3C-like protease inhibitor

Abstract

The present invention provides a 3C-like protease inhibitor represented by formula (I), or a pharmaceutically acceptable salt or ester thereof, stereoisomers or tautomers, racemates, N-oxides, polymorphs, hydrates, solvates, isotope-labeled forms, prodrugs, or metabolites. The present invention also provides methods for preparing the compound, pharmaceutical compositions containing the compound, and the effects of the compound in treating or preventing diseases caused by viral infections. #imgabs0#

Description

[0001] This application claims the following priority:

[0002] CN202210600337.0, application date: May 27, 2022. Technical Field

[0003] The present invention relates to a novel 3C-like protease inhibitor, or a pharmaceutically acceptable salt or ester thereof, stereoisomers or tautomers, racemates, N-oxides, polymorphs, hydrates, solvates, isotope-labeled forms, prodrugs, or metabolites thereof. The present invention also relates to methods for preparing the compound, pharmaceutical compositions containing the compound, and the effects of the compound in treating or preventing diseases caused by viral infections. Background Art

[0004] The novel coronavirus discovered in December 2019 was initially named 2019-nCoV. The World Health Organization (WHO) renamed it COVID-19, and the International Committee on Taxonomy of Viruses later officially named it SARS-CoV-2 based on systematics, taxonomy, and convention. SARS-CoV-2 can cause severe acute respiratory syndrome (SARI) symptoms, including fever, dyspnea, fatigue, and pneumonia.

[0005] Among all known RNA viruses, the largest genome length of coronavirus is between about 26 and 32 kb. In addition to encoding structural proteins, most of the coronavirus genome is also transcribed and translated into polypeptides, which encode proteins necessary for viral replication and gene expression. The main protease (Mpro) of approximately 306 aa is a key enzyme for coronavirus replication. It is also encoded by this polypeptide and is responsible for processing the polypeptide into functional proteins. Mpro has a cleavage site specificity similar to that of the picornavirus 3C protease (3Cpro), and is therefore also called 3C-like protease (3CLpro). Studies have shown that 3CLpro of different coronaviruses is highly conserved in both sequence and 3D structure. These characteristics and their functional importance make 3CLpro a target for the design of anti-coronavirus drugs.

[0006] The role of 3CLpro is to hydrolyze and cleave the expressed peptide chain at the appropriate site, preparing the peptide chain to form a three-dimensional or four-dimensional structure, thereby forming the enzyme required for viral proliferation. During the catalytic process, the enzyme does not change, but the activation energy of the hydrolysis reaction is reduced, thereby accelerating the rate of the hydrolysis reaction. Among them, the sulfhydryl group on the cysteine plays a key role in the entire catalytic hydrolysis process. See Thanigaimalai et al., An Overview of Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) 3CL Protease Inhibitors: Peptidomimetics and Small Molecule Chemotherapy, Journal of Medicinal Chemistry, 59(14): 6595-6628.

[0007] There are public documents on 3CLpro inhibitors in the prior art. For example, WO2021 / 250648A1 discloses a compound currently known as Nirmatrelvir (PF-07321332), which is one of the active ingredients of Paxlovid and, when used in combination with ritonavir, can reduce the risk of death and hospitalization caused by the new coronavirus SARS-CoV-2.

[0008] In addition, WO2021 / 205290A1 also discloses compounds of similar structures, which treat diseases caused by SARS-CoV-2 through pathways mediated by 3C-like protease inhibitors.

[0009] However, the compounds in the prior art all have disadvantages. For example, Parovide also inhibits the CYP3A4 enzyme, which may interfere with the metabolism of other drugs by the enzyme, change the half-life and clearance rate, reduce the efficacy or produce adverse reactions. For example, when a patient takes Parovide and terfenadine at the same time, Parovide inhibits the oxidative metabolism of terfenadine by CYP3A4, causing the concentration of the latter in the patient's body to increase abnormally, causing QT wave prolongation and arrhythmia in the heart. The compounds disclosed in WO2021 / 205290A1 also face the problem of being ineffective when administered orally.

