CN117126137A

CN117126137A - 3C-like protease inhibitors

Info

Publication number: CN117126137A
Application number: CN202310478840.8A
Authority: CN
Inventors: 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
Original assignee: Beijing Wangshi Intelligent Technology Co ltd; Guangzhou National Laboratory
Current assignee: Beijing Wangshi Intelligent Technology Co ltd; Guangzhou National Laboratory
Priority date: 2022-05-27
Filing date: 2023-04-27
Publication date: 2023-11-28
Also published as: WO2023226679A1

Abstract

The present application provides 3C-like protease inhibitors of formula (I), or a pharmaceutically acceptable salt or ester thereof, stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs, hydrates, solvates, isotopic labels, prodrugs or metabolites thereof. The application also provides a preparation method of the compound, a pharmaceutical composition containing the compound and an effect of the compound in treating or preventing diseases caused by virus infection.

Description

3C-like protease inhibitors

The present application requires enjoyment of the following priorities:

CN202210600337.0, filing date: 2022, 5 and 27.

Technical Field

The present application relates to a novel 3C-like protease inhibitor, or a pharmaceutically acceptable salt or ester thereof, a stereoisomer or tautomer, a racemate, a nitrogen oxide, a polymorph, a hydrate, a solvate, an isotopic label, a prodrug or a metabolite thereof. The application also relates to a preparation method of the compound, a pharmaceutical composition containing the compound and an effect of the compound in treating or preventing diseases caused by virus infection.

Background

The new coronavirus found in month 12 of 2019 was initially named 2019-nCoV, which was modified by the World Health Organization (WHO) to be covd-19, after which the international committee for classification of viruses formally named the new coronavirus as SARS-CoV-2 according to systemics, taxonomies and conventions. SARS-CoV-2 can cause Severe Acute Respiratory (SARI) symptoms including fever, dyspnea, debilitation, pneumonia, etc.

Among all known RNA viruses, coronaviruses have a maximum genome length of between about 26 and 32 kb. In addition to encoding structural proteins, a large portion of the coronavirus genome is transcribed and translated into polypeptides encoding proteins necessary for viral replication and gene expression. A major protease (Mpro) of about 306aa length is a key enzyme for coronavirus replication, and is also encoded by the polypeptide and responsible for processing the polypeptide into a functional protein. Mpro has a similar cleavage site specificity as the picornaviral 3C protease (3 Cpro), and is therefore also referred to as 3C-like protease (3 CLpro). Studies have shown that the 3CLpro of different coronaviruses is highly conserved in both sequence and 3D structure. These features and their functional importance make 3CLpro a target for anti-coronavirus drug design.

The function of 3CLpro is to hydrolyse the expressed peptide chain at the appropriate site in preparation for the peptide chain to form a three-dimensional four-dimensional structure to form the enzyme required for viral proliferation. The enzyme is not changed during catalysis but reduces the activation energy of the hydrolysis reaction, thereby accelerating the rate of the hydrolysis reaction, wherein the thiol group on the cysteine plays a critical role throughout the catalytic hydrolysis process, see Thanigaimalai et al, an Overview of Severe Acute Respiratory Syndrome-Coronavir (SARS-CoV) 3CL Protease Inhibitors:Peptidomimetics and Small Molecule Chemotherapy,Journal of Medicinal Chemistry,59 (14): 6595-6628.

There are disclosures in the prior art about 3CLpro inhibitors. For example, WO2021/250648A1 discloses a compound currently known as Nirmatrelvir (PF-07321332) which, as one of the active ingredients of Paxlovid, in combination with ritonavir, is able to reduce the risk of death and hospitalization caused by the novel coronavirus SARS-CoV-2.

Furthermore, WO2021/205290A1 discloses similarly structured compounds which treat SARS-CoV-2 caused disease via a 3C-like protease inhibitor mediated pathway.

However, the compounds of the prior art have disadvantages, such as pampers Luo Weide, and inhibit the CYP3A4 enzyme, so that the situation that metabolism of other medicines by the enzyme is disturbed, half-life and clearance rate are changed, curative effect is reduced or adverse reaction is generated may occur. For example, when the patient takes Pa Luo Weide and terfenadine at the same time, the Pa Luo Weide inhibits the oxidative metabolism of CYP3A4 on terfenadine, so that the concentration of the terfenadine in the patient is abnormally increased, and QT wave prolongation and arrhythmia of the heart are caused. The compounds disclosed in WO2021/205290A1 also face the problem of inefficiency when administered orally.

In addition, it has been reported that the main protease generates a plurality of drug-resistant mutations after pressure screening using the anti-SARS-CoV-2 active molecule ALG-097161. Wherein, the inhibition effect of L50F+E316A+L167F three-point mutation in enzyme activity level test is respectively reduced by 72 times and 93 times; in the virus protection experiments, the EC50 value of Nirmatrelvir was increased 51-fold, see The Substitutions L F, 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. Thus, there is a growing need to develop new 3C-like protease inhibitors.

Disclosure of Invention

The invention takes 3C-like protease as a target point, develops a new class of small molecule inhibitors, and can be used for treating or preventing virus infection.

The compound targets 3C-like protease, has excellent inhibitory activity on the 3C-like protease with P132H mutation, can obviously inhibit the proliferation of SARS-CoV-2, and simultaneously realizes better in vivo stability, lower drug side effect, better pharmacokinetic property and better biological activity of drug-resistant mutation L50F+E166A+L167F main protease generated under the pressure screening of the peptoid anti-SARS-COV-2 drug ALG-097161.

In one aspect, the present invention provides a compound of formula (I), or a pharmaceutically acceptable salt or ester thereof, a stereoisomer or tautomer, a racemate, a nitrogen oxide, a polymorph, a hydrate, a solvate, an isotopic label, a prodrug or a metabolite thereof:

in a second aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention, or a pharmaceutically acceptable salt or ester thereof, a stereoisomer or tautomer, racemate, oxynitride, polymorph, hydrate, solvate, isotopic label, prodrug or metabolite thereof.

In some preferred embodiments according to the present invention, the pharmaceutical composition according to the present invention may optionally further comprise at least one physiologically/pharmaceutically acceptable excipient.

In some preferred embodiments according to the present invention, the pharmaceutical composition according to the present invention may optionally further comprise a pharmaceutically acceptable excipient, such as a carrier, adjuvant or vehicle.

In some preferred embodiments according to the invention, the pharmaceutical composition according to the invention may optionally further comprise an additional active ingredient or therapeutic agent. The additional active ingredient or therapeutic agent is, for example, remdesivir (Remdesivir or GS-5734), lopinavir (Lopinavir), mo Nupi (Molnupiravir), ritonavir (Ritonavir), chloroquine (Chloroquine or Sigma-C6628), hydroxychloroquine and/or interferon alpha.

In some preferred embodiments according to the present invention, the pharmaceutical composition according to the present invention comprises a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt or ester thereof, a stereoisomer or tautomer, a racemate, a nitrogen oxide, a polymorph, a hydrate, a solvate, an isotopic label, a prodrug or a metabolite thereof.

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 an antiviral drug targeting the host.

In some preferred embodiments according to the invention, a compound according to the invention or a pharmaceutically acceptable salt or ester thereof, a stereoisomer or tautomer, racemate, oxynitride, polymorph, hydrate, solvate, isotopic label, prodrug or metabolite thereof, or a pharmaceutical composition thereof, is used for preventing and/or treating a disease, disorder, syndrome and/or disorder selected from the group consisting of: fever, nausea, vomiting, headache, dyspnea, weakness, respiratory tract infection, pneumonia, dysosmia, dysgeusia and complications thereof caused by viral infection, or a combination thereof; preferably, the virus is a coronavirus, preferably an alpha coronavirus and/or a beta coronavirus, more preferably SARS-CoV-2.

According to the present invention, the pharmaceutical composition according to the present invention may be formulated into a dosage form suitable for administration by methods known in the art.

In a third aspect, the present invention provides the use of a compound according to the invention, or a pharmaceutically acceptable salt or ester thereof, a stereoisomer or tautomer, a racemate, a nitrogen oxide, a polymorph, a hydrate, a solvate, an isotopic label, a prodrug or a metabolite thereof, in the manufacture of a medicament.

In some preferred embodiments according to the present invention, the medicament prepared according to the present invention may optionally further comprise an additional active ingredient or therapeutic agent. The additional active ingredient or therapeutic agent is, for example, remdesivir (Remdesivir or GS-5734), lopinavir (Lopinavir), mo Nupi (Molnupiravir), ritonavir (Ritonavir), chloroquine (Chloroquine, sigma-C6628), hydroxychloroquine and/or interferon-alpha.

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 an antiviral drug targeted to the host.

In some preferred embodiments according to the present invention, the medicament prepared according to the present invention is for use in preventing or treating a disease, disorder, syndrome and/or disorder selected from the group consisting of: fever, nausea, vomiting, headache, dyspnea, weakness, respiratory tract infection, pneumonia, dysgeusia and complications thereof caused by viral infection, or a combination thereof; preferably, the virus is a coronavirus, preferably an alpha coronavirus and/or a beta coronavirus, more preferably SARS-CoV-2.

