WO2023226679A1 - 3c-like protease inhibitor - Google Patents

3c-like protease inhibitor Download PDF

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Publication number
WO2023226679A1
WO2023226679A1 PCT/CN2023/091287 CN2023091287W WO2023226679A1 WO 2023226679 A1 WO2023226679 A1 WO 2023226679A1 CN 2023091287 W CN2023091287 W CN 2023091287W WO 2023226679 A1 WO2023226679 A1 WO 2023226679A1
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Prior art keywords
compound
active ingredients
pharmaceutically acceptable
compounds
prodrugs
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PCT/CN2023/091287
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French (fr)
Chinese (zh)
Inventor
张宏波
杨琪
张伟
孙静
石磊
丁康
王虎庭
许庆博
黄博
赵金存
陈新文
彭伟
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广州国家实验室
北京望石智慧科技有限公司
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Publication of WO2023226679A1 publication Critical patent/WO2023226679A1/en

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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
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings

Definitions

  • the present invention relates to a new 3C-like protease inhibitor, or its pharmaceutically acceptable salts or esters, their stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs, hydrated forms compounds, solvates, isotopic labels, prodrugs or metabolites.
  • the present invention also relates to methods for preparing the compounds, pharmaceutical compositions containing the compounds, and the effects of the compounds in treating or preventing diseases caused by viral infections.
  • 2019-nCoV The new coronavirus discovered in December 2019 was initially named 2019-nCoV.
  • the World Health Organization (WHO) renamed it COVID-19.
  • WHO World Health Organization
  • 2019-nCoV The virus was named SARS-CoV-2.
  • SARS-CoV-2 can cause severe acute respiratory (SARI) symptoms, including fever, dyspnea, fatigue, and pneumonia.
  • RNA viruses the maximum genome length of coronaviruses ranges from approximately 26 to 32 kb.
  • Mpro main protease
  • 3Cpro picornavirus 3C protease
  • 3CLpro 3C-like protease
  • 3CLpro The function of 3CLpro is to hydrolyze and cleave the expressed peptide chain at the appropriate site, preparing the peptide chain to form a three-dimensional and four-dimensional structure to form the enzyme required for virus proliferation.
  • the enzyme does not change during the catalytic process, but the activation energy of the hydrolysis reaction is reduced, thereby accelerating the rate of the hydrolysis reaction.
  • the sulfhydryl group on cysteine plays a key role in the entire catalytic hydrolysis process, see Thanigaimalai et al.
  • WO2021/250648A1 discloses a compound currently known as Nirmatrelvir (PF-07321332). As one of the active ingredients of Paxlovid, it can be used in combination with ritonavir to reduce the risk of COVID-19. Risk of death and hospitalization from the virus SARS-CoV-2.
  • WO2021/205290A1 also discloses compounds with similar structures, which treat diseases caused by SARS-CoV-2 through a pathway mediated by 3C-like protease inhibitors.
  • Parovide also inhibits the CYP3A4 enzyme, which may interfere with the enzyme's metabolism of other drugs, change the half-life and clearance rate, reduce efficacy, or produce adverse reactions. situation. For example, when a patient takes parovide and terfenadine at the same time, because parovide inhibits the oxidative metabolism of terfenadine by CYP3A4, the concentration of the latter in the patient's body increases abnormally, causing cardiac QT wave prolongation and arrhythmias. .
  • the compounds disclosed in WO2021/205290A1 also face the problem of being ineffective when administered orally.
  • the present invention uses 3C-like protease as a target and develops a new class of small molecule inhibitors, which can be used to treat or prevent viral infections.
  • the compound of the present invention targets 3C-like protease, has excellent inhibitory activity against 3C-like protease with P132H mutation, and can significantly inhibit the proliferation of SARS-CoV-2. At the same time, it also achieves better in vivo stability and lower drug consumption. Side effects, better pharmacokinetic properties, and better biological activity against the main protease of the drug-resistant mutation L50F+E166A+L167F produced under the pressure screening of the peptidomimetic anti-SARS-COV-2 drug ALG-097161.
  • the invention provides compounds of formula (I), or pharmaceutically acceptable salts or esters thereof, their stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs, Hydrates, solvates, isotopic labels, prodrugs or metabolites:
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the invention or a pharmaceutically acceptable salt or ester thereof, their stereoisomers or tautomers, racemates, nitrogen oxides, etc. substances, polymorphs, hydrates, solvates, isotopic markers, prodrugs or metabolites.
  • the pharmaceutical composition according to the present invention may also optionally comprise at least one physiologically/pharmaceutically acceptable excipient.
  • the pharmaceutical composition according to the present invention may also optionally comprise pharmaceutically acceptable excipients, such as carriers, adjuvants or vehicles.
  • the pharmaceutical compositions according to the invention may also optionally comprise additional active ingredients or therapeutic agents.
  • the additional active ingredient or therapeutic agent is, for example, Remdesivir (Remdesivir or GS-5734), Lopinavir, Molnupiravir, Ritonavir, Chloroquine or Sigma-C6628), hydroxychloroquine and/or alpha-interferon.
  • compositions according to the invention comprise a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt or ester thereof, their stereoisomers or tautomers , racemates, nitrogen oxides, polymorphs, hydrates, solvates, isotope markers, prodrugs or metabolites.
  • the pharmaceutical composition according to the invention is an RNA-dependent RNA polymerase inhibitor, a 3CLpro protease inhibitor, a CYP3A4 inhibitor or a host-targeted antiviral drug.
  • the pharmaceutical composition according to the present invention can be formulated into a dosage form suitable for administration by methods known in the art.
  • the invention provides compounds according to the invention or pharmaceutically acceptable salts or esters thereof, their stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs, Use of hydrates, solvates, isotopic labels, prodrugs or metabolites in the preparation of pharmaceuticals.
  • the medicaments prepared according to the invention may also optionally contain additional active ingredients or therapeutic agents.
  • the additional active ingredient or therapeutic agent is, for example, Remdesivir (Remdesivir or GS-5734), Lopinavir, Molnupiravir, Ritonavir, Chloroquine , Sigma-C6628), hydroxychloroquine and/or alpha-interferon.
  • the drug prepared according to the invention is an RNA-dependent RNA polymerase inhibitor, a 3CLpro protease inhibitor, a CYP3A4 inhibitor or a host-targeted antiviral drug.
  • the medicaments prepared according to the present invention are used to prevent or treat diseases, conditions, syndromes and/or disorders selected from the following group, or for alleviating diseases, conditions selected from the following group , symptoms of syndromes and/or disorders: fever, nausea, vomiting, headache, dyspnea, fatigue, respiratory tract infection, pneumonia, smell and taste disorders and their complications, or combinations thereof caused by viral infection; preferably, the The virus is a coronavirus, preferably alphacoronavirus and/or betacoronavirus, more preferably SARS-CoV-2.
  • the medicine prepared according to the present invention can be further formulated into a dosage form suitable for administration by methods known in the art.
  • the present invention provides a method for treating or preventing diseases, conditions, syndromes and/or disorders caused by viral infection in a subject, comprising administering to the subject a compound of the present invention or a pharmaceutical thereof.
  • the compounds or pharmaceutical compositions of the invention inhibit viral proliferation
  • the compound or pharmaceutical composition of the invention inhibits the activity of viral 3CL protease
  • the 3CL protease has a P132H mutation
  • the virus is a coronavirus, preferably an alphacoronavirus and/or a betacoronavirus, more preferably SARS-CoV-2.
  • the diseases, conditions, syndromes and/or disorders caused by the viral infection are selected from: fever, nausea, vomiting, headache, dyspnea, fatigue, respiratory tract infection, pneumonia, olfactory disorder , dysgeusia and its complications, or combinations thereof;
  • the virus is a coronavirus, preferably alphacoronavirus and/or betacoronavirus, more preferably SARS-CoV-2.
  • coronavirus includes, but is not limited to, the following viruses: HCoV-229E, HCoV-NL63, HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV and/or SARSCoV-2.
  • coronavirus is an alphacoronavirus and/or a betacoronavirus, more preferably a betacoronavirus.
  • the alphacoronavirus is selected from HCoV-229E and HCoV-NL63, preferably HCoV-229E.
  • the betacoronavirus is selected from HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV and SARS-CoV-2, preferably HCoV-OC43 or SARS-CoV-2, more preferably SARS-CoV- 2.
  • treatment refers to reversing, alleviating, inhibiting the progression of, or preventing the disorder or condition to which the term applies, or one or more symptoms of such disorder or condition.
  • noun “treat” refers to the action of the verb treat, as just defined.
  • the term “treating” any disease or condition in some embodiments thereof, means ameliorating the disease or condition (i.e., slowing or arresting or alleviating the development of the disease or at least one clinical symptom thereof). In other embodiments, “treating” or “treating” refers to alleviating or improving at least one physical parameter, including physical parameters that may not be noticeable to the patient.
  • treating refers to modulating a disease or condition physically (eg, stabilizing perceived symptoms) or physiologically (eg, stabilizing body parameters), or both. In other embodiments, “treating” or “treating” refers to preventing or delaying the onset, development, or progression of a disease or disorder.
  • the term "pharmaceutically acceptable salts” means those carboxylate salts and amino acid addition salts of the compounds of the present invention which are suitable for contact with patient tissue within the scope of reliable medical judgment and will not produce undue toxicity, Irritation effects, allergic reactions, etc., commensurate with a reasonable benefit/risk ratio, are effective for their intended use, including (where possible) zwitterionic forms of the compounds of the invention.
  • Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali metal and alkaline earth metal hydroxides or organic amines.
  • metals used as cations are sodium, potassium, magnesium, calcium, etc.
  • suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and procaine.
  • Base addition salts of acidic compounds may be prepared in conventional manner by contacting the free acid form with a sufficient amount of the desired base to form the salt.
  • the free acid can be regenerated by contacting the salt form with the acid and isolating the free acid in the usual manner.
  • the free acid forms differ somewhat from their respective salt forms in certain physical properties, such as solubility in polar solvents, but for the purposes of this invention the salts are nevertheless equivalent to their respective free acids.
  • the salts may be sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates prepared from inorganic acids Salt, chloride, bromide, iodide, acids such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, etc.
  • Representative salts include: hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate Acid, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, Methanesulfonate, glucoheptonate, lactobionate, lauryl sulfonate and isethionate, etc.
  • Salts may also be prepared from organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like.
  • organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like.
  • Representative salts include acetate, propionate, octanoate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, malonate Lenate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, naphthoate, benzenesulfonate, toluenesulfonate, phenylbenzoate Acid, citrate, lactate, maleate, tartrate, methanesulfonate, etc.
  • Pharmaceutically acceptable salts may include alkali and alkaline earth metal based cations such as sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium and amine cations including, but not limited to, ammonium, tetramethyl Ammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. Also contemplated are salts of amino acids, such as arginates, gluconates, galacturonates, etc. (see, for example, Berge SM et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977; 66:1-19 , incorporated by reference).
  • nitrogen oxide means that one or more nitrogen atoms can be oxidized to form N-oxides when the compound contains several nitrogen-containing functional groups.
  • N-oxides are N-oxides of tertiary amines or N-oxides of nitrogen atoms in nitrogen-containing heterocyclic rings.
  • the corresponding nitrogen-containing compounds can be treated with oxidizing agents such as hydrogen peroxide or peracids (eg peroxycarboxylic acid) to form N-oxides.
  • oxidizing agents such as hydrogen peroxide or peracids (eg peroxycarboxylic acid) to form N-oxides.
  • N-oxides can be prepared by the method of L.W. Deady (Syn. Comm. 1977, 7, 509-514), in which the nitrogen-containing compound is reacted with m-chloroperoxybenzoic acid (MCPBA), for example in an inert solvent such as methylene chloride. )reaction.
  • MCPBA m-ch
  • esters refers to an in vivo hydrolyzable ester of a compound containing a hydroxyl or carboxyl group. Such esters are, for example, physiologically/pharmaceutically acceptable esters which hydrolyze in humans or animals to yield the parent alcohol or acid.
  • the compound of formula (I) or (II) of the present invention contains a carboxyl group, which can form a hydrolyzable ester in vivo with an appropriate group.
  • groups include, but are not limited to, alkyl, arylalkyl, etc.
  • Subjects for administration include, but are not limited to: humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young Adults, middle-aged adults or older adults) and/or non-human animals, e.g., mammals, e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and/or dogs.
  • the subject is a human.
  • the subject is a non-human animal.
  • the terms "human,” “patient,” and “human” are used interchangeably herein. "Subject”.
  • treatment includes an action in a subject suffering from a specific disease, disorder or condition that reduces the severity of the disease, disorder or condition, or delays or slows down the disease, disorder or the development of a condition ("therapeutic treatment”), and also includes effects that occur before a subject begins to suffer from a specific disease, disorder or condition ("preventive treatment").
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material in association with a suitable pharmaceutical excipient.
  • the unit dosage form may be a pill, a tablet, a capsule or a lozenge, etc.
  • an "effective amount" of a compound is an amount sufficient to elicit a target biological response.
  • the effective amount of a compound of the present invention may vary depending on factors such as, for example, the biological target, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the condition of the subject. Age health conditions and symptoms.
  • the effective amount includes a therapeutically effective amount and a preventive effective amount.
  • a "therapeutically effective amount" of a compound as used herein is an amount sufficient to provide a therapeutic benefit in treating a disease, disorder, or condition, or to cause one or more symptoms associated with the disease, disorder, or condition The amount to delay or minimize.
  • a therapeutically effective amount of a compound is that amount of therapeutic agent that, when used alone or in combination with other therapies, provides a therapeutic benefit in the treatment of a disease, disorder, or condition.
  • the term "therapeutically effective amount” may include an amount that improves overall treatment, reduces or avoids symptoms or causes of a disease or disorder, or enhances the therapeutic effect of other therapeutic agents.
  • a prophylactically effective amount of a compound as used herein is an amount sufficient to prevent a disease, disorder or condition, or to prevent one or more symptoms associated with a disease, disorder or condition, or to prevent a disease , the amount of recurrence of a disorder or condition.
  • a prophylactically effective amount of a compound is that amount of therapeutic agent that, when used alone or in combination with other agents, provides a prophylactic benefit in preventing a disease, disorder, or condition.
  • the term “prophylactically effective amount” may include an amount that improves overall prophylaxis, or an amount that enhances the prophylactic effect of other prophylactic agents.
  • Combination and related terms refer to the simultaneous or sequential administration of a compound of the invention and another therapeutic agent.
  • the compounds of the present invention may be administered simultaneously or sequentially with other therapeutic agents in separate unit dosage forms, or with other therapeutic agents in a single unit dosage form.
  • compounds of the present invention refer to the following compounds of formula (I), or pharmaceutically acceptable salts or esters thereof, their stereoisomers or tautomers, racemates, and nitrogen oxides. , polymorphs, hydrates, solvates, isotopic markers, prodrugs or metabolites.
  • the present invention relates to compounds of formula (I), or pharmaceutically acceptable salts or esters thereof, their stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs , hydrates, solvates, isotopic labels, prodrugs or metabolites:
  • the compounds of the present invention may contain one or more asymmetric centers and thus may exist in multiple stereoisomeric forms, for example, enantiomeric and/or diastereomeric forms.
  • the compounds of the present invention may be individual enantiomers, diastereomers, or geometric isomers (e.g., cis and trans isomers), or may be in the form of mixtures of stereoisomers, Includes racemic mixtures and mixtures enriched in one or more stereoisomers.
  • the isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or the preferred isomers may be separated by Prepared by asymmetric synthesis.
  • HPLC high pressure liquid chromatography
  • Tautomers are functional group isomers produced by the rapid movement of an atom in a molecule between two positions.
  • a tautomer is a special functional group isomer.
  • a pair of tautomers can interact with each other. conversion, but usually one of the more stable isomers is its main form of existence. The most important examples are the enol and keto tautomers.
  • compounds of the present invention include the following tautomers:
  • solvate refers to a form of a compound or a salt thereof that is combined with a solvent, usually formed by a solvolysis reaction. This physical association may include hydrogen bonding.
  • solvents include water, methanol, ethanol, acetic acid, DMSO, THF, ether, etc.
  • Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric and non-stoichiometric solvates. In some cases, the solvate will be capable of isolating, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid.
  • “Solvate” includes both solution solvates and isolable solvates. Representative solvates include hydrates, ethanolates, and methoxides.
  • hydrate refers to a compound combined with water. Generally, the number of water molecules contained in a hydrate of a compound is related to the hydrate The ratio of the number of molecules in the compound is determined. Thus, a hydrate of a compound may be represented, for example, by the general formula R.xH2O , where R is the compound and x is a number greater than zero.
  • a given compound may form more than one hydrate type, including, for example, monohydrate (x is 1), lower hydrate (x is a number greater than 0 and less than 1), for example, hemihydrate (R ⁇ 0.5H 2 O) and polyhydrates (x is a number greater than 1, for example, dihydrate (R ⁇ 2H 2 O) and hexahydrate (R ⁇ 6H 2 O).
  • the compounds of the invention may be in amorphous or crystalline forms (polymorphs). Furthermore, the compounds of the present invention may exist in one or more crystalline forms. Accordingly, the present invention includes within its scope all amorphous or crystalline forms of the compounds of the invention.
  • polymorph refers to a crystalline form of a compound (or a salt, hydrate or solvate thereof) in a specific crystal packing arrangement. All polymorphs have the same elemental composition. Different crystalline forms often have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optoelectronic properties, stability and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors can lead to the dominance of one crystalline form. Various polymorphs of the compounds can be prepared by crystallization under different conditions.
  • the present invention also includes isotopically labeled compounds (isotopic variants) which are identical to those described in formula (I), except that one or more atoms are surrounded by atoms having an atomic mass or mass number different from that common in nature. replaced.
  • isotopes that may be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 2 H, 3 H, 13 C, 11 C, 14 C, 15 N, 18 respectively. O, 17 O, 31 P, 32 P, 35 S, 18 F and 36 Cl.
  • the isotope-labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by replacing non-isotopes with readily available isotope-labeled reagents when performing the following processes and/or the processes disclosed in the Examples and Preparation Examples. Labeled reagents.
  • prodrugs are also included within the context of the present invention.
  • the term "prodrug” as used herein refers to a compound that is converted in the body to its active form having a medical effect, for example, by hydrolysis in the blood.
  • Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of A.C.S. Symposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, and D. Fleisher, S. Ramon and H. Barbra "Improved oral drug delivery: solubility limitations overcome by the use of prodrugs", Advanced Drug Delivery Reviews (1996) 19 (2) 115-130, each introduced This article serves as a reference.
  • a prodrug is any covalently bonded compound of the invention that releases the parent compound in the body when administered to a patient.
  • Prodrugs are typically prepared by modifying functional groups in a manner such that the modification can be cleaved by conventional manipulations or in vivo to yield the parent compound.
  • Prodrugs include, for example, compounds of the invention in which a hydroxyl, amino or thiol group is bonded to any group that can be cleaved to form a hydroxyl, amino or thiol group when administered to a patient.
  • representative examples of prodrugs include, but are not limited to, acetate/amide, formate/amide and benzoate/amide derivatives of the hydroxyl, thiol and amino functionality of compounds of formula (I).
  • esters such as methyl ester, ethyl ester, etc. can be used.
  • the ester itself may be reactive and/or hydrolyzable under human body conditions.
  • Suitable pharmaceutically acceptable in vivo hydrolyzable ester groups include those that readily break down in the human body to release the parent acid or salt thereof.
  • metabolite refers to a product obtained by metabolism of a specific compound or its salt in the body.
  • the metabolites of a compound can be identified by techniques well known in the art, and its activity can be characterized by assays as described herein. Such products can be obtained by administering compounds through oxidation, reduction, hydrolysis, amidation, deamidation, esterification, delipidation, enzymatic cleavage, etc.
  • the invention includes metabolites of compounds, including metabolites produced by contacting a compound of the invention with a mammal for a period of time sufficient to do so.
  • the present invention also provides pharmaceutical preparations, comprising a therapeutically effective amount of a compound of formula (I) or a therapeutically acceptable salt thereof and a pharmaceutically acceptable salt thereof.
  • acceptable carrier, diluent or excipient All these forms belong to the invention.
  • the invention provides pharmaceutical compositions comprising a compound of the invention (also referred to as an "active ingredient") and a pharmaceutically acceptable excipient.
  • the pharmaceutical compositions comprise an effective amount of a compound of the invention.
  • the pharmaceutical compositions comprise a therapeutically effective amount of a compound of the invention.
  • the pharmaceutical compositions comprise a prophylactically effective amount of a compound of the invention.
  • pharmaceutical composition means a mixture containing one or more compounds described herein, or physiologically/pharmaceutically acceptable salts or prodrugs thereof, and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable salts or prodrugs thereof. Accepted carriers and excipients.
  • the purpose of pharmaceutical compositions is to facilitate administration to living organisms and facilitate the absorption of active ingredients to exert biological activity.
  • physiologically/pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable when administered to humans and generally do not produce allergic or similar inappropriate reactions, such as gastrointestinal upset, dizziness, and the like.
  • carrier refers to a diluent, adjuvant, excipient or matrix with which the compound is administered.
  • These pharmaceutical carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.
  • Water and aqueous solutions, saline solutions and aqueous glucose and glycerol solutions are preferably used as carriers, especially for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
  • compositions of the present invention refer to non-toxic carriers, adjuvants or vehicles that do not destroy the pharmacological activity of the compounds with which they are formulated.
  • Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of the present invention include, but are not limited to, ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins (such as human serum albumin) protein), buffer substances (such as phosphate), glycine, sorbic acid, potassium sorbate, partial glyceride mixture of saturated vegetable fatty acids, water, salt or electrolyte (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate , sodium chloride, zinc salt, silica gel, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene-polyoxypropylene- Block polymers, polyethylene glycols, and
  • Substances that can be used as physiologically/pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, aluminum, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphate, glycine, sorbate Acids, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon, magnesium trisilicate , polyvinylpyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-blocked polymers, lanolin, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives Such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; gum powder; malt; gelatin; talc;
  • compositions of the present invention may be prepared according to the disclosure using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or freeze-drying processes.
  • the dosage form of the medicine of the present invention can be selected according to specific circumstances.
  • Pharmaceutical dosage forms often consist of drug substance, excipients, and a container/closure system.
  • excipients also known as inactive ingredients
  • the type of excipients added to a drug can depend on various factors, such as the physical and chemical properties of the drug, route of administration, and preparation steps.
  • Pharmaceutical excipients exist in the art and include those listed in various pharmacopeias.
  • compositions of the present invention may include one or more physiologically acceptable inactive ingredients that facilitate processing of the active molecules into preparations for pharmaceutical use.
  • Appropriate formulation will depend on the desired route of administration. Routes of administration include intravenous injection, transmucosal or nasal administration, oral administration, etc.
  • the compounds may be formulated in liquid or solid dosage forms and as immediate release or controlled/sustained release preparations. Suitable dosage forms for oral ingestion by individuals include tablets, pills, dragees, hard and soft shell capsules, liquids, gels, syrups, ointments, suspensions and emulsions.
  • Solid oral dosage forms may be obtained using excipients including fillers, disintegrants, binders (dry and wet), dissolution retarder, lubricants, glidants, anti-adhesive agents, cationic exchangers Resins, humectants, antioxidants, preservatives, colorants and flavoring agents. These excipients may be of synthetic or natural origin.
  • excipients examples include cellulose derivatives, citric acid, dicalcium phosphate, gelatin, magnesium carbonate, magnesium lauryl sulfate/sodium lauryl sulfate, mannitol, polyethylene glycol, polyvinylpyrrolidone, silicic acid Salt, silicon dioxide, sodium benzoate, sorbitol, starch, stearic acid or its salts, sugar (i.e. dextrose, sucrose, lactose, etc.), talc, tragacanth mucilage, vegetable oil (hydrogenated) and wax. Ethanol and water can be used as granulation aids.
  • tablets In some cases, it is necessary to coat tablets with, for example, a taste-masking film, an acid-resistant film, or a delayed-release film.
  • Natural and synthetic polymers are often used to coat tablets in combination with colorants, sugars and organic solvents or water to produce dragees.
  • their drug powders, suspensions or solutions may be delivered in compatible hard or soft shell capsules.
  • the therapeutically effective dose can first be estimated using various methods well known in the art. Initial dosages for animal studies may be based on established effective concentrations in cell culture assays. Dosage ranges suitable for humans can be determined, for example, using data obtained from animal studies and cell culture assays. In certain embodiments, the compounds of the present invention can be prepared as a medicament for oral administration.
  • the correct formulation, route of administration, dosage and interval between administrations can be selected according to methods known in the art, taking into account the particularities of the individual condition.