[0010]

[0011] In addition, studies have reported that after pressure screening using the anti-SARS-CoV-2 active molecule ALG-097161, the main protease produced multiple drug-resistant mutations. Among them, the three-point mutation L50F+E166A+L167F reduced the inhibitory effect of Nirmatrelvir and Ensitrelvir by 72 times and 93 times, respectively, in the enzyme activity level test; in the virus protection experiment, the EC50 value of Nirmatrelvir increased by 51 times. See The Substitutions L50F, E166A, and L167F in SARS-CoV-2 3CLpro Are Selected by a Protease Inhibitor In Vitro and Confer Resistance To Nirmatrelvir, mBio 14 (2023) e0281522.10.1128 / mbio.02815-22. Therefore, the need to develop new 3C-like protease inhibitors is becoming increasingly urgent. Summary of the Invention

[0012] The present invention uses 3C-like proteases as targets and develops a new class of small molecule inhibitors that can be used to treat or prevent viral infections.

[0013] The compound of the present invention targets 3C-like proteases, has excellent inhibitory activity against 3C-like proteases with P132H mutations, and can significantly inhibit the proliferation of SARS-CoV-2. At the same time, it also achieves better in vivo stability, lower drug side effects, better pharmacokinetic properties, and better biological activity against the drug-resistant mutation L50F+E166A+L167F main protease produced under pressure screening of the peptidomimetic anti-SARS-COV-2 drug ALG-097161.

[0014] 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 isotopically labeled prodrug or a metabolite thereof:

[0015]

[0016] 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, their stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs, hydrates, solvates, isotopically labeled substances, prodrugs or metabolites.

[0017] 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.

[0018] 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.

[0019] In some preferred embodiments according to the present invention, the pharmaceutical composition according to the present invention may also optionally include another active ingredient or therapeutic agent. The other active ingredient or therapeutic agent is, for example, Remdesivir (Remdesivir or GS-5734), Lopinavir (Lopinavir), Molnupiravir (Molnupiravir), Ritonavir (Ritonavir), Chloroquine (Chloroquine or Sigma-C6628), Hydroxychloroquine and / or alpha-interferon.

[0020] 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.

[0021] In some preferred embodiments according to the present invention, the pharmaceutical composition according to the present invention is an RNA-dependent RNA polymerase inhibitor, a 3CLpro protease inhibitor, a CYP3A4 inhibitor or a host-targeted antiviral drug.

[0022] In some preferred embodiments according to the present invention, the compounds according to the present invention or pharmaceutically acceptable salts or esters thereof, stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs, hydrates, solvates, isotope labels, prodrugs or metabolites thereof, or pharmaceutical compositions thereof, are used to prevent and / or treat diseases, conditions, syndromes and / or disorders selected from the group consisting of, or to alleviate the symptoms of diseases, conditions, syndromes and / or disorders selected from the group consisting of: fever, nausea, vomiting, headache, dyspnea, fatigue, respiratory tract infection, pneumonia, olfactory disturbances, taste disorders and complications thereof, or a combination thereof, caused by viral infection; preferably, the virus is a coronavirus, preferably an alpha coronavirus and / or a beta coronavirus, more preferably SARS-CoV-2.

[0023] According to the present invention, the pharmaceutical composition according to the present invention can be prepared into a dosage form suitable for administration by methods known in the art.

[0024] In a third aspect, the present invention provides the use of a compound according to the present invention or a pharmaceutically acceptable salt or ester thereof, their stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs, hydrates, solvates, isotope labels, prodrugs or metabolites in the preparation of a medicament.

[0025] In some preferred embodiments according to the present invention, the medicine prepared according to the present invention may optionally further comprise another active ingredient or therapeutic agent. The other active ingredient or therapeutic agent is, for example, Remdesivir or GS-5734, Lopinavir, Molnupiravir, Ritonavir, Chloroquine (Sigma-C6628), Hydroxychloroquine and / or α-interferon.

[0026] In some preferred embodiments according to the present invention, the drug prepared according to the present invention is an RNA-dependent RNA polymerase inhibitor, a 3CLpro protease inhibitor, a CYP3A4 inhibitor or a host-targeted antiviral drug.

[0027] In some preferred embodiments of the present invention, the medicament prepared according to the present invention is used to prevent or treat a disease, condition, syndrome and / or disorder selected from the group consisting of, or to alleviate the symptoms of a disease, condition, syndrome and / or disorder selected from the group consisting of: fever, nausea, vomiting, headache, dyspnea, fatigue, respiratory tract infection, pneumonia, olfactory and taste disorders and complications thereof, or a combination thereof, caused by viral infection; preferably, the virus is a coronavirus, preferably an alpha coronavirus and / or a beta coronavirus, more preferably SARS-CoV-2.