According to the present invention, the medicament prepared according to the present invention may be further formulated into a dosage form suitable for administration by methods known in the art.

In a fourth aspect, the invention provides a method of treating or preventing a disease, condition, syndrome and/or disorder caused by a viral infection in a subject, comprising administering to said subject a compound of the invention, or a pharmaceutically acceptable salt or ester thereof, a stereoisomer or tautomer, racemate, oxynitride, polymorph, hydrate, solvate, isotopic label, prodrug or metabolite thereof, or a pharmaceutical composition thereof.

In some preferred embodiments according to the invention, the compounds or pharmaceutical compositions of the invention inhibit viral proliferation;

in another preferred embodiment, the compounds or pharmaceutical compositions of the invention inhibit the activity of viral 3CL protease;

in another preferred embodiment, the 3CL protease has a P132H mutation;

in another preferred embodiment, the virus is a coronavirus, preferably an alpha coronavirus and/or a beta coronavirus, more preferably SARS-CoV-2.

In some preferred embodiments according to the invention, the disease, condition, syndrome and/or disorder caused by the viral infection is selected from: fever, nausea, vomiting, headache, dyspnea, weakness, respiratory tract infections, pneumonia, dysosmia, dysgeusia, and complications thereof, or combinations thereof;

Preferably, the virus is a coronavirus, preferably an alpha coronavirus and/or a beta coronavirus, more preferably SARS-CoV-2.

Those skilled in the art will appreciate that features recited in the various aspects and embodiments of the invention can be freely combined in accordance with them as long as they do not conflict with each other or are incompatible therewith.

Definition of the definition

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.

In one embodiment, the term "coronavirus" is an alpha coronavirus and/or a beta coronavirus, more preferably a beta coronavirus.

In one embodiment, the alpha coronavirus is selected from the group consisting of HCoV-229E and HCoV-NL63, preferably HCoV-229E.

In one embodiment, the beta coronavirus is selected from the group consisting of HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV and SARS-CoV-2, preferably HCoV-OC43 or SARS-CoV-2, more preferably SARS-CoV-2.

The term "treating" as used herein relates to reversing, alleviating, inhibiting the progression or prevention of a disorder or condition to which the term applies, or one or more symptoms of such disorder or condition. The term "treatment" as used herein relates to the action of a verb treatment, the latter as just defined. As used herein, the term "treating" any disease or disorder, in some embodiments refers to ameliorating the disease or disorder (i.e., slowing or preventing or alleviating the progression of the disease or at least one clinical symptom thereof). In other embodiments, "treating" refers to moderating or improving at least one physical parameter, including physical parameters that may not be perceived by the patient. In other embodiments, "treating" refers to modulating a disease or disorder physically (e.g., stabilizing a perceived symptom) or physiologically (e.g., stabilizing a parameter of the body) or both. In other embodiments, "treating" refers to preventing or delaying the onset, or progression of a disease or disorder.

The term "pharmaceutically acceptable salts" as used herein means those carboxylate salts, amino acid addition salts of the compounds of the invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response and the like commensurate with a reasonable benefit/risk ratio, and effective for their intended use, including (if possible) zwitterionic forms of the compounds of the invention.

Pharmaceutically acceptable base addition salts are formed with metals or amines, for example alkali metal and alkaline earth metal hydroxides or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N, N' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and procaine.

The base addition salts of the acidic compounds may be prepared by contacting the free acid form with a sufficient amount of the desired base to form the salt, in a conventional manner. The free acid can be regenerated by contacting the salt form with the acid in a conventional manner, isolating the free acid. The free acid forms differ somewhat in certain physical properties from their respective salt forms, such as solubility in polar solvents, but for the purposes of the present invention, the salts are also equivalent to their respective free acids.

The salt may be a sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide prepared from an inorganic acid, an acid such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, and the like. Representative salts include: hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, mesylate, glucoheptonate, lactobionate, laurylsulfonate, isethionate, and the like. 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 acetates, propionates, octanoates, isobutyrates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzates, dinitrobenzoates, naphthoates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, maleates, tartrates, methanesulfonates, and the like. Pharmaceutically acceptable salts may include cations based on alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Salts of amino acids, such as arginine salts, gluconate salts, galacturonate salts, and the like are also contemplated (see, e.g., berge s.m. et al., "Pharmaceutical Salts," j.pharm.sci.,1977;66:1-19, incorporated herein by reference).

As used herein, the term "nitroxide" refers to the oxidation of 1 or more than 1 nitrogen atom to form an N-oxide when the compound contains several nitrogen-containing functional groups. Specific examples of N-oxides are N-oxides of tertiary amines or N-oxides of nitrogen atoms of nitrogen-containing heterocycles. The corresponding nitrogen-containing compound may be treated with an oxidizing agent such as hydrogen peroxide or a peracid (e.g., peroxycarboxylic acid) to form an N-oxide. In particular, the N-oxides can be prepared by the method L.W.Deady (Syn.Comm.1977, 7, 509-514) in which, for example, a nitrogen-containing compound is reacted with m-chloroperoxybenzoic acid (MCPBA) in an inert solvent such as methylene chloride.

As used herein, the term "ester" refers to an in vivo hydrolysable ester formed from a compound containing a hydroxyl or carboxyl group. Such esters are, for example, physiologically/pharmaceutically acceptable esters which hydrolyze in the human or animal body to give the parent alcohol or acid. The compounds of formula (I) or (II) of the present invention contain a carboxyl group which may form an in vivo hydrolysable ester with a suitable group including, but not limited to, alkyl, arylalkyl, and the like.

The "subject" to be administered includes, but is not limited to: the terms "human," "patient," and "subject" are used interchangeably herein.

"disease," "disorder," "syndrome," and "condition" are used interchangeably herein.

As used herein, unless otherwise indicated, the term "treating" includes an effect that occurs when a subject has a particular disease, disorder, or condition, which reduces the severity of the disease, disorder, or condition, or delays or slows the progression of the disease, disorder, or condition ("therapeutic treatment"), as well as an effect that occurs before the subject begins to have the particular disease, disorder, or condition ("prophylactic treatment").

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.

In general, an "effective amount" of a compound refers to an amount sufficient to elicit a biological response of interest. As will be appreciated by those of ordinary skill in the art, the effective amount of the compounds of the present invention may vary depending on the following factors: for example, biological targets, pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age health and symptoms of the subject. The effective amount includes a therapeutically effective amount and a prophylactically effective amount.

As used herein, unless otherwise indicated, a "therapeutically effective amount" of a compound is an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder, or condition, or to delay or minimize one or more symptoms associated with a disease, disorder, or condition. A therapeutically effective amount of a compound refers to that amount of therapeutic agent, alone or in combination with other therapies, which 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.

As used herein, unless otherwise indicated, a "prophylactically effective amount" of a compound 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 recurrence of a disease, disorder, or condition. A prophylactically effective amount of a compound refers to an amount of a therapeutic agent, alone or in combination with other agents, that 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. For example, the compounds of the invention may be administered simultaneously or sequentially with other therapeutic agents in separate unit dosage forms, or simultaneously with other therapeutic agents in a single unit dosage form.

Detailed description of the preferred embodiments

Herein, "the compound of the present invention" refers to a compound of the following formula (I), or a pharmaceutically acceptable salt or ester thereof, a stereoisomer or tautomer thereof, a racemate, a nitrogen oxide, a polymorph, a hydrate, a solvate, an isotopic label, a prodrug or a metabolite.

Compounds are named herein using standard nomenclature. Compounds having asymmetric centers, it is to be understood (unless otherwise indicated) that all optical isomers and mixtures thereof are encompassed. Furthermore, unless otherwise specified, all isomeric compounds encompassed by the present invention may occur with carbon-carbon double bonds in the form of Z and E. Compounds that exist in different tautomeric forms, one of the compounds is not limited to any particular tautomer, but is intended to encompass all tautomeric forms.

In one embodiment, the present invention relates to a compound of formula (I), or a pharmaceutically acceptable salt or ester thereof, a stereoisomer or tautomer, racemate, oxynitride, polymorph, hydrate, solvate, isotopic label, prodrug or metabolite thereof:

The compounds of the invention may include one or more asymmetric centers and thus may exist in a variety of stereoisomeric forms, for example, enantiomeric and/or diastereomeric forms. For example, the compounds of the 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, including racemic mixtures and mixtures enriched in one or more stereoisomers. The isomers may be separated from the mixtures by methods known to those skilled in the art, including: chiral High Pressure Liquid Chromatography (HPLC), formation and crystallization of chiral salts; alternatively, preferred isomers may be prepared by asymmetric synthesis.

The compounds of the present invention may exist in tautomeric forms. Tautomers are functional group isomers generated by rapid movement of an atom in a molecule at two positions, are special functional group isomers, and a pair of tautomers can be mutually converted, but usually take one isomer which is relatively stable as a main existing form. The most prominent examples are enol and keto tautomers.

For example, the compounds of the present invention comprise the following tautomers:

those skilled in the art will appreciate that the organic compound may form a complex with a solvent in or from which it reacts or from which it precipitates or crystallizes. These complexes are referred to as "solvates". When the solvent is water, the complex is referred to as a "hydrate". The present invention encompasses all solvates of the compounds of the present invention.