  • Suitable formulations for administering the compounds of the invention will be apparent to those of ordinary skill in the art and include, for example, tablets, pills, capsules, suppositories, lozenges, lozenges, solutions (especially injections (subcutaneous, intravenous solution for intramuscular administration) and infusion (injection)), elixir, syrup, cachet, emulsion, inhalation or dispersible powder.
  • the content of one or more pharmaceutically active compounds should range from 0.1 to 90% by weight, preferably from 0.5 to 50% by weight of the composition as a whole, ie an amount sufficient to achieve the dosage ranges specified below. If necessary, the specified dose may be administered several times per day.
  • kits eg, pharmaceutical packaging.
  • Kits provided may include a compound of the invention, other therapeutic agents, and first and second containers (e.g., vials, ampoules, bottles, syringes, and/or dispersible packaging or other) containing the compounds of the invention, other therapeutic agents. suitable container).
  • provided kits may also optionally include a third container containing pharmaceutical excipients for diluting or suspending the compounds of the invention and/or other therapeutic agents.
  • the compound of the invention and the other therapeutic agent provided in the first container and the second container are combined to form a unit dosage form.
  • parenteral administration as used herein includes subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, intraarticular administration, intraarterial administration, intrasynovial administration, intrasternal administration , intracerebrospinal membrane drug administration, intralesional drug administration, and intracranial injection or infusion techniques.
  • an effective amount of a compound provided herein is administered.
  • the amount of compound actually administered can be determined by the physician depending on the circumstances, including the condition being treated, the route of administration chosen, the compound actually administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, etc. .
  • a compound provided herein When used to prevent a disorder described herein, a compound provided herein is administered to a subject at risk of developing the disorder, Typically administered based on a physician's advice and supervision, dosage levels are as described above.
  • Subjects at risk of developing a particular condition generally include subjects with a family history of the condition or those who have been determined by genetic testing or screening to be particularly susceptible to developing the condition.
  • compositions provided herein can also be administered over a long period of time ("chronic administration").
  • Long-term administration refers to the administration of a compound or pharmaceutical composition thereof over a long period of time, for example, 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or administration may be continued indefinitely, For example, the remainder of the subject's life.
  • chronic administration is intended to provide a constant level of the compound in the blood over an extended period of time, eg, within a therapeutic window.
  • a pharmaceutical composition may be administered as a bolus injection, eg, in order to increase the concentration of the compound in the blood to an effective level.
  • the bolus dose depends on the target systemic levels of the active ingredient through the body, e.g., an intramuscular or subcutaneous bolus dose provides a slow release of the active ingredient, whereas a bolus dose delivered directly into the vein (e.g., via an IV drip) ) can be delivered more quickly, allowing the concentration of active ingredients in the blood to quickly increase to effective levels.
  • the pharmaceutical composition may be administered as a continuous infusion, for example, by IV infusion, thereby providing a steady-state concentration of the active ingredient in the subject's body. Additionally, in other embodiments, a bolus dose of the pharmaceutical composition may be administered first, followed by a continuous infusion.
  • Oral compositions may take the form of bulk liquid solutions or suspensions, or bulk powders. More typically, however, the compositions are provided in unit dosage form to facilitate precise dosing. Typical unit dosage forms include prefilled, premeasured ampoules or syringes for liquid compositions, or pills, tablets, capsules, and the like in the case of solid compositions. In such compositions, the compound will generally be a minor component (from about 0.1 to about 50% by weight, or preferably from about 1 to about 40% by weight), with the remainder being various components useful in forming the desired administration form. carriers or excipients and processing aids.
  • a typical regimen is one to five oral doses per day, especially two to four oral doses, typically three oral doses.
  • each dose provides from about 0.01 to about 20 mg/kg of a compound of the invention, with preferred doses each providing from about 0.1 to about 10 mg/kg, especially from about 1 to about 5 mg/kg.
  • a transdermal dose is generally selected in an amount of about 0.01 to about 20% by weight, preferably about 0.1 to about 20% by weight, preferably about 0.1 to about 10% by weight, and more preferably from about 0.5 to about 15% by weight.
  • Injectable dose levels range from about 0.1 mg/kg/hour to at least 10 mg/kg/hour from about 1 to about 120 hours, especially from 24 to 96 hours. To achieve adequate steady state levels, a preload bolus of about 0.1 mg/kg to about 10 mg/kg or more may also be given. For human patients weighing 40 to 80 kg, the maximum total dose should not exceed approximately 2 g/day.
  • Liquid forms suitable for oral administration may include suitable aqueous or non-aqueous carriers as well as buffering agents, suspending and dispersing agents, coloring agents, flavoring agents, and the like.
  • Solid forms may include, for example, any of the following components, or compounds of similar nature: binders, for example, microcrystalline cellulose, tragacanth, or gelatin; excipients, for example, starch or lactose, disintegrants, For example, alginic acid, Primogel or corn starch; lubricant, for example, magnesium stearate; glidant, for example, colloidal silicon dioxide; sweetener, for example, sucrose or saccharin; or flavoring agent, for example, mint, water Methyl glycolate or orange flavoring.
  • binders for example, microcrystalline cellulose, tragacanth, or gelatin
  • excipients for example, starch or lactose, disintegrants, For example, alginic acid, Primogel or corn starch
  • Injectable compositions are typically based on injectable sterile saline or phosphate buffered saline, or other injectable excipients known in the art. As stated previously, in such compositions the active compound is typically a minor component, often about 0.05 to 10% by weight, with the remainder being injectable excipients and the like.
  • Transdermal compositions are typically formulated as topical ointments or creams containing the active ingredients.
  • the active ingredients When formulated as an ointment, the active ingredients are typically combined with a paraffin or water-miscible ointment base.
  • the active ingredient may be formulated as a cream with, for example, an oil-in-water cream base.
  • Such transdermal formulations are well known in the art and often include other ingredients for promoting stable skin penetration of the active ingredient or formulation. All such known transdermal formulations and components are included within the scope provided by this invention.
  • transdermal administration may be achieved using reservoir or porous membrane types, or a variety of solid matrix patches.
  • compositions for oral administration, injection or topical administration are merely representative.
  • Other materials and processing techniques are described in Part 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, which article is incorporated by reference.
  • the compounds of the present invention may also be administered in sustained release form or from a sustained release drug delivery system.
  • sustained release materials can be found in Remington's Pharmaceutical Sciences.
  • the invention also relates to pharmaceutically acceptable formulations of the compounds of the invention.
  • the formulation includes water.
  • the formulation contains a cyclodextrin derivative.
  • the most common cyclodextrins are ⁇ -, ⁇ - and ⁇ -cyclodextrins consisting of 6, 7 and 8 ⁇ -1,4-linked glucose units respectively, optionally including a or multiple substituents including, but not limited to: methylated, hydroxyalkylated, acylated, and sulfoalkyl ether substitutions.
  • the cyclodextrin is a sulfoalkyl ether beta-cyclodextrin, for example, sulfobutyl ether beta-cyclodextrin, also known as Captisol. See, for example, U.S. 5,376,645.
  • the formulation includes hexapropyl-beta-cyclodextrin (eg, in water, 10-50%).
  • 3C-like protease inhibitors For diseases caused by viral infections, the development of 3C-like protease inhibitors can provide therapeutic benefits for a large number of patients.
  • the compounds in the present invention exert therapeutic effects by negatively regulating the activity of 3C-like protease in viruses, especially viruses with P132H mutation in the 3C-like protease.
  • the 3C-like protease inhibitors of the present invention can treat various diseases caused by viral infections and their complications.
  • these compounds can be used to treat the following diseases caused by viral infections: fever, nausea, vomiting, headache, dyspnea, fatigue, respiratory tract infection, pneumonia, smell disorder, taste disorder and its complications, etc.
  • these compounds can be used for the above-mentioned diseases or symptoms caused by SARS-CoV-2 infection.
  • the 3C-like protease inhibitor of the present invention can be combined with other drugs to treat cancer, and contains at least one target drug/viral activity modulator, including Remdesivir (Remdesivir or GS-5734), Lopinavir (Lopinavir) ), Molnupiravir, Ritonavir, chloroquine (Chloroquine or Sigma-C6628), hydroxychloroquine and/or alpha-interferon, etc.
  • Remdesivir Remdesivir or GS-5734
  • Lopinavir Lopinavir
  • Molnupiravir Ritonavir
  • chloroquine Chloroquine or Sigma-C6628
  • hydroxychloroquine and/or alpha-interferon etc.
  • control drugs propranolol and Digoxin were purchased from MCE, and Minoxidil was purchased from China Institute of Food and Drug Control.
  • Caco-2 cells were purchased from American Type Culture Collection (ATCC).
  • FBS medium was purchased from Sigma, DMEM was purchased from Corning Company (Cambridge, MA), non-essential amino acids (NEAA), Hank’s balanced salt solution (HBSS) and trypsin/EDTA were purchased from Thermo Fisher. Penicillin and streptomycin were purchased from Soleba.
  • HTS-96-well Transwell plate and other sterile consumables were purchased from Corning Company.
  • Millicell resistance measurement system was purchased from Millipore. Vision was purchased from Nexcelom Bioscience. Infinite 200 PRO microplate reader was purchased from Tecan. The MTS2/4 orbital shaker was purchased from IKA Labortechnik.
  • Caco-2 was cultured in cell culture flasks.
  • the incubator was set to 37°C, 5% CO 2 , and guaranteed relative humidity of 95%. Cells that reach 70-90% confluence can be used to seed Transwells.
  • the process of replacing the culture medium is as follows. Separate the Transwell chamber from the receiving plate. Discard the medium in the receiving plate first and then discard the medium in the Transwell chamber. Finally, add 75 ⁇ L of fresh culture medium to each chamber and 25 mL of fresh culture medium to the receiving plate. .
  • Caco-2 After 14-18 days of culture, Caco-2 should be fully confluent and complete differentiation. At this time, it can be applied to penetration testing.
  • V A is the volume of the receiving end solution (Ap ⁇ Bl is 0.235mL, Bl ⁇ Ap is 0.075mL), Area is the Transwell-96-well plate membrane area (0.143cm 2 ); time is the incubation time (unit: s ).
  • P app(BA) is the apparent permeability coefficient from the basal end to the top
  • P app(AB) is the apparent permeability coefficient from the top to the basal end.
  • DPX 2 a HepG2 cell line stably transfected with PXR and luciferase systems
  • CellTiter-Fluor TM cell viability assay kit and One-Glo luciferase assay system were purchased from Promega Company (Madison, WI).
  • FBS medium was purchased from Avantor
  • DPX 2 cell culture medium and administration medium were purchased from Puracyp.
  • DPX 2 was cultured in T-75 cell culture flask.
  • the incubator was set to 37°C, 5% CO 2 , and guaranteed relative humidity of 95%. Cells can be used for isolation when they reach 80-90% confluence.
  • Solution configuration Prepare 1. DMSO stock solution of 10mM compound to be tested and 10mM positive control drug rifampicin. The final concentrations of the compounds to be tested were 1 and 10 ⁇ M, respectively, and the final concentration of rifampicin was 10 ⁇ M. The DMSO content of the final system was 0.1%. The blank control is DMSO.
  • the cell plate After 48 hours of drug administration and incubation, the cell plate can be used to test PXR activity.
  • Luciferase activity is determined by RLU/RFU, where RLU represents the average relative luminescence value of three parallel samples of the test compound at each concentration, and RFU represents the average relative fluorescence value of the three parallel samples of the test compound at each concentration.
  • Animal feeding control fast overnight before administration, drink water freely, and provide feed 4 hours after administration.
  • the actual dosing volume was calculated based on the animal's body weight.
  • the LC-MS/MS detection method was applied, and the accompanying standard curve was used to determine the blood drug concentration in the plasma sample.
  • WinNonlin 8.3 (Phoenix TM ) or other similar software will be used to calculate pharmacokinetic parameters. Pharmacokinetic parameters will be calculated if applicable plasma drug concentration-time data are available.
  • Pharmacokinetic data were described by descriptive statistics such as mean, standard deviation, and sample size. Calculations were performed using Microsoft Excel 2013. Other pharmacokinetic parameters and statistical analyzes can also be performed and recorded in the data summary.
  • the assay system (120 ⁇ L) contains 108 ⁇ L main protease (WT or P132H) with a final concentration of 150nM.
  • the protein-free small molecule control group is added with 108 ⁇ L protein diluent.
  • the diluent components are 50mM Tris-HCL PH 7.4 and 1mM EDTA;
  • the negative control NC here is to add 2 ⁇ L of 100% DMSO.
  • the fluorescent substrate is MCA-AVLQSGFR-LYS(DNP)-Lys-NH 2 .
  • the reaction process is as follows: first mix 108 ⁇ L of main protease or protein diluent and 2 ⁇ L of small molecules, add them to a 96-well all-black enzyme plate (Corning costar, #3916), and incubate for 30 minutes. After the incubation is completed, add the fluorescent substrate and quickly start detection on the microplate reader.
  • the microplate reader detection method is:
  • the excitation light wavelength is 320nm
  • the emission light wavelength is 405nm
  • the detection interval is about 15s
  • the total detection time is 20min; the fluorescence value of each reaction well at different times under the detection conditions is recorded, and the fluorescence value recorded in the previous 200s is used for calculation ( Typically 7-10 data points).
  • Inhibition rate (%) (RFU 100% enzyme activity control -RFU sample )/(RFU 100% enzyme activity control -RFU blank control ) ⁇ 100%
  • Inhibition rate (%) (NC initial velocity V 0 - sample initial velocity V 0 )/NC initial velocity V 0 ⁇ 100%;
  • NC enzyme activity is determined as 100%, and the inhibition rate is 0%
  • Inhibition rate (%) (NC initial velocity V 0 - (sample initial velocity V 0 - protein-free small molecule control group V 0 )/NC initial velocity V 0 ⁇ 100% is used to eliminate V 0 (slope ) is a negative value.
  • test sample sample and control substance
  • test process generally uses Formula 2 to optimize the calculation of the inhibition rate.
  • PCR 94°C5min, 94°C30s, 66°C20s, 72°C1min, 30cycles, 72°C10min.
  • Nickel column affinity chromatography column treatment Clean the nickel column with ddH2O first, then use equilibrium buffer to equilibrate the column, combine the filtered bacterial supernatant with the nickel column affinity chromatography gel, and wash with 20mM imidazole.
  • the impurity buffer was used to wash the impurities, and the elution buffer containing 500mM imidazole concentration was used for elution.
  • Reaction buffer 50mM Tris pH 7.4, 1mM EDTA, 0.01% tritonX-100.
  • Protein (108uL) main protease (L50F+E166A+L167F), the final concentration is 500nM, add 108uL reaction buffer to the protein-free small molecule control group;
  • Small molecules (2uL): Compound 1, Comparative Compound 1, Comparative Compound 2 (saved at 50mM), use 100% DMSO to dilute the 50mM concentration of small molecules to 0.06mM, so that the final concentration in the reaction system is 1uM.
  • Gradient dilution of small molecules The above small molecules are diluted 2-fold starting from a concentration of 0.06mM (final concentration 1uM) to the 8th well.
  • the negative control NC here is to add 2uL 100% DMSO.
  • the excitation light wavelength is 320nM
  • the emission light wavelength is 405nM
  • the detection interval is about 15s
  • the total detection time is 20min; the fluorescence values in each reaction well at different times under the detection conditions are recorded, and the fluorescence recorded in the previous 200s is used for calculation. value (typically 7-10 data points)
  • P app (BA) is the apparent permeability coefficient from the basal end to the apical end
  • P app (AB) is the apparent permeability coefficient from the apical end to the basal end.
  • AUC last area under the drug-time curve, to evaluate the degree of drug absorption
  • the above data show that while maintaining excellent inhibitory activity, the compound of the present invention also achieves better stability, lower drug side effects, better pharmacokinetic properties, and is effective against peptoids against SARS-COV- 2.
  • the drug resistance mutation L50F+E166A+L167F main protease produced under the pressure screening of drug ALG-097161 has better biological activity than the comparative compound.
  • the Caco2 permeability experiment characterizes the penetration effect of the drug, and P app (BA) represents the penetration effect from the blood to the small intestine.
  • P app (AB) represents the penetration effect from the small intestine into the blood.
  • the penetration effect of the compound of the present invention from the small intestine into the blood is better than that of the other two comparative compounds, and it is less likely to penetrate from the blood into the small intestine, that is, drug penetration Excellent results.
  • PXR represents a drug interaction. Specifically, PXR activation induces metabolic enzymes (such as CYP3A4). Metabolic enzymes can affect the pharmacokinetics of exogenous and endogenous substances. Overactivation of CYP3A4 enzymes can induce CYP3A4-mediated Anti-tumor drugs, analgesics, etc. accelerate metabolism and affect the efficacy of combined drugs.
  • the PXR value of Compound 1 of the present invention at different concentrations is significantly lower than that of Comparative Compounds 1 and 2, and it has a lower risk of drug interaction and toxic metabolism.
  • the biochemical activity of the main protease of the anti-drug resistance mutation L50F+E166A+L167F of compound 1 is more advantageous than that of comparative compounds 1 and 2.

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Abstract

The present invention provides a 3C-like protease inhibitor represented by formula (I), or a pharmaceutically acceptable salt or ester thereof, and a stereoisomer or tautomer, a racemate, a nitrogen oxide, a polymorph, a hydrate, a solvate, an isotope marker, a prodrug or a metabolite of the pharmaceutically acceptable salt or ester. The present invention additionally provides a preparation method of the chemical compound, a pharmaceutical composition containing the compound, and the effect of the compound in treating or preventing diseases caused by viral infection.

Description

3C样蛋白酶抑制剂3C-like protease inhibitor
本申请要求享受如下优先权：This application claims the following priority rights:
CN202210600337.0，申请日：2022年5月27日。CN202210600337.0, application date: May 27, 2022.
技术领域Technical field
本发明涉及一种新的3C样蛋白酶抑制剂，或其药学上可接受的盐或酯，它们的立体异构体或互变异构体、消旋体、氮氧化物、多晶型、水合物、溶剂合物、同位素标记物、前药或代谢产物。本发明还涉及所述化合物的制备方法、包含所述化合物的药物组合物，以及所述化合物在治疗或预防病毒感染导致的疾病中的作用。The present invention relates to a new 3C-like protease inhibitor, or its pharmaceutically acceptable salts or esters, their stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs, hydrated forms compounds, solvates, isotopic labels, prodrugs or metabolites. The present invention also relates to methods for preparing the compounds, pharmaceutical compositions containing the compounds, and the effects of the compounds in treating or preventing diseases caused by viral infections.
背景技术Background technique
2019年12月发现的新型冠状病毒一开始被命名为2019-nCoV，世界卫生组织(WHO)将其改称为COVID-19，之后国际病毒分类委员会根据***学、分类学和惯例，正式将新型冠状病毒命名为SARS-CoV-2。SARS-CoV-2可引起严重急性呼吸道(SARI)症状，包括发烧、呼吸困难、乏力和肺炎等。The new coronavirus discovered in December 2019 was initially named 2019-nCoV. The World Health Organization (WHO) renamed it COVID-19. Later, the International Committee on Classification of Viruses officially classified the new coronavirus as 2019-nCoV based on systematics, taxonomy and convention. The virus was named SARS-CoV-2. SARS-CoV-2 can cause severe acute respiratory (SARI) symptoms, including fever, dyspnea, fatigue, and pneumonia.
在所有已知的RNA病毒中，冠状病毒的最大基因组长度在约26到32kb之间。除编码结构蛋白外，冠状病毒基因组的大部分也被转录并翻译成多肽，该多肽编码病毒复制和基因表达所必需的蛋白质。约306aa长的主要蛋白酶(Mpro)是冠状病毒复制的关键酶，也由该多肽编码，并负责将该多肽加工为功能蛋白。Mpro具有与微小RNA病毒3C蛋白酶(3Cpro)相似的切割位点特异性，因此也称为3C样蛋白酶(3CLpro)。研究表明，不同的冠状病毒的3CLpro在序列和3D结构方面都高度保守。这些特征及其功能重要性使3CLpro成为抗冠状病毒药物设计的靶标。Among all known RNA viruses, the maximum genome length of coronaviruses ranges from approximately 26 to 32 kb. In addition to encoding structural proteins, much of the coronavirus genome is transcribed and translated into polypeptides that encode proteins necessary for viral replication and gene expression. The approximately 306 aa long main protease (Mpro) is a key enzyme for coronavirus replication and is also encoded by this polypeptide and is responsible for processing this polypeptide into functional proteins. Mpro has a similar cleavage site specificity to picornavirus 3C protease (3Cpro), so it is also called 3C-like protease (3CLpro). Research shows that 3CLpro of different coronaviruses is highly conserved in both sequence and 3D structure. These characteristics and their functional importance make 3CLpro a target for anti-coronavirus drug design.
3CLpro的作用是在适当位点水解切割经表达的肽链，为肽链形成三维四维结构做准备，以形成病毒增殖所需要的酶。在催化过程中酶没有发生改变，但降低了水解反应的活化能，由此加快水解反应的速率，其中，半胱氨酸上的巯基在整个催化水解过程中起关键性作用，参见Thanigaimalai et.al,An Overview of Severe Acute Respiratory Syndrome-Coronavirus(SARS-CoV)3CL Protease Inhibitors:Peptidomimetics and Small Molecule Chemotherapy,Journal of Medicinal Chemistry,59(14):6595-6628。The function of 3CLpro is to hydrolyze and cleave the expressed peptide chain at the appropriate site, preparing the peptide chain to form a three-dimensional and four-dimensional structure to form the enzyme required for virus proliferation. The enzyme does not change during the catalytic process, but the activation energy of the hydrolysis reaction is reduced, thereby accelerating the rate of the hydrolysis reaction. Among them, the sulfhydryl group on cysteine plays a key role in the entire catalytic hydrolysis process, see Thanigaimalai et al. al, An Overview of Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) 3CL Protease Inhibitors: Peptidomimetics and Small Molecule Chemotherapy, Journal of Medicinal Chemistry, 59(14):6595-6628.
现有技术中存在关于3CLpro抑制剂的公开文献。例如WO2021/250648A1中公开了目前被称为Nirmatrelvir的化合物(PF-07321332)，其作为帕罗韦德(Paxlovid)的活性成分之一，与其中的利托那韦联用，能够降低由新型冠状病毒SARS-CoV-2导致的死亡和住院风险。There are published documents on 3CLpro inhibitors in the prior art. For example, WO2021/250648A1 discloses a compound currently known as Nirmatrelvir (PF-07321332). As one of the active ingredients of Paxlovid, it can be used in combination with ritonavir to reduce the risk of COVID-19. Risk of death and hospitalization from the virus SARS-CoV-2.
此外，WO2021/205290A1也公开了类似结构的化合物，其通过3C样蛋白酶抑制剂所介导的途径治疗SARS-CoV-2导致的疾病。In addition, WO2021/205290A1 also discloses compounds with similar structures, which treat diseases caused by SARS-CoV-2 through a pathway mediated by 3C-like protease inhibitors.
然而现有技术的化合物均存在不利之处，例如帕罗韦德同时还抑制CYP3A4酶，从而导致可能出现干扰该酶对其他药物的代谢，使半衰期和清除率发生改变，疗效降低或产生不良反应的情形。例如，患者同时服用帕罗韦德和特非那定时，因帕罗韦德抑制CYP3A4对特非那定的氧化代谢，致使后者在患者体内浓度异常增高，引起心脏的QT波延长和心率失常。而WO2021/205290A1所公开的化合物还面对通过口服给药时无效的问题。
However, existing compounds have disadvantages. For example, Parovide also inhibits the CYP3A4 enzyme, which may interfere with the enzyme's metabolism of other drugs, change the half-life and clearance rate, reduce efficacy, or produce adverse reactions. situation. For example, when a patient takes parovide and terfenadine at the same time, because parovide inhibits the oxidative metabolism of terfenadine by CYP3A4, the concentration of the latter in the patient's body increases abnormally, causing cardiac QT wave prolongation and arrhythmias. . The compounds disclosed in WO2021/205290A1 also face the problem of being ineffective when administered orally.