[0028] According to the present invention, the drug prepared according to the present invention can be further prepared into a dosage form suitable for administration by methods known in the art.

[0029] In a fourth aspect, the present invention provides a method for treating or preventing a disease, condition, syndrome and / or disorder caused by a viral infection in a subject, comprising administering to the subject a compound of the present invention or a pharmaceutically acceptable salt or ester thereof, stereoisomers or tautomers, racemates, nitrogen oxides, polymorphs, hydrates, solvates, isotope labels, prodrugs or metabolites, or a pharmaceutical composition thereof.

[0030] In some preferred embodiments according to the present invention, the compound or pharmaceutical composition of the present invention inhibits viral proliferation;

[0031] In another preferred embodiment, the compound or pharmaceutical composition of the present invention inhibits the activity of viral 3CL protease;

[0032] In another preferred embodiment, the 3CL protease has a P132H mutation;

[0033] In another preferred embodiment, the virus is a coronavirus, preferably an alphacoronavirus and / or a betacoronavirus, more preferably SARS-CoV-2.

[0034] In some preferred embodiments according to the present invention, the disease, condition, syndrome and / or disorder caused by the viral infection is selected from the group consisting of: fever, nausea, vomiting, headache, dyspnea, fatigue, respiratory tract infection, pneumonia, olfactory disorder, taste disorder and complications thereof, or a combination thereof;

[0035] Preferably, the virus is a coronavirus, preferably an alpha coronavirus and / or a beta coronavirus, more preferably SARS-CoV-2.

[0036] Those skilled in the art will understand that the features listed in various aspects and embodiments of the present invention may be freely combined as long as there is no conflict or incompatibility between them.

[0037] definition

[0038] 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.

[0039] In one embodiment, the term "coronavirus" is an alphacoronavirus and / or a betacoronavirus, more preferably a betacoronavirus.

[0040] In one embodiment, the alphacoronavirus is selected from HCoV-229E and HCoV-NL63, preferably HCoV-229E.

[0041] In one embodiment, the beta coronavirus is selected from HCoV-HKU1, HCoV-OC43, SARS-CoV, MERS-CoV and SARS-CoV-2, preferably HCoV-OC43 or SARS-CoV-2, more preferably SARS-CoV-2.

[0042] As used herein, the term "treat" refers to reversing, alleviating, inhibiting the progression of, or preventing the disorder or condition to which the term applies, or one or more symptoms of such disorder or condition. As used herein, the noun "treat" refers to the action of the verb "treat", which is as just defined. In this article, the term "treat" refers to any disease or condition, in some embodiments of which it refers to improving the disease or condition (i.e., slowing down or preventing or alleviating the development of the disease or condition or at least one of its clinical symptoms). In other embodiments, "treat" refers to alleviating or improving at least one physical parameter, including physical parameters that may not be perceived by the patient. In other embodiments, "treat" refers to regulating the disease or condition physically (e.g., stabilizing perceptible symptoms) or physiologically (e.g., stabilizing physical parameters) or both. In other embodiments, "treat" refers to preventing or delaying the onset, occurrence, or worsening of a disease or condition.

[0043] As used herein, the term "pharmaceutically acceptable salt" refers to those carboxylate salts, amino acid addition salts of the compounds of the present invention that are suitable for use in contact with patient tissues within the scope of sound medical judgment, do not produce undue toxicity, irritation, allergic response, etc., are commensurate with a reasonable benefit / risk ratio, and are effective for their intended use, including (where possible) zwitterionic forms of the compounds of the present invention.

[0044] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali metal and alkaline earth metal hydroxides or organic amines. Examples of metals used as cations include sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines include N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, and procaine.

[0045] Base addition salts of acidic compounds can be prepared by contacting the free acid form with a sufficient amount of the desired base in a conventional manner to form the salt. The free acid can be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner. The free acid forms differ somewhat from their respective salt forms in certain physical properties, such as solubility in polar solvents, but for the purposes of this invention, the salts are equivalent to their respective free acids.

[0046] Salts can be sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides prepared from inorganic acids 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, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, methanesulfonate, glucoheptonate, lactobionate, laurylsulfonate, and isethionate, and the like. Salts can also be prepared from organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Representative salts include acetate, propionate, octanoate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, naphthoate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, etc. 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. Also contemplated are salts of amino acids, such as arginate, gluconate, galacturonate, and the like (see, e.g., Berge SM et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977; 66: 1-19, incorporated herein by reference).