The term "solvate" refers to a form of a compound or salt thereof that is bound to a solvent, typically formed by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds described herein may be prepared, for example, in crystalline form, and may be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates. In some cases, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "solvate" includes both solvates in solution and separable solvates. Representative solvates include hydrates, ethanolates and methanolates.

The term "hydrate" refers to a compound that binds to water. Generally, the ratio of the number of water molecules contained in a hydrate of a compound to the number of molecules of the compound in the hydrate is determined. Thus, the hydrates of the compounds can be used, for example, of the formula R x H ₂ O represents, wherein R is the compound, and x is a number greater than 0. 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, e.g., hemihydrate (r.0.5H) ₂ O) and polyhydrates (x is a number greater than 1, e.g., dihydrate (r.2h) ₂ O) and hexahydrate (R.6H) ₂ O)。

The compounds of the present invention may be in amorphous or crystalline form (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 present invention. The term "polymorph" refers to a crystalline form (or salt, hydrate or solvate thereof) of a compound of a particular crystal stacking arrangement. All polymorphs have the same elemental composition. Different crystalline forms typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, optoelectronic properties, stability and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors can lead to a crystalline form predominating. Various polymorphs of a compound can be prepared by crystallization under different conditions.

The invention also includes isotopically-labelled compounds (isotopically-variant) which are identical to those recited in formula (I), but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, respectively, for example ² H、 ³ H、 ¹³ C、 ¹¹ C、 ¹⁴ C、 ¹⁵ N、 ¹⁸ O、 ¹⁷ O、 ³¹ P、 ³² P、 ³⁵ S、 ¹⁸ F and F ³⁶ Cl. The compounds of the invention, prodrugs thereof, and pharmaceutically acceptable salts of the compounds or prodrugs thereof, which contain the isotopes described above and/or other isotopes of other atoms, are within the scope of this invention. Certain isotopically-labeled compounds of the present invention, e.g., for incorporation of a radioisotope (e.g. ³ H and ¹⁴ c) Those useful in drug and/or substrate tissue distribution assays. Tritium, i.e. tritium ³ H and carbon-14 ¹⁴ The C isotopes are particularly preferred because they are easy to prepare and detect. Further, substitution by heavier isotopes, e.g. deuterium, i.e ² H may be preferred in some cases because higher metabolic stability may provide therapeutic benefits, such as extended in vivo half-life or reduced dosage requirements. Isotopically-labelled compounds of formula (I) of the present invention and prodrugs thereof are generally useful in such applications Sample preparation in carrying out the procedures described below and/or in the examples and preparations, the non-isotopically labeled reagent is replaced with a readily available isotopically labeled reagent.

In addition, 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 vivo by hydrolysis, e.g. in blood, into its active form having a medical effect. Pharmaceutically acceptable prodrugs are described in t.higuchi and v.stilla, prodrugs as Novel Delivery Systems, a.c. s.symposium Series vol.14, 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 of which is incorporated herein by reference.

Prodrugs are any covalently bonded compounds of the invention which, when administered to a patient, release the parent compound in vivo. Prodrugs are typically prepared by modifying functional groups in such a way that the modification may be performed by conventional procedures or cleavage in vivo to yield the parent compound. Prodrugs include, for example, compounds of the invention wherein a hydroxy, amino, or sulfhydryl group is bonded to any group that, when administered to a patient, may cleave to form the hydroxy, amino, or sulfhydryl group. Representative examples of prodrugs therefore include, but are not limited to, acetate, formate and benzoate/amide derivatives of hydroxy, mercapto and amino functional groups of compounds of formula (I). In addition, in the case of carboxylic acid (-COOH), esters such as methyl ester, ethyl ester, and the like can be used. The esters themselves may be active and/or may be hydrolysed under in vivo conditions in the human body. Suitable pharmaceutically acceptable in vivo hydrolysable ester groups include those groups which readily decompose in the human body to release the parent acid or salt thereof.

The term "metabolite" refers to a product obtained by metabolizing a specific compound or salt thereof in vivo. The metabolites of a compound may be identified by techniques well known in the art and their activity may be characterized by employing the assay methods as described herein. Such products may be obtained by oxidation, reduction, hydrolysis, amidization, deamination, esterification, degreasing, enzymatic cleavage, etc. of the administered compound. Accordingly, the present invention includes metabolites of compounds, including metabolites produced by contacting a compound of the present invention with a mammal for a period of time sufficient.

The invention also provides a pharmaceutical formulation comprising a therapeutically effective amount of a compound of formula (I) or a therapeutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent or excipient thereof. All of these forms are within the scope of the invention.

Pharmaceutical compositions and kits

In another aspect, the invention provides a pharmaceutical composition comprising a compound of the invention (also referred to as an "active ingredient") and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises an effective amount of a compound of the present invention. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a compound of the invention. In some embodiments, the pharmaceutical composition comprises a prophylactically effective amount of a compound of the present invention.

The term "pharmaceutical composition" means a mixture comprising one or more compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity. The term "physiologically/pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and generally do not produce allergies or similar inappropriate reactions, such as gastrointestinal discomfort, dizziness, etc., when administered to humans. 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 and the like. Water and aqueous solutions saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly injectable solutions. Suitable drug carriers are described in "Remington's Pharmaceutical Sciences" of e.w. martin.

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 co-formulated compounds. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of the invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin.

Substances that may be used as physiologically/pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, 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-block 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; a gum powder; malt; gelatin; talc powder; adjuvants such as cocoa butter and suppository waxes; oils such as peanut oil, cotton seed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycol compounds such as propylene glycol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic salt; ringer's solution; ethanol, phosphate buffer, and other non-toxic suitable lubricants such as sodium lauryl sulfate and magnesium stearate, coloring agents, releasing agents, coating materials, sweetening, flavoring and perfuming agents, preserving and antioxidant agents.

The pharmaceutical compositions of the present invention may be prepared in accordance with the disclosure using any method known to those of skill in the art. For example, conventional mixing, dissolving, granulating, emulsifying, levigating, encapsulating, entrapping or lyophilizing processes.

The dosage form of the medicament of the invention can be selected according to specific conditions. Pharmaceutical dosage forms often consist of a drug, excipients and a container/sealing system. One or more excipients (also known as inactive ingredients) may be added to the compounds of the present invention to improve or promote the manufacture, stability, administration and safety of the drug, and may provide a means to achieve a desired drug release profile. Thus, the type of excipient added to a drug may depend on various factors, such as the physical and chemical characteristics of the drug, the route of administration, and the manufacturing steps. Pharmaceutically acceptable excipients are present in this field and include those listed in the various pharmacopoeias. (see U.S. Pharmacopeia (U.S.Pharmacopeia, USP), japanese Pharmacopeia (Japanese Pharmacopoeia, JP), european Pharmacopeia (European Pharmacopoeia, EP) and British Pharmacopeia (British pharmacopoeia, BP); U.S. food and drug administration (the U.S. food and Drug Administration, www.fda.gov) drug evaluation and research center (Centerfor Drug Evaluation and Research, CEDR) publications, such as Inactive ingredient guide (Inactive Ingredient Guide, 1996); pharmaceutical additives handbook written by Ash (Hand book of Pharmaceutical Additives, 2002), joint information resource company (Synapse Information Resources, inc., endiott NY; etc.).

The pharmaceutical compositions of the present invention may include one or more physiologically acceptable inactive ingredients that facilitate processing of the active molecule into a formulation for pharmaceutical use.

Suitable formulations will depend upon the route of administration desired. The administration route includes 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 release/sustained release formulations. Suitable dosage forms for oral ingestion by an individual include tablets, pills, dragees, hard and soft shell capsules, liquids, gels, syrups, slurries, suspensions and emulsions.

Solid oral dosage forms may be obtained using excipients including fillers, disintegrants, binders (dry and wet), dissolution retarders, lubricants, glidants, anti-sticking agents, cationic exchange resins, wetting agents, antioxidants, preservatives, colorants, and flavoring agents. These excipients may be of synthetic or natural origin. Examples of such excipients include cellulose derivatives, citric acid, dicalcium phosphate, gelatin, magnesium carbonate, magnesium/sodium lauryl sulfate, mannitol, polyethylene glycol, polyvinylpyrrolidone, silicates, silica, sodium benzoate, sorbitol, starch, stearic acid or salts thereof, sugars (i.e., dextrose, sucrose, lactose, etc.), talc, tragacanth, vegetable oils (hydrogenated), and waxes. Ethanol and water may be used as granulation aids. In some cases it may be desirable to coat the tablet with, for example, a taste masking film, a gastric acid resistant film, or a delayed release film. Natural and synthetic polymers are often used in combination with colorants, sugars and organic solvents or water to coat tablets, resulting in dragees. When the capsule is preferred over a tablet, the drug powder, suspension or solution thereof may be delivered in the form of a compatible hard shell or soft shell capsule.

