑)等。
羟基氯喹1944年人工合成，最初用于抗疟治疗，1955年开始用于治疗系统性红斑狼疮(SLE)。与磷酸氯喹相比，由于羟基氯喹带有一个羟基基团，使其在保留原有氯喹药效的同时，毒性比氯喹小一半，具有更好的安全性。
研究发现羟基氯喹除以上已知的临床作用外，在体外表现出对黄病毒、登革病毒的活性。
羟基氯喹对DENV-2感染的细胞的IC 50如下：10.1±1.6μM(A549细胞)；12.9±4.2μM(Hepa1-6细胞)；12.9±1.9μM(WS-1cells.细胞)。在A549细胞系中50-80μM的羟基氯喹可以降低DENV-2病毒滴度100倍。另外羟基氯喹可以抑制DENV-2对J774A.1 巨噬细胞的感染(IC 50＝9.7±1.3μM)。
研究发现羟基氯喹是寨卡病毒NS2B-NS3蛋白酶的抑制剂，对寨卡病毒NS2B-NS3蛋白酶的抑制常数Ki＝92.34±11.91μM。羟基氯喹具有对妊娠期小鼠抵抗ZIKV感染的功效，给药剂量为40mg/kg/天，给药方式为腹腔给药，在ZIKV感染后的第+1天开始给药。结果表明与用PBS处理的对照相比，用羟基氯喹处理的小鼠胎盘持续较低水平的ZIKV感染。
2019新型冠状病毒(2019-nCoV)是以前从未在人类中发现的冠状病毒新毒株。2020年2月11日，国际病毒分类委员会(ICTV)宣布，2019新型冠状病毒(2019-nCoV)的正式分类名为严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2，SARS-CoV-2)。同日，世界卫生组织(WHO)宣布，由这一病毒导致的疾病的正式名称为COVID-19。2019新型冠状病毒感染的症状主要以肺炎为主，依据病情的轻重程度可分为单纯性感染、轻症肺炎、重症肺炎、急性呼吸窘迫综合症、脓毒症、脓毒症休克等。单纯性感染的患者可能有非特异性症状，例如发热、咳嗽、咽痛、鼻塞、乏力、头痛、肌肉疼痛或不适，老年人和免疫抑制者可能会出现非典型症状。轻症肺炎的患者主要以咳嗽、呼吸困难+呼吸急促为主。重症肺炎可见于青少年、成人或儿童，主要症状为呼吸频率增加，严重的呼吸衰竭或呼吸困难，中心型发绀、嗜睡、意识不清或惊厥、抽气等。急性呼吸窘迫综合症的肺部影像为双侧磨玻璃影，但不能完全由积液、大叶渗出或者肺不张或者肺部块影解释，以肺水肿为主要症状。脓毒症患者往往有致命的器官功能障碍，脓毒性休克是最为危重的患者，死亡可能性较高。
目前，针对2019新型冠状病毒(SARS-CoV-2)感染，临床上以支持治疗为主，无特异抗病毒药物可用。
发明内容
本申请目的是发现对冠状病毒特别是SARS-CoV-2有抗病毒活性的药物，可用于其感染引起相关疾病如单纯性感染如发热、咳嗽和咽痛等、肺炎、急性或严重急性呼吸道感染、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克等的救治。
本申请通过创造性的研究发现磷酸氯喹在体外对SARS-CoV-2感染的细胞具有很好的保护作用，其EC 50＝1.13μM，CC 50>100μM,SI>88.5；式I化合物羟基氯喹在人体体内的毒性仅为磷酸氯喹的一半，具有抑制SARS-CoV-2复制的功能，在治疗SARS-CoV-2引起的疾病方面具有很好的治疗效果。
本申请提供具有式Ⅰ或式Ⅱ所示结构的化合物，其几何异构体，及其药物上可接受的盐和/或其溶剂化物或其水合物:
Figure PCTCN2020093989-appb-000002
根据本申请，本申请化合物的可药用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。
根据本申请，式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹可在细胞上抑制病毒复制，减少细胞培养物中病毒核酸载量。
本申请的发明人在经过创造性的发明和研究后，发现了式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹的一些新作用特点：
式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹可以在微摩尔级浓度下降低SARS-CoV-2感染的细胞病毒核酸载量水平。
本申请还涉及含有式Ⅰ所示羟基氯喹和/或式Ⅱ所示氯喹或其几何异构体或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。该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。
本申请的另一个方面涉及药物组合物，其含有本申请化合物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。
本申请还涉及式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物在制备用于治疗SARS-CoV-2引起的疾病或感染(例如呼吸系统疾病(包括但不限于单纯性感染如发热、咳嗽和咽痛等、肺炎、急性或严重急性呼吸道感染、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克等)) 的药物中的用途，