另外，有研究报道，在使用抗SARS-CoV-2活性分子ALG-097161进行压力筛选后，主蛋白酶产生了多个耐药突变。其中L50F+E166A+L167F三点突变在酶活水平测试中，Nirmatrelvir和Ensitrelvir的抑制效果分别降低72倍和93倍；在病毒保护实验中，Nirmatrelvir的EC50值提高了51倍，参见The Substitutions L50F,E166A,and L167F in SARS-CoV-2 3CLpro Are Selected by a Protease Inhibitor In Vitro and Confer Resistance To Nirmatrelvir,mBio 14(2023)e0281522.10.1128/mbio.02815-22。因此，研发新的3C样蛋白酶抑制剂的需求日渐迫切。In addition, studies have reported that after pressure screening using the anti-SARS-CoV-2 active molecule ALG-097161, the main protease produced multiple drug-resistant mutations. Among them, the three point mutations L50F+E166A+L167F reduced the inhibitory effects of Nirmatrelvir and Ensitrelvir by 72 times and 93 times respectively in the enzyme activity level test; in the virus protection experiment, the EC50 value of Nirmatrelvir increased by 51 times, see The Substitutions L50F, E166A, and L167F in SARS-CoV-2 3CLpro Are Selected by a Protease Inhibitor In Vitro and Confer Resistance To Nirmatrelvir, mBio 14(2023)e0281522.10.1128/mbio.02815-22. Therefore, the need to develop new 3C-like protease inhibitors is increasingly urgent.
发明内容Contents of the invention
本发明以3C样蛋白酶作为靶点，研发了一类新的小分子抑制剂，可用于治疗或预防病毒感染。The present invention uses 3C-like protease as a target and develops a new class of small molecule inhibitors, which can be used to treat or prevent viral infections.
本发明化合物靶向3C样蛋白酶，对具有P132H突变的3C样蛋白酶具有优异的抑制活性，能够显著抑制SARS-CoV-2的增殖，同时，还实现了更优的体内稳定性，更低的药物副作用，更优的药代动力学性质，以及针对拟肽类抗SARS-COV-2药物ALG-097161压力筛选下所产生的耐药突变L50F+E166A+L167F主蛋白酶更优的生物活性。The compound of the present invention targets 3C-like protease, has excellent inhibitory activity against 3C-like protease with P132H mutation, and can significantly inhibit the proliferation of SARS-CoV-2. At the same time, it also achieves better in vivo stability and lower drug consumption. Side effects, better pharmacokinetic properties, and better biological activity against the main protease of the drug-resistant mutation L50F+E166A+L167F produced under the pressure screening of the peptidomimetic anti-SARS-COV-2 drug ALG-097161.
在一个方面，本发明提供了式(I)化合物，或其药学上可接受的盐或酯，它们的立体异构体或互变异构体、消旋体、氮氧化物、多晶型、水合物、溶剂合物、同位素标记物、前药或代谢产物：
In one aspect, the invention provides compounds of formula (I), or pharmaceutically acceptable salts or esters thereof, their stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs, Hydrates, solvates, isotopic labels, prodrugs or metabolites:
在第二方面，本发明提供了一种药物组合物，其包含本发明化合物或其药学上可接受的盐或酯，它们的立体异构体或互变异构体、消旋体、氮氧化物、多晶型、水合物、溶剂合物、同位素标记物、前药或代谢产物。In a second aspect, the invention provides a pharmaceutical composition comprising a compound of the invention or a pharmaceutically acceptable salt or ester thereof, their stereoisomers or tautomers, racemates, nitrogen oxides, etc. substances, polymorphs, hydrates, solvates, isotopic markers, prodrugs or metabolites.
在根据本发明的一些优选的实施方案中，根据本发明的药物组合物还可以任选地包含至少一种生理学上/药学上可接受的辅料。In some preferred embodiments according to the present invention, the pharmaceutical composition according to the present invention may also optionally comprise at least one physiologically/pharmaceutically acceptable excipient.
在根据本发明的一些优选的实施方案中，根据本发明的药物组合物还可以任选地包含药学上可接受的赋形剂，例如载体、佐剂或媒介物。In some preferred embodiments according to the present invention, the pharmaceutical composition according to the present invention may also optionally comprise pharmaceutically acceptable excipients, such as carriers, adjuvants or vehicles.
在根据本发明的一些优选的实施方案中，根据本发明的药物组合物还可以任选地包含另外的活性成分或治疗剂。所述另外的活性成分或治疗剂例如是瑞德西韦(Remdesivir或GS-5734)、洛匹那韦(Lopinavir)、莫努匹韦(Molnupiravir)、利托那韦(Ritonavir)、氯喹(Chloroquine或Sigma-C6628)、羟氯喹和/或α-干扰素。 In some preferred embodiments according to the invention, the pharmaceutical compositions according to the invention may also optionally comprise additional active ingredients or therapeutic agents. The additional active ingredient or therapeutic agent is, for example, Remdesivir (Remdesivir or GS-5734), Lopinavir, Molnupiravir, Ritonavir, Chloroquine or Sigma-C6628), hydroxychloroquine and/or alpha-interferon.
在根据本发明的一些优选的实施方案中，根据本发明的药物组合物包含治疗有效量的本发明化合物或其药学上可接受的盐或酯，它们的立体异构体或互变异构体、消旋体、氮氧化物、多晶型、水合物、溶剂合物、同位素标记物、前药或代谢产物。In some preferred embodiments according to the invention, pharmaceutical compositions according to the invention comprise a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt or ester thereof, their stereoisomers or tautomers , racemates, nitrogen oxides, polymorphs, hydrates, solvates, isotope markers, prodrugs or metabolites.
在根据本发明的一些优选的实施方案中，根据本发明的药物组合物是RNA依赖性RNA聚合酶抑制剂、3CLpro蛋白酶抑制剂、CYP3A4抑制剂或靶向宿主的抗病毒药物。In some preferred embodiments according to the invention, the pharmaceutical composition according to the invention is an RNA-dependent RNA polymerase inhibitor, a 3CLpro protease inhibitor, a CYP3A4 inhibitor or a host-targeted antiviral drug.
在根据本发明的一些优选的实施方案中，根据本发明化合物或其药学上可接受的盐或酯，它们的立体异构体或互变异构体、消旋体、氮氧化物、多晶型、水合物、溶剂合物、同位素标记物、前药或代谢产物，或其药物组合物，用于预防和/或治疗选自下组的疾病、病症、症候群和/或紊乱，或者用于缓解选自下组的疾病、病症、症候群和/或紊乱的症状：由病毒感染引起的发热、恶心、呕吐、头痛、呼吸困难、乏力、呼吸道感染、肺炎、嗅觉障碍、味觉障碍及其并发症，或其组合；优选地，所述病毒为冠状病毒，优选为α冠状病毒和/或β冠状病毒，更优选为SARS-CoV-2。In some preferred embodiments according to the present invention, the compounds according to the present invention or pharmaceutically acceptable salts or esters thereof, their stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs forms, hydrates, solvates, isotopic labels, prodrugs or metabolites, or pharmaceutical compositions thereof, for the prevention and/or treatment of diseases, conditions, syndromes and/or disorders selected from the group consisting of, or for Relief of symptoms of diseases, conditions, syndromes and/or disorders selected from the group consisting of fever, nausea, vomiting, headache, dyspnea, fatigue, respiratory infections, pneumonia, anosmia, taste disorders and their complications caused by viral infections , or a combination thereof; preferably, the virus is a coronavirus, preferably alphacoronavirus and/or betacoronavirus, and more preferably SARS-CoV-2.
根据本发明，可以通过本领域已知的方法将根据本发明的药物组合物制成适于给药的剂型。According to the present invention, the pharmaceutical composition according to the present invention can be formulated into a dosage form suitable for administration by methods known in the art.
在第三方面，本发明提供了根据本发明的化合物或其药学上可接受的盐或酯，它们的立体异构体或互变异构体、消旋体、氮氧化物、多晶型、水合物、溶剂合物、同位素标记物、前药或代谢产物在制备药物中的用途。In a third aspect, the invention provides compounds according to the invention or pharmaceutically acceptable salts or esters thereof, their stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs, Use of hydrates, solvates, isotopic labels, prodrugs or metabolites in the preparation of pharmaceuticals.
在根据本发明的一些优选的实施方案中，根据本发明制备的药物还可以任选地包含另外的活性成分或治疗剂。所述另外的活性成分或治疗剂例如是瑞德西韦(Remdesivir或GS-5734)、洛匹那韦(Lopinavir)、莫努匹韦(Molnupiravir)、利托那韦(Ritonavir)、氯喹(Chloroquine,Sigma-C6628)、羟氯喹和/或α-干扰素。In some preferred embodiments according to the invention, the medicaments prepared according to the invention may also optionally contain additional active ingredients or therapeutic agents. The additional active ingredient or therapeutic agent is, for example, Remdesivir (Remdesivir or GS-5734), Lopinavir, Molnupiravir, Ritonavir, Chloroquine , Sigma-C6628), hydroxychloroquine and/or alpha-interferon.
在根据本发明的一些优选的实施方案中，根据本发明制备的药物是RNA依赖性RNA聚合酶抑制剂、3CLpro蛋白酶抑制剂、CYP3A4抑制剂或靶向宿主的抗病毒药物。In some preferred embodiments according to the invention, the drug prepared according to the invention is an RNA-dependent RNA polymerase inhibitor, a 3CLpro protease inhibitor, a CYP3A4 inhibitor or a host-targeted antiviral drug.
在根据本发明的一些优选的实施方案中，根据本发明制备的药物用于预防或治疗选自下组的疾病、病症、症候群和/或紊乱，或者用于缓解选自下组的疾病、病症、症候群和/或紊乱的症状：由病毒感染引起的发热、恶心、呕吐、头痛、呼吸困难、乏力、呼吸道感染、肺炎、嗅觉味觉出现障碍及其并发症，或其组合；优选地，所述病毒为冠状病毒，优选为α冠状病毒和/或β冠状病毒，更优选为SARS-CoV-2。In some preferred embodiments according to the present invention, the medicaments prepared according to the present invention are used to prevent or treat diseases, conditions, syndromes and/or disorders selected from the following group, or for alleviating diseases, conditions selected from the following group , symptoms of syndromes and/or disorders: fever, nausea, vomiting, headache, dyspnea, fatigue, respiratory tract infection, pneumonia, smell and taste disorders and their complications, or combinations thereof caused by viral infection; preferably, the The virus is a coronavirus, preferably alphacoronavirus and/or betacoronavirus, more preferably SARS-CoV-2.
根据本发明，可以通过本领域已知的方法将根据本发明制备的药物进一步制成适于给药的剂型。According to the present invention, the medicine prepared according to the present invention can be further formulated into a dosage form suitable for administration by methods known in the art.
在第四方面，本发明提供了一种在受试者中治疗或预防病毒感染导致的疾病、病症、症候群和/或紊乱的方法，包括向所述受试者给药本发明化合物或其药学上可接受的盐或酯，它们的立体异构体或互变异构体、消旋体、氮氧化物、多晶型、水合物、溶剂合物、同位素标记物、前药或代谢产物，或其药物组合物。In a fourth aspect, the present invention provides a method for treating or preventing diseases, conditions, syndromes and/or disorders caused by viral infection in a subject, comprising administering to the subject a compound of the present invention or a pharmaceutical thereof. Acceptable salts or esters thereof, their stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs, hydrates, solvates, isotopic markers, prodrugs or metabolites, or pharmaceutical compositions thereof.
在根据本发明的一些优选的实施方案中，本发明化合物或药物组合物抑制病毒增殖；In some preferred embodiments according to the invention, the compounds or pharmaceutical compositions of the invention inhibit viral proliferation;
在另一优选的实施方案中，本发明化合物或药物组合物抑制病毒3CL蛋白酶的活性；In another preferred embodiment, the compound or pharmaceutical composition of the invention inhibits the activity of viral 3CL protease;
在另一优选的实施方案中，所述3CL蛋白酶具有P132H突变；In another preferred embodiment, the 3CL protease has a P132H mutation;
在另一优选的实施方案中，所述病毒为冠状病毒，优选为α冠状病毒和/或β冠状病毒，更优选为SARS-CoV-2。In another preferred embodiment, the virus is a coronavirus, preferably an alphacoronavirus and/or a betacoronavirus, more preferably SARS-CoV-2.
在根据本发明的一些优选的实施方案中，所述病毒感染导致的疾病、病症、症候群和/或紊乱选自：发热、恶心、呕吐、头痛、呼吸困难、乏力、呼吸道感染、肺炎、嗅觉障碍、味觉障碍及其并发症，或其组合；In some preferred embodiments according to the present invention, the diseases, conditions, syndromes and/or disorders caused by the viral infection are selected from: fever, nausea, vomiting, headache, dyspnea, fatigue, respiratory tract infection, pneumonia, olfactory disorder , dysgeusia and its complications, or combinations thereof;
优选地，所述病毒为冠状病毒，优选为α冠状病毒和/或β冠状病毒，更优选为SARS-CoV-2。 Preferably, the virus is a coronavirus, preferably alphacoronavirus and/or betacoronavirus, more preferably SARS-CoV-2.
本领域技术人员能够理解，根据本发明的各个方面以及各个实施方案中所列举的特征可以自由组合，只要它们彼此之间不存在冲突或不能兼容的情形。Those skilled in the art can understand that the features listed in various aspects and embodiments of the present invention can be freely combined as long as they do not conflict or be incompatible with each other.
定义definition
术语“冠状病毒”包括但不限于下列病毒：HCoV-229E、HCoV-NL63、HCoV-HKU1、HCoV-OC43、SARS-CoV、MERS-CoV和/或SARSCoV-2。The term "coronavirus" includes, but is not limited to, the following viruses: HCoV-229E, HCoV-NL63, HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV and/or SARSCoV-2.
在一个实施方案中，术语“冠状病毒”为α冠状病毒和/或β冠状病毒、更优选β冠状病毒。In one embodiment, the term "coronavirus" is an alphacoronavirus and/or a betacoronavirus, more preferably a betacoronavirus.
在一个实施方案中，α冠状病毒选自HCoV-229E和HCoV-NL63，优选HCoV-229E。In one embodiment, the alphacoronavirus is selected from HCoV-229E and HCoV-NL63, preferably HCoV-229E.
在一个实施方案中，β冠状病毒选自HCoV-HKU1、HCoV-OC43、SARS-CoV、MERS-CoV和SARS-CoV-2，优选HCoV-OC43或SARS-CoV-2，更优选SARS-CoV-2。In one embodiment, the betacoronavirus is selected from HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV and SARS-CoV-2, preferably HCoV-OC43 or SARS-CoV-2, more preferably SARS-CoV- 2.
本文所用的术语“治疗”涉及逆转、减轻、抑制该术语适用的障碍或病症的进展或者预防之，或者这类障碍或病症的一种或多种症状。本文所用的名词“治疗”涉及动词治疗的动作，后者是如刚才所定义的。在本文中，术语“治疗”任何疾病或病症，在其中一些实施方案中指改善疾病或病症(即减缓或阻止或减轻疾病或其至少一种临床症状的发展)。在另一些实施方案中，“治疗”指缓和或改善至少一种身体参数，包括可能不为患者所察觉的身体参数。在另一些实施方案中，“治疗”指从身体上(例如稳定可察觉的症状)或生理学上(例如稳定身体的参数)或上述两方面调节疾病或病症。在另一些实施方案中，“治疗”指预防或延迟疾病或病症的发作、发生或噁化。As used herein, the term "treatment" refers to reversing, alleviating, inhibiting the progression of, or preventing the disorder or condition to which the term applies, or one or more symptoms of such disorder or condition. As used herein, the noun "treat" refers to the action of the verb treat, as just defined. As used herein, the term "treating" any disease or condition, in some embodiments thereof, means ameliorating the disease or condition (i.e., slowing or arresting or alleviating the development of the disease or at least one clinical symptom thereof). In other embodiments, "treating" or "treating" refers to alleviating or improving at least one physical parameter, including physical parameters that may not be noticeable to the patient. In other embodiments, "treating" or "treating" refers to modulating a disease or condition physically (eg, stabilizing perceived symptoms) or physiologically (eg, stabilizing body parameters), or both. In other embodiments, "treating" or "treating" refers to preventing or delaying the onset, development, or progression of a disease or disorder.
本文所用的术语“药学上可接受的盐”表示本发明化合物的那些羧酸盐、氨基酸加成盐，它们在可靠的医学判断范围内适用于与患者组织接触，不会产生不恰当的毒性、刺激作用、***反应等，与合理的益处/风险比相称，就它们的预期应用而言是有效的，包括(可能的话)本发明化合物的两性离子形式。As used herein, the term "pharmaceutically acceptable salts" means those carboxylate salts and amino acid addition salts of the compounds of the present invention which are suitable for contact with patient tissue within the scope of reliable medical judgment and will not produce undue toxicity, Irritation effects, allergic reactions, etc., commensurate with a reasonable benefit/risk ratio, are effective for their intended use, including (where possible) zwitterionic forms of the compounds of the invention.
药学上可接受的碱加成盐是与金属或胺生成的，例如碱金属与碱土金属氢氧化物或有机胺。用作阳离子的金属的实例有钠、钾、镁、钙等。适合的胺的实例有N,N'-二苄基乙二胺、氯普鲁卡因、胆碱、二乙醇胺、乙二胺、N-甲基葡糖胺和普鲁卡因。Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali metal and alkaline earth metal hydroxides or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, etc. Examples of suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and procaine.
酸性化合物的碱加成盐可以这样制备，按照常规方式使游离酸形式与足量所需的碱接触，生成盐。按照常规方式使盐形式与酸接触，再分离游离酸，可以使游离酸再生。游离酸形式在某些物理性质上多少不同于它们各自的盐形式，例如在极性溶剂中的溶解度，但是出于本发明的目的，盐还是等价于它们各自的游离酸。Base addition salts of acidic compounds may be prepared in conventional manner by contacting the free acid form with a sufficient amount of the desired base to form the salt. The free acid can be regenerated by contacting the salt form with the acid and isolating the free acid in the usual manner. The free acid forms differ somewhat from their respective salt forms in certain physical properties, such as solubility in polar solvents, but for the purposes of this invention the salts are nevertheless equivalent to their respective free acids.
盐可以是从无机酸制备的硫酸盐、焦硫酸盐、硫酸氢盐、亚硫酸盐、亚硫酸氢盐、硝酸盐、磷酸盐、磷酸一氢盐、磷酸二氢盐、偏磷酸盐、焦磷酸盐、氯化物、溴化物、碘化物，酸例如盐酸、硝酸、硫酸、氢溴酸、氢碘酸、磷酸等。代表性盐包括：氢溴酸盐、盐酸盐、硫酸盐、硫酸氢盐、硝酸盐、乙酸盐、草酸盐、戊酸盐、油酸盐、棕榈酸盐、硬脂酸盐、月桂酸盐、硼酸盐、苯甲酸盐、乳酸盐、磷酸盐、甲苯磺酸盐、柠檬酸盐、马来酸盐、富马酸盐、琥珀酸盐、酒石酸盐、萘甲酸盐、甲磺酸盐、葡庚糖酸盐、乳糖酸盐、月桂基磺酸盐和羟乙磺酸盐等。盐也可以是从有机酸制备的，例如脂肪族一元与二元羧酸、苯基取代的烷酸、羟基烷酸、烷二酸、芳香族酸、脂肪族与芳香族磺酸等。代表性盐包括乙酸盐、丙酸盐、辛酸盐、异丁酸盐、草酸盐、丙二酸盐、琥珀酸盐、辛二酸盐、癸二酸盐、富马酸盐、马来酸盐、扁桃酸盐、苯甲酸盐、氯苯甲酸盆、甲基苯甲酸盐、二硝基苯甲酸盐、萘甲酸盐、苯磺酸盐、甲苯磺酸盐、苯乙酸盐、柠檬酸盐、乳酸盐、马来酸盐、酒石酸盐、甲磺酸盐等。药学上可接受的盐可以包括基于碱金属与碱土金属的阳离子，例如钠、锂、钾、钙、镁等，以及无毒的铵、季铵和胺阳离子，包括但不限于铵、四甲基铵、四乙基铵、甲胺、二甲胺、三甲胺、三乙胺、乙胺等。 还涵盖氨基酸的盐，例如精氨酸盐、葡糖酸盐、半乳糖醛酸盐等(例如参见Berge S.M.et al.，"Pharmaceutical Salts,”J.Pharm.Sci.，1977；66:1-19，引入此作为参考)。The salts may be sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates prepared from inorganic acids Salt, chloride, bromide, iodide, acids such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, etc. Representative salts include: hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate Acid, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, Methanesulfonate, glucoheptonate, lactobionate, lauryl sulfonate and isethionate, etc. Salts may also be prepared from organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Representative salts include acetate, propionate, octanoate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, malonate Lenate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, naphthoate, benzenesulfonate, toluenesulfonate, phenylbenzoate Acid, citrate, lactate, maleate, tartrate, methanesulfonate, etc. Pharmaceutically acceptable salts may include alkali and alkaline earth metal based cations such as sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium and amine cations including, but not limited to, ammonium, tetramethyl Ammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. Also contemplated are salts of amino acids, such as arginates, gluconates, galacturonates, etc. (see, for example, Berge SM et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977; 66:1-19 , incorporated by reference).
在本文中，术语“氮氧化物”是指当化合物含几个含氮官能团时，可将1个或大于1个的氮原子氧化形成N-氧化物。N-氧化物的特殊实例是叔胺的N-氧化物或含氮杂环氮原子的N-氧化物。可用氧化剂例如过氧化氢或过酸(例如过氧羧酸)处理相应的含氮化合物而形成N-氧化物。尤其是，N-氧化物可用L.W.Deady的方法制备(Syn.Comm.1977,7,509-514)，其中例如在惰性溶剂例如二氯甲烷中，使含氮化合物与间-氯过氧苯甲酸(MCPBA)反应。As used herein, the term "nitrogen oxide" means that one or more nitrogen atoms can be oxidized to form N-oxides when the compound contains several nitrogen-containing functional groups. Particular examples of N-oxides are N-oxides of tertiary amines or N-oxides of nitrogen atoms in nitrogen-containing heterocyclic rings. The corresponding nitrogen-containing compounds can be treated with oxidizing agents such as hydrogen peroxide or peracids (eg peroxycarboxylic acid) to form N-oxides. In particular, N-oxides can be prepared by the method of L.W. Deady (Syn. Comm. 1977, 7, 509-514), in which the nitrogen-containing compound is reacted with m-chloroperoxybenzoic acid (MCPBA), for example in an inert solvent such as methylene chloride. )reaction.
在本文中，术语“酯”是指含有羟基或羧基的化合物形成的体内可水解的酯。这样的酯是，例如在人或动物体内水解产生母体醇或酸的生理学上/药学上可接受的酯。本发明式(I)或(II)化合物含有羧基，可以与适当的基团形成体内可水解的酯，这样的基团包括，但不限于，烷基、芳基烷基等。As used herein, the term "ester" refers to an in vivo hydrolyzable ester of a compound containing a hydroxyl or carboxyl group. Such esters are, for example, physiologically/pharmaceutically acceptable esters which hydrolyze in humans or animals to yield the parent alcohol or acid. The compound of formula (I) or (II) of the present invention contains a carboxyl group, which can form a hydrolyzable ester in vivo with an appropriate group. Such groups include, but are not limited to, alkyl, arylalkyl, etc.
给药的“受试者”包括但不限于：人(即，任何年龄组的男性或女性，例如，儿科受试者(例如，婴儿、儿童、青少年)或成人受试者(例如，年轻的成人、中年的成人或年长的成人)和/或非人的动物，例如，哺乳动物，例如，灵长类(例如，食蟹猴、恒河猴)、牛、猪、马、绵羊、山羊、啮齿动物、猫和/或狗。在一些实施方案中，受试者是人。在一些实施方案中，受试者是非人动物。本文可互换使用术语“人”、“患者”和“受试者”。"Subjects" for administration include, but are not limited to: humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young Adults, middle-aged adults or older adults) and/or non-human animals, e.g., mammals, e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and/or dogs. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human," "patient," and "human" are used interchangeably herein. "Subject".
“疾病”、“障碍”、“紊乱”、“症候群”和“病症”在本文中可互换地使用。"Disease," "disorder," "disorder," "syndrome" and "condition" are used interchangeably herein.
除非另作说明，否则，本文使用的术语“治疗”包括受试者患有具体疾病、障碍或病症时所发生的作用，它降低疾病、障碍或病症的严重程度，或延迟或减缓疾病、障碍或病症的发展(“治疗性治疗”)，还包括在受试者开始患有具体疾病、障碍或病症之前发生的作用(“预防性治疗”)。Unless otherwise indicated, the term "treatment" or "treatment" as used herein includes an action in a subject suffering from a specific disease, disorder or condition that reduces the severity of the disease, disorder or condition, or delays or slows down the disease, disorder or the development of a condition ("therapeutic treatment"), and also includes effects that occur before a subject begins to suffer from a specific disease, disorder or condition ("preventive treatment").