The therapeutically effective dose may be estimated first using various methods well known in the art. The initial dose used for animal studies may be based on the established effective concentration in the cell culture assay. The dosage range suitable for the human body can be determined, for example, using data obtained from animal studies and cell culture assays. In certain embodiments, the compounds of the present invention may be prepared as medicaments for oral administration.

The correct formulation, route of administration, dosage and interval of administration may be selected in consideration of the particularities of the individual condition according to methods known in the art.

Suitable formulations for administration of the compounds of the present invention will be apparent to those of ordinary skill in the art and include, for example, tablets, pills, capsules, suppositories, troches, lozenges, solutions (particularly solutions for injection (subcutaneous, intravenous, intramuscular) and infusion (injectable)), elixirs, syrups, cachets, emulsions, inhalants or dispersible powders. The amount of the one or more pharmaceutically active compounds should be in the range of 0.1 to 90wt%, preferably 0.5 to 50wt% of the composition as a whole, i.e. in an amount sufficient to achieve the dosage ranges specified below. The prescribed dose may be administered several times per day, if necessary.

The invention also includes kits (e.g., pharmaceutical packages). Kits provided can include a compound of the invention, other therapeutic agent, and first and second containers (e.g., vials, ampoules, bottles, syringes, and/or dispersible packages or other suitable containers) containing a compound of the invention, other therapeutic agent. In some embodiments, the provided kits may also optionally include a third container containing pharmaceutically acceptable excipients for diluting or suspending the compounds of the invention and/or other therapeutic agents. In some embodiments, the compounds of the invention and other therapeutic agents provided in the first and second containers are combined to form one unit dosage form.

Administration of drugs

The pharmaceutical compositions provided herein may be administered by a number of routes including, but not limited to: oral, parenteral, inhalation, topical, rectal, nasal, buccal, vaginal, by implantation or other means of administration. For example, parenteral administration as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intramuscularly, and intracranial injection or infusion techniques.

Typically, an effective amount of a compound provided herein is administered. The amount of the compound actually administered may be determined by a physician, according to the circumstances involved, including the condition being treated, the route of administration selected, the compound actually administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, and the like.

When used to prevent a disorder of the present invention, a subject at risk of developing the disorder is administered a compound provided herein, typically based on physician recommendations and administered under the supervision of a physician, at a dosage level as described above. Subjects at risk for developing a particular disorder generally include subjects having a family history of the disorder, or those subjects determined by genetic testing or screening to be particularly susceptible to developing the disorder.

The pharmaceutical compositions provided herein may also be administered chronically ("chronically"). Chronic administration refers to administration of a compound or pharmaceutical composition thereof over a prolonged period of time, e.g., 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or may continue administration indefinitely, e.g., for 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 a prolonged period of time, e.g., within a therapeutic window.

Various methods of administration may be used to further deliver the pharmaceutical compositions of the present invention. For example, in some embodiments, the pharmaceutical composition may be administered as a bolus, e.g., in order to increase the concentration of the compound in the blood to an effective level. Bolus doses depend on the targeted systemic level of active ingredient through the body, e.g., intramuscular or subcutaneous bolus doses cause slow release of the active ingredient, whereas bolus injections delivered directly to veins (e.g., by IV intravenous drip) can be delivered more rapidly, causing the concentration of the active ingredient in the blood to rise rapidly to effective levels. In other embodiments, the pharmaceutical composition may be administered in the form of a continuous infusion, for example, by IV intravenous drip, thereby providing a steady state concentration of the active ingredient in the subject's body. Furthermore, in other embodiments, a bolus dose of the pharmaceutical composition may be administered first, followed by 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 in order to facilitate accurate dosing. Typical unit dosage forms include pre-filled, pre-measured ampoules or syringes of liquid compositions, or in the case of solid compositions, pills, tablets, capsules and the like. In such compositions, the compound is typically a minor component (about 0.1 to about 50 wt.%, or preferably about 1 to about 40 wt.%) with the remainder being various carriers or excipients and processing aids useful for forming the desired administration form.

For oral doses, a typical regimen is one to five oral doses per day, especially two to four oral doses, typically three oral doses. Using these modes of dosing, each dose provides from about 0.01 to about 20mg/kg of a compound of the invention, with preferred doses each providing from about 0.1 to about 10mg/kg, especially from about 1 to about 5mg/kg.

In order to provide similar blood levels to, or lower than, the use of an injected dose, a transdermal dose is typically selected in an amount of about 0.01 to about 20% by weight, preferably about 0.1 to about 10% by weight, and more preferably about 0.5 to about 15% by weight.

From about 1 to about 120 hours, especially 24 to 96 hours, the injection dosage level is in the range of about 0.1 mg/kg/hour to at least 10 mg/kg/hour. To achieve adequate steady state levels, a preloaded bolus of about 0.1mg/kg to about 10mg/kg or more may also be administered. For human patients of 40 to 80kg, the maximum total dose cannot exceed about 2 g/day.

Liquid forms suitable for oral administration may include suitable aqueous or nonaqueous carriers, buffers, suspending and dispersing agents, colorants, flavors, and the like. Solid forms may include, for example, any of the following components, or compounds having similar properties: binders, for example microcrystalline cellulose, gum tragacanth or gelatin; excipients, for example starch or lactose, disintegrants, for example alginic acid, primogel or corn starch; lubricants, for example, magnesium stearate; glidants, for example, colloidal silicon dioxide; sweeteners, for example, sucrose or saccharin; or a flavoring agent, for example, peppermint, methyl salicylate, or orange flavoring.

Injectable compositions are typically based on sterile saline or phosphate buffered saline for injectable use, or other injectable excipients known in the art. As previously mentioned, in such compositions, the active compound is typically a minor component, often about 0.05 to 10% by weight, the remainder being an injectable excipient or the like.

Transdermal compositions are typically formulated as topical ointments or creams containing the active ingredient. When formulated as ointments, the active ingredients are typically combined with a paraffinic or a 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 typically include other components for enhancing stable skin penetration of the active ingredient or formulation. All such known transdermal formulations and compositions are included within the scope provided by the present invention.

The compounds of the invention may also be administered via a transdermal device. Transdermal administration may thus be achieved using a reservoir (reservoir) or porous membrane type, or a variety of solid matrix patches.

The above components of the compositions for oral administration, injection or topical administration are merely representative. Other materials and processing techniques, etc. are set forth in Remington's Pharmaceutical Sciences,17th edition,1985,Mack Publishing Company,Easton,Pennsylvania, section 8, incorporated herein by reference.

The compounds of the present invention may also be administered in sustained release form, or from a sustained release delivery system. A description of representative 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. In one embodiment, the formulation comprises water. In another embodiment, the formulation comprises 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 one or more substituents on the linked sugar moiety, including but not limited to: methylated, hydroxyalkylated, acylated and sulfoalkyl ether substitutions. In some embodiments, the cyclodextrin is a sulfoalkyl ether β -cyclodextrin, e.g., sulfobutyl ether β -cyclodextrin, also known as Captisol. See, for example, U.S.5,376,645. In some embodiments, the formulation comprises hexapropyl- β -cyclodextrin (e.g., 10-50% in water).

Indication of disease

For diseases caused by viral infections, the development of 3C-like protease inhibitors may provide therapeutic benefit to a large number of patients. The compounds of the invention exert therapeutic effects by down-regulating the activity of 3C-like proteases within viruses, particularly viruses in which the 3C-like protease has a P132H mutation.

In some embodiments, the 3C-like protease inhibitors of the invention can treat a variety of diseases and complications thereof caused by viral infection.

More specifically, these compounds are useful in the treatment of diseases caused by viral infections: fever, nausea, vomiting, headache, dyspnea, weakness, respiratory tract infection, pneumonia, dysosmia, dysgeusia and complications thereof.

More specifically, these compounds are useful for the above-described diseases or conditions caused by SARS-CoV-2 infection.

Combination drug

The 3C-like protease inhibitors of the invention may be used in combination with other agents to treat cancer, and comprise at least one target agent/viral activity modulator, including Remdeivir (Remdeivir or GS-5734), lopinavir (Lopinavir), mo Nupi (Molnupiravir), ritonavir (Ritonavir), chloroquine (Chloroquine or Sigma-C6628), hydroxychloroquine and/or alpha-interferon, and the like.

Examples

The compounds and methods of preparation of the present disclosure will be described in further detail below in conjunction with the specific examples. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All technical solutions realized based on the present disclosure are included in the scope of the present disclosure.

Unless otherwise indicated, the experimental methods used in the following examples are all conventional in the art; reagents, materials, instruments, equipment, and the like used in the examples described below are all commercially available.

Example 1

Synthesis of Compound 1

Synthesis of intermediate 1-2

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 (200 mL) and washed with saturated sodium bicarbonate solution (100 mL x 2), the organic phase was separated and dried over anhydrous sodium sulfate, filtered. The filtrate was concentrated under reduced pressure to give intermediate 1-2 (10 g) as a brown solid, which was used directly in the next reaction.