Figure PCTCN2020093989-appb-000003
本申请还涉及式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物在制备作为SARS-CoV-2抑制剂的药物中的用途，或者在制备用于抑制SARS-CoV-2在细胞(例如哺乳动物细胞)中复制或繁殖的药物中的用途，
Figure PCTCN2020093989-appb-000004
本申请还涉及药物组合物在制备作为SARS-CoV-2抑制剂的药物中的用途，或者在制备用于抑制SARS-CoV-2在细胞(例如哺乳动物细胞)中复制或繁殖的药物中的用途，
其中所述药物组合物包含式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物，
Figure PCTCN2020093989-appb-000005
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。
本申请还涉及药物组合物在制备用于治疗SARS-CoV-2引起的疾病或感染(例如呼吸系统疾病(包括但不限于单纯性感染如发热、咳嗽和咽痛等、肺炎、急性呼吸道感染或严重急性呼吸道感染、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克等))的药物中的用途，其中所述药物组合物包含式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物，
Figure PCTCN2020093989-appb-000006
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。
本申请还涉及一种在有需要的哺乳动物中治疗和/或预防疾病或病毒感染的方法或者在有需要的哺乳动物中抑制SARS-CoV-2复制或繁殖的方法，该方法包括给有需要的哺乳动物施用治疗和/或预防有效量的式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物或治疗和/或预防有效量的包含式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物的药物组合物，
Figure PCTCN2020093989-appb-000007
其中所述的疾病包括SARS-CoV-2引起的疾病(例如呼吸系统疾病(包括但不限于单纯性感染如发热、咳嗽和咽痛等、肺炎、急性呼吸道感染或严重急性呼吸道感染(SARI)、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克等))，所述的病毒感染包括由包括SARS-CoV-2引起的感染。
本申请还涉及式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物或包含式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物的药物组合物，其作为SARS-CoV-2抑制剂，或者用于抑制SARS-CoV-2在细胞(例如哺乳动物细胞)中复制或繁殖，
Figure PCTCN2020093989-appb-000008
优选地，所述药物组合物中还包含药学上可接受的载体或辅料。
本申请还涉及式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物或包含式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物的药物组合物，其用于治疗SARS-CoV-2引起的疾病或感染(例如呼吸系统疾病(包括但不限于单纯性感染如发 热、咳嗽和咽痛等、肺炎、急性呼吸道感染或严重急性呼吸道感染、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克等))，
Figure PCTCN2020093989-appb-000009
优选地，所述药物组合物中还包含药学上可接受的载体或辅料。
在某些实施方案中，式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹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，
所述药学上可接受的盐包括但不限于：所述化合物的钠盐，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。
在某些实施方案中，式Ⅱ所示氯喹的药学上可接受的盐为硫酸氯喹。
在某些实施方案中，式Ⅱ所示氯喹的药学上可接受的盐为磷酸氯喹。
在某些实施方案中，本申请所述的SARS-CoV-2引起的疾病为COVID-19。
本申请中，所用术语“2019新型冠状病毒(2019-nCoV)”的正式分类名为严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2，SARS-CoV-2)。