[0047] As used herein, the term "nitrogen oxide" refers to a compound containing several nitrogen-containing functional groups in which one or more nitrogen atoms can be oxidized to form an N-oxide. Specific examples of N-oxides are N-oxides of tertiary amines or N-oxides of nitrogen atoms of nitrogen-containing heterocyclic rings. The corresponding nitrogen-containing compound can be treated with an oxidizing agent such as hydrogen peroxide or a peracid (e.g., a peroxycarboxylic acid) to form an N-oxide. In particular, N-oxides can be prepared using the method of LW Deady (Syn. Comm. 1977, 7, 509-514), wherein, for example, the nitrogen-containing compound is reacted with m-chloroperoxybenzoic acid (MCPBA) in an inert solvent such as dichloromethane.

[0048] As used herein, the term "ester" refers to an in vivo hydrolyzable ester formed by a compound containing a hydroxyl or carboxyl group. Such esters are, for example, physiologically / pharmaceutically acceptable esters that hydrolyze in the human or animal body to produce the parent alcohol or acid. The compounds of formula (I) or (II) of the present invention contain a carboxyl group and can form in vivo hydrolyzable esters with suitable groups, including, but not limited to, alkyl groups, arylalkyl groups, and the like.

[0049] "Subjects" to be administered include, but are not limited to, humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or older adults) and / or non-human animals, e.g., mammals, e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), cows, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human," "patient," and "subject" are used interchangeably herein.

[0050] "Disease," "disorder," "disorder," "syndrome," and "condition" are used interchangeably herein.

[0051] As used herein, and unless otherwise indicated, the term "treating" includes actions that occur while a subject has a particular disease, disorder, or condition that reduces the severity of, or delays or slows the development of, the disease, disorder, or condition ("therapeutic treatment"), as well as actions that occur before a subject develops a particular disease, disorder, or condition ("prophylactic treatment").

[0052] The term "unit dosage form" refers to physically discrete units suitable as unit dosages for human subjects and other mammals, each unit containing a predetermined amount of active material, and a suitable pharmaceutical excipient. For example, a unit dosage form can be a pill, a tablet, a capsule, or a lozenge, etc.

[0053] Generally, an "effective amount" of a compound is an amount sufficient to elicit the desired biological response. As will be appreciated by those skilled in the art, the effective amount of a compound of the invention can vary depending on factors such as the biological target, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, health, and symptoms of the subject. An effective amount includes both a therapeutically effective amount and a prophylactically effective amount.

[0054] As used herein, and unless otherwise specified, 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 the disease, disorder, or condition. A therapeutically effective amount of a compound refers to an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a disease, disorder, or condition. The term "therapeutically effective amount" can include an amount that improves overall treatment, reduces or avoids symptoms or causes of a disease or condition, or enhances the therapeutic effects of other therapeutic agents.

[0055] As used herein, unless otherwise specified, a "prophylactically effective amount" of a compound is an amount sufficient to prevent a disease, disorder, or condition, or an amount sufficient to prevent one or more symptoms associated with a disease, disorder, or condition, or an amount to prevent the 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" can include an amount that improves overall prevention, or an amount that enhances the prophylactic effect of other prophylactic agents.

[0056] "Combination" and related terms refer to the simultaneous or sequential administration of a compound of the invention and other therapeutic agents. For example, a compound of the invention can be administered simultaneously or sequentially with the other therapeutic agents in separate unit dosage forms, or can be administered simultaneously with the other therapeutic agents in a single unit dosage form. Specific implementation plan

[0057] As used herein, the term "compound of the present invention" refers to the compound of formula (I) below, or a pharmaceutically acceptable salt or ester thereof, a stereoisomer or tautomer, racemate, nitrogen oxide, polymorph, hydrate, solvate, isotope label, prodrug or metabolite thereof.

[0058] Herein, compounds are named using standard nomenclature. For compounds with asymmetric centers, it should be understood that (unless otherwise specified) all optical isomers and mixtures thereof are encompassed. In addition, unless otherwise specified, all isomeric compounds encompassed by the present invention may occur in both Z and E forms with carbon-carbon double bonds. Compounds that exist in different tautomeric forms are not limited to any particular tautomer, but are intended to encompass all tautomeric forms.