LCMS(ESI)m/z:198.0[M+H] ⁺ 。

Synthesis of intermediates 1-3

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. The reaction mixture was stirred overnight at room temperature, washed with water (200 mL) and saturated sodium chloride solution (200 mL), the organic phase was separated and dried over anhydrous sodium sulfate, and filtered. 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 give intermediate 1-3 (4.93 g, yield 46.1%) as a yellow solid.

LCMS(ESI)m/z:212.0[M+H] ⁺ 。

Synthesis of intermediates 1-4

A saturated ammonium chloride solution (100 mL) and reduced iron powder (3.91 g,0.07 mol) were added to a solution of intermediates 1-3 (4.93 g,0.02 mol) in absolute ethanol (100 mL) at room temperature. Stir at 80 ℃ overnight. After cooling, the mixture was filtered, and the filtrate was concentrated under reduced pressure, followed by dilution with ethyl acetate (100 mL). The organic phase was separated and dried over anhydrous sodium sulfate and filtered. 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 give intermediate 1-4 (3.4 g, yield 80.9%) as a brown solid.

LCMS(ESI)m/z:182.2[M+H] ⁺ 。

Synthesis of intermediates 1-6

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) and 1, 8-diazabicyclo [5.4.0] undec-7-ene (10.52 g,0.069 mol) at 0deg.C. 1, 8-diazabicyclo [5.4.0] undec-7-ene (10.52 g,0.069 mol) and N, N-carbonyldiimidazole (10.51 g,0.065 mol) were added at 0deg.C for 5 hours with stirring. Stirring at room temperature for 24 hours. The pH was adjusted to 3-4 with 1N HCl, extracted with ethyl acetate (200 mL x 3), the organic phases combined, washed with saturated sodium chloride solution (100 mL), the organic phase separated and dried over anhydrous sodium sulfate, filtered. The filtrate was concentrated under reduced pressure, and the crude product was purified by column chromatography (silica gel, dichloromethane: ethyl acetate=5:1) to give intermediate 1-6 (5.23 g, yield 45.1%) as a white solid.

LCMS(ESI)m/z:252.0[M+Na] ⁺ 。

Synthesis of intermediates 1-7

1- (bromomethyl) -2,4, 5-trifluorobenzene (8.46 g,37.6 mmol) was added to a mixture of 1-6 (5.75 g,25.0 mmol), potassium carbonate (6.93 g,50.0 mmol) and acetonitrile (30 mL) and stirred at 85deg.C for 16 hours. Cooled, water (100 mL), ethyl acetate extracted (200 mL x 3) were added, the organic phases were combined and washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered and the filtrate concentrated under reduced pressure, and the crude product was purified by column chromatography (silica gel, petroleum ether: ethyl acetate=2:1) to give intermediate 1-7 (8.6 g, yield: 89%) as a white solid.

LCMS(ESI)m/z:318.0[M+H-56] ⁺ 。

Synthesis of intermediates 1-8

Intermediate 1-7 (6.7 g,17.9 mmol) was dissolved in dichloromethane and trifluoroacetic acid (30/30 mL) and stirred at room temperature for 6 hours. Concentrated under reduced pressure to give 1-8 (7.8 g) as a white solid which was used directly in the next reaction.

LCMS(ESI)m/z:318.0[M+H] ⁺ 。

Synthesis of intermediates 1-9

A mixture of intermediate 1-8 (160 mg,0.5 mmol), pyridin-3-ylboronic acid (93 mg,0.75 mmol), copper acetate (92 mg,0.5 mmol), 4-dimethylaminopyridine (247 mg,2.0 mmol), pyridine (100 mg,1.2 mmol) and dioxane (10 mL) was stirred under oxygen sphere at 100deg.C for 16 hours. Cooled, water (30 mL) was added, extracted with ethyl acetate (3 x 30 mL), the combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the crude product was purified by column chromatography (silica gel, dichloromethane: methanol=20:1) to give intermediate 1-9 (120 mg, yield: 40%) as a white solid.

LCMS(ESI)m/z:395.0[M+H] ⁺ 。

Synthesis of 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

To a solution of intermediate 1-9 (120 mg,0.3 mmol) in tetrahydrofuran (20 mL) was added sequentially lithium bis (trimethylsilyl) amide (0.6mmol,0.6mL,1.0M in THF) and intermediate 1-4 (67 mg,0.37 mmol) at 0deg.C. Stirring was maintained at 0deg.C for 2 hours. Saturated ammonium chloride solution (30 mL) was quenched, ethyl acetate (3 x 30 mL) was extracted, the combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered and the filtrate concentrated under reduced pressure, and the crude product was purified by high pressure chromatography (Gemini-C18 150x21.2mm,5 μm, acetonitrile-water (0.1% formic acid), gradient: 30% -60%) to give white solid 1 (14.5 mg, yield: 9%).

LCMS(ESI)m/z:513.9[M+H] ⁺ 。

¹ H NMR(400MHz,DMSO-d ₆ ,DCl in D ₂ O)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)。

Synthesis of comparative Compound 1

The intermediates 1-8 and 1-4 were synthesized by the synthetic method of the reference compound 1.

Synthesis of intermediate 2-2

To a solution of 1-8 (250 mg,0.79 mmol) in N, N-dimethylformamide (5 mL) was added triethylamine (390 mg,3.94 mmol), 2-1 (324 mg,2.37 mmol), copper acetate (242 mg,1.35 mmol) and molecular sieve (300 mg) at room temperature. The mixed solution was stirred at 60 ℃ and oxygen flow for 16 hours. After the reaction was completed, filtration was performed, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, dichloromethane: methanol=30:1) to give compound 2-2 (145.5 mg, yield: 45.1%) as a yellow oil.

LCMS(ESI)m/z:409.1[M+H] ⁺ 。

Synthesis of comparative Compound 1

To a solution of 2-2 (106 mg,0.26 mmol) in tetrahydrofuran (2 mL) was added 1-4 (56.3 mg,0.31 mmol) under an ice bath, nitrogen blanket, lithium bis (trimethylsilyl) amide (0.65 mL) was added at 0deg.C and stirred for 2 hours. After the reaction was completed, the mixed solution was quenched with water (10 mL), extracted with ethyl acetate (10 mL x 3), and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product is purified by high pressure chromatography (column: -Gemini-C18 150x21.2mm,5 μm. Flow term: ACN- -H) ₂ O(0.05％NH ₃ .H ₂ O). Gradient: 35-40.) to give comparative compound 1 (49.5 mg, yield) as a white solid: 36.1%).

LCMS(ESI)m/z:528.1[M+H] ⁺ 。

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).

Biological examples

In carrying out the biological examples below, compound 1 of the test compounds and comparative compound 1 were prepared by example 1, comparative compound 2 was prepared by the method described in Y Unoh et al (J.Med. Chem.2022,65,9,6499-6512, DOI:10.1021/acs. Jmedchem.2c 00117).

1. Caco-2 cell model for evaluating bidirectional permeability of compound

1.1 instruments and materials

1) The control agents propranolol and Digoxin were purchased from MCE and Minoxidil from the national food and drug verification institute.

2) Caco-2 cells were purchased from the American Type Culture Collection (ATCC).

3) FBS medium was purchased from Sigma, DMEM was purchased from Corning corporation (Cambridge, MA), nonessential amino acids (NEAA), hank's Balanced Salt Solution (HBSS) and trypsin/EDTA were all purchased from sameimers. Penicillin and streptomycin were purchased from soribao.

4) HTS-96 well Transwell plates and other sterile consumables are available from Corning corporation.

5) The Millicell resistance measurement system was purchased from Millipore.Vision was purchased from Nexcelom Bioscience. Infinite 200PRO microplate reader was purchased from Tecan. MTS2/4orbital platforms were purchased from IKA Labortechnik.

1.2 design of experiments

1.2.1 cell culture and seed plates

1) Using high sugar (4.5 g/L) DMEM medium containing L-glutamine, 10% fetal bovine serum, 0.1mg/mL streptomycin and 100 units of penicillin were added for cell culture.

2) Caco-2 was cultured in cell culture flasks. The incubator was set at 37℃with 5% CO ₂ Ensuring the relative humidity to be 95 percent. The cell confluence reaches 70-90% and can be used for inoculation of Transwell.

3) Before cell seeding, 50. Mu.L of cell culture medium was added to each well of the Transwell upper chamber, and 25mL of cell culture medium was added to the lower plate. The plates were placed at 37℃with 5% CO ₂ After incubation in the incubator for 1 hour, it can be used to inoculate cells.

4) After cell digestion, the cell suspension was pipetted into a round bottom centrifuge tube and centrifuged at 120g for 5 min.

5) Cells were resuspended using medium at a final concentration of 6.86×105cells/mL. The cell suspension was added to the upper chamber of a 96-well Transwell plate at 50. Mu.L per well and the final inoculation density was 2.4X105 cells/cm ² 。

6) The medium was changed 48 hours after inoculation, and the culture was continued for 14-18 days with one medium change every other day.

7) The medium exchange procedure was as follows, the Transwell cells were separated from the receiving plate, medium was discarded from the receiving plate and then from the Transwell cells, and finally 75. Mu.L fresh medium was added to each cell and 25mL fresh medium was added to the receiving plate.