术语“单位剂型”是指适合作为人类受试者和其他哺乳动物的单位剂量的物理离散单位，每个单位含有预定量的活性物质，以及合适的药物赋形剂。例如，单位剂型可以是一个丸剂、一个片剂、一个胶囊或者一个锭剂等等。The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material in association with a suitable pharmaceutical excipient. For example, the unit dosage form may be a pill, a tablet, a capsule or a lozenge, etc.
通常，化合物的“有效量”是指足以引起目标生物反应的数量。正如本领域普通技术人员所理解的那样，本发明化合物的有效量可以根据下列因素而改变：例如，生物学目标、化合物的药代动力学、所治疗的疾病、给药模式以及受试者的年龄健康情况和症状。有效量包括治疗有效量和预防有效量。Generally, an "effective amount" of a compound is an amount sufficient to elicit a target biological response. As will be understood by those of ordinary skill in the art, the effective amount of a compound of the present invention may vary depending on factors such as, for example, the biological target, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the condition of the subject. Age health conditions and symptoms. The effective amount includes a therapeutically effective amount and a preventive effective amount.
除非另作说明，否则，本文使用的化合物的“治疗有效量”是在治疗疾病、障碍或病症的过程中足以提供治疗益处的量，或使与疾病、障碍或病症有关的一或多种症状延迟或最小化的量。化合物的治疗有效量是指单独使用或与其它疗法联用时，治疗剂的量，它在治疗疾病、障碍或病症的过程中提供治疗益处。术语“治疗有效量”可以包括改善总体治疗、降低或避免疾病或病症的症状或病因、或增强其它治疗剂的治疗效果的量。Unless otherwise specified, a "therapeutically effective amount" of a compound as used herein is an amount sufficient to provide a therapeutic benefit in treating a disease, disorder, or condition, or to cause one or more symptoms associated with the disease, disorder, or condition The amount to delay or minimize. A therapeutically effective amount of a compound is that amount of therapeutic agent that, when used alone or in combination with other therapies, provides a therapeutic benefit in the treatment of a disease, disorder, or condition. The term "therapeutically effective amount" may include an amount that improves overall treatment, reduces or avoids symptoms or causes of a disease or disorder, or enhances the therapeutic effect of other therapeutic agents.
除非另作说明，否则，本文使用的化合物的“预防有效量”是足以预防疾病、障碍或病症的量，或足以预防与疾病、障碍或病症有关的一或多种症状的量，或防止疾病、障碍或病症复发的量。化合物的预防有效量是指单独使用或与其它药剂联用时，治疗剂的量，它在预防疾病、障碍或病症的过程中提供预防益处。术语“预防有效量”可以包括改善总体预防的量，或增强其它预防药剂的预防效果的量。Unless otherwise stated, a "prophylactically effective amount" of a compound as used herein is an amount sufficient to prevent a disease, disorder or condition, or to prevent one or more symptoms associated with a disease, disorder or condition, or to prevent a disease , the amount of recurrence of a disorder or condition. A prophylactically effective amount of a compound is that amount of therapeutic agent that, when used alone or in combination with other agents, provides a prophylactic benefit in preventing a disease, disorder, or condition. The term "prophylactically effective amount" may include an amount that improves overall prophylaxis, or an amount that enhances the prophylactic effect of other prophylactic agents.
“组合”以及相关术语是指同时或依次给药本发明化合物和其它治疗剂。例如，本发明化合物可以与其它治疗剂以分开的单位剂型同时或依次给药，或与其它治疗剂一起在单一单位剂型中同时给药。"Combination" and related terms refer to the simultaneous or sequential administration of a compound of the invention and another therapeutic agent. For example, the compounds of the present invention may be administered simultaneously or sequentially with other therapeutic agents in separate unit dosage forms, or with other therapeutic agents in a single unit dosage form.
具体实施方案Specific implementation plan
本文中，“本发明化合物”指的是以下的式(I)化合物，或其药学上可接受的盐或酯，它们的立体异构体或互变异构体、消旋体、氮氧化物、多晶型、水合物、溶剂合物、同位素标记物、前药或代谢产物。 Herein, "compounds of the present invention" refer to the following compounds of formula (I), or pharmaceutically acceptable salts or esters thereof, their stereoisomers or tautomers, racemates, and nitrogen oxides. , polymorphs, hydrates, solvates, isotopic markers, prodrugs or metabolites.
本文中，化合物使用标准的命名法命名。具有非对称中心的化合物，应该明白(除非另有说明)所有的光学异构体及其混合物均包含在内。此外，除非另有规定，本发明所包括的所有异构体化合物与碳碳双键可能以Z和E的形式出现。在不同的互变异构形式存在的化合物，一个所述化合物并不局限于任何特定的互变异构体，而是旨在涵盖所有的互变异构形式。In this article, compounds are named using standard nomenclature. For compounds having asymmetric centers, it should be understood that (unless otherwise stated) all optical isomers and mixtures thereof are included. Furthermore, unless otherwise specified, all isomeric compounds encompassed by the present invention with carbon-carbon double bonds may appear in the Z and E forms. Compounds exist in different tautomeric forms, and a said compound is not limited to any particular tautomeric form, but is intended to encompass all tautomeric forms.
在一个实施方案中，本发明涉及式(I)化合物，或其药学上可接受的盐或酯，它们的立体异构体或互变异构体、消旋体、氮氧化物、多晶型、水合物、溶剂合物、同位素标记物、前药或代谢产物：
In one embodiment, the present invention relates to compounds of formula (I), or pharmaceutically acceptable salts or esters thereof, their stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs , hydrates, solvates, isotopic labels, prodrugs or metabolites:
本发明化合物可包括一个或多个不对称中心，且因此可以存在多种立体异构体形式，例如，对映异构体和/或非对映异构体形式。例如，本发明化合物可为单独的对映异构体、非对映异构体或几何异构体(例如顺式和反式异构体)，或者可为立体异构体的混合物的形式，包括外消旋体混合物和富含一种或多种立体异构体的混合物。异构体可通过本领域技术人员已知的方法从混合物中分离，所述方法包括：手性高压液相色谱法(HPLC)以及手性盐的形成和结晶；或者优选的异构体可通过不对称合成来制备。The compounds of the present invention may contain one or more asymmetric centers and thus may exist in multiple stereoisomeric forms, for example, enantiomeric and/or diastereomeric forms. For example, the compounds of the present invention may be individual enantiomers, diastereomers, or geometric isomers (e.g., cis and trans isomers), or may be in the form of mixtures of stereoisomers, Includes racemic mixtures and mixtures enriched in one or more stereoisomers. The isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or the preferred isomers may be separated by Prepared by asymmetric synthesis.
本发明化合物可以互变异构体形式存在。互变异构体为因分子中某一原子在两个位置迅速移动而产生的官能团异构体，互变异构体是一种特殊的官能团异构体，一对互变异构体可以互相转换，但通常以比较稳定的一种异构体为其主要的存在形式。最主要的例子为烯醇式和酮式互变异构体。The compounds of the present invention may exist in tautomeric forms. Tautomers are functional group isomers produced by the rapid movement of an atom in a molecule between two positions. A tautomer is a special functional group isomer. A pair of tautomers can interact with each other. conversion, but usually one of the more stable isomers is its main form of existence. The most important examples are the enol and keto tautomers.
例如，本发明化合物包含如下互变异构体：
For example, compounds of the present invention include the following tautomers:
本领域技术人员将理解，有机化合物可以与溶剂形成复合物，其在该溶剂中发生反应或从该溶剂中沉淀或结晶出来。这些复合物称为“溶剂合物”。当溶剂是水时，复合物称为“水合物”。本发明涵盖了本发明化合物的所有溶剂合物。Those skilled in the art will understand that organic compounds can form complexes with solvents, react in the solvent, or precipitate or crystallize out of the solvent. These complexes are called "solvates". When the solvent is water, the complex is called a "hydrate." This invention encompasses all solvates of the compounds of the invention.
术语“溶剂合物”是指通常由溶剂分解反应形成的与溶剂相结合的化合物或其盐的形式。这个物理缔合可包括氢键键合。常规溶剂包括水、甲醇、乙醇、乙酸、DMSO、THF、***等。本文所述的化合物可制备成，例如，结晶形式，且可被溶剂化。合适的溶剂合物包括药学上可接受的溶剂合物且进一步包括化学计量的溶剂合物和非化学计量的溶剂合物。在一些情况下，所述溶剂合物将能够分离，例如，当一或多个溶剂分子掺入结晶固体的晶格中时。“溶剂合物”包括溶液状态的溶剂合物和可分离的溶剂合物。代表性的溶剂合物包括水合物、乙醇合物和甲醇合物。The term "solvate" refers to a form of a compound or a salt thereof that is combined with a solvent, usually formed by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, ether, etc. The compounds described herein can be prepared, for example, in crystalline form, and can be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric and non-stoichiometric solvates. In some cases, the solvate will be capable of isolating, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid. "Solvate" includes both solution solvates and isolable solvates. Representative solvates include hydrates, ethanolates, and methoxides.
术语“水合物”是指与水相结合的化合物。通常，包含在化合物的水合物中的水分子数与该水合物 中该化合物分子数的比率确定。因此，化合物的水合物可用例如通式R·x H2O代表，其中R是该化合物，和x是大于0的数。给定化合物可形成超过一种水合物类型，包括，例如，单水合物(x为1)、低级水合物(x是大于0且小于1的数，例如，半水合物(R·0.5H2O)和多水合物(x为大于1的数，例如，二水合物(R·2H2O)和六水合物(R·6H2O)。The term "hydrate" refers to a compound combined with water. Generally, the number of water molecules contained in a hydrate of a compound is related to the hydrate The ratio of the number of molecules in the compound is determined. Thus, a hydrate of a compound may be represented, for example, by the general formula R.xH2O , where R is the compound and x is a number greater than zero. A given compound may form more than one hydrate type, including, for example, monohydrate (x is 1), lower hydrate (x is a number greater than 0 and less than 1), for example, hemihydrate (R·0.5H 2 O) and polyhydrates (x is a number greater than 1, for example, dihydrate (R·2H 2 O) and hexahydrate (R·6H 2 O).
本发明化合物可以是无定形或结晶形式(多晶型)。此外，本发明化合物可以以一种或多种结晶形式存在。因此，本发明在其范围内包括本发明化合物的所有无定形或结晶形式。术语“多晶型物”是指特定晶体堆积排列的化合物的结晶形式(或其盐、水合物或溶剂合物)。所有的多晶型物具有相同的元素组成。不同的结晶形式通常具有不同的X射线衍射图、红外光谱、熔点、密度、硬度、晶体形状、光电性质、稳定性和溶解度。重结晶溶剂、结晶速率、贮存温度和其他因素可导致一种结晶形式占优。化合物的各种多晶型物可在不同的条件下通过结晶制备。The compounds of the invention may be in amorphous or crystalline forms (polymorphs). Furthermore, the compounds of the present invention may exist in one or more crystalline forms. Accordingly, the present invention includes within its scope all amorphous or crystalline forms of the compounds of the invention. The term "polymorph" refers to a crystalline form of a compound (or a salt, hydrate or solvate thereof) in a specific crystal packing arrangement. All polymorphs have the same elemental composition. Different crystalline forms often have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optoelectronic properties, stability and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors can lead to the dominance of one crystalline form. Various polymorphs of the compounds can be prepared by crystallization under different conditions.
本发明还包括同位素标记的化合物(同位素变体)，它们等同于式(I)所述的那些，但一个或多个原子被原子质量或质量数不同于自然界常见的原子质量或质量数的原子所代替。可以引入本发明化合物中的同位素的实例包括氢、碳、氮、氧、磷、硫、氟和氯的同位素，分别例如2H、3H、13C、11C、14C、15N、18O、17O、31P、32P、35S、18F和36Cl。含有上述同位素和/或其它原子的其它同位素的本发明化合物、其前体药物和所述化合物或所述前体药物的药学上可接受的盐都属于本发明的范围。某些同位素标记的本发明化合物、例如引入放射性同位素(例如3H和14C)的那些可用于药物和/或底物组织分布测定。氚、即3H和碳-14、即14C同位素是特别优选的，因为它们容易制备和检测。进而，被更重的同位素取代，例如氘、即2H，由于代谢稳定性更高可以提供治疗上的益处，例如延长体内半衰期或减少剂量需求，因而在有些情况下可能是优选的。同位素标记的本发明式(I)化合物及其前体药物一般可以这样制备，在进行下述流程和/或实施例与制备例所公开的工艺时，用容易得到的同位素标记的试剂代替非同位素标记的试剂。The present invention also includes isotopically labeled compounds (isotopic variants) which are identical to those described in formula (I), except that one or more atoms are surrounded by atoms having an atomic mass or mass number different from that common in nature. replaced. Examples of isotopes that may be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 2 H, 3 H, 13 C, 11 C, 14 C, 15 N, 18 respectively. O, 17 O, 31 P, 32 P, 35 S, 18 F and 36 Cl. Compounds of the invention containing the above-mentioned isotopes and/or other isotopes of other atoms, prodrugs thereof and pharmaceutically acceptable salts of the compounds or prodrugs are within the scope of the present invention. Certain isotopically labeled compounds of the present invention, such as those incorporating radioactive isotopes (eg, 3 H and 14 C), may be used in drug and/or substrate tissue distribution assays. Tritium, ie 3 H, and carbon-14, ie 14 C isotopes are particularly preferred because they are easy to prepare and detect. Furthermore, substitution with heavier isotopes, such as deuterium, i.e. 2 H, may be preferred in some cases as greater metabolic stability may provide therapeutic benefits, such as increased half-life in vivo or reduced dosage requirements. The isotope-labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by replacing non-isotopes with readily available isotope-labeled reagents when performing the following processes and/or the processes disclosed in the Examples and Preparation Examples. Labeled reagents.
此外，前药也包括在本发明的上下文内。本文所用的术语“前药”是指在体内通过例如在血液中水解转变成其具有医学效应的活性形式的化合物。药学上可接受的前药描述于T.Higuchi和V.Stella，Prodrugs as Novel Delivery Systems，A.C.S.Symposium Series的Vol.14，Edward B.Roche，ed.，Bioreversible Carriers in Drug Design，American Pharmaceutical Association and Pergamon Press，1987，以及D.Fleisher、S.Ramon和H.Barbra“Improved oral drug delivery：solubility limitations overcome by the use of prodrugs”，Advanced Drug Delivery Reviews(1996)19(2)115-130，每篇引入本文作为参考。Furthermore, prodrugs are also included within the context of the present invention. The term "prodrug" as used herein refers to a compound that is converted in the body to its active form having a medical effect, for example, by hydrolysis in the blood. Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of A.C.S. Symposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, and D. Fleisher, S. Ramon and H. Barbra "Improved oral drug delivery: solubility limitations overcome by the use of prodrugs", Advanced Drug Delivery Reviews (1996) 19 (2) 115-130, each introduced This article serves as a reference.
前药为任何共价键合的本发明化合物，当将这种前药给予患者时，其在体内释放母体化合物。通常通过修饰官能团来制备前药，修饰是以使得该修饰可以通过常规操作或在体内裂解产生母体化合物的方式进行的。前药包括，例如，其中羟基、氨基或巯基与任意基团键合的本发明化合物，当将其给予患者时，可以裂解形成羟基、氨基或巯基。因此，前药的代表性实例包括(但不限于)式(I)化合物的羟基、巯基和氨基官能团的乙酸酯/酰胺、甲酸酯/酰胺和苯甲酸酯/酰胺衍生物。另外，在羧酸(-COOH)的情况下，可以使用酯，例如甲酯、乙酯等。酯本身可以是有活性的和/或可以在人体体内条件下水解。合适的药学上可接受的体内可水解的酯基包括容易在人体中分解而释放母体酸或其盐的那些基团。A prodrug is any covalently bonded compound of the invention that releases the parent compound in the body when administered to a patient. Prodrugs are typically prepared by modifying functional groups in a manner such that the modification can be cleaved by conventional manipulations or in vivo to yield the parent compound. Prodrugs include, for example, compounds of the invention in which a hydroxyl, amino or thiol group is bonded to any group that can be cleaved to form a hydroxyl, amino or thiol group when administered to a patient. Accordingly, representative examples of prodrugs include, but are not limited to, acetate/amide, formate/amide and benzoate/amide derivatives of the hydroxyl, thiol and amino functionality of compounds of formula (I). In addition, in the case of carboxylic acid (-COOH), esters such as methyl ester, ethyl ester, etc. can be used. The ester itself may be reactive and/or hydrolyzable under human body conditions. Suitable pharmaceutically acceptable in vivo hydrolyzable ester groups include those that readily break down in the human body to release the parent acid or salt thereof.
术语“代谢产物”是指具体的化合物或其盐在体内通过代谢作用所得到的产物。一个化合物的代谢产物可以通过所属领域公知的技术来进行鉴定，其活性可以通过如本发明所描述的那样采用试验的方法进行表征。这样的产物可以是通过给药化合物经过氧化，还原，水解，酰氨化，脱酰氨作用，酯化，脱脂作用，酶裂解等等方法得到。相应地，本发明包括化合物的代谢产物，包括将本发明的化合物与哺乳动物充分接触一段时间所产生的代谢产物。The term "metabolite" refers to a product obtained by metabolism of a specific compound or its salt in the body. The metabolites of a compound can be identified by techniques well known in the art, and its activity can be characterized by assays as described herein. Such products can be obtained by administering compounds through oxidation, reduction, hydrolysis, amidation, deamidation, esterification, delipidation, enzymatic cleavage, etc. Accordingly, the invention includes metabolites of compounds, including metabolites produced by contacting a compound of the invention with a mammal for a period of time sufficient to do so.
本发明还提供药物制剂，包含治疗有效量的式(I)化合物或其治疗学上可接受的盐和其药学上可接 受的载体、稀释剂或赋形剂。所有这些形式都属于本发明。The present invention also provides pharmaceutical preparations, comprising a therapeutically effective amount of a compound of formula (I) or a therapeutically acceptable salt thereof and a pharmaceutically acceptable salt thereof. acceptable carrier, diluent or excipient. All these forms belong to the invention.
药物组合物和试剂盒Pharmaceutical compositions and kits
在另一方面，本发明提供了药物组合物，其包含本发明化合物(还称为“活性组分”)和药学上可接受的赋形剂。在一些实施方案中，所述药物组合物包含有效量的本发明化合物。在一些实施方案中，所述药物组合物包含治疗有效量的本发明化合物。在一些实施方案中，所述药物组合物包含预防有效量的本发明化合物。In another aspect, the invention provides pharmaceutical compositions comprising a compound of the invention (also referred to as an "active ingredient") and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical compositions comprise an effective amount of a compound of the invention. In some embodiments, the pharmaceutical compositions comprise a therapeutically effective amount of a compound of the invention. In some embodiments, the pharmaceutical compositions comprise a prophylactically effective amount of a compound of the invention.
术语“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/药学上可接受的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/药学上可接受的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。术语“生理学上/药学上可接受的”是指当给人施用时生理上可耐受的并且一般不产生过敏或相似不适当的反应，例如肠胃不适、眩晕等的分子实体和组合物。术语“载体”指与所述化合物一同施用的稀释剂、辅剂、赋形剂或基质。这些药物载体可以是无菌液体，例如水和油类，包括石油、动物、植物或合成来源的，例如花生油、大豆油、矿物油、芝麻油等。水和水性溶液盐水溶液和水性葡萄糖与甘油溶液优选用作载体、特别是可注射溶液。适宜的药物载体描述于E.W.Martin的“Remington’s Pharmaceutical Sciences”中。The term "pharmaceutical composition" means a mixture containing one or more compounds described herein, or physiologically/pharmaceutically acceptable salts or prodrugs thereof, and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable salts or prodrugs thereof. Accepted carriers and excipients. The purpose of pharmaceutical compositions is to facilitate administration to living organisms and facilitate the absorption of active ingredients to exert biological activity. The term "physiologically/pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable when administered to humans and generally do not produce allergic or similar inappropriate reactions, such as gastrointestinal upset, dizziness, and the like. The term "carrier" refers to a diluent, adjuvant, excipient or matrix with which the compound is administered. These pharmaceutical carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water and aqueous solutions, saline solutions and aqueous glucose and glycerol solutions are preferably used as carriers, especially for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.
用于本发明的药学上可接受的赋形剂是指不会破坏一起调配的化合物的药理学活性的无毒载剂、佐剂或媒剂。可以用于本发明组合物中的药学上可接受的载剂、佐剂或媒剂包括(但不限于)离子交换剂、氧化铝、硬脂酸铝、卵磷脂、血清蛋白(如人类血清白蛋白)、缓冲物质(如磷酸盐)、甘氨酸、山梨酸、山梨酸钾、饱和植物脂肪酸的偏甘油酯混合物、水、盐或电解质(如硫酸鱼精蛋白)、磷酸氢二钠、磷酸氢钾、氯化钠、锌盐、硅胶、三硅酸镁、聚乙烯吡咯烷酮、基于纤维素的物质、聚乙二醇、羧甲基纤维素钠、聚丙烯酸酯、蜡、聚乙烯-聚氧丙烯-嵌段聚合物、聚乙二醇以及羊毛脂。Pharmaceutically acceptable excipients for use in the present invention refer to non-toxic carriers, adjuvants or vehicles that do not destroy the pharmacological activity of the compounds with which they are formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of the present invention include, but are not limited to, ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins (such as human serum albumin) protein), buffer substances (such as phosphate), glycine, sorbic acid, potassium sorbate, partial glyceride mixture of saturated vegetable fatty acids, water, salt or electrolyte (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate , sodium chloride, zinc salt, silica gel, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene-polyoxypropylene- Block polymers, polyethylene glycols, and lanolin.
可作为生理学上/药学上可接受辅料的物质包括，但并不限于，离子交换剂，铝，硬脂酸铝，卵磷脂，血清蛋白，如人血清蛋白，缓冲物质如磷酸盐，甘氨酸，山梨酸，山梨酸钾，饱和植物脂肪酸的部分甘油酯混合物，水，盐或电解质，如硫酸鱼精蛋白，磷酸氢二钠，磷酸氢钾，氯化钠，锌盐，胶体硅，三硅酸镁，聚乙烯吡咯烷酮，聚丙烯酸脂，蜡，聚乙烯-聚氧丙烯-阻断聚合体，羊毛脂，糖，如乳糖，葡萄糖和蔗糖；淀粉如玉米淀粉和土豆淀粉；纤维素和它的衍生物如羧甲基纤维素钠，乙基纤维素和乙酸纤维素；树胶粉；麦芽；明胶；滑石粉；辅料如可可豆脂和栓剂蜡状物；油如花生油，棉子油，红花油，麻油，橄榄油，玉米油和豆油；二醇类化合物，如丙二醇和聚乙二醇；酯类如乙基油酸酯和乙基月桂酸酯；琼脂；缓冲剂如氢氧化镁和氢氧化铝；海藻酸；无热原的水；等渗盐；林格(氏)溶液；乙醇，磷酸缓冲溶液，和其他无毒的合适的润滑剂如月桂硫酸钠和硬脂酸镁，着色剂，释放剂，包衣衣料，甜味剂，调味剂和香料，防腐剂和抗氧化剂。Substances that can be used as physiologically/pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, aluminum, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphate, glycine, sorbate Acids, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon, magnesium trisilicate , polyvinylpyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-blocked polymers, lanolin, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives Such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; gum powder; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil , sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and hydroxide Aluminum; alginic acid; pyrogen-free water; isotonic salts; Ringer's solution; ethanol, phosphate buffered solutions, and other nontoxic suitable lubricants such as sodium lauryl sulfate and magnesium stearate, colorants, Release agents, coatings, sweeteners, flavors and fragrances, preservatives and antioxidants.
本发明的药物组合物可根据公开的内容使用本领域技术人员已知的任何方法来制备。例如，常规混合、溶解、造粒、乳化、磨细、包封、包埋或冻干工艺。Pharmaceutical compositions of the present invention may be prepared according to the disclosure using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or freeze-drying processes.
