WO2023222018A1 - Eidd-1931或其衍生物在治疗肠道病毒感染方面的应用 - Google Patents
Eidd-1931或其衍生物在治疗肠道病毒感染方面的应用 Download PDFInfo
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- 125000003187 heptyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
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- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
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- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
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- 238000002156 mixing Methods 0.000 description 1
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- HTNPEHXGEKVIHG-ZJTJHKMLSA-N molnupiravir Chemical compound CC(C)C(=O)OC[C@H]1O[C@H](C(O)C1O)N1C=C\C(NC1=O)=N\O HTNPEHXGEKVIHG-ZJTJHKMLSA-N 0.000 description 1
- 229940075124 molnupiravir Drugs 0.000 description 1
- 125000002757 morpholinyl group Chemical group 0.000 description 1
- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 1
- 125000001298 n-hexoxy group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])O* 0.000 description 1
- 125000003935 n-pentoxy group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])O* 0.000 description 1
- 125000000740 n-pentyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000003506 n-propoxy group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])O* 0.000 description 1
- 125000001971 neopentyl group Chemical group [H]C([*])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
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- 229910052760 oxygen Inorganic materials 0.000 description 1
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- 229940049954 penicillin Drugs 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 229940075930 picrate Drugs 0.000 description 1
- OXNIZHLAWKMVMX-UHFFFAOYSA-M picrate anion Chemical compound [O-]C1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-M 0.000 description 1
- 125000004193 piperazinyl group Chemical group 0.000 description 1
- 125000003386 piperidinyl group Chemical group 0.000 description 1
- IUGYQRQAERSCNH-UHFFFAOYSA-M pivalate Chemical compound CC(C)(C)C([O-])=O IUGYQRQAERSCNH-UHFFFAOYSA-M 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
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- 230000000069 prophylactic effect Effects 0.000 description 1
- 125000000719 pyrrolidinyl group Chemical group 0.000 description 1
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- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 235000011076 sorbitan monostearate Nutrition 0.000 description 1
- 239000001587 sorbitan monostearate Substances 0.000 description 1
- 229940035048 sorbitan monostearate Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 229940086735 succinate Drugs 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 230000003319 supportive effect Effects 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
- 125000001973 tert-pentyl group Chemical group [H]C([H])([H])C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000003718 tetrahydrofuranyl group Chemical group 0.000 description 1
- 125000005958 tetrahydrothienyl group Chemical group 0.000 description 1
- 125000004305 thiazinyl group Chemical group S1NC(=CC=C1)* 0.000 description 1
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical class CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
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Classifications
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
-
- 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
Definitions
- the present invention relates to the use of compounds represented by formula I, their geometric isomers or their pharmaceutically acceptable salts, solvates and/or hydrates and pharmaceutical compositions containing the above compounds for the treatment of enterovirus infections.
- EIDD-1931 (compound of formula I, where R is hydroxyl) is the active form of the marketed drug monapiravir (Molnupiravir, EIDD-2801, compound of formula I, where R is isobutyrate group).
- EIDD-1931 is effective against chikungunya virus (CHIKV), Venezuelan equine encephalitis virus (VEEV), respiratory syncytial virus (RSV), hepatitis C virus (HCV), norovirus (NV), influenza A virus ( IAV), influenza B virus (IBV), Ebola virus (EBOV), Middle East respiratory syndrome coronavirus (MERS-CoV) and new coronavirus (SARS-CoV-2) all have good inhibitory effects.
- CHCV Chikungunya virus
- VEEV Venezuelan equine encephalitis virus
- RSV respiratory syncytial virus
- HCV hepatitis C virus
- NV norovirus
- influenza A virus IAV
- EIDD-2801 Monaprevir
- EIDD-2801 is an oral dosage form of EIDD-2801.
- EIDD-2801 preventive medication can prevent lung damage caused by MERS-CoV and SARS-CoV infection.
- EIDD-2801 When EIDD-2801 is given within 12 hours or 24 hours after infection, it can significantly reduce the lung damage of mice. Extent of injury and weight loss.
- EIDD-2801 has been approved for marketing in many countries such as the United States and the United Kingdom for the treatment of COVID-19.
- Enteroviruses belong to the genus Enterovirus in the family Picornaviridae and are a type of single-stranded positive-strand RNA virus, which mainly include poliovirus, echovirus, coxsackievirus and new enteroviruses (including EV-A71, EV -D68 etc.).
- Typical symptoms of enterovirus infection include hand, foot and mouth disease, herpangina, aseptic encephalitis, meningitis, poliomyelitis, etc.
- Symptoms of diseases caused by enterovirus infections vary, including mild fever that can heal on its own, but a few develop into severe illness or even death.
- clinical treatment is mostly symptomatic and supportive, and no specific antiviral drugs are available.
- the purpose of the present invention is to discover drugs with antiviral activity against enteroviruses, which can be used to cause related diseases such as hand, foot and mouth disease, viral angina, aseptic meningitis, brainstem encephalitis, myocarditis, neurogenic disease, etc. Treatment of pulmonary edema and central nervous system infections.
- the present invention has found that the compound represented by formula I, its geometric isomer or its pharmaceutically acceptable salt, solvate and/or hydrate has the function of inhibiting enterovirus replication and can be used to treat enterovirus-induced disease.
- the present invention relates to the use of the compound represented by formula I, its geometric isomer or its pharmaceutically acceptable salt, solvate and/or hydrate in the preparation of medicaments for the treatment of diseases or infections caused by enteroviruses,
- R is a hydroxyl group, a hydrogen atom, an ester group, an alkoxy group, a phenyl group, a heterocyclyl group or an alkyl group.
- the present invention also relates to the use of the compound represented by Formula I, its geometric isomers or its pharmaceutically acceptable salts, solvates and/or hydrates in the preparation of drugs as enterovirus inhibitors.
- the present invention also relates to the use of the compound represented by Formula I, its geometric isomers or its pharmaceutically acceptable salts, solvates and/or hydrates in the preparation of medicaments for inhibiting the replication or reproduction of enteroviruses in cells. uses in.
- the present invention also relates to the use of a pharmaceutical composition in the preparation of medicaments for the treatment of diseases or infections caused by enteroviruses, wherein the pharmaceutical composition contains the compound represented by formula I, its geometric isomers or its pharmaceutical properties. Acceptable salts, solvates and/or hydrates.
- the present invention also relates to the use of a pharmaceutical composition in the preparation of medicaments as enterovirus inhibitors, wherein the pharmaceutical composition contains the compound of formula I, its geometric isomers or pharmaceutically acceptable salts thereof, Solvates and/or Hydrates.
- the present invention also relates to the use of a pharmaceutical composition in the preparation of medicaments for inhibiting the replication or propagation of enteroviruses in cells, wherein the pharmaceutical composition contains the compound represented by formula I, its geometric isomer or its medicament Acceptable salts, solvates and/or hydrates above.
- the present invention also relates to the compound represented by formula I, its geometric isomer or its pharmaceutically acceptable salt, solvate and/or hydrate, which is used to treat diseases or infections caused by enteroviruses.
- the present invention also relates to the compound represented by formula I, its geometric isomer or its pharmaceutically acceptable salt, solvate and/or hydrate, which is used as an enterovirus inhibitor.
- the present invention also relates to the compound represented by formula I, its geometric isomer or its pharmaceutically acceptable salt, solvate and/or hydrate, which is used to inhibit the replication or propagation of enterovirus in cells.
- the present invention also relates to a pharmaceutical composition for treating diseases or infections caused by enteroviruses, wherein the pharmaceutical composition contains the compound of formula I, its geometric isomer or a pharmaceutically acceptable salt thereof, Solvates and/or Hydrates.
- the present invention also relates to a pharmaceutical composition for use as an enterovirus inhibitor, wherein the pharmaceutical composition contains the compound of formula I, its geometric isomers or its pharmaceutically acceptable salts, solvates and/or or hydrate.
- the present invention also relates to a pharmaceutical composition for inhibiting the replication or propagation of enteroviruses in cells, wherein the pharmaceutical composition contains the compound of formula I, its geometric isomer or a pharmaceutically acceptable salt thereof , solvates and/or hydrates.
- the present invention also relates to a method for treating diseases or infections caused by enteroviruses in a mammal in need, which method includes administering to the mammal in need a therapeutically effective amount of a compound represented by the formula I, its geometry Pharmaceutical compositions of isomers or pharmaceutically acceptable salts, solvates and/or hydrates thereof or compounds represented by formula I, geometric isomers thereof or pharmaceutically acceptable salts, solvates and/or hydrates thereof or hydrate.
- the present invention also relates to a method for inhibiting enterovirus replication or reproduction in a mammal in need, which method includes administering to the mammal in need an effective amount of a compound represented by the formula I and its geometric isomer. Or a pharmaceutical composition of a pharmaceutically acceptable salt, solvate and/or hydrate thereof or the compound represented by Formula I, a geometric isomer thereof or a pharmaceutically acceptable salt, solvate and/or hydrate thereof .