本申请中，所用术语“2019新型冠状病毒(2019-nCoV)引起的疾病”的正式名称为COVID-19。
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。
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。
本申请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。
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，所述氯喹或羟基氯喹或其几何异构体、其药物上可接受的盐、其溶剂化物和/或其水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，所述氯喹或羟基氯喹或其几何异构体、其药物上可接受的盐、其溶剂化物和/或其水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，所述氯喹或羟基氯喹或其几何异构体、其药物上可接受的盐、其溶剂化物和/或其水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，所述氯喹或羟基氯喹或其几何异构体、其药物上可接受的盐、其溶剂化物和/或其水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，所述氯喹或羟基氯喹或其几何异构体、其药物上可接受的盐、其溶剂化物和/或其水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，所述氯喹或羟基氯喹或其几何异构体、其药物上可接受的盐、其溶剂化物和/或其水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。
所述氯喹或羟基氯喹或其几何异构体、其药物上可接受的盐、其溶剂化物和/或其水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。
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。
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，所述氯喹或羟基氯喹或其几何异构体、其药物上可接受的盐、其溶剂化物和/或其水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，药物的活性强度，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0.001-100mg/kg体重/天。
附图说明
图1.磷酸氯喹对SARS-CoV-2病毒感染的vero-E6细胞的有效性和安全性实验结果，其中(A)为磷酸氯喹在细胞感染SARS-CoV-2病毒48h后的半数有效浓度(μM)和对细胞的半数致死浓度(μM)度；(B)为磷酸氯喹处理感染SARS-CoV-2病毒的细胞后的免疫荧光图。
图2.磷酸氯喹和羟基氯喹体外(vero-E6细胞)抗SARS-CoV-2有效性和安全性研究结果，其中(A)为磷酸氯喹和羟基氯喹细胞毒性及半数致死浓度；(B)为MOI＝0.01时，磷酸氯喹和羟基氯喹抗病毒活性及半数抑制浓度；(C)为MOI＝0.02时，磷酸氯喹和羟基氯喹抗病毒活性及半数抑制浓度；(D)为MOI＝0.2时，磷酸氯喹和羟基氯喹抗病毒活性及半数抑制浓度；(E)为MOI＝0.8时，磷酸氯喹和羟基氯喹抗病毒活性及半数抑制浓度。
图3.磷酸氯喹和羟基氯喹抗病毒机制研究结果，其中(A)为磷酸氯喹和羟基氯喹处理后，细胞中病毒颗粒与早期内含体或溶酶体共定位定量分析结果；(B)为代表性共 定位的共聚焦显微图。
具体实施方式
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。以下对至少一个示例性实施例的描述实际上仅仅是说明性的，不作为对本申请及其应用或使用的任何限制。基于本申请中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
下面实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用材料或设备未注明生产厂商者，均为可通过购买获得的常规产品。
实施例1：磷酸氯喹降低SARS-CoV-2感染的细胞病毒核酸载量实验
(1)药物处理感染病毒的细胞
将Vero E6细胞(购自ATCC，货号1586)接种至24孔板，培养24h；然后进行病毒感染，具体的，用2％细胞维持液(配方为：将FBS(购自Gibco公司，货号16000044)按照2％的体积比加入MEM(购自Gibco公司，货号10370021)，即为2％细胞维持液)将SARS-CoV-2(2019-nCoV)病毒(nCoV-2019BetaCoV/Wuhan/WIV04/2019株，由中国科学院武汉病毒研究所保存)稀释成相应浓度，然后加入24孔板中使每孔含有病毒量为100TCID 50。接下来，用2％细胞维持液将磷酸氯喹和羟基氯喹(磷酸氯喹购自Sigma-Aldrich公司，货号C6628；羟基氯喹购自MCE公司，货号HY-B1370)分别稀释成相应浓度，加入到对应的孔中，使药物最终浓度分别为50μM、16.67μM、5.56μM、1.85μM、0.62μM、0.21μM、0.068μM，然后放入37℃、5％CO 2孵育箱继续培养48h，细胞对照组只加不含有任何受试药物的2％细胞维持液。