1.2.2 evaluation of cell monolayer membrane integrity

1) Caco-2 should be fully pooled and differentiated after 14-18 days of culture. At this time, it can be applied to a penetration test.

2) The single layer film resistance was measured with a resistance meter (Millipore, USA) and the resistance per well was recorded.

3) After the measurement, the Transwell plates were returned to the incubator.

4) Calculating the resistance value: measurement of resistance value (ohms). Times.membrane area (cm) ² ) TEER value (ohms cm) ² ) If TEER value<230ohms·cm ² The well cannot be used for penetration testing.

1.2.3 preparation of solutions

1) 1L of buffer (HBSS, 10mM HEPES,pH 7.4) was prepared, 2.38g of HEPES,0.35g of sodium hydrogencarbonate were weighed out respectively, and dissolved by adding 900mL of pure water, then 100mL of 10 XHBSS was added and stirred uniformly, the pH was adjusted to 7.4, and finally filtration was performed.

2) DMSO stock solutions of the test subjects were prepared. A 1mM DMSO stock of control was prepared. The mixture was diluted with buffer to give 5. Mu.M working solution. The test sample was diluted with a buffer solution to obtain 5. Mu.M working solution. The DMSO content of the final system was 0.5%.

3) Preparing a dosing end solution:

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4) Preparing a receiving end solution:

1.2.4 drug penetration test

1) The Caco-2 Transwell plates were removed from the incubator. The cell monolayer was rinsed twice with buffer and incubated at 37℃for 30 min.

2) To determine the compound transport rate from tip to base, 125. Mu.L of test compound and control solution were added to each well (tip) of the plug-in plate, 235. Mu.L of HBSS (10mM HEPES,pH 7.4) buffer was added to each well (base) of the test compound receiving plate, and 235. Mu.L of HBSS (10mM HEPES,pH 7.4) buffer was added to each well (base) of the control solution receiving plate. Transfer of 50. Mu.L of sample from the apical solution was detected as a 0 minute apical sample by adding 200. Mu.L of acetonitrile containing internal standard (100 nM alprazolam, 200nM labetalol, 200nM caffeine and 2. Mu.M tyrosol).

3) To determine the rate of compound transport from substrate end to tip end, 285 μl of test compound and control solution were added to each well of the receiving plate (substrate end), 75 μl of HBSS (10mM HEPES,pH 7.4) buffer was added to each well of the test compound solution-inserted plate (tip end), and 75 μl of HBSS (10mM HEPES,pH 7.4) buffer was added to each well of the control solution-inserted plate (tip end). Transfer of 50. Mu.L of sample from the basal end solution was detected as a 0 minute basal end-administered sample by adding 200. Mu.L of acetonitrile containing an internal standard (100 nM alprazolam, 200nM labetalol, 200nM caffeine and 2. Mu.M tyrosol).

4) Incubate in a CO2 incubator at 37℃for 2 hours.

5) After the end of the transport experiment, 50. Mu.L of the sample was transferred from the dosing end (top end in the direction Ap. Fwdarw.Bl. End in the direction Bl. Fwdarw.Ap. End in the substrate end) to 200. Mu.L of acetonitrile containing the internal standard (100 nM alprazolam, 200nM labetalol, 200nM caffeine and 2. Mu.M tyrosol). Transfer 50. Mu.L of the sample from the receiving end (Ap. Fwdarw. Bl. Sup. Base end, bl. Fwdarw. Ap. Top end) to 200. Mu.L of acetonitrile containing the internal standard (100 nM alprazolam, 200nM labetalol, 200nM caffeine and 2. Mu.M tyrosol). Vortex for 5 minutes. Quenched samples 3220g for each time point were centrifuged for 30 min. Transfer 100 μl of supernatant from each sample to a 96-well loading plate while adding 100 μl of purified water to the corresponding well. The sample analysis plate was vortexed and subjected to LC/MS analysis. All incubation samples were double-parallel.

6) Fluorescence values were measured at the end of the two hour transfer experiment, 10mM fluorescence Huang Chubei solution was prepared with water, and diluted to 100. Mu.M with transfer buffer solution. 100. Mu.L of the fluorogenic yellow solution was added to the Transwell chamber (top), 300. Mu.L of the transport buffer solution was added to the base end, and incubated in a CO2 incubator at 37℃for 30 minutes. 80. Mu.L of solution was removed directly from the tip and base end (using the base outer hole) and transferred to a new 96-well plate. Cell fluorescence (membrane integrity) was measured with an enzyme-labeled instrument with excitation wavelength of 485nM and emission wavelength of 530nM.

1.3 data analysis

Data calculations were all performed using Excel. The peak area was calculated from the ion chromatography results. Apparent permeability coefficient (P) of the Compound _app Units: cm/s.times.10 ^-6 ) Calculated using the following formula:

in the formula: v (V) _A For the volume of the receiving side solution (Ap. Fwdarw.Bl is 0.235mL, bl. Fwdarw.Ap is 0.075 mL), area is the Transwell-96 well plate membrane Area (0.143 cm) ² ) The method comprises the steps of carrying out a first treatment on the surface of the time is the incubation time (unit: s).

P _app(B-A) Is the apparent permeability coefficient from the base end to the top end; p (P) _app(A-B) Is the apparent permeability coefficient from the top end to the bottom end of the substrate.

2. In vitro evaluation of the activation potential of PXR of DPX2 cells

2.1 materials and reagents

1)DPX ₂ (a HepG2 cell line stably transfected with PXR and luciferase systems) cells were derived from Puracyp Inc (Carlsbad, calif.).

2) The positive control agent rifampicin was purchased from Sigma company (st.louis, MO).

3)CellTiter-Fluor ^TM Cell viability assay kit and One-Glo luciferase assay system were purchased from Promega corporation (Madison, wis.). FBS medium was purchased from Avantor, DPX ₂ Cell culture medium and dosing medium were purchased from Puracyp corporation.

4) Reagent and consumable information

Name of the name	Suppliers of goods	Goods number	Lot number
				Puracyp Dosing Medium	Puracyp	D-500-100	-
Puracyp Culture Medium	Puracyp	C-500-100	-
				FBS (fetal bovine serum)	Avantor	76294-180	234D19
CellTiter-Fluor ^TM Cell Viability Assay kit	Promega	G6081	-
				One-Glo Luciferase Assay System	Promega	E6120	-

2.2 design of experiments

2.2.1 cell culture and seed plates

1)DPX ₂ Preparation of the culture Medium and the administration Medium 45ml of fetal bovine serum was added to each of 450ml of the culture Medium and the administration Medium.

2)DPX ₂ Cultured in T-75 cell culture flasks. The incubator was set at 37℃with 5% CO ₂ Ensuring the relative humidity to be 95 percent. Cell confluence of 80-90% can be used for separation.

3) After washing the cells cultured in the T-75 cell culture flask with 10mL PBS, they were aspirated, 3mL trypsin was added and incubated at 37 ℃ for about 5 minutes or until the cells were completely isolated and floating, followed by the addition of excess serum-containing medium to inactivate trypsin and terminate digestion.

4) After cell digestion, the cell suspension was aspirated and transferred to a conical bottom centrifuge tube and centrifuged at 150g for 5 minutes. Cells were resuspended using medium at a final concentration of 4.5X10 ⁵ cells/mL. The cell suspension was added to 384 well culture plates at 25 μl per well, the inoculated plates were placed in an incubator for 24 hours, and then the cell plates were available for PXR experimental study.

2.2.2 incubation of test Compounds

1) Solution preparation: DMSO stock solutions of 1, 10mM test compound and 10mM positive control rifampicin were prepared. The final concentrations of the test compounds were 1 and 10. Mu.M, respectively, and the final concentration of rifampicin was 10. Mu.M. The DMSO content of the final system was 0.1%. The blank control is DMSO.

2) The cell plates were removed from the incubator and 25nL of the blank control solution, the solution containing the test compound, and the solution containing the positive control drug were added directly, three of each set being arranged in parallel. The dosed cell plates were transferred to a cell incubator for additional incubation for 48 hours.

3) The cell structure and monolayer integrity were checked prior to testing to ensure compliance with the study requirements.

Assay for 2.2.3PXR Activity

1) After 48 hours of drug administration incubation, the cell plates were used for testing PXR activity.

2) Allowing CellTiter-Fluor to stand ^TM The cell viability assay kit and ONE-Glo luciferase assay reagent reached room temperature. mu.L of GF-AFC substrate was taken into an assay buffer container (10 mL) to form the 2 Xreagent, and then 10mL of PBS was added to dilute to 1 Xreagent. The ONE-Glo luciferase assay substrate was transferred to ONE-Glo luciferase assay buffer.

3) The DPX 2-dosed incubation cell plates were removed from the incubator and the medium in the cell plates was discarded. Will contain 1 XCellTiter-Fluor ^TM The cell viability assay reagents were poured into sterile loading cells and the cell plates were incubated in a 37℃CO2 incubator for 30 minutes using a multichannel pipettor with 25. Mu.L of reagents added to each well.

4) 384-well cell plates were removed from the cell incubator, cooled briefly to ambient temperature, and the fluorescence of the cells was measured with an microplate reader, with excitation at 400nM and emission at 505nM.