- the compound represented by Formula I, its geometric isomer or its pharmaceutically acceptable salt, solvate and/or hydrate is used to treat intestinal tract An effective amount for the disease or infection caused by the virus is present.
- compositions of the present invention further comprise pharmaceutically acceptable carriers or excipients.
- the pharmaceutical composition of the present invention is a solid preparation, injection, topical preparation, spray, liquid preparation or compound preparation.
- diseases or infections caused by enteroviruses of the invention include, but are not limited to, hand, foot and mouth disease, viral anginitis, aseptic meningitis, brainstem encephalitis, myocarditis, neurogenic Pulmonary edema and central nervous system infection.
- cells described herein are mammalian cells.
- mammals of the invention include bovines, equids, ovines, porcines, canines, felines, rodents, primates, such as Human, cat, dog or pig.
- pharmaceutically acceptable salts of the compound represented by Formula I of the present invention include inorganic or organic acid salts thereof, as well as inorganic or organic alkali salts, and the present invention relates to all forms of the above-mentioned salts.
- alkyl group mentioned in the present invention refers to a saturated linear or branched chain monovalent hydrocarbon group, preferably having 1 to 8 carbon atoms, such as 1 to 6, 1 to 4 or 1 to 3 carbon atoms.
- alkyl groups described in the present invention include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, tert-pentyl Base, neopentyl, hexyl, heptyl, octyl, etc.
- the alkyl group of the present invention is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.
- the alkyl group of the present invention is methyl, ethyl, n-propyl, isopropyl or n-butyl.
- ester group mentioned in the present invention refers to R 1 -C(O)-O-, wherein R 1 is selected from the alkyl group as described in the present invention.
- ester groups described in the present invention include, but are not limited to, CH 3 -C(O)-O-, CH 3 -CH 2 -C(O)-O-, (CH 3 ) 2 -CH -C(O)-O-, CH 3 -(CH 2 ) 2 -C(O)-O- or CH 3 -(CH 2 ) 3 -C(O)-O-, etc.
- the alkoxy group described in the present invention refers to the group R 2 -O-, wherein R 2 is an alkyl group as described in the present invention.
- the alkoxy groups described in the present invention include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy base, n-pentyloxy group, n-hexyloxy group, 1,2-dimethylbutoxy group, etc.
- alkoxy groups described in the present invention include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy or n-butoxy.
- heterocyclyl group mentioned in the present invention refers to a group containing one, two or more heteroatoms independently selected from N, O and S and having 3-8, 5-8, 3-6 or 5-6 carbon atoms. Atoms of cycloalkyl.
- heterocyclyl include, but are not limited to, tetrahydrofuryl, tetrahydrothienyl, pyrrolidinyl, piperazinyl, thiazinyl, piperidinyl, morpholinyl, and the like.
- R in Formula I of the present invention is hydroxyl, hydrogen atom, R 1 -C(O)-O-, C 1-8 alkoxy, phenyl, heterocyclyl or C 1- 8 straight chain or branched chain alkyl group, wherein R 1 is C 1-8 straight chain or branched chain alkyl group.
- R in Formula I of the present invention is hydroxyl, hydrogen atom, R 1 -C(O)-O-, C 1-6 alkoxy, phenyl, heterocyclyl or C 1- 6 linear or branched alkyl, wherein R 1 is C 1-6 linear or branched alkyl.
- R in Formula I of the present invention is hydroxyl, hydrogen atom, R 1 -C(O)-O-, C 1-4 alkoxy, phenyl, heterocyclyl or C 1- 4 linear or branched alkyl, wherein R 1 is C 1-4 linear or branched alkyl.
- R in Formula I of the present invention is hydroxyl, hydrogen atom, methyl, ethyl, n-propyl, isopropyl, n-butyl, methoxy, ethoxy, n-propoxy base, isopropoxy, n-butoxy, phenyl, CH 3 -C(O)-O-, CH 3 -CH 2 -C(O)-O-, (CH 3 ) 2 -CH-C(O )-O-, CH 3 -(CH 2 ) 2 -C(O)-O- or CH 3 -(CH 2 ) 3 -C(O)-O-.
- R in Formula I of the present invention is hydroxyl or (CH 3 ) 2 -CH-C(O)-O-(isobutyrate).
- R in Formula I of the present invention is R 1 -C(O)-O-, wherein R 1 is a C 1-8 linear or branched alkyl group. In certain embodiments, R in Formula I of the present invention is R 1 -C(O)-O-, wherein R 1 is a C 1-6 linear or branched alkyl group. In certain embodiments, R in Formula I of the present invention is R 1 -C(O)-O-, wherein R 1 is a C 1-4 linear or branched alkyl group.
- R in Formula I of the present invention is C 1-8 alkoxy. In certain embodiments, R in Formula I of the present invention is C 1-6 alkoxy. In certain embodiments, R in Formula I of the present invention is C 1-4 alkoxy.
- R in Formula I of the present invention is heterocyclyl.
- R in Formula I of the present invention is a C 1-8 linear or branched alkyl group. In certain embodiments, R in Formula I of the present invention is a C 1-6 linear or branched alkyl group. In certain embodiments, R in Formula I of the present invention is a C 1-4 linear or branched alkyl group.
- R in Formula I of the present invention is hydroxyl
- R in Formula I of the present invention is a hydrogen atom.
- R in Formula I of the present invention is methyl
- R in Formula I of the present invention is ethyl
- R in Formula I of the present invention is n-propyl.
- R in Formula I of the present invention is isopropyl.
- R in Formula I of the present invention is n-butyl.
- R in Formula I of the present invention is methoxy
- R in Formula I of the present invention is ethoxy.
- R in Formula I of the present invention is n-propoxy.
- R in Formula I of the present invention is isopropoxy
- R in Formula I of the present invention is n-butoxy.
- R in Formula I of the present invention is phenyl
- R in Formula I of the present invention is CH 3 -C(O)-O-.
- R in Formula I of the present invention is CH 3 -CH 2 -C(O)-O-.
- R in Formula I of the present invention is (CH 3 ) 2 -CH-C(O)-O-.
- R in Formula I of the present invention is CH 3 -(CH 2 ) 2 -C(O)-O-.
- R in Formula I of the present invention is CH 3 -(CH 2 ) 3 -C(O)-O-.
- the compound of Formula I of the present invention is EIDD-2801.
- the compound of Formula I of the present invention is EIDD-1931.
- the pharmaceutical composition of the present invention can be prepared in various forms according to different administration routes.
- 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, and intramuscular administration. , intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or by means of an explanted reservoir. Among them, oral, intraperitoneal or intravenous administration is preferred.
- the compound represented by formula I of the present invention When administered orally, the compound represented by formula I of the present invention, its geometric isomer or its pharmaceutically acceptable salt, solvate and/or hydrate can be prepared into any orally acceptable preparation form, including but not Limited to tablets, capsules, aqueous solutions or aqueous suspensions.
- commonly used carriers for tablets include lactose and corn starch, and lubricants such as magnesium stearate can also be added.
- Commonly used diluents for capsule formulations include lactose and dried corn starch.
- Aqueous suspension formulations usually consist of the active ingredient mixed with suitable emulsifying and suspending agents. If desired, some sweetening, flavoring or coloring agents may be added to the above oral preparation forms.
- the compound represented by formula I of the present invention When used for rectal administration, the compound represented by formula I of the present invention, its geometric isomer or its pharmaceutically acceptable salt, solvate and/or hydrate can generally be made into the form of a suppository by combining the drug with a Prepared by mixing appropriate non-irritating excipients.
- the excipient is solid at room temperature and melts at rectal temperature to release the drug.
- excipients include cocoa butter, beeswax and polyethylene glycols.
- the compound represented by formula I of the present invention When used locally, especially when treating affected surfaces or organs easily accessible by topical application, such as eyes, skin or lower intestinal neurological diseases, the compound represented by formula I of the present invention, its geometric isomer or its pharmaceutical Acceptable salts, solvates and/or hydrates can be formulated into different topical formulations according to different affected areas or organs, as detailed below:
- the compound of formula I of the present invention When applied topically to the eye, the compound of formula I of the present invention, its geometric isomer or its pharmaceutically acceptable salt, solvate and/or hydrate can be formulated into a micronized suspension or solution.
- the carrier used is isotonic sterile saline with a certain pH, in which preservatives such as benzyl alkoxide chloride may or may not be added.
- the compounds can also be formulated in the form of ointments such as petroleum jelly.
- the compound represented by Formula I of the present invention When applied topically to the skin, the compound represented by Formula I of the present invention, its geometric isomers or its pharmaceutically acceptable salts, solvates and/or hydrates can be prepared into appropriate ointments, lotions or cream preparations Forms in which the active ingredient is suspended or dissolved in one or more carriers.