本发明药物的剂型可根据具体情况进行选择。药物剂型常常由药物、赋形剂和容器/密封***组成。可将一种或多种赋形剂(又称为非活性成分)添加到本发明的化合物中来改善或促进药物的制造、稳定性、给药和安全性，并且可提供获得所需药物释放曲线的方法。因此，添加到药物中的赋形剂类型可视各种因素而定，例如药物的物理和化学特性、给药途径和制备步骤。在该领域中存在药用赋形剂并且包括各种药典中所列的那些。(参见美国药典(U.S.Pharmacopeia，USP)、日本药典(Japanese Pharmacopoeia，JP)、欧洲药典(European Pharmacopoeia，EP)和英国药典(British pharmacopoeia，BP)；美国食品与药品管理局(the U.S.Food and Drug Administration，www.fda.gov)药物评价与研究中心(Centerfor Drug Evaluation and Research，CEDR)出版物，例如《非活性组分指南》(Inactive Ingredient  Guide，1996)；Ash编写的《药物添加剂手册》(Hand book of Pharmaceutical Additives，2002)，联合信息资源公司(Synapse Information Resources,Inc.,Endicott NY；etc.)。The dosage form of the medicine of the present invention can be selected according to specific circumstances. Pharmaceutical dosage forms often consist of drug substance, excipients, and a container/closure system. One or more excipients (also known as inactive ingredients) can be added to the compounds of the invention to improve or facilitate the manufacture, stability, administration and safety of the drug and to provide for desired drug release curve method. Therefore, the type of excipients added to a drug can depend on various factors, such as the physical and chemical properties of the drug, route of administration, and preparation steps. Pharmaceutical excipients exist in the art and include those listed in various pharmacopeias. (See the United States Pharmacopeia (USP), Japanese Pharmacopoeia (JP), European Pharmacopoeia (EP), and British Pharmacopoeia (BP); the USFood and Drug Administration, www.fda.gov) Center for Drug Evaluation and Research (CEDR) publications, such as Inactive Ingredient Guide, 1996); "Hand book of Pharmaceutical Additives" (2002) by Ash, Synapse Information Resources, Inc., Endicott NY; etc.
本发明的药物组合物可包括一种或一种以上生理学上可接受的非活性成分，这些非活性成分会促进活性分子被加工成用于医药用途的制剂。Pharmaceutical compositions of the present invention may include one or more physiologically acceptable inactive ingredients that facilitate processing of the active molecules into preparations for pharmaceutical use.
适当的制剂视所需的给药途径而定。给药途径包括静脉注射、经粘膜或鼻给药、口服给药等。对于口服给药来说，化合物可配制成液体或固体剂型并作为速释或控释/缓释制剂。用于个体口服摄取的合适剂型包括片剂、药丸、糖衣药丸、硬壳和软壳胶囊、液体、凝胶、糖浆、膏剂、悬浮液和乳液。Appropriate formulation will depend on the desired route of administration. Routes of administration include intravenous injection, transmucosal or nasal administration, oral administration, etc. For oral administration, the compounds may be formulated in liquid or solid dosage forms and as immediate release or controlled/sustained release preparations. Suitable dosage forms for oral ingestion by individuals include tablets, pills, dragees, hard and soft shell capsules, liquids, gels, syrups, ointments, suspensions and emulsions.
固体口服剂型可使用赋形剂获得，所述赋形剂包括填充剂、崩解剂、粘合剂(干和湿)、溶解延缓剂、润滑剂、助流剂、抗粘剂、阳离子***换树脂、湿润剂、抗氧化剂、防腐剂、着色剂和调味剂。这些赋形剂可为合成或天然来源。所述赋形剂的实例包括纤维素衍生物、柠檬酸、磷酸二钙、明胶、碳酸镁、月桂基硫酸镁/月桂基硫酸钠、甘露糖醇、聚乙二醇、聚乙烯吡咯烷酮、硅酸盐、二氧化硅、苯甲酸钠、山梨糖醇、淀粉、硬脂酸或其盐、糖(即右旋糖、蔗糖、乳糖等)、滑石、西黄蓍胶浆、植物油(氢化)和蜡。乙醇和水可用作造粒助剂。在某些情况下，需要用例如掩味膜、抗胃酸膜或延缓释放膜来涂覆片剂。常常将天然和合成的聚合物与着色剂、糖和有机溶剂或水组合用于涂覆片剂，从而产生糖衣药丸。当胶囊优于片剂时，可以用兼容的硬壳或软壳胶囊形式递送其药物粉末、悬浮液或溶液。Solid oral dosage forms may be obtained using excipients including fillers, disintegrants, binders (dry and wet), dissolution retarder, lubricants, glidants, anti-adhesive agents, cationic exchangers Resins, humectants, antioxidants, preservatives, colorants and flavoring agents. These excipients may be of synthetic or natural origin. Examples of the excipients include cellulose derivatives, citric acid, dicalcium phosphate, gelatin, magnesium carbonate, magnesium lauryl sulfate/sodium lauryl sulfate, mannitol, polyethylene glycol, polyvinylpyrrolidone, silicic acid Salt, silicon dioxide, sodium benzoate, sorbitol, starch, stearic acid or its salts, sugar (i.e. dextrose, sucrose, lactose, etc.), talc, tragacanth mucilage, vegetable oil (hydrogenated) and wax. Ethanol and water can be used as granulation aids. In some cases, it is necessary to coat tablets with, for example, a taste-masking film, an acid-resistant film, or a delayed-release film. Natural and synthetic polymers are often used to coat tablets in combination with colorants, sugars and organic solvents or water to produce dragees. When capsules are preferred over tablets, their drug powders, suspensions or solutions may be delivered in compatible hard or soft shell capsules.
治疗有效剂量可首先使用本领域中熟知的各种方法来估算。用于动物研究的初始剂量可基于细胞培养测定中所确立的有效浓度。适合于人体的剂量范围例如可使用从动物研究和细胞培养测定所获得的数据来确定。在某些实施方案中，可以将本发明的化合物制备为用于口服的药剂。The therapeutically effective dose can first be estimated using various methods well known in the art. Initial dosages for animal studies may be based on established effective concentrations in cell culture assays. Dosage ranges suitable for humans can be determined, for example, using data obtained from animal studies and cell culture assays. In certain embodiments, the compounds of the present invention can be prepared as a medicament for oral administration.
可根据本领域中已知的方法，考虑个体状况的特殊性来选择正确的制剂、给药途径、剂量和给药间隔时间。The correct formulation, route of administration, dosage and interval between administrations can be selected according to methods known in the art, taking into account the particularities of the individual condition.
用于给予本发明化合物的合适制剂将对于本领域普通技术人员而言是显而易见的，并且包括例如片剂、丸剂、胶囊、栓剂、锭剂、糖锭剂、溶液(特别是注射(皮下、静脉内、肌内)和输注(注射剂)用溶液)、酏剂、糖浆、扁囊剂、乳液、吸入剂或可分散粉剂。一种或多种药物活性化合物的含量的范围应该是作为整体的组合物的0.1至90wt％、优选0.5至50wt％，即，其量足以实现以下指定的剂量范围。如有必要，指定的剂量可每天给药若干次。Suitable formulations for administering the compounds of the invention will be apparent to those of ordinary skill in the art and include, for example, tablets, pills, capsules, suppositories, lozenges, lozenges, solutions (especially injections (subcutaneous, intravenous solution for intramuscular administration) and infusion (injection)), elixir, syrup, cachet, emulsion, inhalation or dispersible powder. The content of one or more pharmaceutically active compounds should range from 0.1 to 90% by weight, preferably from 0.5 to 50% by weight of the composition as a whole, ie an amount sufficient to achieve the dosage ranges specified below. If necessary, the specified dose may be administered several times per day.
本发明还包括试剂盒(例如，药物包装)。所提供的试剂盒可以包括本发明化合物、其它治疗剂，以及含有本发明化合物、其它治疗剂的第一和第二容器(例如，小瓶、安瓿瓶、瓶、注射器和/或可分散包装或其它合适的容器)。在一些实施方案中，提供的试剂盒还可以任选包括第三容器，其含有用于稀释或悬浮本发明化合物和/或其它治疗剂的药用赋形剂。在一些实施方案中，提供在第一容器和第二容器中的本发明化合物和其它治疗剂组合形成一个单位剂型。The present invention also includes kits (eg, pharmaceutical packaging). Kits provided may include a compound of the invention, other therapeutic agents, and first and second containers (e.g., vials, ampoules, bottles, syringes, and/or dispersible packaging or other) containing the compounds of the invention, other therapeutic agents. suitable container). In some embodiments, provided kits may also optionally include a third container containing pharmaceutical excipients for diluting or suspending the compounds of the invention and/or other therapeutic agents. In some embodiments, the compound of the invention and the other therapeutic agent provided in the first container and the second container are combined to form a unit dosage form.
给药Give medication
本发明提供的药物组合物可以通过许多途径给药，包括但不限于：口服给药、肠胃外给药、吸入给药、局部给药、直肠给药、鼻腔给药、口腔给药、***给药、通过植入剂给药或其它给药方式。例如，本文使用的肠胃外给药包括皮下给药、皮内给药、静脉内给药、肌肉内给药、关节内给药、动脉内给药、滑膜腔内给药、胸骨内给药、脑脊髓膜内给药、病灶内给药、和颅内的注射或输液技术。The pharmaceutical composition provided by the present invention can be administered through many routes, including but not limited to: oral administration, parenteral administration, inhalation administration, topical administration, rectal administration, nasal administration, buccal administration, vaginal administration drugs, administered via implants or other methods of administration. For example, parenteral administration as used herein includes subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, intraarticular administration, intraarterial administration, intrasynovial administration, intrasternal administration , intracerebrospinal membrane drug administration, intralesional drug administration, and intracranial injection or infusion techniques.
通常，给予有效量的本文所提供的化合物。按照有关情况，包括所治疗的病症、选择的给药途径、实际给予的化合物、个体患者的年龄、体重和响应、患者症状的严重程度，等等，可以由医生确定实际上给予的化合物的量。Typically, an effective amount of a compound provided herein is administered. The amount of compound actually administered can be determined by the physician depending on the circumstances, including the condition being treated, the route of administration chosen, the compound actually administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, etc. .
当用于预防本发明所述病症时，给予处于形成所述病症危险之中的受试者本文所提供的化合物， 典型地基于医生的建议并在医生监督下给药，剂量水平如上所述。处于形成具体病症的危险之中的受试者，通常包括具有所述病症的家族史的受试者，或通过遗传试验或筛选确定尤其对形成所述病症敏感的那些受试者。When used to prevent a disorder described herein, a compound provided herein is administered to a subject at risk of developing the disorder, Typically administered based on a physician's advice and supervision, dosage levels are as described above. Subjects at risk of developing a particular condition generally include subjects with a family history of the condition or those who have been determined by genetic testing or screening to be particularly susceptible to developing the condition.
还可以长期给予本文所提供的药物组合物(“长期给药”)。长期给药是指在长时间内给予化合物或其药物组合物，例如，3个月、6个月、1年、2年、3年、5年等等，或者可无限期地持续给药，例如，受试者的余生。在一些实施方案中，长期给药意欲在长时间内在血液中提供所述化合物的恒定水平，例如，在治疗窗内。The pharmaceutical compositions provided herein can also be administered over a long period of time ("chronic administration"). Long-term administration refers to the administration of a compound or pharmaceutical composition thereof over a long period of time, for example, 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or administration may be continued indefinitely, For example, the remainder of the subject's life. In some embodiments, chronic administration is intended to provide a constant level of the compound in the blood over an extended period of time, eg, within a therapeutic window.
可以使用各种给药方法，进一步递送本发明的药物组合物。例如，在一些实施方案中，可以推注给药药物组合物，例如，为了使化合物在血液中的浓度提高至有效水平。推注剂量取决于通过身体的活性组分的目标全身性水平，例如，肌内或皮下的推注剂量使活性组分缓慢释放，而直接递送至静脉的推注(例如，通过IV静脉滴注)能够更加快速地递送，使得活性组分在血液中的浓度快速升高至有效水平。在其它实施方案中，可以以持续输液形式给予药物组合物，例如，通过IV静脉滴注，从而在受试者身体中提供稳态浓度的活性组分。此外，在其它实施方案中，可以首先给予推注剂量的药物组合物，而后持续输液。Various methods of administration may be used to further deliver the pharmaceutical compositions of the present invention. For example, in some embodiments, a pharmaceutical composition may be administered as a bolus injection, eg, in order to increase the concentration of the compound in the blood to an effective level. The bolus dose depends on the target systemic levels of the active ingredient through the body, e.g., an intramuscular or subcutaneous bolus dose provides a slow release of the active ingredient, whereas a bolus dose delivered directly into the vein (e.g., via an IV drip) ) can be delivered more quickly, allowing the concentration of active ingredients in the blood to quickly increase to effective levels. In other embodiments, the pharmaceutical composition may be administered as a continuous infusion, for example, by IV infusion, thereby providing a steady-state concentration of the active ingredient in the subject's body. Additionally, in other embodiments, a bolus dose of the pharmaceutical composition may be administered first, followed by a continuous infusion.
口服组合物可以采用散装液体溶液或混悬剂或散装粉剂形式。然而，更通常，为了便于精确地剂量给药，以单位剂量形式提供所述组合物。典型的单位剂量形式包括液体组合物的预装填的、预先测量的安瓿或注射器，或者在固体组合物情况下的丸剂、片剂、胶囊剂等。在这种组合物中，所述化合物通常为较少的组分(约0.1至约50重量％，或优选约1至约40重量％)，剩余部分为对于形成所需给药形式有用的各种载体或赋形剂以及加工助剂。Oral compositions may take the form of bulk liquid solutions or suspensions, or bulk powders. More typically, however, the compositions are provided in unit dosage form to facilitate precise dosing. Typical unit dosage forms include prefilled, premeasured ampoules or syringes for liquid compositions, or pills, tablets, capsules, and the like in the case of solid compositions. In such compositions, the compound will generally be a minor component (from about 0.1 to about 50% by weight, or preferably from about 1 to about 40% by weight), with the remainder being various components useful in forming the desired administration form. carriers or excipients and processing aids.
对于口服剂量，代表性的方案是，每天一个至五个口服剂量，尤其是两个至四个口服剂量，典型地是三个口服剂量。使用这些剂量给药模式，每个剂量提供大约0.01至大约20mg/kg的本发明化合物，优选的剂量各自提供大约0.1至大约10mg/kg，尤其是大约1至大约5mg/kg。For oral dosing, a typical regimen is one to five oral doses per day, especially two to four oral doses, typically three oral doses. Using these dosing modes, each dose provides from about 0.01 to about 20 mg/kg of a compound of the invention, with preferred doses each providing from about 0.1 to about 10 mg/kg, especially from about 1 to about 5 mg/kg.
为了提供与使用注射剂量类似的血液水平，或比使用注射剂量更低的血液水平，通常选择透皮剂量，数量为大约0.01至大约20％重量，优选大约0.1至大约20％重量，优选大约0.1至大约10％重量，且更优选大约0.5至大约15％重量。In order to provide similar blood levels, or lower blood levels than with injectable doses, a transdermal dose is generally selected in an amount of about 0.01 to about 20% by weight, preferably about 0.1 to about 20% by weight, preferably about 0.1 to about 10% by weight, and more preferably from about 0.5 to about 15% by weight.
从大约1至大约120小时，尤其是24至96小时，注射剂量水平在大约0.1mg/kg/小时至至少10mg/kg/小时的范围。为了获得足够的稳定状态水平，还可以给予大约0.1mg/kg至大约10mg/kg或更多的预载推注。对于40至80kg的人类患者来说，最大总剂量不能超过大约2g/天。Injectable dose levels range from about 0.1 mg/kg/hour to at least 10 mg/kg/hour from about 1 to about 120 hours, especially from 24 to 96 hours. To achieve adequate steady state levels, a preload bolus of about 0.1 mg/kg to about 10 mg/kg or more may also be given. For human patients weighing 40 to 80 kg, the maximum total dose should not exceed approximately 2 g/day.
适于口服给药的液体形式可包括合适的水性或非水载体以及缓冲剂、悬浮剂和分散剂、着色剂、调味剂，等等。固体形式可包括，例如，任何下列组份，或具有类似性质的化合物：粘合剂，例如，微晶纤维素、黄蓍胶或明胶；赋形剂，例如，淀粉或乳糖，崩解剂，例如，褐藻酸、Primogel或玉米淀粉；润滑剂，例如，硬脂酸镁；助流剂，例如，胶体二氧化硅；甜味剂，例如，蔗糖或糖精；或调味剂，例如，薄荷、水杨酸甲酯或橙味调味剂。Liquid forms suitable for oral administration may include suitable aqueous or non-aqueous carriers as well as buffering agents, suspending and dispersing agents, coloring agents, flavoring agents, and the like. Solid forms may include, for example, any of the following components, or compounds of similar nature: binders, for example, microcrystalline cellulose, tragacanth, or gelatin; excipients, for example, starch or lactose, disintegrants, For example, alginic acid, Primogel or corn starch; lubricant, for example, magnesium stearate; glidant, for example, colloidal silicon dioxide; sweetener, for example, sucrose or saccharin; or flavoring agent, for example, mint, water Methyl glycolate or orange flavoring.
可注射的组合物典型地基于可注射用的无菌盐水或磷酸盐缓冲盐水，或本领域中已知的其它可注射的赋形剂。如前所述，在这种组合物中，活性化合物典型地为较少的组分，经常为约0.05至10％重量，剩余部分为可注射的赋形剂等。Injectable compositions are typically based on injectable sterile saline or phosphate buffered saline, or other injectable excipients known in the art. As stated previously, in such compositions the active compound is typically a minor component, often about 0.05 to 10% by weight, with the remainder being injectable excipients and the like.
典型地将透皮组合物配制为含有活性组分的局部软膏剂或乳膏剂。当配制为软膏剂时，活性组分典型地与石蜡或可与水混溶的软膏基质组合。或者，活性组分可与例如水包油型乳膏基质一起配制为乳膏剂。这种透皮制剂是本领域中公知的，且通常包括用于提升活性组分或制剂的稳定的皮肤渗透的其它组份。所有这种已知的透皮制剂和组份包括在本发明提供的范围内。 Transdermal compositions are typically formulated as topical ointments or creams containing the active ingredients. When formulated as an ointment, the active ingredients are typically combined with a paraffin or water-miscible ointment base. Alternatively, the active ingredient may be formulated as a cream with, for example, an oil-in-water cream base. Such transdermal formulations are well known in the art and often include other ingredients for promoting stable skin penetration of the active ingredient or formulation. All such known transdermal formulations and components are included within the scope provided by this invention.
本发明化合物还可通过经皮装置给予。因此，经皮给药可使用贮存器(reservoir)或多孔膜类型、或者多种固体基质的贴剂实现。The compounds of the present invention may also be administered via transdermal devices. Thus, transdermal administration may be achieved using reservoir or porous membrane types, or a variety of solid matrix patches.
用于口服给予、注射或局部给予的组合物的上述组份仅仅是代表性的。其它材料以及加工技术等阐述于Remington's Pharmaceutical Sciences,17th edition,1985,Mack Publishing Company,Easton,Pennsylvania的第8部分中，本文以引用的方式引入该文献。The foregoing components of compositions for oral administration, injection or topical administration are merely representative. Other materials and processing techniques are described in Part 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, which article is incorporated by reference.
本发明化合物还可以以持续释放形式给予，或从持续释放给药***中给予。代表性的持续释放材料的描述可在Remington's Pharmaceutical Sciences中找到。The compounds of the present invention may also be administered in sustained release form or from a sustained release drug delivery system. Descriptions of representative sustained release materials can be found in Remington's Pharmaceutical Sciences.
本发明还涉及本发明化合物的药学上可接受的制剂。在一个实施方案中，所述制剂包含水。在另一个实施方案中，所述制剂包含环糊精衍生物。最常见的环糊精为分别由6、7和8个α-1,4-连接的葡萄糖单元组成的α-、β-和γ-环糊精，其在连接的糖部分上任选包括一个或多个取代基，其包括但不限于：甲基化的、羟基烷基化的、酰化的和磺烷基醚取代。在一些实施方案中，所述环糊精为磺烷基醚β-环糊精，例如，磺丁基醚β-环糊精，也称作Captisol。参见，例如，U.S.5,376,645。在一些实施方案中，所述制剂包括六丙基-β-环糊精(例如，在水中，10-50％)。The invention also relates to pharmaceutically acceptable formulations of the compounds of the invention. In one embodiment, the formulation includes water. In another embodiment, the formulation contains a cyclodextrin derivative. The most common cyclodextrins are α-, β- and γ-cyclodextrins consisting of 6, 7 and 8 α-1,4-linked glucose units respectively, optionally including a or multiple substituents including, but not limited to: methylated, hydroxyalkylated, acylated, and sulfoalkyl ether substitutions. In some embodiments, the cyclodextrin is a sulfoalkyl ether beta-cyclodextrin, for example, sulfobutyl ether beta-cyclodextrin, also known as Captisol. See, for example, U.S. 5,376,645. In some embodiments, the formulation includes hexapropyl-beta-cyclodextrin (eg, in water, 10-50%).
适应症Indications
对于被病毒感染导致的疾病，开发3C样蛋白酶抑制剂可以为大量的肿病人提供治疗获益。本发明中的化合物通过负调节病毒内3C样蛋白酶的活性发挥治疗作用，尤其是3C样蛋白酶具有P132H突变的病毒。For diseases caused by viral infections, the development of 3C-like protease inhibitors can provide therapeutic benefits for a large number of patients. The compounds in the present invention exert therapeutic effects by negatively regulating the activity of 3C-like protease in viruses, especially viruses with P132H mutation in the 3C-like protease.
在一些实施方案中，本发明的3C样蛋白酶抑制剂可以治疗由病毒感染导致的多种疾病及其并发症。In some embodiments, the 3C-like protease inhibitors of the present invention can treat various diseases caused by viral infections and their complications.
更具体地说，这些化合物可用于治疗病毒感染导致的如下疾病：发热、恶心、呕吐、头痛、呼吸困难、乏力、呼吸道感染、肺炎、嗅觉障碍、味觉障碍及其并发症等。More specifically, these compounds can be used to treat the following diseases caused by viral infections: fever, nausea, vomiting, headache, dyspnea, fatigue, respiratory tract infection, pneumonia, smell disorder, taste disorder and its complications, etc.
更具体地说，这些化合物可用于SARS-CoV-2感染导致的上述疾病或症状。More specifically, these compounds can be used for the above-mentioned diseases or symptoms caused by SARS-CoV-2 infection.
联合用药Combination medication
本发明所述的3C样蛋白酶抑制剂可以与其他药物联合治疗癌症，至少包含一种靶点药物/病毒活性调节剂，包括瑞德西韦(Remdesivir或GS-5734)、洛匹那韦(Lopinavir)、莫努匹韦(Molnupiravir)、利托那韦(Ritonavir)、氯喹(Chloroquine或Sigma-C6628)、羟氯喹和/或α-干扰素等。The 3C-like protease inhibitor of the present invention can be combined with other drugs to treat cancer, and contains at least one target drug/viral activity modulator, including Remdesivir (Remdesivir or GS-5734), Lopinavir (Lopinavir) ), Molnupiravir, Ritonavir, chloroquine (Chloroquine or Sigma-C6628), hydroxychloroquine and/or alpha-interferon, etc.
实施例Example
下文将结合具体实施例对本公开的化合物和制备方法做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明的内容所实现的技术方案均涵盖在本发明旨在保护的范围内。The compounds and preparation methods of the present disclosure will be described in further detail below with reference to specific examples. It should be understood that the following examples are only illustrative and explain the present invention and should not be construed as limiting the scope of the present invention. All technical solutions implemented based on the content of the present invention are covered by the scope of protection intended by the present invention.
除非另外指明，否则下述实施例中所使用的实验方法均为本领域常规方法；下述实施例中所使用的试剂、原料、仪器、设备等，均可从商业途径获得。Unless otherwise specified, the experimental methods used in the following examples are routine methods in the art; the reagents, raw materials, instruments, equipment, etc. used in the following examples can all be obtained from commercial sources.
实施例1Example 1
化合物1的合成
Synthesis of Compound 1
中间体1-2的合成Synthesis of intermediate 1-2
冰浴下将亚硝酸钠(4.44g，0.06mol)的水溶液(10mL)加入到中间体1-1(10g，0.05mol)的冰醋酸(100mL)溶液中并混合。将混合溶液在室温下搅拌6个小时，然后将反应混合物浓缩并向其中加入乙酸乙酯(200mL)稀释，用饱和碳酸氢钠溶液(100mL*2)洗涤，分离出有机相并用无水硫酸钠干燥，过滤。滤液减压浓缩得棕色固体状中间体1-2(10g)，直接用于下一步反应。An aqueous solution (10 mL) of sodium nitrite (4.44 g, 0.06 mol) was added to a solution of intermediate 1-1 (10 g, 0.05 mol) in glacial acetic acid (100 mL) under ice bath and mixed. The mixed solution was stirred at room temperature for 6 hours, then the reaction mixture was concentrated and diluted with ethyl acetate (200mL), washed with saturated sodium bicarbonate solution (100mL*2), the organic phase was separated and washed with anhydrous sodium sulfate Dry and filter. The filtrate was concentrated under reduced pressure to obtain brown solid intermediate 1-2 (10 g), which was directly used in the next reaction.