5) ONE-Glo assay reagents were poured into sterile loading wells and 25. Mu.L of reagent was added directly to each well. The cell plate was gently shaken and the solution was mixed well. After incubation for 5 minutes at room temperature, the chemiluminescent value of each well was read using an enzyme-labeled instrument.

2.2.4 data analysis

1) Data calculations were all performed using Excel.

2) Luciferase activity was determined by RLU/RFU, where RLU represents the average relative luminescence value of the test compound at each concentration for three parallel samples and RFU represents the average relative luminescence value of the test compound at each concentration for three parallel samples.

3) The fold of activation is calculated by the following formula:

the activation times of the compounds according to the invention are shown in Table 2.

4) The calculation formula of the positive control percentage is as follows:

％Positive control＝(Fold activation _{test compound} /Fold activation _{positive control compound} )*100％

3. mouse intravenous oral pharmacokinetics

1. Administration information

Animal feeding control: the feed was administered after 4 hours after overnight fast and free drinking. Frequency of administration: the experiment was dosed once on the day.

The actual dosing volume was calculated from the animal body weight.

2. Acquisition time

Acceptable time frame for blood sample collection

3. Blood sample collection and processing procedure

4. Evaluation during in vivo experiment

5. Information of experimental animal

6. Sample analysis

And (3) determining the blood concentration in the plasma sample by using an LC-MS/MS detection method and using a follow-up standard curve.

For plasma assay results, winNonlin 8.3 (Phoenix) ^TM ) Or other similar software. If applicable, the pharmacokinetic parameters will be calculated as plasma drug concentration-time data.

Pharmacokinetic data are described by descriptive statistics such as mean, standard deviation and sample size. Calculations were performed with Microsoft Excel 2013. Other pharmacokinetic parameters and statistical analyses can also be performed and recorded in the data summary.

4. Enzymatic Activity assay

The biochemical experiment adopts a fluorescence resonance energy transfer experiment (FRET) test method.

The assay system (120. Mu.L) contained 108. Mu.L of the main protease (WT or P132H) at a final concentration of 150nM, and a small-molecule control group without protein was added with 108. Mu.L of protein diluent consisting of 50mM Tris-HCl pH 7.4,1mM EDTA;

contains 10. Mu.L of substrate, and the final concentration of the substrate is 20. Mu.M; the negative control NC was 2. Mu.L of 100% DMSO added to the sample containing 2. Mu.L of small molecules to be tested at different concentrations (3-fold gradient dilution with 100% DMSO as the diluent).

A substrate: the fluorogenic substrate is MCA-AVLQSFGFR-LYS (DNP) -Lys-NH ₂ 。

The other components of the reaction system were reaction buffer 50mM Tris-HCl pH 7.4,1mM EDTA.

The reaction process is as follows: mu.L of the main protease or protein dilution was mixed with 2. Mu.L of small molecules and added to 96 Kong Quanhei ELISA plates (Corning costar, # 3916) and incubated for 30min. And adding a fluorogenic substrate after incubation is finished, and rapidly starting detection on an enzyme-labeled instrument.

The detection method of the enzyme-labeled instrument comprises the following steps:

in the dynamic 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 values of each reaction well at different times under the detection conditions were recorded, and the fluorescence values recorded for the first 200s (typically 7-10 data points) were taken during the calculation.

The experimental data processing and calculating process comprises the following steps:

first, the Slope of fluorescence value versus time for each time of different reaction wells [ Slope (fluorescence value RFU: time s) is calculated and recorded as the initial reaction rate V ₀ . Initial velocity V ₀ Slope (RFU within 200 s: time s).

Initial enzyme reaction rate V with different drug concentrations ₀ Determining the inhibition ratio (formula one, formula two, and formula two optimization) of the representative compound to the main protease by the ratio of the initial reaction rate of the enzyme with the control group, and calculating IC by using a GraphPad Prism nonlinear fitting curve ₅₀ Values.

Equation one: inhibition (%) = (RFU) _{100% enzyme Activity control} -RFU _{Sample of} )/(RFU _{100% enzyme Activity control} -RFU _{Blank control} )×100％

Formula II: inhibition (%) = (NC initial velocity V ₀ Sample initial velocity V ₀ ) NC initial velocity V ₀ ×100％；

NC enzyme activity was 100%, and inhibition was 0%

And (3) optimizing a formula II: inhibition (%) = (NC initial velocity V ₀ - (sample initial velocity V) ₀ Protein-free small molecule control V ₀ ) NC initial velocity V ₀ X 100% for V elimination ₀ The problem of negative value (slope).

Half inhibition concentration IC50 values and inhibition curves were fitted using GraphPad Prism software non-linear fit curves { Nonlinear regression (cut fit) - [ inhibitor ] vs. response-Variable slope (four parameters) }.

Note that in this test, each test sample (sample and control) was repeated three times in parallel in the group, and three biological repeated experiments were performed, and standard deviation analysis was performed by the biological repeated experiments. The test procedure generally uses equation two to optimally calculate the inhibition rate.

5. anti-SARS-COV-2

Experimental materials

Cell line: vero E6 (ATCC, CRL-1586), caco-2 (ATCC, HTB-37) and Calu-3 (ATCC, HTB-55).

Virus strain: 2019-nCoV-WIV04 (IVCAS 6.7512)

Dose of infection: moi=0.01

(II) Experimental methods

1) 100 mu L of the mixture containing 2X 10 ⁴ Inoculating the cells into a 96-well plate, and placing the cells in a constant temperature and humidity incubator at 37 ℃ for overnight culture;

2) After 20 hours of cell attachment, the culture broth was pipetted off and 100. Mu.L of culture broth containing the indicated concentration of test compound +CP-100356 was added to each well. 8 dilutions were set for each test compound, 3-4 duplicate wells were set for each dilution, and DMSO-treated and normal cell groups were set simultaneously. In addition to the normal cell group, the wells were incubated with a complete culture medium containing 0.01MOI virus in a 37℃incubator at constant temperature and humidity for 72 hours.

3) Cytopathic rate, inhibition = (1-disease rate of test compound group) ×100% was recorded using a full field cell scanner 72 hours after infection.

4) According to the inhibition rate result, four-parameter fitting calculation of EC ₅₀ 。

6. Anti-drug-resistant mutation L50F+E316A+L167F SARS-COV-2

Construction of (one) prokaryotic expression vector

1) Mutant primer design and synthesis

The Primer software was used to design a mutant Primer for L50F, E166A+L167F, which was synthesized by the company of Biotechnology (Shanghai) Co., ltd, and the Primer sequences are shown in the following table:

2) Cloning and transformation of mutant plasmids

The wild type main protease plasmid is used as a template, a Fast Mutagenesis System single-point mutation kit (purchased from TRAN company) is adopted for PCR amplification, and after the amplified product is digested with DMT restriction enzyme to methylate the plasmid template, the amplified product is transformed into competent cells with degradation methylate plasmid. The specific operation is as follows:

PCR system and conditions

a)PCR:94℃5min，94℃30s，66℃20s，72℃1min，30cycles，72℃10min。

b) Digestion of PCR products: 1uL DMT enzyme was added to the PCR product, and incubated at 37℃for 1 hour after mixing.

c) Conversion: adding 8uL digested product into 50uL competent cells, mixing well, and ice-bathing for 30min; heat shock in a 42 ℃ water bath for 45s, and immediately standing on ice for 3min; adding 250uL of antibiotic-free LB medium which is balanced to room temperature, and culturing for 1 hour at 200rpm and 37 ℃; 100uL of the bacterial liquid is evenly spread on a plate containing resistance, and is cultured overnight at 37 ℃.

d) Monoclonal identification: single colony is selected and sent to a biological engineering (Shanghai) stock company for sequencing; and after the sequencing is correct, extracting plasmids, and converting the plasmids into BL21 competent cells to perform protein expression.

Expression and purification of (II) muteins

1) Prokaryotic expression of proteins

Transforming the successfully constructed mutant plasmid into BL21 (DE 3) competent cells, selecting single colony the next day, and performing amplification culture at 37 ℃ until bacterial liquid D ₆₀₀ The nanometer value reaches 0.6 to 0.8, and IPTG with the final concentration of 0.5mol/L is added for overnight induction expression.

Ni-NAT affinity chromatography purification and molecular sieve purification of protein

Buffer solution:

a) Affinity chromatography base buffer: 50mM Tris,500mM Nacl,10% glycerol, ph=8.0;

b) Molecular sieve buffer: 25mM Heps,150mM Nacl,pH =7.4.

Collecting bacterial liquid precipitate expressed overnight, centrifuging at 18000rpm for 40min after high pressure crushing treatment, and collecting supernatant for Ni-NAT affinity chromatography purification. Nickel column affinity chromatography column treatment: the nickel column is firstly washed by ddH2O, then the column is balanced by using a balancing buffer solution, filtered bacterial liquid supernatant is combined with nickel column affinity chromatography gel, eluting is carried out by using an eluting buffer solution containing 20mM imidazole concentration, and eluting is carried out by using an eluting buffer solution containing 500mM imidazole concentration.