- the carriers that can be used for ointments include, but are not limited to: mineral oil, liquid petroleum jelly, white petroleum jelly, propylene glycol, polyoxyethylene, polyoxypropylene, 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, hexadecene aromatic alcohol, 2-octyldodecanol, benzyl alcohol and water.
- the compound represented by formula I of the present invention When used locally in the lower intestine, the compound represented by formula I of the present invention, its geometric isomer or its pharmaceutically acceptable salt, solvate and/or hydrate can be made into a rectal suppository preparation as described above or a suitable in the form of an enema, or as a topical transdermal patch.
- the compound represented by Formula I of the present invention can also be administered in the form of sterile injection preparations, including sterile injection water or oil suspensions, or sterile injectable solutions.
- sterile injection preparations including sterile injection water or oil suspensions, or sterile injectable solutions.
- carriers and solvents that can be used include water, Ringer's solution and isotonic sodium chloride solution.
- sterile fixed oils may be used as solvents or suspending media, such as mono- or diglycerides.
- the drugs in various dosage forms mentioned above can be prepared according to conventional methods in the pharmaceutical field.
- the term "effective amount” refers to an amount sufficient to achieve a desired therapeutic or prophylactic effect, for example, an amount to achieve a reduction in symptoms associated with the disease to be treated, such as a disease or infection caused by an enterovirus. , or an amount effective to prevent, arrest or delay the occurrence of a disease (e.g., a disease or infection caused by, for example, an enterovirus). Determining such effective amounts is within the ability of those skilled in the art.
- treatment is intended to alleviate, lessen, ameliorate or eliminate the disease state or disorder targeted. If the subject receives a therapeutic amount of the compound of Formula I, its geometric isomer or its pharmaceutically acceptable salt, solvate and/or hydrate or the pharmaceutical composition according to the methods described herein, A subject is successfully “treated” if the subject exhibits an observable and/or detectable reduction or improvement in one or more indications and symptoms. It should also be understood that treatment of a disease state or disorder includes not only complete treatment, but also includes less than complete treatment but achieving some biologically or medically relevant results.
- the dosage and method of use of the compound of formula I, its geometric isomer or its pharmaceutically acceptable salt, solvate and/or hydrate according to the present invention depend on many factors, including the age of the patient, Body weight, gender, natural health, nutritional status, activity intensity of the compound, duration of administration, metabolic rate, severity of illness, and the subjective judgment of the treating physician.
- the preferred dosage is between 0.01-1000mg/kg body weight/day.
- the compound represented by formula I, its geometric isomer or its pharmaceutically acceptable salt, solvate and/or hydrate according to the present invention can inhibit enterovirus replication on cells and reduce viral nucleic acid load in cell culture. quantity.
- the compound represented by formula I according to the present invention has one or more of the following biological activities:
- FIG 1 shows the structural formulas of compounds EIDD-1931 and EIDD-2801.
- FIG. 2 shows the CPE protective effects of compounds EIDD-1931 and EIDD-2801 on EV-A71 virus in different cell lines.
- the results show that EIDD-1931 and EIDD-2801 are effective in multiple cell lines infected by EV-A71 virus. It can inhibit the cytopathic effects caused by viruses in a dose-dependent manner.
- Figure 3 shows the experimental results of compounds EIDD-1931 and EIDD-2801 inhibiting EV-A71 viral RNA replication and infectious virus particle formation. The results show that both EIDD-1931 and EIDD-2801 inhibit EV-A71 in a dose-dependent manner. Replication of viral RNA (B and D), while also inhibiting the production of infectious virus particles (A and C).
- Figure 4 shows the experimental results of compounds EIDD-1931 and EIDD-2801 inhibiting the synthesis of EV-A71 virus VP1 protein. The results show that both EIDD-1931 and EIDD-2801 can inhibit the production of EV-A71 virus VP1 protein.
- Figure 5 shows the experimental results of the mechanism of action of compound EIDD-1931 against EV-A71 virus. The results show that EIDD-1931 exerts an antiviral effect during the replication stage of EV-A71 virus.
- Figure 6 shows the protective effect of compounds EIDD-1931 and EIDD-2801 on lethal infection in 1-day-old ICR suckling mice.
- the results show that both EIDD-1931 and EIDD-2801 can improve the efficiency of EV-A71-infected 1-day-old ICR suckling mice. survival rate.
- Figure 7 shows that both compounds EIDD-1931 and EIDD-2801 can reduce the viral load in tissues and organs of EV-A71-infected 1-day-old ICR suckling mice.
- Example 1 In vitro anti-EV-A71 virus activity evaluation experiment of compounds EIDD-1931 and EIDD-2801 based on cytopathic effect
- the human liver cancer cells (Huh7cell) used in the experiment were purchased from the National Experimental Cell Resource Sharing Platform.
- Human rhabdomyosarcoma cells (RD cells) and African green monkey kidney cells (Vero cells) were purchased from ATCC (product numbers: CCL-136, CCL-81 respectively).
- the above cells were cultured in a cell culture incubator at 37°C and 5% CO2 .
- the complete culture medium used for cell growth was supplemented with 10% FBS (purchased from Gibco Company, product number: 16000044) and penicillin and streptomycin double antibodies (purchased from Gibco Company). , Catalog No.: 2321152) DMEM high-glucose medium (purchased from Gibco Company, Catalog No.: 2367374).
- the maintenance medium used for cell culture was DMEM high-glucose medium supplemented with 2% FBS and penicillin-streptomycin antibodies.
- EV-A71 virus H strain was purchased from ATCC (catalog number: VR-1432).
- Compounds EIDD-1931 and EIDD-2801 were purchased from Shanghai Taosu Biochemical Technology Co., Ltd. (item numbers: T8498 and T8309 respectively), and their structural formulas are shown in Figure 1.
- Cell-Titer Luminescent Cell Viability detection solution was purchased from Promega Company (product number: G7572).
- the microplate reader was purchased from Molecular Devices, model SpectraMax M5.
- Huh7 cells, RD cells, and Vero cells were seeded into a 96-well plate with white walls and transparent bottom at a density of approximately 1.0 ⁇ 10 4 per well, and cultured at 37°C and 5% CO 2 for 24 hours. Then the EV-A71 virus was diluted with cell maintenance solution and added to a 96-well plate to a final concentration of 100TCID 50 . At the same time, the test compounds EIDD-2801 and EIDD-1931 were diluted with cell maintenance solution and added to the 96-well plate. The final concentrations are 100 ⁇ M, 33 ⁇ M, 11 ⁇ M, 0.37 ⁇ M, 0.12 ⁇ M, 0.04 ⁇ M, 0.01 ⁇ M and 0.005 ⁇ M.
- the formula for calculating the virus inhibition rate of the compound to be tested is:
- Inhibition rate (%) (experimental group - average value of virus group) / (average value of cell control group - average value of virus group) ⁇ 100
- the experimental plan is as follows:
- Huh7 cells, RD cells, and Vero cells were seeded into a 96-well plate with white walls and transparent bottom at a density of 1.0 ⁇ 10 4 per well, and cultured at 37°C and 5% CO 2 for 24 hours.
- the compounds to be tested EIDD-1931 and EIDD-2801 were diluted with cell maintenance solution and then added to the 96-well plate to make the final concentrations 100 ⁇ M, 33 ⁇ M, 11 ⁇ M, 0.37 ⁇ M, 0.12 ⁇ M, 0.04 ⁇ M, 0.01 ⁇ M and 0.005 ⁇ M respectively. .
- the inhibition rate calculation formula for each dilution of the compound to be tested is:
- Inhibition rate (%) (average value of cell control group - experimental group)/average value of cell control group ⁇ 100
- EIDD-1931 and EIDD-2801 can dose-dependently inhibit the cytopathic effects caused by the virus in multiple cell lines infected with EV-A71 virus.
- the EC 50 value, CC 50 value and SI of EIDD-1931 on RD cells are 5.13 ⁇ 0.56 ⁇ M, 80.47 ⁇ 0.02 ⁇ M and 15.69 respectively, and the EC50 value, CC50 value and SI on Vero cells are 7.04 ⁇ 0.38 ⁇ M respectively. , 14.07 ⁇ 0.43 ⁇ M and 2.0, the EC 50 value, CC 50 value and SI on Huh7 cells were 4.43 ⁇ 0.33 ⁇ M, 34.09 ⁇ 0.06 ⁇ M and 7.69 respectively.