LCMS(ESI)m/z:198.0[M+H]+。LCMS(ESI)m/z:198.0[M+H] + .
中间体1-3的合成Synthesis of intermediates 1-3
室温下向中间体1-2(10g，0.05mol)的乙酸乙酯(200mL)溶液中加入三甲基氧鎓四氟硼酸盐(11.34g，0.08mol)。室温搅拌过夜，将反应混合物用水(200mL)和饱和氯化钠溶液(200mL)洗涤，分离出有机相并用无水硫酸钠干燥，过滤。滤液减压浓缩，粗品柱层析纯化(硅胶，石油醚:乙酸乙酯＝4:1)得黄色固体状中间体1-3(4.93g，产率46.1％)。To a solution of intermediate 1-2 (10 g, 0.05 mol) in ethyl acetate (200 mL) was added trimethyloxonium tetrafluoroborate (11.34 g, 0.08 mol) at room temperature. Stir at room temperature overnight, wash the reaction mixture with water (200 mL) and saturated sodium chloride solution (200 mL), separate the organic phase, dry over anhydrous sodium sulfate, and filter. The filtrate was concentrated under reduced pressure, and the crude product was purified by column chromatography (silica gel, petroleum ether:ethyl acetate=4:1) to obtain yellow solid intermediate 1-3 (4.93g, yield 46.1%).
LCMS(ESI)m/z:212.0[M+H]+。LCMS(ESI)m/z:212.0[M+H] + .
中间体1-4的合成Synthesis of intermediates 1-4
室温下将饱和氯化铵溶液(100mL)和还原铁粉(3.91g，0.07mol)加入中间体1-3(4.93g，0.02mol)的无水乙醇(100mL)溶液中。80℃搅拌过夜。冷却后过滤，滤液减压浓缩，加入乙酸乙酯(100mL)稀释。用水(100mL*2)洗涤，分离出有机相并用无水硫酸钠干燥，过滤。滤液减压浓缩，粗品经柱层析纯化(硅胶，石油醚:乙酸乙酯＝1:1)得棕色固体状中间体1-4(3.4g，产率80.9％)。Saturated ammonium chloride solution (100 mL) and reduced iron powder (3.91 g, 0.07 mol) were added to a solution of intermediate 1-3 (4.93 g, 0.02 mol) in absolute ethanol (100 mL) at room temperature. Stir overnight at 80°C. After cooling, filter, and the filtrate was concentrated under reduced pressure, and diluted with ethyl acetate (100 mL). Wash with water (100mL*2), separate the organic phase, dry with anhydrous sodium sulfate, and filter. The filtrate was concentrated under reduced pressure, and the crude product was purified by column chromatography (silica gel, petroleum ether: ethyl acetate = 1:1) to obtain brown solid intermediate 1-4 (3.4 g, yield 80.9%).
LCMS(ESI)m/z:182.2[M+H]+。LCMS(ESI)m/z:182.2[M+H] + .
中间体1-6的合成Synthesis of intermediates 1-6
0℃下向中间体1-5(10.0g，0.054mol)的N,N-二甲基甲酰胺(60mL)溶液中加入2-异氰酸-2-甲基丙烷(5.62g，0.057mol)和1,8-二氮杂双环[5.4.0]十一碳-7-烯(10.52g，0.069mol)。室温搅拌5小时，0℃下加入1,8-二氮杂双环[5.4.0]十一碳-7-烯(10.52g，0.069mol)和N,N-羰基二咪唑(10.51g，0.065 mol)。室温搅拌24小时。用1N HCl调pH至3-4，用乙酸乙酯萃取(200mL*3)，合并有机相，用饱和氯化钠溶液(100mL)洗涤，分离出有机相并且用无水硫酸钠干燥，过滤。滤液减压浓缩，粗品经柱层析纯化(硅胶，二氯甲烷:乙酸乙酯＝5:1)得白色固体状中间体1-6(5.23g,产率45.1％)。To a solution of intermediate 1-5 (10.0 g, 0.054 mol) in N,N-dimethylformamide (60 mL) was added 2-isocyanato-2-methylpropane (5.62 g, 0.057 mol) at 0°C. and 1,8-diazabicyclo[5.4.0]undec-7-ene (10.52g, 0.069mol). Stir at room temperature for 5 hours, then add 1,8-diazabicyclo[5.4.0]undec-7-ene (10.52g, 0.069mol) and N,N-carbonyldiimidazole (10.51g, 0.065 mol). Stir at room temperature for 24 hours. Adjust pH to 3-4 with 1N HCl, extract with ethyl acetate (200 mL*3), combine the organic phases, wash with saturated sodium chloride solution (100 mL), separate the organic phase, dry over anhydrous sodium sulfate, and filter. The filtrate was concentrated under reduced pressure, and the crude product was purified by column chromatography (silica gel, dichloromethane:ethyl acetate=5:1) to obtain white solid intermediate 1-6 (5.23g, yield 45.1%).
LCMS(ESI)m/z:252.0[M+Na]+。LCMS(ESI)m/z:252.0[M+Na] + .
中间体1-7的合成Synthesis of intermediates 1-7
将1-(溴甲基)-2,4,5-三氟苯(8.46g,37.6mmol)加入1-6(5.75g,25.0mmol)，碳酸钾(6.93g,50.0mmol)和乙腈(30mL)混合物中，85℃搅拌16小时。冷却，加入水(100mL)，乙酸乙酯萃取(200mL*3)，合并有机相并用饱和食盐水(100mL)洗涤，经无水硫酸钠干燥，过滤并减压浓缩滤液，粗品经柱层析纯化(硅胶，石油醚:乙酸乙酯＝2:1)得白色固体状中间体1-7(8.6g,产率:89％)。Add 1-(bromomethyl)-2,4,5-trifluorobenzene (8.46g, 37.6mmol), 1-6 (5.75g, 25.0mmol), potassium carbonate (6.93g, 50.0mmol) and acetonitrile (30mL ) mixture, stir at 85°C for 16 hours. Cool, add water (100mL), extract with ethyl acetate (200mL*3), combine the organic phases and wash with saturated brine (100mL), dry over anhydrous sodium sulfate, filter and concentrate the filtrate under reduced pressure, the crude product is purified by column chromatography (Silica gel, petroleum ether: ethyl acetate = 2:1) White solid intermediate 1-7 (8.6 g, yield: 89%) was obtained.
LCMS(ESI)m/z:318.0[M+H-56]+。LCMS(ESI)m/z:318.0[M+H-56] + .
中间体1-8的合成Synthesis of intermediates 1-8
将中间体1-7(6.7g,17.9mmol)溶于二氯甲烷和三氟乙酸(30/30mL)中，室温搅拌6小时。减压浓缩得白色固体1-8(7.8g)，直接用于下一步反应。Intermediate 1-7 (6.7g, 17.9mmol) was dissolved in dichloromethane and trifluoroacetic acid (30/30mL), and stirred at room temperature for 6 hours. Concentrate under reduced pressure to obtain white solid 1-8 (7.8g), which was directly used in the next reaction.
LCMS(ESI)m/z:318.0[M+H]+。LCMS(ESI)m/z:318.0[M+H] + .
中间体1-9的合成Synthesis of intermediates 1-9
将中间体1-8(160mg,0.5mmol)，吡啶-3-基硼酸(93mg,0.75mmol),醋酸铜(92mg,0.5mmol)，4-二甲氨基吡啶(247mg,2.0mmol),吡啶(100mg,1.2mmol)和二氧六环(10mL)的混合物在氧气球保护下在100℃搅拌16小时。冷却，加入水(30mL)，用乙酸乙酯萃取(3*30mL)，合并有机相用饱和食盐水(30mL)洗涤,无水硫酸钠干燥，过滤并减压浓缩，粗品经柱层析纯化(硅胶，二氯甲烷：甲醇＝20:1)得白色固体状中间体1-9(120mg，产率：40％)。Intermediate 1-8 (160mg, 0.5mmol), pyridin-3-ylboronic acid (93mg, 0.75mmol), copper acetate (92mg, 0.5mmol), 4-dimethylaminopyridine (247mg, 2.0mmol), pyridine ( A mixture of 100 mg, 1.2 mmol) and dioxane (10 mL) was stirred at 100°C for 16 hours under the protection of an oxygen sphere. Cool, add water (30mL), extract with ethyl acetate (3*30mL), wash the combined organic phases with saturated brine (30mL), dry over anhydrous sodium sulfate, filter and concentrate under reduced pressure, and the crude product is purified by column chromatography ( Silica gel, dichloromethane: methanol = 20:1) to obtain white solid intermediate 1-9 (120 mg, yield: 40%).
LCMS(ESI)m/z:395.0[M+H]+。LCMS(ESI)m/z:395.0[M+H] + .
化合物1(6-(6-氯-2-甲基-2H-吲唑-5-基)氨基)-3-(吡啶-3-基)-1-(2,4,5-三氟苄基)-1,3,5-三嗪-2,4(1H,3H)-二酮)的合成Compound 1(6-(6-chloro-2-methyl-2H-indazol-5-yl)amino)-3-(pyridin-3-yl)-1-(2,4,5-trifluorobenzyl )-1,3,5-Triazine-2,4(1H,3H)-dione) Synthesis
0℃下，向中间体1-9(120mg,0.3mmol)的四氢呋喃(20mL)溶液中依次加入双(三甲基硅基)氨基锂(0.6mmol,0.6mL,1.0M in THF)和中间体1-4(67mg,0.37mmol)。保持0℃搅拌2小时。饱和氯化铵溶液(30mL)淬灭，乙酸乙酯(3*30mL)萃取，合并有机相用饱和食盐水(30mL)洗涤，用无水硫酸钠干燥，过滤并减压浓缩滤液，粗品经高压制备色谱纯化(Gemini-C18 150x 21.2mm，5μm，乙腈-水(0.1％甲酸)，梯度：30％-60％)得白色固体1(14.5mg,产率:9％)。To a solution of intermediate 1-9 (120 mg, 0.3 mmol) in tetrahydrofuran (20 mL) at 0°C, lithium bis(trimethylsilyl)amide (0.6 mmol, 0.6 mL, 1.0 M in THF) and the intermediate were added successively 1-4 (67 mg, 0.37 mmol). Keep stirring at 0°C for 2 hours. Quench with saturated ammonium chloride solution (30 mL), extract with ethyl acetate (3*30 mL), wash the combined organic phases with saturated brine (30 mL), dry over anhydrous sodium sulfate, filter and concentrate the filtrate under reduced pressure, and the crude product is treated with high pressure Preparative chromatography purification (Gemini-C18 150x 21.2mm, 5μm, acetonitrile-water (0.1% formic acid), gradient: 30%-60%) gave white solid 1 (14.5mg, yield: 9%).
LCMS(ESI)m/z:513.9[M+H]+。LCMS(ESI)m/z:513.9[M+H] + .
1H NMR(400MHz,DMSO-d6,DCl in D2O)δ9.17(d,J＝2.0Hz,1H),9.05(d,J＝5.5Hz,1H),8.77–8.74(m,1H),8.54(s,1H),8.32-8.27(m,1H),7.88–7.82(m,2H),7.64–7.56(m,2H),5.34(s,2H),4.21(s,3H)。 1 H NMR (400MHz, DMSO-d 6 , DCl in D 2 O) δ9.17 (d, J = 2.0 Hz, 1H), 9.05 (d, J = 5.5 Hz, 1H), 8.77–8.74 (m, 1H ),8.54(s,1H),8.32-8.27(m,1H),7.88–7.82(m,2H),7.64–7.56(m,2H),5.34(s,2H),4.21(s,3H).
对比化合物1的合成
Synthesis of Comparative Compound 1
参照化合物1的合成方法合成中间体1-8以及中间体1-4。 Intermediate 1-8 and intermediate 1-4 were synthesized according to the synthesis method of compound 1.
中间体2-2的合成Synthesis of intermediate 2-2
常温下向1-8(250mg，0.79mmol)的N,N-二甲基甲酰胺(5mL)溶液中加入三乙胺(398mg，3.94mmol)，2-1(324mg，2.37mmol)，醋酸铜(242mg，1.35mmol)和分子筛(300mg)。混合溶液在60℃并且氧气流通下搅拌16小时。反应完成后，过滤，滤液减压浓缩。粗品用柱层析(硅胶，二氯甲烷:甲醇＝30:1)提纯得黄色油状化合物2-2(145.5mg，产率：45.1％)。To a solution of 1-8 (250 mg, 0.79 mmol) in N,N-dimethylformamide (5 mL), triethylamine (398 mg, 3.94 mmol), 2-1 (324 mg, 2.37 mmol), and copper acetate were added at room temperature. (242mg, 1.35mmol) and molecular sieve (300mg). The mixed solution was stirred at 60° C. with oxygen flow for 16 hours. After the reaction was completed, it was filtered and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, dichloromethane:methanol=30:1) to obtain yellow oily compound 2-2 (145.5 mg, yield: 45.1%).
LCMS(ESI)m/z:409.1[M+H]+。LCMS(ESI)m/z:409.1[M+H] + .
对比化合物1的合成Synthesis of Comparative Compound 1
冰浴下向2-2(106mg，0.26mmol)的四氢呋喃(2mL)溶液中加入1-4(56.3mg，0.31mmol)，氮气保护，0℃下加入双(三甲基硅基)氨基锂(0.65mL)并搅拌2小时。反应完成后，混合溶液加入水(10mL)淬灭，乙酸乙酯(10mL*3)萃取，合并有机相用无水硫酸钠干燥，过滤，滤液减压浓缩。粗品由高压制备色谱(色谱柱:-Gemini-C18 150x 21.2mm，5μm。流动项:ACN--H2O(0.05％NH3.H2O)。梯度:35-40.)纯化得到白色固体对比化合物1(49.5mg，产率：36.1％)。1-4 (56.3 mg, 0.31 mmol) was added to a solution of 2-2 (106 mg, 0.26 mmol) in tetrahydrofuran (2 mL) under ice bath, under nitrogen protection, and lithium bis(trimethylsilyl)amide (lithium bis(trimethylsilyl)amide) was added at 0°C. 0.65mL) and stir for 2 hours. After the reaction was completed, the mixed solution was quenched by adding water (10 mL), extracted with ethyl acetate (10 mL*3), the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by high-pressure preparative chromatography (Column: -Gemini-C18 150x 21.2mm, 5μm. Flow term: ACN--H 2 O (0.05% NH 3 .H 2 O). Gradient: 35-40.) to obtain a white solid. Comparative compound 1 (49.5 mg, yield: 36.1%).
LCMS(ESI)m/z:528.1[M+H]+。LCMS(ESI)m/z:528.1[M+H] + .
1H NMR(400MHz,CD3OD)δ8.43-8.32(m,2H),8.16(s,1H),7.70(s,2H),7.60(dd,J＝18.4,8.2Hz,1H),7.55-7.05(m,2H),5.34(s,2H),4.17(s,3H),2.39(s,3H). 1 H NMR (400MHz, CD 3 OD) δ8.43-8.32 (m, 2H), 8.16 (s, 1H), 7.70 (s, 2H), 7.60 (dd, J = 18.4, 8.2Hz, 1H), 7.55 -7.05(m,2H),5.34(s,2H),4.17(s,3H),2.39(s,3H).
生物学实施例Biological Examples
在进行以下生物学实施例时，待测化合物中的化合物1和对比化合物1通过实施例1制备，对比化合物2参考Y Unoh等人(J.Med.Chem.2022,65,9,6499–6512,DOI:10.1021/acs.jmedchem.2c00117)中描述的方法进行制备。When performing the following biological examples, Compound 1 and Comparative Compound 1 among the compounds to be tested were prepared according to Example 1, and Comparative Compound 2 was prepared with reference to Y Unoh et al. (J. Med. Chem. 2022, 65, 9, 6499-6512 , DOI: 10.1021/acs.jmedchem.2c00117).
一、Caco-2细胞模型评价化合物的双向渗透率1. Caco-2 cell model to evaluate the bidirectional permeability of compounds
1.1仪器设备及材料1.1 Instruments, equipment and materials
1)对照药propranolol和Digoxin购自MCE，Minoxidil购自中国食品药品检定研究院。1) The control drugs propranolol and Digoxin were purchased from MCE, and Minoxidil was purchased from China Institute of Food and Drug Control.
2)Caco-2细胞购自美国典型菌种保藏中心(ATCC)。2) Caco-2 cells were purchased from American Type Culture Collection (ATCC).
3)FBS培养基购自Sigma，DMEM购自Corning公司(Cambridge,MA)，非必需氨基酸(NEAA)，Hank’s平衡盐溶液(HBSS)和胰蛋白酶/EDTA均购自赛默飞世尔。青霉素、链霉素购自索莱宝。3) FBS medium was purchased from Sigma, DMEM was purchased from Corning Company (Cambridge, MA), non-essential amino acids (NEAA), Hank’s balanced salt solution (HBSS) and trypsin/EDTA were purchased from Thermo Fisher. Penicillin and streptomycin were purchased from Soleba.
4)HTS-96孔Transwell板和其他无菌耗材购自Corning公司。4) HTS-96-well Transwell plate and other sterile consumables were purchased from Corning Company.
5)Millicell电阻测定***购自Millipore。Vision购自Nexcelom Bioscience。Infinite 200 PRO酶标仪购自Tecan。MTS2/4 orbital摇床购自IKA Labortechnik。5) Millicell resistance measurement system was purchased from Millipore. Vision was purchased from Nexcelom Bioscience. Infinite 200 PRO microplate reader was purchased from Tecan. The MTS2/4 orbital shaker was purchased from IKA Labortechnik.
1.2试验设计1.2 Experimental design
1.2.1细胞培养和种板1.2.1 Cell culture and seed plates
1)使用含L-谷氨酰胺的高糖(4.5g/L)DMEM培养基，添加10％胎牛血清、0.1mg/mL链霉素和100单位的青霉素用于细胞培养。1) Use high-glucose (4.5g/L) DMEM medium containing L-glutamine, add 10% fetal calf serum, 0.1mg/mL streptomycin and 100 units of penicillin for cell culture.
2)Caco-2培养于细胞培养瓶。培养箱设置为37℃、5％CO2、保证相对湿度95％。细胞汇合度达到70‐90％时可用于接种Transwell。2) Caco-2 was cultured in cell culture flasks. The incubator was set to 37°C, 5% CO 2 , and guaranteed relative humidity of 95%. Cells that reach 70-90% confluence can be used to seed Transwells.
3)细胞接种前，向Transwell上室每孔中加入50μL细胞培养基，下层培养板内加入25mL细胞培养基。将培养板置于37℃,5％CO2培养箱内孵育1小时后可用于接种细胞。3) Before cell seeding, add 50 μL of cell culture medium to each well in the upper chamber of the Transwell, and add 25 mL of cell culture medium to the lower culture plate. Place the culture plate in a 37°C, 5% CO2 incubator and incubate it for 1 hour before seeding cells.
4)细胞消化后，吸取细胞混悬液转移至圆底离心管，于120g离心5分钟。4) After cell digestion, transfer the cell suspension to a round-bottomed centrifuge tube and centrifuge at 120g for 5 minutes.
5)使用培养基重悬细胞，终浓度为6.86×105cells/mL。将细胞悬液以50μL每孔加入到96孔Transwell培养板上室中，最终接种密度为2.4×105cells/cm2。5) Resuspend the cells in culture medium to a final concentration of 6.86×105 cells/mL. The cell suspension was added into the chamber of the 96-well Transwell culture plate at 50 μL per well, and the final seeding density was 2.4×105 cells/cm 2 .
6)接种后48小时开始换液，培养14-18天，隔一天换一次培养基。 6) Start changing the medium 48 hours after inoculation, culture for 14-18 days, and change the medium every other day.
7)更换培养基过程如下，将Transwell小室与接收板分开，先弃掉接收板中培养基然后再弃掉Transwell小室培养基，最后每个小室加入75μL新鲜培养基，接收板加入25mL新鲜培养基。7) The process of replacing the culture medium is as follows. Separate the Transwell chamber from the receiving plate. Discard the medium in the receiving plate first and then discard the medium in the Transwell chamber. Finally, add 75 μL of fresh culture medium to each chamber and 25 mL of fresh culture medium to the receiving plate. .
1.2.2细胞单层膜完整性的评价1.2.2 Evaluation of cell monolayer membrane integrity
1)Caco-2经过14-18天培养后，应完全汇合并完成分化。此时，可应用于穿透试验。1) After 14-18 days of culture, Caco-2 should be fully confluent and complete differentiation. At this time, it can be applied to penetration testing.
2)用电阻仪(Millipore，USA)测量单层膜电阻，记录每孔电阻。2) Use a resistance meter (Millipore, USA) to measure the resistance of the single layer film and record the resistance of each hole.
3)测定结束后，将Transwell培养板放回培养箱。3) After the measurement, put the Transwell culture plate back into the incubator.
4)电阻值的计算：测定电阻值(ohms)×膜面积(cm2)＝TEER值(ohms·cm2)，若TEER值<230ohms·cm2，则该孔不能用于穿透试验。4) Calculation of resistance value: Measure resistance value (ohms) × membrane area (cm 2 ) = TEER value (ohms·cm 2 ). If the TEER value is <230 ohms·cm 2 , the hole cannot be used for the penetration test.
1.2.3溶液配制1.2.3 Solution preparation
1)配制1L缓冲液(HBSS，10mM HEPES，pH 7.4)，分别称取2.38g HEPES，0.35g碳酸氢钠，加900mL纯水让其溶解，然后加100mL 10×HBSS搅拌均匀，调pH至7.4，最后过滤。1) Prepare 1L buffer solution (HBSS, 10mM HEPES, pH 7.4), weigh 2.38g HEPES and 0.35g sodium bicarbonate respectively, add 900mL pure water to dissolve, then add 100mL 10×HBSS, stir evenly, and adjust the pH to 7.4 , and finally filtered.
2)配制受试物的DMSO储备液。配制对照药的1mM DMSO储备液。用缓冲液稀释得到5μM工作液。受试物用缓冲液稀释液得到5μM工作液。最终体系的DMSO含量为0.5％。2) Prepare a DMSO stock solution of the test substance. Prepare a 1mM DMSO stock solution of the control drug. Dilute with buffer to obtain a 5 μM working solution. The test substance was diluted with buffer to obtain a 5 μM working solution. The DMSO content of the final system was 0.5%.
3)制备给药端溶液：
3) Prepare the drug-end solution:
4)制备接收端溶液：
4) Prepare the receiving end solution:
1.2.4药物穿透试验1.2.4 Drug penetration test
1)从培养箱中取出Caco-2Transwell培养板。使用缓冲液润洗细胞单层膜两次，37℃条件下孵育30分钟。1) Remove the Caco-2 Transwell culture plate from the incubator. Rinse the cell monolayer twice with buffer and incubate at 37°C for 30 minutes.
2)测定化合物由顶端到基底端的转运速率时，向***式培养板每孔(顶端)加入125μL待测化合物及对照药溶液，待测化合物接收板(基底端)每孔加入235μL HBSS(10mM HEPES,pH 7.4)缓冲液，对照药溶液接收板(基底端)每孔加入235μL HBSS(10mM HEPES,pH 7.4)缓冲液。从顶端溶液中转移50μL样品加到200μL含内标的乙腈(100nM阿普***,200nM拉贝洛尔,200nM咖啡因和2μM酪洛芬)作为0分钟顶端给药样品进行检测。2) When measuring the transport rate of compounds from the top to the basal end, add 125 μL of the test compound and control drug solution to each well of the insert culture plate (top), and add 235 μL of HBSS (10mM HEPES) to each well of the test compound receiving plate (basal end). , pH 7.4) buffer, add 235μL HBSS (10mM HEPES, pH 7.4) buffer to each well of the control drug solution receiving plate (basal end). Transfer 50 μL sample from the apical solution and add to 200 μL acetonitrile containing internal standard (100 nM alprazolam, 200 nM labetalol, 200 nM caffeine and 2 μM catoprofen) as a 0-minute apical administration sample for detection.