(2)RNA提取
RNA提取试剂盒购自Qiagen公司，货号74106。下述RNA提取步骤中所涉及的耗材(离心柱、无RNA酶的2ml收集管等)及试剂(RLT、RW1、RPE、无RNA酶水等)均为试剂盒的组成部分。下述提取步骤均为试剂盒说明书所推荐的步骤。
1)取受试培养板的上清液100μL，加入到无核酸酶EP管(购自Axygen，货号mct-150-c)中，然后每孔加入350μL Buffer RLT，用移液枪吹吸混匀使其充分裂解后，离心取上清；
2)向1)中所得上清液加入等体积的70％乙醇，混匀；
3)将上述2)中所得混合液转入无RNA酶的离心柱中，12000rpm离心15s，弃废液；
4)加入700μL Buffer RW1，12000rpm离心15s清洗离心柱，弃废液；
5)加入500μL Buffer RPE，12000rpm离心15s清洗离心柱，弃废液；
6)加入500μL Buffer RPE，12000rpm离心2min清洗离心柱，弃废液，然后将离心柱整体转移至步骤7的新的无RNA酶的2ml收集管中；
7)换新的无RNA酶的2ml收集管，12000rpm离心1min，干燥离心柱，然后将离心柱整体转移至步骤8的1.5ml收集管中；
8)换上新的1.5ml收集管，放入步骤7中干燥后的离心柱，并向离心柱中加入30μl不含RNA酶的水，12000rpm离心2min，洗脱液即含有相应的RNA，加入RNA酶抑制剂(购自NEB公司，货号M0314L)，用Nano Drop(购自Thermo scientific，型号Nano Drop One)检测各RNA浓度。
(3)RNA反转录
实验采用TaKaRa公司生产的反转录试剂盒(PrimeScript TM RT reagent Kit with gDNA Eraser，货号RR047Q)进行RNA反转录，步骤如下。
①gDNA去除：收集各实验组RNA样品，分别取1μg进行反转录。首先，向各实验组RNA中加入2μl 5×gDNA Eraser Buffer，用RNase Free水补足反应体系至10μl，充分混匀，42℃水浴2min去除样品中可能存在的g DNA；
②逆转录：向①所得样品中加入适量的酶和引物Mix及反应缓冲液，用RNase Free水补足体积至20μl，37℃水浴反应15min，之后投入85℃水中5sec，即可转录得到cDNA。
(4)Real-time PCR
采用荧光定量PCR检测原病毒液每毫升所含拷贝数。
采用TB Green Premix(Takara，Cat#RR820A)混好反应体系，在StepOne Plus Real-time PCR仪(品牌：ABI)进行扩增反应和读数。计算原病毒液每毫升所含拷贝数。步骤如下：
①首先建立标准品：将质粒pMT-RBD(质粒由中国科学院武汉病毒研究所保存)稀释成5×10 8copies/μL,5×10 7copies/μL,5×10 6copies/μL,5×10 5copies/μL,5×10 4copies/μL,5×10 3copies/μL,5×10 2copies/μL。取2μL标准品或cDNA模板用于qPCR反应。
②实验过程中所用引物序列如下(均为5’-3’方向表示)：
RBD-qF:CAATGGTTTAACAGGCACAGG
RBD-qR:CTCAAGTGTCTGTGGATCACG
③反应程序如下：
预变性：95℃ 5分钟；
循环参数：95℃ 15秒，54℃ 15秒，72℃30秒，共40个循环。
(5)药物对细胞毒性测试
药物对细胞毒性的检测利用CCK-8试剂盒(Beoytime)测定。具体步骤如下：
①96孔板中接种1×10 4个Vero-E6细胞，37℃培养8小时。
②将药物用DMSO稀释到合适的母液浓度，再用含2％FBS(购自Gibco公司，货号16000044)的MEM(购自Gibco公司，货号10370021)培养基稀释到与药物处理同样的浓度，弃96孔板中原培养基，取100μL含药物的MEM培养基加入到细胞中，每个浓度做三个复孔。设置阴性对照(细胞孔中加DMSO和培养基，而不加药物)和空白对照(不含细胞，加DMSO和培养基)。加药完毕，将细胞置于37℃培养48小时。
③向待测孔中加入20μL CCK-8溶液(Beoytime)，轻轻混匀，不要产生气泡，37℃继续培养2小时。在酶标仪(购自Molecular Devices公司，型号SpectraMax M5)上读取OD 450，将读数带入以下公式，计算细胞活性：
细胞活性(％)＝(A (药物处理组)-A (空白对照))/(A (阴性对照)-A (空白对照))×100％
其中A为酶标仪读数。
(6)实验结果
病毒增殖抑制实验的结果显示，受试化合物磷酸氯喹在50μM、16.67μM、5.56μM的浓度下，均能够有效抑制感染上清中SARS-CoV-2病毒基因组的复制。