- the EC 50 value, CC 50 value, and SI of EIDD-2801 on RD cells were 70.12 ⁇ 4.40 ⁇ M, >100 ⁇ M, and >1.43 respectively, and the EC 50 value, CC 50 value, and SI on Vero cells were 88.52 ⁇ 3.18 ⁇ M, respectively. , >100 ⁇ M and >1.13, the EC 50 value, CC 50 value and SI on Huh7 cells are 35.64 ⁇ 0.47 ⁇ M, >100 ⁇ M and >2.81 respectively.
- Example 2 Experiment on the inhibition of EV-A71 viral RNA replication and the production of infectious virus particles in the supernatant by compounds EIDD-1931 and EIDD-2801
- the Mini kit was purchased from Qiagen (Cat. No. 74106), the One Step PrimeScript TM RT-PCR kit was purchased from Takara (Cat. No.: RR47Q), and the standard plasmid and RT-PCR primers of the EV-A71H strain were purchased from Sangon Bioengineering. (Shanghai) Co., Ltd.
- the final concentrations of EIDD-1931 are 20 ⁇ M, 10 ⁇ M, 5 ⁇ M, 2.5 ⁇ M, and 1.25 ⁇ M, and the final concentrations of EIDD-2801 are 200 ⁇ M, 100 ⁇ M, 50 ⁇ M, 25 ⁇ M, and 12.5 ⁇ M.
- Incubate for 1.5 hours at 37°C, 5% CO2 then discard the supernatant, add 1 mL of diluent of the compound to be tested to the corresponding well in each well, continue to culture for 30 hours, collect the supernatant when the cells begin to show lesions, 8000 rpm for 5 min Centrifuge, aliquot, and store at -80°C for later use.
- Inhibition rate (%) (RNA copy number in drug-treated group)/(RNA copy number in cell-infected group) ⁇ 100
- RD cells were seeded in a 96-well plate at a density of approximately 1.0 ⁇ 10 4 per well and cultured for 24 hours at 37°C and 5% CO 2 . Then discard the cell culture medium and add 150 ⁇ L of cell maintenance solution to each well. Take the supernatant from each group obtained in 1, dilute it with cell maintenance solution (starting from 10 -1 , dilute 10 times, a total of 8 gradients), and then inoculate the diluted supernatant into a 96-well plate, 50 ⁇ L/well , 4 duplicate wells for each dilution, and 50 ⁇ L of cell maintenance solution was added to the cell control group. The culture was continued for 5 days, and then the TCID 50 value of each sample was calculated using the Reed-Muench method.
- compounds EIDD-1931 and EIDD-2801 both inhibited the replication of EV-A71 viral RNA in a dose-dependent manner (B and D), and also inhibited the production of infectious virus particles (A and C). .
- Example 3 Compounds EIDD-1931 and EIDD-2801 inhibit EV-A71 viral protein synthesis experiment
- mouse anti-EV-A71VP1 antibody primary antibody
- Mouse anti-alpha tubulin antibody and HRP-conjugated goat anti-mouse IgG antibody were purchased from Abcam Company (Product No.: ab7291, ab205719 respectively)
- Alexa Fluor 647 donkey anti-mouse IgG (H+L) secondary antibody and nuclear dye Hoechst 33342 were purchased from Invitrogen Company (Product No.: A31571, H21492 respectively)
- RIPA lysis buffer, Skimmed milk powder, BCA method protein quantification kit, and Super Ecl ultrasensitive luminescent liquid were all purchased from Prilite (item numbers: P1622, P1511, C1053, and P1050).
- RD cells were seeded in a 12-well plate at a density of approximately 1.0 ⁇ 10 5 per well and cultured for 24 hours at 37°C and 5% CO 2 . Then use cell maintenance solution to dilute the EV-A71 virus to the corresponding concentration, add it to a 12-well plate so that the amount of virus contained in each well is 0.1 MOI, and use the cell maintenance solution to dilute EIDD-1931 and EIDD-2801 to the corresponding concentration, and add it to In a 12-well plate, the final concentrations of EIDD-1931 and EIDD-2801 were 10 ⁇ M, 5 ⁇ M, 2.5 ⁇ M, 1.25 ⁇ M, 0.63 ⁇ M and 100 ⁇ M, 50 ⁇ M, 25 ⁇ M, 12.5 ⁇ M, and 6.3 ⁇ M, respectively.
- Adherent cells are fully lysed with cell lysis buffer (RIPA) (100 ⁇ L/well) to obtain whole cell protein. After measuring the protein concentration with the BCA protein quantification kit, add the corresponding volume of 5 ⁇ SDS Loading Buffer, 100°C Boil for 10 minutes to fully denature, and then use 10% SDS-PAGE to electrophores at 80V/120V for 2 hours to separate proteins of different sizes. The protein and PVDF membrane were transferred at 300mA current for 90 minutes to allow the membrane to be fully transferred.
- RIPA cell lysis buffer
- the membrane was cut into a suitable size according to the molecular weight of the target protein, and blocked with 5% skimmed milk powder diluted in TBST on a room temperature shaker for 1 hour, and then added
- the primary antibodies diluted in blocking solution: anti-EV-A71VP1 protein antibody (1:500 dilution) and internal reference Tublin antibody (1:5000 dilution) were incubated overnight at 4°C on a shaker.
- HRP-labeled goat anti-mouse IgG antibody (1:5000 dilution
- color photography was taken using ultrasensitive chemiluminescence method.
- the concentration of EIDD-1931 used in the experiment was 10 ⁇ M, the concentration of positive compound NITD008 was 2 ⁇ M, and the infectious dose of EV-A71 virus was 0.01 MOI.
- the specific steps are: seed RD cells in a 12-well plate at a density of approximately 4.0 ⁇ 10 5 per well, and culture at 37°C and 5% CO 2 for 24 hours. Subsequently, the test compound EIDD-1931, positive compound NITD008 and EV-A71 virus were diluted to corresponding concentrations with cell maintenance solution, and then the pre-diluted compounds or viruses were added according to the time points shown in A in Figure 5.
- RNA extraction method After continuing to culture for 30 hours, the cell supernatant was discarded, intracellular RNA was extracted, and intracellular viral RNA load was detected using qRT-PCR.
- the intracellular RNA extraction method, qRT-PCR system, and reaction conditions were the same as previously described.
- the 1-day-old ICR suckling mice used in the experiment were SPF grade and purchased from Beijing Vitong Lihua Experimental Animal Technology Co., Ltd. The source was clear and they passed the inspection.
- mice Ten litters of 1-day-old ICR female mice were randomly divided into 10 groups, namely virus control group (group 2), EIDD-1931 administration group (group 4), EIDD-2801 administration group (group 4), EIDD-1931 and The dosages of EIDD-2801 are 200mg/kg, 66.6mg/kg, 22.2mg/kg and 7.41mg/kg.
- Each mouse was intraperitoneally challenged with 10 6 PFU, and the first intraperitoneal administration was given 4 hours later.
- the virus control group was given drug solvent, and then administered once a day for 7 consecutive days. The mice were weighed and recorded every day. survival and weight changes.
- EIDD-1931 and EIDD-2801 showed a dose-dependent protective effect on ICR suckling mice inoculated with a lethal dose of EV-A71 virus.
- EIDD-1931 can completely protect 1-day-old ICR suckling mice from a lethal dose of EV-71 virus challenge at doses of 200 mg/kg and 66.6 mg/kg, and can protect 1-day-old ICR mice at a dose of 22.2 mg/kg.
- the survival protection rate of suckling mice was 60%. The mice that survived at these three dosage concentrations were able to maintain good weight gain, while the mice in the control or low-dose group (7.41mg/kg) died within 12 days ( A and B in Figure 6).
- Example 6 Compounds EIDD-1931 and EIDD-2801 inhibit viral load in tissues of suckling mice infected with EV-A71 virus.
- TRIzol reagent was purchased from Invitrogen, product number is 15596026.
- the 1-day-old ICR suckling mice used in the experiment were SPF grade and purchased from Beijing Vitong Lihua Experimental Animal Technology Co., Ltd. The source was clear and they passed the inspection.
- mice Three litters of 1-day-old ICR female mice were randomly divided into 3 groups, namely virus control group, EIDD-1931 administration group (200 mg/kg), and EIDD-2801 administration group (200 mg/kg). Each mouse was intraperitoneally challenged with 10 6 PFU, and the first intraperitoneal administration was given 4 hours later. The virus control group was given drug solvent, and then administered once a day for 4 consecutive days. On the fourth day, the mice were The mice were euthanized, and the brain, heart, small intestine, liver, hind limb muscles and lung tissues of the mice were collected. Tissue RNA was extracted using TRIzol reagent, and then qRT-PCR was used to quantitatively analyze the viral RNA in each tissue.
- EIDD-1931 and EIDD-2801 were effective against EV-A71 virus AH08/06 strain, EV-D68 virus STL-2014-12 strain, CV-A6 TW-2007-00141 strain, and CV-A16 190-D1
- the CPE protection experiments of strains were all measured using RD cells.