And collecting the supernatant, sediment, fluid and protein samples eluted by various concentration gradients of the crushed bacterial liquid, boiling the protein samples, and performing SDS-PAGE electrophoresis to verify the expression condition of the protein. The eluted proteins were concentrated using a 30KD ultrafiltration tube at 3500rpm at 4deg.C.

The protein was concentrated to a standard loading volume (about 0.5 ml) of the AKTA PURIFIER molecular sieve and further purified using GE Healthcare product Superdex 200 increment. Collecting the protein sample purified by the molecular sieve, concentrating to 10mg/ml, and sub-packaging and freezing at-80 ℃ for standby.

(III) fluorescence resonance energy transfer experiment test (FRET) Ki value

1) Reaction buffer:50mM Tris pH 7.4,1mM EDTA,0.01%tritonX-100.

2) Test system

Protein (108 uL): the main protease (L50F+E316A+L167F) with a final concentration of 500nM and a small molecule control group without protein was added with 108uL reaction buffer;

substrate (10 uL): fluorogenic substrate MCA-AVLQ-SGFR-Lys (Dnp) -Lys-NH2 (24 mM preservation), 24mM substrate was diluted with DMSO to 0.06mM and 0.12mM so that its final concentration in the reaction system was 5uM and 10uM, respectively.

Small molecule (2 uL): compound 1, comparative compound 2 (50 mM preservation), small molecules at 50mM concentration were diluted to 0.06mM with 100% DMSO so that their final concentration in the reaction system was 1uM. Gradient dilution of small molecules: the small molecules were diluted 2-fold from a concentration of 0.06mM (final concentration 1 uM) to the 8 th well. The negative control NC here was 2uL of 100% DMSO added.

3) Reaction procedure

108uL of the main protease or reaction buffer was mixed with 2uL of small molecules and added to 96 Kong Quanhei ELISA plates (Corning costar, # 3916) and incubated for 30min. And adding a fluorogenic substrate after incubation is finished, and rapidly starting detection on an enzyme-labeled instrument.

4) Layout of the plates:

5) Detection method of enzyme-labeled instrument

In the dynamic 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; recording the fluorescence values of different times in each reaction well under the detection condition, and taking the fluorescence values recorded in the first 200s (generally 7-10 data points) during calculation

6) Data analysis

a) Slope calculation: the slope was calculated from the fluorescence values within 200s before selection, V at each small molecule concentration was obtained by subtracting the slope from the blank, and Dixon mapping was performed by GraphPad prism8.0.1 after 1/V calculation.

b) Dixon mapping Ki: testing, under different substrate concentrations, the slope 1/V of a cluster of straight lines drawn under different fixed I is plotted with the corresponding I, and the intersection point of the straight lines obtained by fitting under different substrate concentrations is the-Ki.

The structures and data comparisons of the inventive and comparative compounds 1-2 are shown in tables 1 and 2 below.

TABLE 1

TABLE 2

Note that:

Caco2：P _app(B-A) from base end to topApparent permeability coefficient; p (P) _app(A-B) To apparent permeability coefficient from top to bottom

IV: intravenous injection

PO: oral administration

T _1/2 : half-life period

C _max : peak concentration of blood medicine

AUC _last : area under the curve of drug time, evaluate the drug absorption degree

F%: bioavailability of the active ingredients

PXR: receptor activation induction

The data show that the compound of the invention realizes better stability, lower drug side effect, better pharmacokinetic property and better biological activity compared with the comparison compound for drug-resistant mutation L50F+E166A+L167F main protease generated by pressure screening of the peptoid anti-SARS-COV-2 drug ALG-097161 while maintaining excellent inhibitory activity.

Specifically, caco2 permeation experiments characterize the permeation effects of drugs, P _app(B-A) Represents the osmotic effect from blood into the small intestine, P _app(A-B) The permeation effect from the small intestine into the blood is shown, and the permeation effect of the compound of the present invention from the small intestine into the blood is superior to that of the other two comparative compounds, and is less likely to permeate from the blood into the small intestine, i.e., the drug permeation effect is excellent.

In terms of PK, the half-life of the compound 1, whether intravenous or oral, is higher than that of the comparative compound 1 and the comparative compound 2, which indicates that the elimination rate of the drug in vivo is lower and the in vivo stability is more excellent. And in the peak concentration (C) _max ) With consistent AUC _last The values were significantly higher than the remaining two compounds, indicating that compound 1 had excellent absorption and in vivo exposure. In addition, compound 1 has more excellent bioavailability and has more excellent pharmacokinetic properties than the other two compounds.

PXR indicates drug interactions, specifically, PXR activation induces metabolic enzymes (e.g., CYP3 A4) that can affect the pharmacokinetics of exogenous and endogenous substances, and excessive activation of CYP3A4 enzymes induces accelerated metabolism of CYP3 A4-mediated antineoplastic agents, analgesics, etc., affecting the efficacy of the combination. The PXR value of the compound 1 is obviously lower than that of the comparative compounds 1 and 2 at different concentrations, and the compound 1 has lower risks of drug interaction and toxic metabolism.

The biochemical activity of the drug-resistant mutant L50F+E316A+L167F main protease of compound 1 is more advantageous than that of the comparison compounds 1 and 2.

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt or ester thereof, a stereoisomer or tautomer, racemate, oxynitride, polymorph, hydrate, solvate, isotopic label, prodrug or metabolite thereof:

2. A pharmaceutical composition comprising a compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt or ester thereof, a stereoisomer or tautomer, racemate, oxynitride, polymorph, hydrate, solvate, isotopic label, prodrug or metabolite thereof, and optionally at least one physiologically/pharmaceutically acceptable auxiliary material;

optionally, the pharmaceutical composition further comprises other active ingredients;

preferably, the other active ingredient is selected from: remdesivir (Remdesivir or GS-5734), lopinavir (Lopinavir), mo Nupi (Molnupiravir), ritonavir (Ritonavir), chloroquine (Chloroquine or Sigma-C6628), hydroxychloroquine or alpha-interferon;

preferably, the pharmaceutical composition is an RNA-dependent RNA polymerase inhibitor, a 3CLpro protease inhibitor, a CYP3A4 inhibitor or an antiviral drug targeted to the host.

3. A kit comprising a compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt or ester thereof, stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs, hydrates, solvates, isotopic labels, prodrugs or metabolites, and other active ingredients;

Preferably, the other active ingredient is selected from: ritonavir, lopinavir, mo Nupi, ritonavir, chloroquine, hydroxychloroquine or alpha-interferon.

4. Use of a compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt or ester thereof, stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs, hydrates, solvates, isotopic labels, prodrugs or metabolites, and optionally other active ingredients, in the manufacture of a medicament for the treatment or prophylaxis of viral infections;

preferably, the medicament is for the treatment or prophylaxis of a disease, condition, syndrome and/or disorder caused by a viral infection;

preferably, the other active ingredient is present in the medicament and the compound is present in the same unit dosage form as the other active ingredient;

preferably, the other active ingredient is present in the medicament and the compound and other active ingredient are present in separate unit dosage forms;

5. Use of a compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt or ester thereof, stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs, hydrates, solvates, isotopic labels, prodrugs or metabolites thereof for the manufacture of a medicament for use in combination with other active ingredients for the treatment or prophylaxis of viral infections;

6. Use of other active ingredients in the manufacture of a medicament for use in combination with a compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt or ester thereof, stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs, hydrates, solvates, isotopic labels, prodrugs or metabolites thereof for the treatment or prevention of viral infections;

7. A method of treating or preventing a viral infection in a subject comprising administering to the subject a compound of formula (I) of claim 1, or a pharmaceutically acceptable salt or ester thereof, a stereoisomer or tautomer, racemate, oxynitride, polymorph, hydrate, solvate, isotopic label, prodrug or metabolite thereof, or a pharmaceutical composition of claim 2, or a kit of claim 3;

Preferably, the method is a method of treating or preventing a disease, disorder, syndrome and/or disorder caused by a viral infection;

8. A compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt or ester thereof, a stereoisomer or tautomer, racemate, oxynitride, polymorph, hydrate, solvate, isotopic label, prodrug or metabolite thereof, or a pharmaceutical composition according to claim 2 or a kit according to claim 3, for use in the treatment or prophylaxis of a viral infection;

preferably, the compounds, pharmaceutical compositions or kits are used for the treatment or prevention of diseases, conditions, syndrome and/or disorders caused by viral infections;

9. Use according to claims 4-6 or a method according to claim 7 or a compound or pharmaceutical composition according to claim 8, wherein the compound or pharmaceutical composition inhibits viral proliferation;

Preferably, the compound or pharmaceutical composition inhibits the activity of viral 3CL protease;

preferably, the 3CL protease has a P132H mutation;

10. The use of claims 4-6 or the method of claim 7 or the use of a compound or pharmaceutical composition of claim 8, wherein the disease, condition, syndrome and/or disorder caused by the viral infection is selected from the group consisting of: fever, nausea, vomiting, headache, dyspnea, weakness, respiratory tract infections, pneumonia, dysosmia, dysgeusia, and complications thereof, or combinations thereof;