[0059] 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, a racemate, a nitrogen oxide, a polymorph, a hydrate, a solvate, an isotopically labeled prodrug or a metabolite thereof:

[0060]

[0061] The compounds of the present invention may include one or more asymmetric centers and may therefore exist in a variety of stereoisomeric forms, for example, enantiomers and / or diastereomeric forms. For example, the compounds of the present invention may be individual enantiomers, diastereomers, or geometric isomers (e.g., cis and trans isomers), or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis.

[0062] The compounds of the present invention may exist as tautomers. Tautomers are functional group isomers resulting from the rapid shift of an atom between two positions in a molecule. Tautomers are a special type of functional group isomer. A pair of tautomers can convert between each other, but the more stable isomer usually predominates. The most prominent examples are the enol and keto tautomers.

[0063] For example, the compounds of the present invention include the following tautomers:

[0064]

[0065] Those skilled in the art will appreciate that organic compounds can form complexes with solvents in which they react or from which they precipitate or crystallize. 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.

[0066] The term "solvate" refers to a form of a compound or its salt that is associated with 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 can be prepared, for example, in crystalline form and can 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 separation, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid. "Solvate" includes solvates in solution and separable solvates. Representative solvates include hydrates, ethanolates, and methanolates.

[0067] The term "hydrate" refers to a compound that is combined with water. Generally, the ratio of the number of water molecules contained in the hydrate of a compound to the number of molecules of the compound in the hydrate is determined. Therefore, a hydrate of a compound can be represented by the general formula R·xH2O, for example, where R is the compound and x is a number greater than 0. A given compound can form more than one type of hydrate, including, for example, monohydrates (x is 1), lower hydrates (x is a number greater than 0 and less than 1, for example, hemihydrates (R·0.5H2O) and polyhydrates (x is a number greater than 1, for example, dihydrates (R·2H2O) and hexahydrates (R·6H2O).

[0068] The compounds of the present invention can be in amorphous or crystalline form (polymorph). In addition, the compounds of the present invention can exist in one or more crystalline forms. Therefore, the present invention includes all amorphous or crystalline forms of the compounds of the present invention within its scope. The term "polymorph" refers to the crystalline form (or its salt, hydrate or solvate) of a compound with a specific crystal packing arrangement. All polymorphs have the same elemental composition. Different crystalline forms typically have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, photoelectric properties, stability and solubility. Recrystallization solvent, crystallization rate, storage temperature and other factors can cause one crystalline form to dominate. The various polymorphs of a compound can be prepared by crystallization under different conditions.

[0069] The present invention also includes isotopically labeled compounds (isotopic variants) which are identical to those described in formula (I) but for which 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 introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, 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 36 Cl. Compounds of the present invention containing the above-mentioned isotopes and / or other isotopes of other atoms, their prodrugs and pharmaceutically acceptable salts of the compounds or prodrugs are within the scope of the present invention. Certain isotopically labeled compounds of the present invention, such as those in which radioactive isotopes (e.g. 3 H and 14 C) can be used in drug and / or substrate tissue distribution assays. 3 H and carbon-14, i.e.14 C isotopes are particularly preferred because they are easy to prepare and detect. 2 H, because greater metabolic stability can provide therapeutic benefits, such as prolonged in vivo half-life or reduced dosage requirements, and thus may be preferred in some cases. Isotopically labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents when carrying out the processes disclosed in the following schemes and / or the Examples and Preparations.

[0070] In addition, prodrugs are also included in the context of the present invention. The term "prodrug" as used herein refers to a compound that is converted in vivo, for example by hydrolysis in the blood, into its active form that has a medical effect. Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of the ACS Symposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, and D. Fleisher, S. Ramon and H. Barbra, "Improved oral drug delivery: solubility limitations overcome by the use of prodrugs", Advanced Drug Delivery Reviews (1996) 19 (2) 115-130, each of which is incorporated herein by reference.