- the CPE protection experiment of EIDD-1931 and EIDD-2801 against CV-B3 Nancy strain was measured using Vero cells.
- the specific experimental method is the same as Example 1.
- EIDD-1931 can inhibit the infection of a variety of enteroviruses, and its EC 50 values are all less than 20 ⁇ M, while EIDD-2801 has lower inhibitory activity against EV-V68, CV-A16 and CV-B3, and is less effective against CV-
- the EC50 value of A6 is greater than 100 ⁇ M.
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Abstract
本发明涉及式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物及含有此化合物的药物组合物在制备用于治疗肠道病毒引起的疾病或感染的药物中的用途。
Description
本申请是以CN申请号为202210549687.9，申请日为2022年5月20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。
本发明涉及式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物及含上述化合物的药物组合物用于治疗肠道病毒感染方面的用途。
EIDD-1931(式I化合物，其中R为羟基)是已上市药物莫那匹韦(Molnupiravir，EIDD-2801，式I化合物，其中R为异丁酸酯基)的活性形式。EIDD-1931对基孔肯雅病毒(CHIKV)、委内瑞拉马脑炎病毒(VEEV)、呼吸道合胞病毒(RSV)、丙型肝炎病毒(HCV)、诺如病毒(NV)、甲型流感病毒(IAV)、乙型流感病毒(IBV)、埃博拉病毒(EBOV)以及中东呼吸综合征冠状病毒(MERS-CoV)和新型冠状病毒(SARS-CoV-2)等均有良好的抑制效果。莫那匹韦(EIDD-2801)是EIDD-2801的一种口服剂型。小鼠体内实验结果表明，EIDD-2801预防用药可以防止MERS-CoV和SARS-CoV感染所致的肺损伤，在感染后12小时或24小时内给予EIDD-2801治疗时，可显著降低小鼠肺损伤和体重减轻的程度。目前，EIDD-2801已在美国、英国等多个国家获批上市，用于新冠肺炎的治疗。
肠道病毒属于小RNA病毒科肠道病毒属，是一类单股正链RNA病毒，主要包括脊髓灰质炎病毒、埃可病毒、柯萨奇病毒和新型肠道病毒(包括EV-A71、EV-D68等)。肠道病毒感染的典型症状包括手足口病、疱疹性咽峡炎、无菌性脑炎、脑膜炎、脊髓灰质炎等。肠道病毒感染引起的疾病症状多种多样，包括可以自愈的轻微发热等，也有少数发展成重症甚至死亡。目前，针对肠道病毒感染，临床上多以对症治疗和支持治疗为主，无特异性抗病毒药物可用。
发明内容
本发明的目的是发现对肠道病毒有抗病毒活性的药物，可用于其感染引起相关疾病如手足口病、病毒性咽峡炎、无菌性脑膜炎、脑干脑炎、心肌炎、神经源性肺水肿和中枢神经系统感染等的救治。本发明通过创造性的研究发现式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物具有抑制肠道病毒复制的功能，可用于治疗肠道病毒引起的疾病。
本发明涉及式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物在制备用于治疗肠道病毒引起的疾病或感染的药物中的用途，
其中R为羟基、氢原子、酯基、烷氧基、苯基、杂环基或烷基。
本发明还涉及所述式I所示的化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物在制备作为肠道病毒抑制剂的药物中的用途。
本发明还涉及所述式I所示的化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物在制备用于抑制肠道病毒在细胞中复制或繁殖的药物中的用途。
本发明还涉及药物组合物在制备用于治疗肠道病毒引起的疾病或感染的药物中的用途，其中所述药物组合物包含所述式Ⅰ所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物。
本发明还涉及药物组合物在制备作为肠道病毒抑制剂的药物中的用途，其中所述药物组合物包含所述式Ⅰ所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物。
本发明还涉及药物组合物在制备用于抑制肠道病毒在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所述式Ⅰ所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物。
本发明还涉及所述式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物，其用于治疗肠道病毒引起的疾病或感染。
本发明还涉及所述式I所示的化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物，其用作肠道病毒抑制剂。
本发明还涉及所述式I所示的化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物，其用于抑制肠道病毒在细胞中复制或繁殖。
本发明还涉及药物组合物，其用于治疗肠道病毒引起的疾病或感染，其中所述药物组合物包含所述式Ⅰ所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物。
本发明还涉及药物组合物，其用作肠道病毒抑制剂，其中所述药物组合物包含所述式Ⅰ所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物。
本发明还涉及药物组合物，其用于抑制肠道病毒在细胞中复制或繁殖，其中所述药物组合物包含所述式Ⅰ所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物。
本发明还涉及一种在有需要的哺乳动物中治疗肠道病毒引起的疾病或感染的方法，该方法包括给有需要的哺乳动物施用治疗有效量的包含所述式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物的药物组合物或所述式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物。
本发明还涉及一种在有需要的哺乳动物中抑制肠道病毒复制或繁殖的方法，该方法包括给有需要的哺乳动物施用有效量的包含所述式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物的药物组合物或所述式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物。
在某些实施方案中，本发明所述的药物组合物中，所述式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物以治疗肠道病毒引起的疾病或感染的有效量存在。
在某些实施方案中，本发明所述的药物组合物还包含药学上可接受的载体或辅料。
在某些实施方案中，本发明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。
在某些实施方案中，本发明所述的肠道病毒引起的疾病或感染包括但不限于手足口病、病毒性咽峡炎、无菌性脑膜炎、脑干脑炎、心肌炎、神经源性肺水肿和中枢神经系统感染。
在某些实施方案中，本发明所述的细胞为哺乳动物细胞。在某些实施方案中，本发明所述哺乳动物包括牛科动物、马科动物、羊亚科动物、猪科动物、犬科动物、猫科动物、啮齿类动物、灵长类动物，例如是人、猫、狗或猪。
在某些实施方案中，本发明所述式I所示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，本发明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，氢溴酸盐，氢碘酸盐，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。
本发明中所述的烷基是指饱和的直链或支链一价烃基，优选具有1－8个碳原子，例如具有1－6，1－4或1－3个碳原子。在某些实施方案中，本发明所述的烷基包括但不限于甲基，乙基，正丙基，异丙基，正丁基，异丁基，叔丁基，正戊基，叔戊基，新戊基，己基，庚基，辛基等。
在某些实施方案中，本发明所述的烷基为甲基，乙基，正丙基，异丙基，正丁基，异丁基或叔丁基。
在某些实施方案中，本发明所述的烷基为甲基，乙基，正丙基，异丙基或正丁基。
本发明中所述的酯基是指R1-C(O)-O-，其中R1选自如本发明所述的烷基。在某些实施方案中，本发明所述的酯基包括但不限于CH3-C(O)-O-、CH3-CH2-C(O)-O-、(CH3)2-CH-C(O)-O-、CH3-(CH2)2-C(O)-O-或CH3-(CH2)3-C(O)-O-等。
本发明中所述的烷氧基是指基团R2-O-，其中R2为如本发明所述的烷基。在某些实施方案中，本发明所述的烷氧基包括但不限于甲氧基，乙氧基，正丙氧基，异丙氧基，正丁氧基，叔丁氧基，仲丁氧基，正戊氧基，正己氧基，1,2-二甲基丁氧基等。在某些实施方案中，本发明所述的烷氧基包括但不限于甲氧基，乙氧基，正丙氧基，异丙氧基或正丁氧基。