细胞毒性(参见图1)结果显示，在所有受试浓度下，受试化合物磷酸氯喹的处理均未改变细胞活力，即受试化合物在所有浓度下对细胞均无毒性作用。
经计算，磷酸氯喹的半数有效浓度(half-maximal effective concentration，EC 50)为1.13μM，对细胞的半数致死浓度(half-cytotoxic concentration，CC 50)大于100μM，选择系数(selectivity index，SI)大于88.5)。结果显示，磷酸氯喹在低微摩尔浓度下能有效阻断SARS-CoV-2病毒感染，并显示出较高的SI。
实施例2：磷酸氯喹和羟基氯喹在4种不同感染滴度(MOI)下降低SARS-CoV-2感染的细胞病毒核酸载量实验
(1)药物处理
将Vero E6细胞接种至24孔板，培养24h，然后进行病毒感染。设置四组不同感染剂量，分别为0.01MOI，0.02MOI，0.2MOI和0.8MOI。用2％细胞维持液将SARS-CoV-2(2019-nCoV)病毒稀释成相应浓度，然后加入至24孔板中，使每孔中的细胞病毒载量达到设定的感染剂量。再用2％细胞维持液将磷酸氯喹和羟基氯喹分别稀释成相应浓度，加入到对应的孔中，使药物最终浓度分别为50μM、16.67μM、5.56μM、1.85μM、0.62μM、0.21μM、0.068μM，然后放入37℃、5％CO 2孵育箱继续培养48h，细胞对照组只加不含有任何受试药物的2％细胞维持液。
(2)RNA提取
参照实施例1中步骤(2)记载的方法进行RNA提取。
(3)RNA反转录
参照实施例1中步骤(3)记载的方法进行RNA反转录。
(4)Real-time PCR
参照实施例1中步骤(4)记载的方法，用荧光定量PCR检测原病毒液每毫升所含拷贝数。
(5)药物对细胞毒性测试
参照实施例1中步骤(5)记载的方法测试药物对细胞的毒性。
(6)实验结果
细胞毒性(参见图2中的A)结果显示，在受试浓度为100μM下时，受试化合物的处理均未改变细胞活力，即受试化合物在该浓度下对细胞均无毒性作用。经计算，羟基氯喹的CC 50大于249.50μM,磷酸氯喹的CC 50大于273.20μM。
病毒增殖抑制实验的结果显示，在MOI为0.01、0.02、0.2和0.8情况下，受试化合物均能够有效抑制感染上清中SARS-CoV-2病毒基因组的复制(参见图2中的B-E)。不同MOI下，受试化合物的EC 50如下：
MOI＝0.01
羟基氯喹：EC 50＝4.51μM；磷酸氯喹：EC 50＝2.71μM；
MOI＝0.02
羟基氯喹：EC 50＝4.06μM；磷酸氯喹：EC 50＝3.81μM；
MOI＝0.2
羟基氯喹：EC 50＝17.31μM；磷酸氯喹：EC 50＝7.14μM；
MOI＝0.8
羟基氯喹：EC 50＝12.96μM；磷酸氯喹：EC 50＝7.36μM。
实施例3：磷酸氯喹和羟基氯喹抑制SARS-CoV-2病毒侵入机制实验
(1)实验方法
1)用含有2％FBS的MEM培养液将磷酸氯喹或羟氯喹配制成50μM的含药培养液，然后利用上述含药培养液处理veroE6细胞，处理时间为1h；
2)在4℃下使veroE6细胞与SARS-CoV-2病毒结合1h(MOI＝10)；
3)用PBS(购自Gibco公司，货号C10010500BT)清洗2次，除去未结合的病毒颗粒，添加新鲜预热的含有2％FBS的MEM培养液，并在37℃下孵育90min；
4)固定细胞，并用抗病毒NP蛋白的抗体(红色)(由中国科学院武汉病毒研究所保存)和抗早期内吞体蛋白EEA的抗体(绿色)(购自Cell Signaling Technology公司，货号48453)或抗溶酶体蛋白LAMP的抗体(绿色)(购自Cell Signaling Technology公司，货号3243)对细胞进行免疫荧光染色；细胞核用Hoechst染色(蓝色)(购自Invitrogen公司，货号H21492)；
5)对每组中细胞进行病毒颗粒与内吞体共定位量化分析，统计共定位率。
(2)实验结果
免疫荧光分析(IFA)和共聚焦显微镜分析结果显示(参见图3)，在磷酸氯喹或羟基氯喹处理后，病毒颗粒与早期内吞体(EES)共定位率显著增多(磷酸氯喹为35.3％，羟基氯喹为29.2％；P<0.001)，而与溶酶体(ELs)的共定位率显著降低(磷酸氯喹为2.4％，羟基氯喹为0.03％；P<0.001)，表明了磷酸氯喹或羟基氯喹通过阻止SARS-CoV-2病毒从早期内吞体到溶酶体的运输过程抑制病毒感染。
最后应当说明的是：以上实施例仅用以说明本申请的技术方案而非对其限制；尽管参照较佳实施例对本申请进行了详细的说明，所属领域的普通技术人员应当理解：依然可以对本申请的具体实施方式进行修改或者对部分技术特征进行等同替换；而不脱离本申请技术方案的精神，其均应涵盖在本申请请求保护的技术方案范围当中。

Claims (9)

  1. 式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物在制备用于治疗SARS-CoV-2引起的疾病或感染(例如呼吸系统疾病(包括但不限于单纯性感染如发热、咳嗽和咽痛等、肺炎、急性或严重急性呼吸道感染、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克等))的药物中的用途，