[0071] A prodrug is any covalently bonded compound of the present invention that releases the parent compound in vivo when such a prodrug is administered to a patient. Prodrugs are typically prepared by modifying functional groups in such a way that the modification can be cleaved to produce the parent compound by conventional manipulation or in vivo. Prodrugs include, for example, compounds of the present invention in which a hydroxyl, amino, or sulfhydryl group is bonded to any group that, when administered to a patient, can be cleaved to form a hydroxyl, amino, or sulfhydryl group. Thus, representative examples of prodrugs include, but are not limited to, acetate / amide, formate / amide, and benzoate / amide derivatives of the hydroxyl, sulfhydryl, and amino functional groups of compounds of formula (I). Additionally, in the case of carboxylic acids (-COOH), esters such as methyl esters, ethyl esters, and the like can be used. Esters themselves can be active and / or can be hydrolyzed under human in vivo conditions. Suitable pharmaceutically acceptable in vivo hydrolyzable ester groups include those that readily decompose in the human body to release the parent acid or its salt.

[0072] The term "metabolite" refers to a product resulting from the in vivo metabolism of a specific compound or salt thereof. Metabolites of a compound can be identified using techniques known in the art, and their activity can be characterized using assays such as those described herein. Such products can be obtained by subjecting the compound to oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic cleavage, and the like. Accordingly, the present invention encompasses metabolites of the compound, including metabolites produced by contacting a compound of the invention with a mammal for a sufficient period of time.

[0073] The present invention also provides a pharmaceutical preparation 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 belong to the present invention.

[0074] Pharmaceutical compositions and kits

[0075] In another aspect, the present invention provides pharmaceutical compositions comprising a compound of the present 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 present invention. In some embodiments, the pharmaceutical composition comprises a prophylactically effective amount of a compound of the present invention.

[0076] The term "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, together with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity. The term "physiologically / pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and generally do not produce allergic or similar untoward 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, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water and aqueous saline solutions and aqueous dextrose and glycerol solutions are preferred as carriers, particularly injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin.

[0077] Pharmaceutically acceptable excipients used in the present invention refer to non-toxic carriers, adjuvants or vehicles that do not destroy the pharmacological activity of the compound formulated together. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the compositions of the present invention include (but are not limited to) ion exchangers, alumina, 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, silica gel, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and lanolin.

[0078] Physiologically / pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, aluminum, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as 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-blocking polymers, lanolin, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as carboxymethylcellulose. sodium cellulose, ethyl cellulose and cellulose acetate; gum powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed 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; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol, phosphate buffered solution, and other non-toxic suitable lubricants such as sodium lauryl sulfate and magnesium stearate, colorants, release agents, coatings, sweeteners, flavorings and fragrances, preservatives and antioxidants.

[0079] The pharmaceutical compositions of the present invention can be prepared according to the disclosed content using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or lyophilizing processes.

[0080] The dosage form of the medicine of the present invention can be selected according to specific circumstances. Pharmaceutical dosage forms are usually composed of medicine, excipients and container / sealing system. One or more excipients (also known as inactive ingredients) can be added to the compound of the present invention to improve or promote the manufacture, stability, administration and safety of the medicine, and the method for obtaining the required drug release curve can be provided. Therefore, the excipient type added to the medicine can be determined according to various factors, such as the physical and chemical properties, route of administration and preparation steps of the medicine. In this field, there are pharmaceutical excipients and include those listed in various pharmacopoeias. (See US Pharmacopeia (USP), Japanese Pharmacopoeia (JP), European Pharmacopoeia (EP), and British Pharmacopoeia (BP); the US Food and Drug Administration (www.fda.gov), Center for Drug Evaluation and Research (CEDR) publications, e.g., Inactive Ingredient Guide (1996); Hand book of Pharmaceutical Additives, Ash, 2002, Synapse Information Resources, Inc., Endicott NY; etc.).

[0081] The pharmaceutical compositions of the present invention may include one or more physiologically acceptable inactive ingredients that facilitate processing of the active molecules into preparations for pharmaceutical use.

[0082] The appropriate formulation depends on the desired route of administration. Routes of administration include intravenous injection, transmucosal or nasal administration, oral administration, and the like. For oral administration, the compound can be formulated into liquid or solid dosage forms and as immediate release or controlled / slow release formulations. Suitable dosage forms for oral ingestion by an individual include tablets, pills, dragees, hard and soft shell capsules, liquids, gels, syrups, ointments, suspensions, and emulsions.