本发明中所述的杂环基是指包含一个、两个或多个独立地选自N，O和S的杂原子的具有3－8，5－8，3－6或5－6个碳原子的环烷基。“杂环基”的典型实例包括但不限于四氢呋喃基，四氢噻吩基，吡咯烷基，哌嗪基，噻嗪基，哌啶基和吗啉基等。
在某些实施方案中，本发明所述式I中R为羟基、氢原子、R1-C(O)-O-、C1-8烷氧基、苯基、杂环基或C1-8直链或支链烷基，其中R1为C1-8直链或支链烷基。
在某些实施方案中，本发明所述式I中R为羟基、氢原子、R1-C(O)-O-、C1-6烷氧基、苯基、杂环基或C1-6直链或支链烷基，其中R1为C1-6直链或支链烷基。
在某些实施方案中，本发明所述式I中R为羟基、氢原子、R1-C(O)-O-、C1-4烷氧基、苯基、杂环基或C1-4直链或支链烷基，其中R1为C1-4直链或支链烷基。
在某些实施方案中，本发明所述式I中R为羟基、氢原子、甲基、乙基、正丙基、异丙基、正丁基、甲氧基、乙氧基、正丙氧基、异丙氧、正丁氧基、苯基、CH3-C(O)-O-、CH3-CH2-C(O)-O-、(CH3)2-CH-C(O)-O-、CH3-(CH2)2-C(O)-O-或CH3-(CH2)3-C(O)-O-。
在某些实施方案中，本发明所述式I中R为羟基或(CH3)2-CH-C(O)-O-(异丁酸酯基)。
在某些实施方案中，本发明所述式I中R为R1-C(O)-O-，其中R1为C1-8直链或支链烷基。在某些实施方案中，本发明所述式I中R为R1-C(O)-O-，其中R1为C1-6直链或支链烷基。在某些实施方案中，本发明所述式I中R为R1-C(O)-O-，其中R1为C1-4直链或支链烷基。
在某些实施方案中，本发明所述式I中R为C1-8烷氧基。在某些实施方案中，本发明所述式I中R为C1-6烷氧基。在某些实施方案中，本发明所述式I中R为C1-4烷氧基。
在某些实施方案中，本发明所述式I中R为杂环基。
在某些实施方案中，本发明所述式I中R为C1-8直链或支链烷基。在某些实施方案中，本发明所述式I中R为C1-6直链或支链烷基。在某些实施方案中，本发明所述式I中R为C1-4直链或支链烷基。
在某些实施方案中，本发明所述式I中R为羟基。
在某些实施方案中，本发明所述式I中R为氢原子。
在某些实施方案中，本发明所述式I中R为甲基。
在某些实施方案中，本发明所述式I中R为乙基。
在某些实施方案中，本发明所述式I中R为正丙基。
在某些实施方案中，本发明所述式I中R为异丙基。
在某些实施方案中，本发明所述式I中R为正丁基。
在某些实施方案中，本发明所述式I中R为甲氧基。
在某些实施方案中，本发明所述式I中R为乙氧基。
在某些实施方案中，本发明所述式I中R为正丙氧基。
在某些实施方案中，本发明所述式I中R为异丙氧。
在某些实施方案中，本发明所述式I中R为正丁氧基。
在某些实施方案中，本发明所述式I中R为苯基。
在某些实施方案中，本发明所述式I中R为CH3-C(O)-O-。
在某些实施方案中，本发明所述式I中R为CH3-CH2-C(O)-O-。
在某些实施方案中，本发明所述式I中R为(CH3)2-CH-C(O)-O-。
在某些实施方案中，本发明所述式I中R为CH3-(CH2)2-C(O)-O-。
在某些实施方案中，本发明所述式I中R为CH3-(CH2)3-C(O)-O-。
在某些实施方案中，本发明所述式I所示化合物为EIDD-2801。
在某些实施方案中，本发明所述式I所示化合物为EIDD-1931。
本发明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。
根据本发明，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，本发明所述式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或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，本发明所述式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或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，本发明所述式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或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，本发明所述式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或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，本发明所述式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或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，本发明所述式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或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。
本发明所述式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或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。
如本发明中所使用的，术语“有效量”是指足以实现所需治疗或预防效果的量，例如，实现减轻与待治疗疾病(例如肠道病毒引起的疾病或感染)相关的症状的量，或者能够有效预防、阻止或延迟疾病(例如例如肠道病毒引起的疾病或感染)的发生的量。测定这样的有效量在本领域技术人员的能力范围之内。
如本发明中所使用的，术语“治疗”目的是缓解、减轻、改善或消除所针对的疾病状态或病症。如果受试者按照本文所述方法接受了治疗量的所述式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物或所述药物组合物，该受试者一种或多种指征和症状表现出可观察到的和/或可检测出的降低或改善，则受试者被成功地“治疗”了。还应当理解，所述的疾病状态或病症的治疗不仅包括完全地治疗，还包括未达到完全地治疗，但实现了一些生物学或医学相关的结果。
另外需要指出，本发明所述的式I化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物的使用剂量和使用方法取决于诸多因素，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。优选的使用剂量介于0.01－1000mg/kg体重/天。
本发明所述的式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物可在细胞上抑制肠道病毒复制，减少细胞培养物中病毒核酸载量。
本发明所述的式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物具有以下一种或多种生物活性：
1)抗肠道病毒活性，
2)增强或提高肠道病毒感染细胞的细胞活力或细胞存活率。
本发明所述的式I所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物的上述生物活性是通过下列的至少一种途径实现的：
A、抑制肠道病毒感染细胞的CPE水平。
B、抑制肠道病毒RNA复制和感染性病毒颗粒的形成。
C、抑制肠道病毒蛋白的产生。
D、在病毒复制阶段发挥抗病毒作用。
图1示出了化合物EIDD-1931和EIDD-2801的结构式。
图2示出了化合物EIDD-1931和EIDD-2801在不同细胞系中对EV-A71病毒的CPE保护效果，结果显示EIDD-1931和EIDD-2801在EV-A71病毒感染的多个细胞系中均能剂量依赖性抑制病毒所致的细胞病变效应。
图3示出了化合物EIDD-1931和EIDD-2801抑制EV-A71病毒RNA复制和感染性病毒颗粒形成的实验结果，结果显示EIDD-1931和EIDD-2801均以剂量依赖性的方式抑制EV-A71病毒RNA的复制(B和D)，同时也抑制了感染性病毒颗粒的产生(A和C)。
图4示出了化合物EIDD-1931和EIDD-2801抑制EV-A71病毒VP1蛋白合成的实验结果，结果显示EIDD-1931和EIDD-2801均能抑制EV-A71病毒VP1蛋白的产生。
图5示出了化合物EIDD-1931抗EV-A71病毒作用机制的实验结果，结果显示EIDD-1931在EV-A71病毒的复制阶段发挥抗病毒作用。
图6示出了化合物EIDD-1931和EIDD-2801对1日龄ICR乳鼠致死性感染的保护效果，结果显示EIDD-1931和EIDD-2801均能提高EV-A71感染的1日龄ICR乳鼠的生存率。
图7示出了化合物EIDD-1931和EIDD-2801均能降低EV-A71感染的1日龄ICR乳鼠的组织器官的病毒载量。
下面结合本发明的具体实施例来进一步说明本发明的实质性内容，应理解，以下实施例仅用于说明本发明，但并不以此来限定本发明的保护范围。下面实施例中未注明具体条件者，按照常规条件或制造商建议的进行。所用药品或试剂未注明生产商者，均为可以通过市购获得的常规产品。
虽然以下实施例中所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细描述。本领域技术人员清楚，如果未特别注明，下面实施例中所用的材料和操作方法是本领域公知的。
实施例1化合物EIDD-1931和EIDD-2801基于细胞病变效应的体外抗EV-A71病毒活性评价实验
(1)实验材料