3)测定化合物由基底端到顶端的转运速率时，向接收板(基底端)每孔加入285μL待测化合物及对照药溶液，向待测化合物溶液***式培养板每孔(顶端)加入75μL HBSS(10mM HEPES,pH 7.4)缓冲液，向对照药溶液***式培养板每孔(顶端)加入75μL HBSS(10mM HEPES,pH 7.4)缓冲液。从基底端溶液中转移50μL样品加到200μL含内标的乙腈(100nM阿普***,200nM拉贝洛尔,200nM咖啡因和2μM酪洛芬)作为0分钟基底端给药样品进行检测。3) When measuring the transport rate of compounds from the basal end to the top, add 285 μL of the test compound and control drug solution to each well of the receiving plate (basal end), and add 75 μL of HBSS to each well (top) of the test compound solution insert culture plate. (10mM HEPES, pH 7.4) buffer, add 75 μL HBSS (10mM HEPES, pH 7.4) buffer to each well (top) of the control drug solution insert culture plate. Transfer 50 μL of sample from the basal end solution to 200 μL of acetonitrile containing internal standards (100 nM alprazolam, 200 nM labetalol, 200 nM caffeine and 2 μM typrofen) as a 0-minute basal end administration sample for detection.
4)37℃CO2培养箱中孵育2小时。4) Incubate in 37°C CO2 incubator for 2 hours.
5)转运实验结束后，从加药端(Ap→Bl方向取顶端，Bl→Ap方向取基底端)转移50μL样品到200μL含内标的乙腈(100nM阿普***,200nM拉贝洛尔,200nM咖啡因和2μM酪洛芬)。从接收端(Ap→Bl方向取基底端，Bl→Ap方向取顶端)转移50μL样品到加到200μL含内标的乙腈(100nM阿普***,200nM拉贝洛尔,200nM咖啡因和2μM酪洛芬)。涡旋5分钟。每个时间点的淬灭样品3220g离心30分钟。转移每个样品的上清液100μL到一个96孔上样盘中，同时在相对应的孔中加入100μL纯水。样品分析盘涡旋混匀后进行LC/MS/MS分析。所有孵育样品都做双平行。5) After the transfer experiment, transfer 50 μL of sample from the dosing end (take the top end in the direction of Ap → Bl, and take the basal end in the direction of Bl → Ap) to 200 μL of acetonitrile containing internal standard (100 nM alprazolam, 200 nM labetalol, 200 nM caffeine and 2 μM Tyroprofen). Transfer 50 μL of sample from the receiving end (take the basal end in the direction of Ap → Bl, take the top in the direction of Bl → Ap) to 200 μL of acetonitrile containing internal standards (100 nM alprazolam, 200 nM labetalol, 200 nM caffeine and 2 μM Tyrol). Finn). Vortex for 5 minutes. Quenched samples at each time point were centrifuged at 3220<i>g for 30 min. Transfer 100 μL of the supernatant of each sample to a 96-well sample plate, and add 100 μL of pure water to the corresponding well. Vortex and mix the sample analysis plate before performing LC/MS/MS analysis. All incubated samples were run in duplicate.
6)两个小时转运实验结束后测量荧光值，用水准备10mM荧光黄储备液，并且用转运缓冲溶 液稀释至100μM。Transwell小室(顶端)加入100μL荧光黄溶液，基底端加入300μL转运缓冲溶液，37℃，CO2培养箱中孵育30分钟。从顶端与基底端直接取出80μL溶液(使用基底外侧孔)并且转移到新的96孔板中。用酶标仪测量细胞荧光值(检测膜完整性)，激发波长为485nM，发射波长为530nM。6) After the two-hour transfer experiment, measure the fluorescence value, prepare a 10mM Lucifer Yellow stock solution with water, and dissolve it with transfer buffer. The solution was diluted to 100 μM. Add 100 μL of Lucifer Yellow solution to the Transwell chamber (top), add 300 μL of transport buffer solution to the basal end, and incubate in a CO 2 incubator at 37°C for 30 minutes. Remove 80 μL of solution directly from the apical and basal ends (using the basolateral wells) and transfer to a new 96-well plate. Use a microplate reader to measure cell fluorescence (to detect membrane integrity), with an excitation wavelength of 485nM and an emission wavelength of 530nM.
1.3数据分析1.3 Data analysis
数据计算均使用Excel进行。峰面积由离子色谱结果计算得出。化合物的表观渗透系数(Papp，单位：cm/s×10-6)用以下公式计算得出：
Data calculations were performed using Excel. Peak areas were calculated from ion chromatography results. The apparent permeability coefficient of the compound (P app , unit: cm/s×10 -6 ) is calculated using the following formula:
公式中：VA为接收端溶液的体积(Ap→Bl是0.235mL，Bl→Ap是0.075mL)，Area为Transwell-96孔板膜面积(0.143cm2)；time为孵育时间(单位：s)。In the formula: V A is the volume of the receiving end solution (Ap→Bl is 0.235mL, Bl→Ap is 0.075mL), Area is the Transwell-96-well plate membrane area (0.143cm 2 ); time is the incubation time (unit: s ).
Papp(B-A)为由基底端到顶端的表观渗透系数；Papp(A-B)为由顶端到基底端的表观渗透系数。P app(BA) is the apparent permeability coefficient from the basal end to the top; P app(AB) is the apparent permeability coefficient from the top to the basal end.
二、体外评估DPX2细胞的PXR的激活潜力2. In vitro evaluation of the activation potential of PXR in DPX2 cells
2.1材料及试剂2.1 Materials and reagents
1)DPX2(一种稳定转染PXR和荧光素酶***的HepG2细胞系)细胞来源于Puracyp Inc(Carlsbad,CA)。1) DPX 2 (a HepG2 cell line stably transfected with PXR and luciferase systems) cells were obtained from Puracyp Inc (Carlsbad, CA).
2)阳性对照药利福平购自Sigma公司(St.Louis,MO)。2) The positive control drug rifampicin was purchased from Sigma (St. Louis, MO).
3)CellTiter-FluorTM细胞活力测定试剂盒和One-Glo荧光素酶测定***购自Promega公司(Madison,WI)。FBS培养基购自Avantor，DPX2细胞的培养基和给药培养基购自Puracyp公司。3) CellTiter-Fluor TM cell viability assay kit and One-Glo luciferase assay system were purchased from Promega Company (Madison, WI). FBS medium was purchased from Avantor, and DPX 2 cell culture medium and administration medium were purchased from Puracyp.
4)试剂和耗材信息
4) Reagents and consumables information
2.2试验设计2.2 Experimental design
2.2.1细胞培养和种板2.2.1 Cell culture and seed plates
1)DPX2培养基和给药培养基的配置，450ml的培养基和给药培养基分别各加45ml的胎牛血清。1) Configuration of DPX 2 medium and dosing medium, add 45 ml of fetal bovine serum to each of 450 ml of culture medium and dosing medium.
2)DPX2培养于T-75细胞培养瓶。培养箱设置为37℃、5％CO2、保证相对湿度95％。细胞汇合度达到80-90％时可用于分离。2) DPX 2 was cultured in T-75 cell culture flask. The incubator was set to 37°C, 5% CO 2 , and guaranteed relative humidity of 95%. Cells can be used for isolation when they reach 80-90% confluence.
3)用10mL PBS冲洗T-75细胞培养瓶中培养的细胞后吸出，加入3mL胰蛋白酶，并在37℃下孵育约5分钟或直到细胞完全分离并漂浮，随后添加过量的含血清培养基来灭活胰蛋白酶终止消化。3) Rinse the cells cultured in the T-75 cell culture flask with 10 mL of PBS and aspirate, add 3 mL of trypsin, and incubate at 37°C for about 5 minutes or until the cells are completely detached and floating, and then add excess serum-containing medium. Inactivate trypsin to terminate digestion.
4)细胞消化后，吸取细胞混悬液转移至锥形底离心管，于150g离心5分钟。使用培养基重悬细胞，终浓度为4.5×105cells/mL。将细胞悬液以25μL每孔加入到384孔培养板中，将接种板放入培养箱中孵育24小时，随后细胞板可用于PXR实验研究。4) After cell digestion, transfer the cell suspension to a conical bottom centrifuge tube and centrifuge at 150g for 5 minutes. Resuspend the cells in culture medium to a final concentration of 4.5×10 5 cells/mL. Add the cell suspension to the 384-well culture plate at 25 μL per well, place the seeded plate in the incubator and incubate for 24 hours, and then the cell plate can be used for PXR experimental research.
2.2.2待测化合物的孵育 2.2.2 Incubation of compounds to be tested
1)溶液配置：配制1、10mM待测化合物和10mM阳性对照药利福平的DMSO储备液。待测化合物的最终浓度分别为1、10μM，利福平的终浓度为10μM。最终体系的DMSO含量为0.1％。空白对照是DMSO。1) Solution configuration: Prepare 1. DMSO stock solution of 10mM compound to be tested and 10mM positive control drug rifampicin. The final concentrations of the compounds to be tested were 1 and 10 μM, respectively, and the final concentration of rifampicin was 10 μM. The DMSO content of the final system was 0.1%. The blank control is DMSO.
2)从培养箱中取出细胞板，直接加入25nL空白对照溶液、含待测化合物溶液、含阳性对照药溶液，每组设置三个平行。加药后的细胞板转移至细胞培养箱继续孵育48小时。2) Take out the cell plate from the incubator, directly add 25nL blank control solution, solution containing the compound to be tested, and solution containing positive control drug, and set three parallels for each group. After adding the drug, the cell plate was transferred to the cell culture incubator and continued to incubate for 48 hours.
3)在测试之前检查细胞结构和单层完整性，确保其符合研究要求。3) Check the cell structure and monolayer integrity before testing to ensure it meets the research requirements.
2.2.3 PXR活性的测试2.2.3 Testing of PXR activity
1)给药孵育48小时后，细胞板可用于PXR活性的测试。1) After 48 hours of drug administration and incubation, the cell plate can be used to test PXR activity.
2)使CellTiter-FluorTM细胞活力测定试剂盒和ONE-Glo荧光素酶测定试剂达到室温。取10μL GF-AFC底物到测定缓冲液容器(10mL)中以形成2×试剂，然后加入10mL PBS稀释至1×试剂。将ONE-Glo荧光素酶测定底物转移到ONE-Glo荧光素酶测定缓冲液中。2) Let the CellTiter-Fluor TM cell viability assay kit and ONE-Glo luciferase assay reagent reach room temperature. Take 10 μL of GF-AFC substrate into the assay buffer container (10 mL) to make a 2× reagent, then add 10 mL of PBS to dilute to a 1× reagent. Transfer ONE-Glo Luciferase Assay Substrate to ONE-Glo Luciferase Assay Buffer.
3)从培养箱中取出DPX2的加药孵育细胞板，弃去细胞板中的培养基。将含有1×CellTiter-FluorTM细胞活力测定试剂倒入无菌加样槽中，使用多通道移液器，每孔加入25μL的试剂，将细胞板放入37℃CO2培养箱中孵育30分钟。3) Take out the DPX2 drug-incubated cell plate from the incubator and discard the culture medium in the cell plate. Pour the cell viability assay reagent containing 1×CellTiter-Fluor TM into a sterile loading tank, use a multi-channel pipette to add 25 μL of reagent to each well, and place the cell plate in a 37°C CO 2 incubator and incubate for 30 minutes. .
4)从细胞培养箱中取出384孔细胞板，短暂冷却至环境温度，用酶标仪测量细胞荧光值，激发波长为400nM，发射波长为505nM。4) Take out the 384-well cell plate from the cell culture incubator, briefly cool it to ambient temperature, and measure the cell fluorescence value with a microplate reader. The excitation wavelength is 400nM and the emission wavelength is 505nM.
5)将ONE-Glo测定试剂倒入无菌加样槽中，在每个孔中直接加入25μL试剂。轻摇细胞板至孔中溶液混匀。在室温下孵育5分钟后，使用酶标仪读取每个孔的化学发光数值。5) Pour the ONE-Glo assay reagent into the sterile sample tank and add 25 μL of reagent directly into each well. Gently shake the cell plate to mix the solution in the wells. After incubation for 5 minutes at room temperature, read the chemiluminescence value of each well using a microplate reader.
2.2.4数据分析2.2.4 Data analysis
1)数据计算均使用Excel进行。1) Data calculations are all performed using Excel.
2)荧光素酶活性由RLU/RFU确定，其中RLU表示测试化合物在每个浓度下三个平行样品的平均相对发光值，RFU表示测试化合物在每个浓度下三个平行样品的平均相对荧光值。2) Luciferase activity is determined by RLU/RFU, where RLU represents the average relative luminescence value of three parallel samples of the test compound at each concentration, and RFU represents the average relative fluorescence value of the three parallel samples of the test compound at each concentration. .
3)激活的倍数由如下公式计算：
3) The activation multiple is calculated by the following formula:
本发明化合物的激活倍数记载于表2。The activation multiples of the compounds of the present invention are reported in Table 2.
4)阳性对照百分比的计算公式为：
％Positive control＝(Fold activationtest compound/Fold activationpositive control compound)*100％4) The formula for calculating the percentage of positive control is:
%Positive control＝(Fold activation test compound /Fold activation positive control compound )*100%
三、小鼠静脉口服药代动力学3. Intravenous and oral pharmacokinetics in mice
1、给药信息
1. Medication information
动物饲喂控制：给药前过夜禁食，自由饮水，给药4小时后给予饲料。Animal feeding control: fast overnight before administration, drink water freely, and provide feed 4 hours after administration.
给药频率：实验当天单次给药。Frequency of administration: Single administration on the day of the experiment.
实际给药体积依据动物体重计算而得。The actual dosing volume was calculated based on the animal's body weight.
2、采集时间
2. Collection time
血样采集可接受的时间范围
Acceptable time frame for blood sample collection
3、血液样品的采集与处理过程
3. Blood sample collection and processing process
4、在体实验期评价
4. Evaluation during the in vivo experiment period
5、实验动物信息
5. Experimental animal information
6、样品分析6. Sample analysis
应用LC-MS/MS检测方法，应用随行标准曲线测定血浆样品中血药浓度。The LC-MS/MS detection method was applied, and the accompanying standard curve was used to determine the blood drug concentration in the plasma sample.
对血浆测定结果，将用WinNonlin 8.3(PhoenixTM)或其它类似软件进行药代动力学参数计算。如有适用的血浆药物浓度-时间数据，将计算药代动力学参数。For plasma measurement results, WinNonlin 8.3 (Phoenix ™ ) or other similar software will be used to calculate pharmacokinetic parameters. Pharmacokinetic parameters will be calculated if applicable plasma drug concentration-time data are available.
药代动力学数据通过描述性统计学如平均值、标准偏差和样本量进行描述。用Microsoft Excel 2013进行计算。也可进行其他药代动力学参数和统计学分析，并记录在数据总结中。 Pharmacokinetic data were described by descriptive statistics such as mean, standard deviation, and sample size. Calculations were performed using Microsoft Excel 2013. Other pharmacokinetic parameters and statistical analyzes can also be performed and recorded in the data summary.
四、酶学活性测定4. Determination of enzymatic activity
生化实验采用荧光共振能量迁移实验(FRET)测试方法。Biochemical experiments adopt fluorescence resonance energy transfer experiment (FRET) testing method.
测定体系(120μL)中含有108μL主蛋白酶(WT或P132H)，终浓度为150nM，无蛋白的小分子对照组加入108μL蛋白稀释液，稀释液组分为50mM Tris-HCL PH 7.4，1mM EDTA；The assay system (120μL) contains 108μL main protease (WT or P132H) with a final concentration of 150nM. The protein-free small molecule control group is added with 108μL protein diluent. The diluent components are 50mM Tris-HCL PH 7.4 and 1mM EDTA;
含有10μL底物，底物终浓度20μM；含有2μL不同浓度的待测小分子(3倍梯度稀释，稀释溶剂为100％DMSO)，此处阴性对照NC为加入2μL 100％DMSO。Contains 10 μL of substrate, the final concentration of the substrate is 20 μM; contains 2 μL of small molecules to be tested at different concentrations (3-fold gradient dilution, dilution solvent is 100% DMSO). The negative control NC here is to add 2 μL of 100% DMSO.
底物：荧光底物为MCA-AVLQSGFR-LYS(DNP)-Lys-NH2。Substrate: The fluorescent substrate is MCA-AVLQSGFR-LYS(DNP)-Lys-NH 2 .
反应体系其他组分为反应缓冲液50mM Tris-HCL pH 7.4,1mM EDTA。The other components of the reaction system are reaction buffer 50mM Tris-HCL pH 7.4, 1mM EDTA.
反应过程为：先将108μL主蛋白酶或者蛋白稀释液与2μL小分子混合加入96孔全黑酶标板(康宁costar，#3916)孵育30min。待孵育结束加入荧光底物，迅速在酶标仪开始检测。The reaction process is as follows: first mix 108 μL of main protease or protein diluent and 2 μL of small molecules, add them to a 96-well all-black enzyme plate (Corning costar, #3916), and incubate for 30 minutes. After the incubation is completed, add the fluorescent substrate and quickly start detection on the microplate reader.
酶标仪检测方法为：The microplate reader detection method is:
动力学监测过程，激发光波长320nm，发射光波长405nm，检测间隔约15s，总检测时长20min；记录该检测条件下各反应孔的不同时间的荧光值，计算时取前200s记录的荧光值(一般为7-10个数据点)。During the kinetic monitoring process, the excitation light wavelength is 320nm, the emission light wavelength is 405nm, the detection interval is about 15s, and the total detection time is 20min; the fluorescence value of each reaction well at different times under the detection conditions is recorded, and the fluorescence value recorded in the previous 200s is used for calculation ( Typically 7-10 data points).
本实验数据处理计算过程为：The data processing and calculation process of this experiment is:
首先，计算不同反应孔的各时间荧光值对时间的斜率[Slope(荧光值RFU：时间s)，记为反应初始速率V0。初速度V0＝Slope(200s内RFU：时间s)。First, calculate the slope of the fluorescence value of different reaction wells at each time versus time [Slope (fluorescence value RFU: time s), recorded as the initial reaction rate V 0 . Initial velocity V 0 =Slope (RFU within 200s: time s).
使用不同药物浓度对酶初反应速率V0与对照组酶初反应速率的比值确定代表性化合物对主蛋白酶的抑制率(公式一，公式二，以及公式二优化)，再使用GraphPad Prism非线性拟合曲线计算IC50值。Use the ratio of the initial reaction rate V 0 of the enzyme at different drug concentrations to the initial reaction rate of the enzyme in the control group to determine the inhibitory rate of the representative compound on the main protease (formula 1, formula 2, and formula 2 optimization), and then use GraphPad Prism nonlinear simulation Calculate the IC 50 value from the combined curve.
公式一：抑制率(％)＝(RFU100％酶活性对照-RFU样品)/(RFU100％酶活性对照-RFU空白对照)×100％Formula 1: Inhibition rate (%) = (RFU 100% enzyme activity control -RFU sample )/(RFU 100% enzyme activity control -RFU blank control ) × 100%
公式二：抑制率(％)＝(NC初速度V0-样品初速度V0)/NC初速度V0×100％；Formula 2: Inhibition rate (%) = (NC initial velocity V 0 - sample initial velocity V 0 )/NC initial velocity V 0 ×100%;
NC酶活定为100％，抑制率为0％NC enzyme activity is determined as 100%, and the inhibition rate is 0%
公式二优化：抑制率(％)＝(NC初速度V0-(样品初速度V0-无蛋白的小分子对照组V0)/NC初速度V0×100％用来消除V0(斜率)为负值的问题。Formula 2 Optimization: Inhibition rate (%) = (NC initial velocity V 0 - (sample initial velocity V 0 - protein-free small molecule control group V 0 )/NC initial velocity V 0 ×100% is used to eliminate V 0 (slope ) is a negative value.
使用GraphPad Prism软件非线性拟合曲线{Nonlinear regression(curve fit)-[inhibitor]vs.response-Variable slope(four parameters)}拟合出半数抑制浓度IC50值以及抑制曲线。Use GraphPad Prism software nonlinear fitting curve {Nonlinear regression (curve fit)-[inhibitor] vs. response-Variable slope (four parameters)} to fit the half inhibitory concentration IC50 value and inhibition curve.
注:本测试中每个测试样品(样品以及对照品)均组内三次平行重复，并进行三次生物重复实验，并以生物重复实验进行标准差分析。测试过程一般使用公式二优化计算抑制率。Note: In this test, each test sample (sample and control substance) was repeated three times in parallel within the group, and three biological replicate experiments were performed, and the standard deviation analysis was performed based on the biological replicate experiment. The test process generally uses Formula 2 to optimize the calculation of the inhibition rate.
五、抗SARS-COV-25. Anti-SARS-COV-2
(一)实验材料(1) Experimental materials
细胞系：非洲绿猴肾细胞Vero E6(ATCC，CRL-1586),人结直肠腺癌细胞Caco-2(ATCC,HTB-37)和人肺腺癌细胞Calu-3(ATCC,HTB-55)。Cell lines: African green monkey kidney cells Vero E6 (ATCC, CRL-1586), human colorectal adenocarcinoma cells Caco-2 (ATCC, HTB-37) and human lung adenocarcinoma cells Calu-3 (ATCC, HTB-55) .
病毒株：2019-nCoV-WIV04(IVCAS 6.7512)Virus strain: 2019-nCoV-WIV04(IVCAS 6.7512)
感染剂量：MOI＝0.01Infectious dose: MOI=0.01
(二)实验方法(2) Experimental methods
1)将100μL含2×104细胞接种至96孔板中，放置于37℃恒温恒湿培养箱过夜培养；1) Inoculate 100 μL of 2 × 10 4 cells into a 96-well plate and place it in a 37°C constant temperature and humidity incubator for overnight culture;
2)细胞贴壁20小时后吸弃培养液，每孔分别加入100μL含指定浓度测试化合物+CP-100356的培养液。每种测试化合物设置8个稀释度，每个稀释度设置3～4个复孔，同时设置DMSO处理组 和正常细胞组。除正常细胞组外，其它孔加入含0.01MOI病毒完全培养液，37℃恒温恒湿培养箱孵育72小时。2) After the cells have adhered for 20 hours, aspirate the culture medium and add 100 μL of culture medium containing the specified concentration of test compound + CP-100356 to each well. Set up 8 dilutions for each test compound, set up 3 to 4 duplicate wells for each dilution, and set up a DMSO treatment group at the same time and normal cell groups. Except for the normal cell group, complete virus culture medium containing 0.01 MOI was added to other wells and incubated in a 37°C constant temperature and humidity incubator for 72 hours.
3)在感染后72小时使用全视野细胞扫描仪记录细胞病变率，抑制率＝(1－测试化合物组的病变率)×100％。3) Use a full-field cell scanner to record the cell lesion rate 72 hours after infection, and the inhibition rate = (1 - the lesion rate of the test compound group) × 100%.
4)根据抑制率结果，进行四参数拟合计算EC50。4) Based on the inhibition rate results, perform four-parameter fitting to calculate EC 50 .
六、抗耐药突变L50F+E166A+L167F SARS-COV-26. Anti-drug resistance mutation L50F+E166A+L167F SARS-COV-2
(一)原核表达载体的构建(1) Construction of prokaryotic expression vector
1)突变引物设计与合成1) Design and synthesis of mutation primers
运用Primer软件设计L50F、E166A+L167F的突变引物，引物由生工生物工程(上海)股份有限公司合成，引物序列下表所示：
Use Primer software to design mutation primers for L50F and E166A+L167F. The primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The primer sequences are shown in the table below:
2)突变质粒的克隆与转化2) Cloning and transformation of mutant plasmid
以野生型的主蛋白酶质粒为模板，采用Fast Mutagenesis System单点突变试剂盒(购于TRAN公司)进行PCR扩增，扩增产物用DMT限制性内切酶消化甲基化质粒模板后，转化至具有降解甲基化质粒的感受态细胞。具体操作如下：Using the wild-type main protease plasmid as the template, use the Fast Mutagenesis System single point mutation kit (purchased from TRAN Company) for PCR amplification. After the amplified product is digested with DMT restriction endonuclease, the methylated plasmid template is transformed into Competent cells with degrading methylation plasmid. The specific operations are as follows:
PCR体系与条件
PCR system and conditions
a)PCR:94℃5min，94℃30s，66℃20s，72℃1min，30cycles，72℃10min。a) PCR: 94℃5min, 94℃30s, 66℃20s, 72℃1min, 30cycles, 72℃10min.
b)PCR产物的消化：加1uL DMT酶于PCR产物中，混匀后37℃孵育1小时。b) Digestion of PCR product: Add 1uL DMT enzyme to the PCR product, mix and incubate at 37°C for 1 hour.
c)转化：加入8uL消化后的产物于50uL感受态细胞中，混匀后冰浴30min；42℃水浴热激45s，立即置于冰上静置3min；加入250uL平衡至室温的无抗LB培养基，200rpm、37℃培养1小时；取100uL菌液均匀涂布在含抗性的平板上，37℃培养过夜。c) Transformation: Add 8uL of the digested product to 50uL of competent cells, mix well and then incubate on ice for 30 minutes; heat shock in a 42°C water bath for 45 seconds, immediately place on ice and let stand for 3 minutes; add 250uL of anti-antibody LB culture that has been equilibrated to room temperature. Base, incubate at 200 rpm and 37°C for 1 hour; take 100uL bacterial liquid and spread it evenly on the plate containing the resistance, and incubate at 37°C overnight.
d)单克隆鉴定：挑选单菌落送至生工生物工程(上海)股份有限公司进行测序；测序正确后提取质粒转化至BL21感受态细胞进行蛋白的表白。d) Single clone identification: Select a single colony and send it to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing; after sequencing is correct, the plasmid is extracted and transformed into BL21 competent cells for protein expression.