[0083] Solid oral dosage forms can be obtained using excipients including fillers, disintegrants, binders (dry and wet), dissolution retardants, lubricants, glidants, antiadherents, cationic exchange resins, wetting agents, antioxidants, preservatives, colorants, and flavorings. These excipients can be synthetic or natural origin. Examples of the excipient include cellulose derivatives, citric acid, dicalcium phosphate, gelatin, magnesium carbonate, magnesium lauryl sulfate / sodium lauryl sulfate, mannitol, polyethylene glycol, polyvinyl pyrrolidone, silicates, silicon dioxide, sodium benzoate, sorbitol, starch, stearic acid or its salt, sugar (i.e., dextrose, sucrose, lactose, etc.), talc, mucilage of tragacanth, vegetable oil (hydrogenated), and wax. Ethanol and water can be used as granulation aids. In some cases, tablets may need to be coated with, for example, a taste masking film, a gastric acid resistant film, or a delayed-release film. Natural and synthetic polymers are often used to coat tablets in combination with colorants, sugars, and organic solvents or water to produce dragees. When capsules are preferred over tablets, drug powders, suspensions, or solutions thereof can be delivered in compatible hard or soft shell capsules.

[0084] The therapeutically effective dose can first be estimated using various methods well known in the art. The initial dose used in animal studies can be based on the effective concentration established in cell culture assays. The dosage range suitable for humans can be determined, for example, using data obtained from animal studies and cell culture assays. In certain embodiments, the compounds of the present invention can be prepared as a medicament for oral administration.

[0085] The correct formulation, administration route, dosage and dosing interval can be selected according to methods known in the art, taking into account the particularities of the individual situation.

[0086] Suitable formulations for administering the compounds of the present invention will be readily apparent to those skilled in the art and include, for example, tablets, pills, capsules, suppositories, lozenges, troches, solutions (particularly solutions for injection (subcutaneous, intravenous, intramuscular) and infusion (injection)), elixirs, syrups, cachets, emulsions, inhalants, or dispersible powders. The content of the one or more pharmaceutically active compounds should be in the range of 0.1 to 90% by weight, preferably 0.5 to 50% by weight, of the composition as a whole, i.e., an amount sufficient to achieve the dosage range specified below. If necessary, the specified dosage may be administered several times a day.

[0087] The present invention also includes kits (e.g., pharmaceutical packaging). The kits provided may include a compound of the invention, other therapeutic agents, and first and second containers (e.g., vials, ampoules, bottles, syringes, and / or dispersible packaging or other suitable containers) containing the compound of the invention and other therapeutic agents. In some embodiments, the kit provided may also optionally include a third container containing a pharmaceutical excipient for diluting or suspending the compound of the invention and / or other therapeutic agents. In some embodiments, the compound of the invention and other therapeutic agents provided in the first and second containers are combined to form a unit dosage form.

[0088] Drug administration

[0089] Pharmaceutical compositions provided by the invention can be administered by many routes, including but not limited to: oral administration, parenteral administration, inhalation administration, topical administration, rectal administration, nasal administration, oral administration, vaginal administration, administration by implant or other modes of administration. For example, parenteral administration used herein includes subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, intraarticular administration, intraarterial administration, intrasynovial administration, intrasternal administration, intrathecal administration, intralesional administration, and intracranial injection or infusion technology.

[0090] Typically, an effective amount of the compounds provided herein is administered. The amount of compound actually administered can be determined by a physician based on the relevant circumstances, 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.

[0091] When used to prevent the conditions described herein, the compounds provided herein are administered to a subject at risk of developing the condition, typically based on the advice and under the supervision of a physician, at dosage levels as described above. Subjects at risk of developing a particular condition typically include those with a family history of the condition, or those identified by genetic testing or screening as being particularly susceptible to developing the condition.

[0092] The pharmaceutical compositions provided herein can also be administered long-term ("chronic administration"). Long-term administration refers to administration of a compound or pharmaceutical composition thereof over an extended period of time, e.g., 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or administration can continue indefinitely, e.g., for the remainder of the subject's life. In some embodiments, long-term administration is intended to provide a constant level of the compound in the blood over an extended period of time, e.g., within the therapeutic window.

[0093] Various methods of administration can be used to further deliver the pharmaceutical composition of the present invention. For example, in some embodiments, the pharmaceutical composition can be administered by injection, for example, in order to increase the concentration of the compound in the blood to an effective level. The bolus dose depends on the target systemic level of the active ingredient by the body, for example, an intramuscular or subcutaneous bolus dose slowly releases the active ingredient, and the bolus (for example, by IV intravenous drip) delivered directly to the vein can be delivered more quickly so that the concentration of the active ingredient in the blood is rapidly increased to an effective level. In other embodiments, the pharmaceutical composition can be given in a continuous infusion form, for example, by IV intravenous drip, so as to provide a steady-state concentration of the active ingredient in the subject's body. In addition, in other embodiments, the pharmaceutical composition of the bolus dose can be first given, and then continuous infusion.