实验过程中所用人肝癌细胞(Huh7cell)购买于国家实验细胞资源共享平台。人横纹肌瘤细胞(RD cell)和非洲绿猴肾细胞(Vero cell)均购买于ATCC(货号分别为：CCL-136、CCL-81)。上述细胞培养于37℃，5％CO2的细胞培养箱中，细胞生长所用完全培养基为添加10％FBS(购自Gibco公司，货号：16000044)和青链霉素双抗(购自Gibco公司，货号：2321152)的DMEM高糖培养基(购自Gibco公司，货号：2367374)。细胞培养所用维持培养基为添加2％FBS和青链霉素双抗的DMEM高糖培养基。EV-A71病毒H株购买于ATCC(货号：VR-1432)。化合物EIDD-1931和EIDD-2801购买于上海陶素生化科技有限公司(货号分别为：T8498和T8309)，结构式如图1所示。Cell-TiterLuminescent Cell Viability检测液购买于Promega公司(货号：G7572)。酶标仪购买于Molecular Devices公司，型号为SpectraMax M5。
(2)实验方案如下：
将Huh7细胞、RD细胞、Vero细胞以每孔约1.0×104的密度接种于白壁透明底的96孔板，在37℃、5％CO2条件下培养24小时。随后用细胞维持液稀释EV-A71病毒，加入96孔板中，使其终浓度为100TCID50，同时将待测化合物EIDD-2801和EIDD-1931用细胞维持液稀释，加入96孔板中，使其终浓度为100μM、33μM、11μM、0.37μM、0.12μM、0.04μM、0.01μM和0.005μM。3天后弃上清，加入稀释好的Cell-TiterLuminescent Cell Viability检测液，避光震荡裂解5min，静置3min，最后用酶标仪对各孔的发光值进行测定。
待测化合物对病毒抑制率计算公式为：
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
(3)化合物EIDD-1931和EIDD-2801对3种不同细胞系的细胞毒性实验
实验方案如下：
将Huh7细胞、RD细胞、Vero细胞以每孔1.0×104的密度接种于白壁透明底的96孔板，在37℃、5％CO2条件下培养24小时。用细胞维持液倍比稀释待测化合物EIDD-1931和EIDD-2801，然后加入96孔板，使其终浓度分别为100μM、33μM、11μM、0.37μM、0.12μM、0.04μM、0.01μM和0.005μM。3天后弃上清，加入稀释好的Cell-TiterLuminescent Cell Viability检测液，避光震荡裂解5min，静置3min，最后用酶标仪对各孔的发光值进行测定。
待测化合物各稀释度的抑制率计算公式为：
抑制率(％)＝(细胞对照组平均值-实验组)/细胞对照组平均值×100
(4)数据分析
利用Origin9.0软件对抑制率-浓度进行S型曲线拟合，计算待测化合物的半数抑制有效浓度(half-maximal effective concentration,EC50)。利用同样方法计算待测化合物的半数毒性浓度(half-cytotoxic concentrations,CC50)，并根据EC50及CC50计算选择指数(Select index,SI)SI＝CC50/EC50。
(5)实验结果
实验结果如图2所示，EIDD-1931和EIDD-2801在EV-A71病毒感染的多个细胞系中均能剂量依赖性抑制病毒所致的细胞病变效应。EIDD-1931在RD细胞上的EC50值、CC50值、SI分别为5.13±0.56μM、80.47±0.02μM和15.69，在Vero细胞上的EC50值、CC50值、SI分别7.04±0.38μM、14.07±0.43μM和2.0，在Huh7细胞上的EC50值、CC50值、SI分别4.43±0.33μM、34.09±0.06μM和7.69。而EIDD-2801在RD细胞上的EC50值、CC50值、SI分别为70.12±4.40μM、>100μM和>1.43，在Vero细胞上的EC50值、CC50值、SI分别88.52±3.18μM、>100μM和>1.13，在Huh7细胞上的EC50值、CC50值、SI分别35.64±0.47μM、>100μM和>2.81。
实施例2化合物EIDD-1931和EIDD-2801抑制EV-A71病毒RNA复制与上清中感染性病毒颗粒产生实验
(1)实验材料
部分实验材料同上，额外需要的实验材料如下：Mini试剂盒购自Qiagen公司(货号74106)，One Step PrimeScriptTM RT-PCR试剂盒购自Takara公司(货号：RR47Q)，EV-A71H株的标品质粒及RT-PCR引物均由生工生物工程(上海)股份有限公司合成。
(2)实验方法：
①将RD细胞以每孔约4.0×105的密度接种于12孔板，在37℃、5％CO2条件下培养24小时。随后用细胞维持液将EV-A71病毒稀释成0.1MOI，加入12孔板中，同时用细胞维持液将待测化合物EIDD-1931和EIDD-2801稀释成相应浓度，加入到12孔板中，使EIDD-1931的最终浓度为20μM、10μM、5μM、2.5μM和1.25μM，EIDD-2801的最终浓度为200μM、100μM、50μM、25μM和12.5μM。37℃，5％CO2条件下孵育1.5小时，随后弃上清，每孔补加1mL待测化合物稀释液至相应孔中，继续培养30小时，待细胞开始出现病变时收集上清，8000rpm 5min离心、分装，于-80℃保存待用。
②RNA提取
30h后，按照Mini试剂盒说明书提取细胞内总RNA。
③qRT-PCR检测
1)用重组EV-A71线性化质粒制备标准品：根据线性化质粒浓度和分子量计算标准品拷贝数，并将其精确稀释为-1，-2，-3，-4，-5，-6，-7，-8，-9……直至拷贝数低于荧光定量PCR仪检测限。标准品制备完成后将各浓度标准品分装，于-80℃冻存待用。
2)用One Step PrimeScriptTM RT-PCR试剂盒检测样品中EV-A71病毒的RNA载量。实验过程中所用引物和探针序列如下：
EV71-RT-F：5'-CCAATCTCAGCGGCTTGGAG-3'
EV71-RT-R:5'-CACTCAAGCTCTACCGGCAC-3'
EV71-RT-Probe:FAM-TCCAATCGATGGCTGCTCACCTGCGT-BHQ1.
3)反应程序如下：
反转录：42℃5分钟；
预变性：95℃3分钟；
信号采集：95℃20秒，59℃1分钟，共45个循环。
抑制率(％)＝(药物处理组RNA拷贝数)/(细胞感染组RNA拷贝数)×100
④TCID50检测
将RD细胞以每孔约1.0×104的密度接种于96孔板，在37℃、5％CO2条件下培养24小时。随后弃细胞培养液，每孔补加150μL细胞维持液。取①中所得各组上清，用细胞维持液进行倍比稀释(从10-1开始，10倍稀释，共8个梯度)，然后将稀释后的上清接种于96孔板，50μL/孔，每个稀释度4个复孔，细胞对照组补加50μL细胞维持液。继续培养5天，然后用Reed-Muench法计算每个样品的TCID50值。
(3)统计学分析
利用方差分析(ANOVA)进行统计学显著性的计算。数据以平均值±标准差的形式来体现。p<0.05表示具有统计学差异。
(4)实验结果
如图3所示，化合物EIDD-1931和EIDD-2801均以剂量依赖性的方式抑制EV-A71病毒RNA的复制(B和D)，同时也抑制了感染性病毒颗粒的产生(A和C)。
实施例3化合物EIDD-1931和EIDD-2801抑制EV-A71病毒蛋白合成实验
(1)实验材料
部分实验材料同上，额外需要的实验材料如下：鼠抗EV-A71VP1抗体(一抗)由厦门大学程通教授赠送，Mouse anti-alpha tubulin抗体和HRP-conjugated goat anti-mouse IgG antibody均购自Abcam公司(货号分别为：ab7291、ab205719)，Alexa Fluor 647 donkey anti-mouse IgG(H+L)二抗和细胞核染料Hoechst 33342均购自Invitrogen公司(货号分别为：A31571、H21492)，RIPA裂解液、脱脂奶粉、BCA法蛋白定量试剂盒、Super Ecl超敏发光液均购买自普利莱(货号分别为：P1622、P1511、C1053和P1050)。
(2)免疫荧光(IF)实验
将Vero细胞以每孔约1.0×104的密度接种于96孔底透黑板，在37℃、5％CO2条件下培养24小时。随后用细胞维持液将EV-A71病毒稀释成相应浓度，接入96孔板中(MOI＝1)，同时加入预先用细胞维持液稀释好的待测化合物EIDD-1931和EIDD-2801，使其终浓度分别为10μM、3μM和100μM、30μM，继续培养16小时后弃液，每孔加入100μL 4％甲醛固定30分钟，弃固定液，加PBS缓冲液，进行抗体孵育标记后成像。
(3)Western blot实验
将RD细胞以每孔约1.0×105的密度接种于12孔板，在37℃、5％CO2条件下培养24小时。随后用细胞维持液将EV-A71病毒稀释成相应浓度，加入12孔板中使每孔含有病毒量为0.1MOI，同时用细胞维持液将EIDD-1931和EIDD-2801稀释成相应浓度，加入到12孔板中，使EIDD-1931和EIDD-2801的终浓度分别为10μM、5μM、2.5μM、1.25μM、0.63μM和100μM、50μM、25μM、12.5μM、6.3μM。继续培养24h，随后弃上清，收集细胞内蛋白，用Western blot进行分析。具体步骤如下：贴壁细胞用细胞裂解液(RIPA)充分裂解(100μL/孔)，获得全细胞蛋白，用BCA蛋白定量试剂盒测定蛋白浓度后，加入相应体积的5×SDS Loading Buffer，100℃煮沸10分钟，使其充分变性，然后用10％的SDS-PAGE在80V/120V下电泳2h使不同大小的蛋白分离。蛋白与PVDF膜在300mA电流下转膜90min，使其充分转膜，转膜完成后，根据目的蛋白分子量将膜裁剪成合适大小，用TBST稀释的5％脱脂奶粉室温摇床封闭1小时，加入封闭液稀释的一抗：抗EV-A71VP1蛋白抗体(1:500稀释)和内参Tublin抗体(1:5000稀释)，4℃摇床孵育过夜。然后加入HRP标记的山羊抗小鼠IgG抗体(1:5000稀释)，室温摇床孵育1小时。最后通过超敏化学发光法进行显色拍照。
(4)实验结果
实验结果如图4所示，EIDD-1931和EIDD-2801均能抑制EV-A71病毒VP1蛋白的产生，随着待测化合浓度的降低，抑制效果逐渐减弱，具有剂量依赖性。