(二)突变蛋白的表达与纯化(2) Expression and purification of mutant proteins
1)蛋白的原核表达1) Prokaryotic expression of protein
将构建成功的突变质粒转化至BL21(DE3)感受态细胞，次日挑选单菌落，37℃扩大培养至菌液D600纳米值达到0.6～0.8，加入终浓度为0.5mol/L的IPTG过夜诱导表达。Transform the successfully constructed mutant plasmid into BL21 (DE3) competent cells. Select a single colony the next day and expand the culture at 37°C until the D 600 nm value of the bacterial solution reaches 0.6-0.8. Add IPTG with a final concentration of 0.5 mol/L for overnight induction. Express.
蛋白的Ni-NAT亲和层析纯化与分子筛纯化Ni-NAT affinity chromatography purification and molecular sieve purification of proteins
缓冲液：Buffer:
a)亲和层析基础缓冲液：50mM Tris，500mM Nacl，10％甘油，pH＝8.0； a) Affinity chromatography basic buffer: 50mM Tris, 500mM Nacl, 10% glycerol, pH=8.0;
b)分子筛缓冲液：25mM Heps，150mM Nacl，pH＝7.4。b) Molecular sieve buffer: 25mM Heps, 150mM Nacl, pH=7.4.
收集过夜表达的菌液沉淀，高压破碎处理后以18000rpm离心40min，收集上清进行Ni-NAT亲和层析纯化。镍柱亲和层析柱处理：将镍柱先使用ddH2O清洗，再使用平衡缓冲液平衡柱子，将过滤后的菌液上清与镍柱亲和层析凝胶结合，使用含20mM咪唑浓度洗杂缓冲液进行洗杂，使用含500mM咪唑浓度洗脱缓冲液进行洗脱。Collect the precipitate of the bacterial solution expressed overnight, perform high-pressure disruption and centrifuge at 18,000 rpm for 40 min, and collect the supernatant for Ni-NAT affinity chromatography purification. Nickel column affinity chromatography column treatment: Clean the nickel column with ddH2O first, then use equilibrium buffer to equilibrate the column, combine the filtered bacterial supernatant with the nickel column affinity chromatography gel, and wash with 20mM imidazole. The impurity buffer was used to wash the impurities, and the elution buffer containing 500mM imidazole concentration was used for elution.
收集破碎后的菌液上清、沉淀、流穿液、各浓度梯度洗脱下来的蛋白样品，煮沸后进行SDS-PAGE电泳验证蛋白的表达情况。将洗脱下来的蛋白使用截留分子量为30KD的超滤管以3500rpm 4℃进行浓缩。Collect the supernatant, precipitate, flow-through fluid, and protein samples eluted from each concentration gradient after the disruption, and perform SDS-PAGE electrophoresis after boiling to verify the protein expression. The eluted protein was concentrated using an ultrafiltration tube with a molecular weight cutoff of 30KD at 3500 rpm and 4°C.
将蛋白浓缩至AKTA PURIFIER分子筛的标准上样体积(约0.5ml)，利用GE Healthcare产品Superdex 200 Increase对蛋白进行进一步纯化。收集分子筛纯化后的蛋白样浓缩至10mg/ml，分装冻存于-80℃备用。Concentrate the protein to the standard loading volume of AKTA PURIFIER molecular sieve (about 0.5ml), and use GE Healthcare product Superdex 200 Increase to further purify the protein. Collect the protein samples purified by molecular sieve and concentrate to 10 mg/ml, aliquot and freeze at -80°C for later use.
(三)荧光共振能量迁移实验测试(FRET)测Ki值(3) Fluorescence resonance energy transfer experimental test (FRET) to measure Ki value
1)反应buffer：50mM Tris pH 7.4,1mM EDTA,0.01％tritonX-100。1) Reaction buffer: 50mM Tris pH 7.4, 1mM EDTA, 0.01% tritonX-100.
2)测试体系2) Test system
蛋白(108uL)：主蛋白酶(L50F+E166A+L167F)，终浓度为500nM，无蛋白的小分子对照组加入108uL反应buffer；Protein (108uL): main protease (L50F+E166A+L167F), the final concentration is 500nM, add 108uL reaction buffer to the protein-free small molecule control group;
底物(10uL)：荧光底物MCA-AVLQ-SGFR-Lys(Dnp)-Lys-NH2(24mM保存)，用DMSO将24mM底物稀释至0.06mM和0.12mM,使得其在反应体系中的终浓度分别为5uM和10uM。Substrate (10uL): Fluorescent substrate MCA-AVLQ-SGFR-Lys(Dnp)-Lys-NH2 (stored at 24mM). Use DMSO to dilute 24mM substrate to 0.06mM and 0.12mM, so that its final concentration in the reaction system The concentrations are 5uM and 10uM respectively.
小分子(2uL)：化合物1、对比化合物1、对比化合物2(50mM保存)，用100％DMSO将50mM浓度的小分子稀释至0.06mM，使得其在反应体系中的终浓度为1uM。梯度稀释小分子：上述小分子从浓度为0.06mM(终浓度1uM)开始2倍倍比稀释至第8孔。此处阴性对照NC为加入2uL 100％DMSO。Small molecules (2uL): Compound 1, Comparative Compound 1, Comparative Compound 2 (saved at 50mM), use 100% DMSO to dilute the 50mM concentration of small molecules to 0.06mM, so that the final concentration in the reaction system is 1uM. Gradient dilution of small molecules: The above small molecules are diluted 2-fold starting from a concentration of 0.06mM (final concentration 1uM) to the 8th well. The negative control NC here is to add 2uL 100% DMSO.
3)反应过程3) reaction process
先将108uL主蛋白酶或者反应buffer与2uL小分子混合加入96孔全黑酶标板(康宁costar，#3916)孵育30min。待孵育结束加入荧光底物，迅速在酶标仪开始检测。First, mix 108uL of main protease or reaction buffer and 2uL of small molecules and add it to a 96-well black enzyme plate (Corning costar, #3916) and incubate for 30 minutes. After the incubation is completed, add the fluorescent substrate and quickly start detection on the microplate reader.
4)板的布局：
4) Board layout:
5)酶标仪检测方法5) Microplate reader detection method
动力学检测过程，激发光波长320nM，发射光波长405nM，检测间隔约15s，总检测时长为20min；记录该检测条件下各反应孔中的不同时间的荧光值，计算时取前200s记录的荧光值(一般为7-10个数据点)During the kinetic detection process, the excitation light wavelength is 320nM, the emission light wavelength is 405nM, the detection interval is about 15s, and the total detection time is 20min; the fluorescence values in each reaction well at different times under the detection conditions are recorded, and the fluorescence recorded in the previous 200s is used for calculation. value (typically 7-10 data points)
6)数据分析 6)Data analysis
a)斜率计算：选取前200s内的荧光值计算斜率，减掉空白组斜率后得出每个小分子浓度下的V，计算1/V后通过GraphPad Prism8.0.1进行Dixon作图。a) Slope calculation: Select the fluorescence value within the first 200s to calculate the slope. Subtract the slope of the blank group to obtain V at each small molecule concentration. After calculating 1/V, perform Dixon plotting through GraphPad Prism8.0.1.
b)Dixon作图求Ki：测试在不同的两个底物浓度下，从不同固定I下所作一簇直线的斜率1/V与相应的I作图，不同底物浓度下拟合所得直线交点即为-Ki。b) Dixon plot to find Ki: test under two different substrate concentrations, plot the slope 1/V of a cluster of straight lines drawn under different fixed I with the corresponding I, and fit the intersection points of the straight lines under different substrate concentrations. That is -Ki.
本发明化合物和对比化合物1-2的结构和数据对比如下表1和表2所示。A comparison of the structures and data of the compounds of the present invention and comparative compounds 1-2 is shown in Table 1 and Table 2 below.
表1
Table 1
表2
Table 2
注：Note:
Caco2：Papp(B-A)为由基底端到顶端的表观渗透系数；Papp(A-B)为由顶端到基底端的表观渗透系数Caco2: P app (BA) is the apparent permeability coefficient from the basal end to the apical end; P app (AB) is the apparent permeability coefficient from the apical end to the basal end.
IV：静脉注射IV: intravenous injection
PO：口服PO: Orally
T1/2：半衰期T 1/2 : half-life
Cmax：血药峰浓度 Cmax : peak plasma concentration
AUClast：药时曲线下面积，评价药物吸收程度AUC last : area under the drug-time curve, to evaluate the degree of drug absorption
F％：生物利用度F%: Bioavailability
PXR：受体激活诱导PXR: Induction of receptor activation
上述数据表明，本发明化合物在保持优异的抑制活性的同时，还实现了更优的稳定性，更低的药物副作用，更优的药代动力学性质，以及针对拟肽类抗SARS-COV-2药物ALG-097161压力筛选下所产生的耐药突变L50F+E166A+L167F主蛋白酶相比对比化合物更优的生物活性。The above data show that while maintaining excellent inhibitory activity, the compound of the present invention also achieves better stability, lower drug side effects, better pharmacokinetic properties, and is effective against peptoids against SARS-COV- 2. The drug resistance mutation L50F+E166A+L167F main protease produced under the pressure screening of drug ALG-097161 has better biological activity than the comparative compound.
具体来说，Caco2渗透性实验表征的是药物的渗透效果，Papp(B-A)表示从血液到小肠中的渗透效果， Papp(A-B)表示从小肠到血液中的渗透效果，本发明化合物从小肠到血液中的渗透效果优于另外两种对比化合物，且更不容易从血液中渗透到小肠中，即，药物渗透效果优异。Specifically, the Caco2 permeability experiment characterizes the penetration effect of the drug, and P app (BA) represents the penetration effect from the blood to the small intestine. P app (AB) represents the penetration effect from the small intestine into the blood. The penetration effect of the compound of the present invention from the small intestine into the blood is better than that of the other two comparative compounds, and it is less likely to penetrate from the blood into the small intestine, that is, drug penetration Excellent results.
在PK方面，化合物1的半衰期，无论是静脉注射还是口服，其半衰期均高于对比化合物1及对比化合物2，表明药物在体内的消除速度更低，具有更加优异的体内稳定性。而且在药物达峰浓度(Cmax)保持一致的情况下，其AUClast数值明显高于其余两个化合物，表明化合物1具有优异的吸收以及体内暴露量。此外，化合物1具有更加优异的生物利用度，相较于其余两个化合物具有更优异的药代动力学性质。In terms of PK, the half-life of Compound 1, whether administered intravenously or orally, is higher than that of Comparative Compound 1 and Comparative Compound 2, indicating that the elimination rate of the drug in the body is lower and it has better in vivo stability. Moreover, when the peak drug concentration (C max ) remains consistent, its AUC last value is significantly higher than the other two compounds, indicating that compound 1 has excellent absorption and in vivo exposure. In addition, compound 1 has better bioavailability and better pharmacokinetic properties than the other two compounds.
PXR表示药物的相互作用，具体来说，PXR活化诱导代谢酶(例如CYP3A4)，代谢酶可以影响外源性物质和内源性物质的药代动力学，CYP3A4酶的过度活化会诱导CYP3A4介导的抗肿瘤药、止痛药等加速代谢，影响联合用药的药效。本发明化合物1在不同浓度下的PXR值都明显低于对比化合物1和2，具有较低的药物相互作用风险及毒性代谢风险。PXR represents a drug interaction. Specifically, PXR activation induces metabolic enzymes (such as CYP3A4). Metabolic enzymes can affect the pharmacokinetics of exogenous and endogenous substances. Overactivation of CYP3A4 enzymes can induce CYP3A4-mediated Anti-tumor drugs, analgesics, etc. accelerate metabolism and affect the efficacy of combined drugs. The PXR value of Compound 1 of the present invention at different concentrations is significantly lower than that of Comparative Compounds 1 and 2, and it has a lower risk of drug interaction and toxic metabolism.
化合物1的抗耐药突变L50F+E166A+L167F主蛋白酶的生化活性较对比化合物1和2更具有优势。 The biochemical activity of the main protease of the anti-drug resistance mutation L50F+E166A+L167F of compound 1 is more advantageous than that of comparative compounds 1 and 2.

Claims (10)

  1. 式(I)化合物，或其药学上可接受的盐或酯，它们的立体异构体或互变异构体、消旋体、氮氧化物、多晶型、水合物、溶剂合物、同位素标记物、前药或代谢产物：
    Compounds of formula (I), or pharmaceutically acceptable salts or esters thereof, their stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs, hydrates, solvates, isotopes Markers, prodrugs or metabolites:
  2. 药物组合物，其包含权利要求1的式(I)化合物，或其药学上可接受的盐或酯，它们的立体异构体或互变异构体、消旋体、氮氧化物、多晶型、水合物、溶剂合物、同位素标记物、前药或代谢产物，和任选地包含至少一种生理学上/药学上可接受的辅料；Pharmaceutical compositions comprising the compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt or ester thereof, their stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs forms, hydrates, solvates, isotopic labels, prodrugs or metabolites, and optionally contains at least one physiologically/pharmaceutically acceptable excipient;
    任选地，所述药物组合物还包含其它活性成分；Optionally, the pharmaceutical composition further contains other active ingredients;
    优选地，所述其它活性成分选自：瑞德西韦(Remdesivir或GS-5734)、洛匹那韦(Lopinavir)、莫努匹韦(Molnupiravir)、利托那韦(Ritonavir)、氯喹(Chloroquine或Sigma-C6628)、羟氯喹或α-干扰素；Preferably, the other active ingredients are selected from: Remdesivir (Remdesivir or GS-5734), Lopinavir, Molnupiravir, Ritonavir, Chloroquine or Sigma-C6628), hydroxychloroquine or alpha-interferon;
    优选地，所述药物组合物是RNA依赖性RNA聚合酶抑制剂、3CLpro蛋白酶抑制剂、CYP3A4抑制剂或靶向宿主的抗病毒药物。Preferably, the pharmaceutical composition is an RNA-dependent RNA polymerase inhibitor, a 3CLpro protease inhibitor, a CYP3A4 inhibitor or a host-targeted antiviral drug.
  3. 试剂盒，其包含权利要求1的式(I)化合物，或其药学上可接受的盐或酯，它们的立体异构体或互变异构体、消旋体、氮氧化物、多晶型、水合物、溶剂合物、同位素标记物、前药或代谢产物，和其它活性成分；A kit comprising the compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt or ester thereof, their stereoisomers or tautomers, racemates, nitrogen oxides, and polymorphs. , hydrates, solvates, isotopic markers, prodrugs or metabolites, and other active ingredients;
    优选地，所述其它活性成分选自：瑞德西韦、洛匹那韦、莫努匹韦、利托那韦、氯喹、羟氯喹或α-干扰素。Preferably, the other active ingredients are selected from: remdesivir, lopinavir, monupivir, ritonavir, chloroquine, hydroxychloroquine or alpha-interferon.
  4. 根据权利要求1的式(I)化合物或其药学上可接受的盐或酯，它们的立体异构体或互变异构体、消旋体、氮氧化物、多晶型、水合物、溶剂合物、同位素标记物、前药或代谢产物，和任选地其它活性成分，在制备用于治疗或预防病毒感染的药物中的用途；The compound of formula (I) according to claim 1 or its pharmaceutically acceptable salt or ester, their stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs, hydrates, solvents The use of compounds, isotopic labels, prodrugs or metabolites, and optionally other active ingredients, in the preparation of medicaments for the treatment or prevention of viral infections;
    优选地，所述药物用于治疗或预防病毒感染导致的疾病、病症、症候群和/或紊乱；Preferably, the medicament is used to treat or prevent diseases, conditions, syndromes and/or disorders caused by viral infections;
    优选地，所述药物中存在其它活性成分，并且所述化合物与其它活性成分存在于同一单位剂型中；Preferably, other active ingredients are present in the medicament, and the compound is present in the same unit dosage form as the other active ingredients;
    优选地，所述药物中存在其它活性成分，并且所述化合物和其它活性成分存在于分开的单位剂型中；Preferably, other active ingredients are present in the medicament and the compound and other active ingredients are present in separate unit dosage forms;
    优选地，所述其它活性成分选自：瑞德西韦、洛匹那韦、莫努匹韦、利托那韦、氯喹、羟氯喹或α-干扰素。Preferably, the other active ingredients are selected from: remdesivir, lopinavir, monupivir, ritonavir, chloroquine, hydroxychloroquine or alpha-interferon.
  5. 根据权利要求1的式(I)化合物或其药学上可接受的盐或酯，它们的立体异构体或互变异构体、消旋体、氮氧化物、多晶型、水合物、溶剂合物、同位素标记物、前药或代谢产物在制备与其它活性成分组合使用的用于治疗或预防病毒感染的药物中的用途；The compound of formula (I) according to claim 1 or its pharmaceutically acceptable salt or ester, their stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs, hydrates, solvents The use of compounds, isotopic labels, prodrugs or metabolites in the preparation of medicaments for use in combination with other active ingredients for the treatment or prevention of viral infections;
    优选地，所述药物用于治疗或预防病毒感染导致的疾病、病症、症候群和/或紊乱；Preferably, the medicament is used to treat or prevent diseases, conditions, syndromes and/or disorders caused by viral infections;
    优选地，所述其它活性成分选自：瑞德西韦、洛匹那韦、莫努匹韦、利托那韦、氯喹、羟氯喹或α-干扰素。Preferably, the other active ingredients are selected from: remdesivir, lopinavir, monupivir, ritonavir, chloroquine, hydroxychloroquine or alpha-interferon.
  6. 其它活性成分在制备与根据权利要求1的式(I)化合物或其药学上可接受的盐或酯，它们的立 体异构体或互变异构体、消旋体、氮氧化物、多晶型、水合物、溶剂合物、同位素标记物、前药或代谢产物组合使用的用于治疗或预防病毒感染的药物中的用途；Other active ingredients in preparation with the compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt or ester thereof, their immediate Combinations of isomers or tautomers, racemates, nitrogen oxides, polymorphs, hydrates, solvates, isotopic markers, prodrugs or metabolites for the treatment or prevention of viral infections Use in medicines;
    优选地，所述药物用于治疗或预防病毒感染导致的疾病、病症、症候群和/或紊乱；Preferably, the medicament is used to treat or prevent diseases, conditions, syndromes and/or disorders caused by viral infections;
    优选地，所述其它活性成分选自：瑞德西韦、洛匹那韦、莫努匹韦、利托那韦、氯喹、羟氯喹或α-干扰素。Preferably, the other active ingredients are selected from: remdesivir, lopinavir, monupivir, ritonavir, chloroquine, hydroxychloroquine or alpha-interferon.
  7. 一种在受试者中治疗或预防病毒感染的方法，包括向所述受试者给药权利要求1的式(I)化合物，或其药学上可接受的盐或酯，它们的立体异构体或互变异构体、消旋体、氮氧化物、多晶型、水合物、溶剂合物、同位素标记物、前药或代谢产物，或权利要求2的药物组合物，或权利要求3的试剂盒；A method of treating or preventing viral infection in a subject, comprising administering to the subject a compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt or ester thereof, their stereoisomers isomers or tautomers, racemates, nitrogen oxides, polymorphs, hydrates, solvates, isotopic markers, prodrugs or metabolites, or the pharmaceutical composition of claim 2, or claim 3 test kit;
    优选地，所述方法为治疗或预防病毒感染导致的疾病、病症、症候群和/或紊乱的方法；Preferably, the method is a method of treating or preventing diseases, conditions, syndromes and/or disorders caused by viral infection;
    优选地，所述其它活性成分选自：瑞德西韦、洛匹那韦、莫努匹韦、利托那韦、氯喹、羟氯喹或α-干扰素。Preferably, the other active ingredients are selected from: remdesivir, lopinavir, monupivir, ritonavir, chloroquine, hydroxychloroquine or alpha-interferon.
  8. 权利要求1的式(I)化合物，或其药学上可接受的盐或酯，它们的立体异构体或互变异构体、消旋体、氮氧化物、多晶型、水合物、溶剂合物、同位素标记物、前药或代谢产物，或权利要求2的药物组合物或权利要求3的试剂盒，其用于治疗或预防病毒感染；The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt or ester thereof, their stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs, hydrates, solvents Compounds, isotope labels, prodrugs or metabolites, or the pharmaceutical composition of claim 2 or the kit of claim 3, which is used to treat or prevent viral infections;
    优选地，所述化合物、药物组合物或试剂盒用于治疗或预防病毒感染导致的疾病、病症、症候群和/或紊乱；Preferably, the compound, pharmaceutical composition or kit is used to treat or prevent diseases, conditions, syndromes and/or disorders caused by viral infections;
    优选地，所述其它活性成分选自：瑞德西韦、洛匹那韦、莫努匹韦、利托那韦、氯喹、羟氯喹或α-干扰素。Preferably, the other active ingredient is selected from: remdesivir, lopinavir, monupivir, ritonavir, chloroquine, hydroxychloroquine or alpha-interferon.
  9. 权利要求4-6的用途或权利要求7的方法或权利要求8的化合物或药物组合物的用途，其中，所述化合物或药物组合物抑制病毒增殖；The use of claims 4-6 or the method of claim 7 or the use of the compound or pharmaceutical composition of claim 8, wherein the compound or pharmaceutical composition inhibits viral proliferation;
    优选地，所述化合物或药物组合物抑制病毒3CL蛋白酶的活性；Preferably, the compound or pharmaceutical composition inhibits the activity of viral 3CL protease;
    优选地，所述3CL蛋白酶具有P132H突变；Preferably, the 3CL protease has a P132H mutation;
    优选地，所述病毒为冠状病毒，优选为α冠状病毒和/或β冠状病毒，更优选为SARS-CoV-2。Preferably, the virus is a coronavirus, preferably alphacoronavirus and/or betacoronavirus, more preferably SARS-CoV-2.
  10. 权利要求4-6的用途或权利要求7的方法或权利要求8的化合物或药物组合物的用途，其中，所述病毒感染导致的疾病、病症、症候群和/或紊乱选自：发热、恶心、呕吐、头痛、呼吸困难、乏力、呼吸道感染、肺炎、嗅觉障碍、味觉障碍及其并发症，或其组合；The use of claims 4-6 or the method of claim 7 or the use of the compound or pharmaceutical composition of claim 8, wherein the disease, illness, syndrome and/or disorder caused by the viral infection is selected from: fever, nausea, Vomiting, headache, dyspnea, fatigue, respiratory tract infection, pneumonia, smell disorder, taste disorder and their complications, or combinations thereof;
    优选地，所述病毒为冠状病毒，优选为α冠状病毒和/或β冠状病毒，更优选为SARS-CoV-2。 Preferably, the virus is a coronavirus, preferably alphacoronavirus and/or betacoronavirus, more preferably SARS-CoV-2.
PCT/CN2023/091287 2022-05-27 2023-04-27 3c-like protease inhibitor WO2023226679A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114507221A (en) * 2022-04-21 2022-05-17 北京科翔中升医药科技有限公司 Triazine compound and application thereof in preparation of antiviral drugs
CN115038696A (en) * 2021-04-14 2022-09-09 盐野义制药株式会社 Triazine derivatives having virus proliferation inhibitory activity and pharmaceutical composition containing the same
CN115504968A (en) * 2022-11-21 2022-12-23 歌礼生物科技（杭州）有限公司 Triazine derivatives

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115038696A (en) * 2021-04-14 2022-09-09 盐野义制药株式会社 Triazine derivatives having virus proliferation inhibitory activity and pharmaceutical composition containing the same
CN114507221A (en) * 2022-04-21 2022-05-17 北京科翔中升医药科技有限公司 Triazine compound and application thereof in preparation of antiviral drugs
CN115504968A (en) * 2022-11-21 2022-12-23 歌礼生物科技（杭州）有限公司 Triazine derivatives

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
UNOH, YUTO ET AL.: "Discovery of S-217622, a Non-Covalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19", JOURNAL OF MEDICINAL CHEMISTRY, vol. 65, no. 9, 30 March 2022 (2022-03-30), XP093007183, ISSN: 0022-2623, DOI: 10.1021/acs.jmedchem.2c00117 *

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