    Figure PCTCN2020093989-appb-100001
  2. 式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物在制备作为SARS-CoV-2抑制剂的药物中的用途，或者在制备用于抑制SARS-CoV-2在细胞(例如哺乳动物细胞)中复制或繁殖的药物中的用途，
    Figure PCTCN2020093989-appb-100002
  3. 药物组合物在制备作为SARS-CoV-2抑制剂的药物中的用途，或者在制备用于抑制SARS-CoV-2在细胞(例如哺乳动物细胞)中复制或繁殖的药物中的用途，
    其中所述药物组合物包含式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物，
    Figure PCTCN2020093989-appb-100003
    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。
  4. 药物组合物在制备用于治疗SARS-CoV-2引起的疾病或感染(例如呼吸系统疾病(包括但不限于单纯性感染如发热、咳嗽和咽痛等、肺炎、急性呼吸道感染或严重急性呼吸道感染、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克等))的药物中的用途，其中所述药物组合物包含式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物，
    Figure PCTCN2020093989-appb-100004
    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。
  5. 一种在有需要的哺乳动物中治疗和/或预防疾病或病毒感染的方法或者在有需要的哺乳动物中抑制SARS-CoV-2复制或繁殖的方法，该方法包括给有需要的哺乳动物施用治疗和/或预防有效量的式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物或治疗和/或预防有效量的包含式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物的药物组合物，
    Figure PCTCN2020093989-appb-100005
    其中所述的疾病包括SARS-CoV-2引起的疾病(例如呼吸系统疾病(包括但不限于单纯性感染如发热、咳嗽和咽痛等、肺炎、急性呼吸道感染或严重急性呼吸道感染(SARI)、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克等))，所述的病毒感染包括由包括SARS-CoV-2引起的感染。
  6. 式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物或包含式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物的药物组合物，其作为SARS-CoV-2抑制剂，或者用于抑制SARS-CoV-2在细胞(例如哺乳动物细胞)中复制或繁殖，
    Figure PCTCN2020093989-appb-100006
    优选地，所述药物组合物中还包含药学上可接受的载体或辅料。
  7. 式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物或包含式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹，或其几何异构体或其药物上可接受的盐和/或其溶剂化物和/或其水合物的药物组合物，其用于治疗SARS-CoV-2引起的疾病或感染(例如呼吸系统疾病(包括但不限于单纯性感染如发热、咳嗽和咽痛等、肺炎、急性呼吸道感染或严重急性呼吸道感染、低氧性呼吸衰竭及急性呼吸窘迫综合征、脓毒症和脓毒性休克等))，
    Figure PCTCN2020093989-appb-100007
    优选地，所述药物组合物中还包含药学上可接受的载体或辅料。
  8. 权利要求1-4所述的用途，权利要求5所述的方法或权利要求6或7所述的化合物或组合物，其中式Ⅰ所示羟基氯喹或式Ⅱ所示氯喹的药学上可接受的盐包括包括其无机或有机酸盐，以及无机或有机碱盐，
    例如，所述药学上可接受的盐包括但不限于：所述化合物的钠盐，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，例如硫酸氯喹，例如磷酸氯喹。
  9. 权利要求1-4所述的用途，权利要求5所述的方法或权利要求6或7所述的化合物或组合物，其中所述的SARS-CoV-2引起的疾病为COVID-19。
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