实施例4化合物EIDD-1931抗EV-A71病毒作用机制验证实验
(1)试验材料
本部分实验材料同上。
(2)时序实验
实验中所用EIDD-1931的浓度为10μM，阳性化合物NITD008的浓度为2μM，EV-A71病毒的感染剂量为0.01MOI。具体步骤为：将RD细胞以每孔约4.0×105的密度接种于12孔板，在37℃、5％CO2条件下培养24小时。随后用细胞维持液将待测化合物EIDD-1931、阳性化合物NITD008和EV-A71病毒稀释成相应浓度，然后按照图5中A所示时间点加入预先稀释好的化合物或病毒。继续培养30小时后弃细胞上清，提取细胞内RNA，用qRT-PCR法对细胞内病毒RNA载量进行检测。细胞内RNA提取方法及qRT-PCR体系，反应条件均同前描述。
(2)统计学分析
利用方差分析(ANOVA)进行统计学显著性的计算。数据以平均值±标准差的形式来体现。p<0.05表示具有统计学差异。
(3)实验结果
实验结果如图5所示，化合物EIDD-1931主要在EV-A71病毒的复制阶段发挥抗病毒作用。在EV-A71病毒感染RD细胞的不同阶段加入EIDD-1931后检测细胞内EV-A71病毒RNA载量显示，EIDD-1931主要在病毒复制阶段发挥作用，对病毒的吸附、进入无明显抑制。
实施例5化合物EIDD-1931和EIDD-2801对1日龄ICR乳鼠致死性感染的保护实验
(1)实验材料
本部分所用实验材料同上。
(2)小鼠品系
实验过程中所用1日龄ICR乳鼠为SPF级，由北京维通利华实验动物技术有限公司购入，来源明确，经检验合格。
(3)实验步骤
将10窝1日龄ICR子母鼠随机分成10组，即病毒对照组(2组)、EIDD-1931给药组(4组)、EIDD-2801给药组(4组)，EIDD-1931和EIDD-2801的给药剂量均为200mg/kg、66.6mg/kg、22.2mg/kg和7.41mg/kg。每只小鼠经腹腔攻毒106PFU，4小时后进行第一次腹腔给药，病毒对照组给予药物溶剂，之后每天给药1次，连续给药7天，每天称量并记录小鼠的生存及体重变化情况。
(4)统计学分析
利用Log-Rank进行生存曲线统计学显著性的计算。p<0.05表示具有统计学差异。
(5)实验结果
实验结果如图6所示，化合物EIDD-1931和EIDD-2801对致死剂量EV-A71病毒接种的ICR乳鼠表现出剂量依赖的保护效果。EIDD-1931在200mg/kg和66.6mg/kg的剂量下能够完全保护1日龄的ICR乳鼠免受致死剂量的EV-71病毒攻击，在22.2mg/kg的剂量下对1日龄的ICR乳鼠有60％的存活保护率，这三个给药浓度下存活的小鼠都能够保持良好的体重增长，而对照组或低剂量组(7.41mg/kg)的小鼠在12天内死亡(图6中A和B)。EIDD-2801在200mg/kg和66.6mg/kg剂量下对1日龄的ICR乳鼠的保护率分别为100％和67％，而22.2mg/k和7.41mg/kg两个剂量组与对照组一样，在13天内全部死亡(图6中C和D)。
实施例6化合物EIDD-1931和EIDD-2801抑制EV-A71病毒感染乳鼠组织中病毒载量实验
(1)实验材料
部分实验材料同上，额外需要的实验材料如下：TRIzol试剂购自Invitrogen，货号为15596026。
(2)小鼠品系
实验过程中所用1日龄ICR乳鼠为SPF级，由北京维通利华实验动物技术有限公司购入，来源明确，经检验合格。
(3)ICR乳鼠组织中EV-A71病毒载量的检测
将3窝1日龄ICR子母鼠随机分成3组，即病毒对照组、EIDD-1931给药组(200mg/kg)、EIDD-2801给药组(200mg/kg)。每只小鼠经腹腔攻毒106PFU，4小时后进行第一次腹腔给药，病毒对照组给予药物溶剂，之后每天给药1次，连续给药4天，并于第四天将小鼠安乐死，收集小鼠的脑、心、小肠、肝、后肢肌肉及肺组织，使用TRIzol试剂提取组织RNA，之后用qRT-PCR对各组织中的病毒RNA进行定量分析。
(4)统计学分析
利用非配对t检验进行统计学显著性分析。p<0.05表示具有统计学差异。
(5)实验结果
如图7所示，化合物EIDD-1931和EIDD-2801治疗后显著降低了小鼠脑、心、小肠、肝、后肢肌肉及肺等组织脏器中的病毒载量。上述结果表明，EIDD-1931和EIDD-2801具有体内抗EV-A71病毒活性。
实施例7化合物EIDD-1931和EIDD-2801广谱抗肠道病毒活性评价实验
(1)实验材料
部分实验材料同上，额外如要的材料如下：EV-A71 AH08/06株、CV-B3 Nancy株均于本室保存，EV-D68 STL-2014–12株、CV-A6 TW-2007-00141株以及CV-A16 190-D1株均由厦门大学程通教授赠送.。
(2)EIDD-1931和EIDD-2801对各种肠道病毒所致细胞病变效应(CPE)的保护实验
在这部分实验中，EIDD-1931和EIDD-2801对EV-A71病毒AH08/06株，EV-D68病毒STL-2014-12株，CV-A6 TW-2007-00141株，CV-A16 190-D1株的CPE保护实验均用RD细胞测定。EIDD-1931和EIDD-2801对CV-B3 Nancy株的CPE保护实验用Vero细胞测定。具体实验方法同实施例1。
(3)实验结果
实验结果表明：EIDD-1931能够抑制多种肠道病毒的感染，其EC50值均小于20μM，而EIDD-2801对EV-V68、CV-A16和CV-B3的抑制活性较低，对CV-A6的EC50值大于100μM。
表1.EIDD-1931和EIDD-2801的广谱抗肠道病毒活性
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
Claims (8)
- 式I所示的化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物在制备用于治疗肠道病毒引起的疾病或感染(包括但不限于手足口病、病毒性咽峡炎、无菌性脑膜炎、脑干脑炎、心肌炎、神经源性肺水肿和中枢神经系统感染)的药物中的用途，
其中R为羟基、氢原子、酯基、烷氧基、苯基、杂环基或烷基。 - 药物组合物在制备用于治疗肠道病毒引起的疾病或感染(例如手足口病、病毒性咽峡炎、无菌性脑膜炎、脑干脑炎、心肌炎、神经源性肺水肿和中枢神经系统感染)的药物中的用途，其中所述药物组合物包含式Ⅰ所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物，
其中R为羟基、氢原子、酯基、烷氧基、苯基、杂环基或烷基，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。 - 药物组合物在制备作为肠道病毒抑制剂的药物中的用途，其中所述药物组合物包含式Ⅰ所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物，
其中R为羟基、氢原子、酯基、烷氧基、苯基、杂环基或烷基，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。 - 药物组合物在制备用于抑制肠道病毒在细胞(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所示化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物，
其中R为羟基、氢原子、酯基、烷氧基、苯基、杂环基或烷基，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。 - 式I所示的化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物在制备作为肠道病毒抑制剂的药物中的用途，
其中R为羟基、氢原子、酯基、烷氧基、苯基、杂环基或烷基。 - 式I所示的化合物、其几何异构体或其药物上可接受的盐、溶剂化物和/或水合物在制备用于抑制肠道病毒在细胞(例如哺乳动物细胞)中复制或繁殖的药物中的用途，
其中R为羟基、氢原子、酯基、烷氧基、苯基、杂环基或烷基。 - 权利要求4或6所述的用途，其中，所述药物是通过下列的至少一种途径实现的：A、抑制肠道病毒感染细胞的CPE水平；B、抑制肠道病毒RNA复制和感染性病毒颗粒的形成；C、抑制肠道病毒VP1蛋白的产生；D、在病毒吸附后阶段发挥抗病毒作用。
- 权利要求1至6任一项所述的用途，其中R为羟基、氢原子、R1-C(O)-O-、C1- 8烷氧基、苯基、杂环基或C1-8直链或支链烷基，其中R1为C1-8直链或支链烷基；优选地，R为羟基、氢原子、R1-C(O)-O-、C1-6烷氧基、苯基、杂环基或C1-6直链或支链烷基，其中R1为C1-6直链或支链烷基；优选地，R为羟基、氢原子、R1-C(O)-O-、C1-4烷氧基、苯基、杂环基或C1-4直链或支链烷基，其中R1为C1-4直链或支链烷基；优选地，R为羟基、氢原子、甲基、乙基、正丙基、异丙基、正丁基、甲氧基、乙氧基、正丙氧基、异丙氧、正丁氧基、苯基、CH3-C(O)-O-、CH3-CH2-C(O)-O-、(CH3)2-CH-C(O)-O-、CH3-(CH2)2-C(O)-O-或CH3-(CH2)3-C(O)-O-；优选地，R为羟基或(CH3)2-CH-C(O)-O-。
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| CA3173931A1 (en) * | 2020-04-03 | 2021-10-07 | Felix Frueh | Viral entry inhibitors and rna polymerase inhibitors |
| CN113893260A (zh) * | 2021-11-29 | 2022-01-07 | 山东省农业科学院畜牧兽医研究所 | 莫纳皮拉韦作为抗牛感染病毒活性成分的应用 |
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