[0094] Oral compositions can be in the form of bulk liquid solutions or suspensions or bulk powders. However, more generally, in order to facilitate accurate dosing, the compositions are provided in unit dosage form. Typical unit dosage forms include pre-filled, pre-measured ampoules or syringes of liquid compositions, or pills, tablets, capsules, etc. in the case of solid compositions. In such compositions, the compound is typically a relatively small component (about 0.1 to about 50% by weight, or preferably about 1 to about 40% by weight), with the remainder being various carriers or excipients and processing aids useful for forming the desired dosage form.

[0095] For oral dosage, a representative regimen is one to five oral doses per day, particularly two to four oral doses, typically three oral doses. Using these dosage administration modes, each dose provides about 0.01 to about 20 mg / kg of the compound of the invention, with preferred doses each providing about 0.1 to about 10 mg / kg, particularly about 1 to about 5 mg / kg.

[0096] To provide blood levels similar to, or lower than, those obtained with an injectable 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 20% by weight, preferably about 0.1 to about 10% by weight, and more preferably about 0.5 to about 15% by weight.

[0097] From about 1 to about 120 hours, and particularly from 24 to 96 hours, the injected dose 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 preload bolus of about 0.1 mg / kg to about 10 mg / kg or more may also be administered. For a 40 to 80 kg human patient, the maximum total dose may not exceed about 2 g / day.

[0098] Liquid forms suitable for oral administration may include a suitable aqueous or non-aqueous carrier and buffers, suspending and dispersing agents, colorants, flavorings, etc. Solid forms may include, for example, any of the following components, or compounds of a similar nature: binders such as microcrystalline cellulose, tragacanth, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavorings such as peppermint, methyl salicylate, or orange flavor.

[0099] Injectable compositions are typically based on sterile saline or phosphate buffered saline for injection, 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, with the remainder being injectable excipients and the like.

[0100] Typically, transdermal compositions are formulated as topical ointments or creams containing the active ingredient. When formulated as an ointment, the active ingredient is typically combined with a paraffin or water-miscible ointment base. Alternatively, the active ingredient can be formulated into a cream together with, for example, an oil-in-water cream base. Such transdermal formulations are well known in the art and typically include other components that enhance the stable skin penetration of the active ingredient or formulation. All such known transdermal formulations and components are included within the scope provided by the present invention.

[0101] The compounds of the present invention may also be administered by transdermal means.Thus, transdermal administration may be achieved using patches of the reservoir or porous membrane type, or various solid matrices.

[0102] The above components for oral administration, injection or topical administration are representative only. Other materials and processing techniques are described in Part 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, which is incorporated herein by reference.

[0103] The compounds of the invention can also be administered in sustained release form or from a sustained release delivery system. Descriptions of representative sustained release materials can be found in Remington's Pharmaceutical Sciences.

[0104] The present invention also relates to pharmaceutically acceptable formulations of the compounds of the present 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, which optionally include one or more substituents on the linked sugar portion, including but not limited to: methylated, hydroxyalkylated, acylated, and sulfoalkyl ether substitutions. In some embodiments, the cyclodextrin is a sulfoalkyl ether β-cyclodextrin, for example, sulfobutyl ether β-cyclodextrin, also known as Captisol. See, for example, US5,376,645. In some embodiments, the formulation includes hexapropyl-β-cyclodextrin (e.g., in water, 10-50%).

[0105] Indications

[0106] For diseases caused by viral infection, the development of 3C-like protease inhibitors can provide therapeutic benefits to a large number of patients. The compounds of the present invention exert their therapeutic effects by negatively regulating the activity of 3C-like proteases in viruses, especially those with viruses that have a P132H mutation in the 3C-like protease.

[0107] In some embodiments, the 3C-like protease inhibitors of the present invention can be used to treat various diseases caused by viral infection and their complications.

[0108] More specifically, these compounds can be used to treat the following diseases caused by viral infection: fever, nausea, vomiting, headache, dyspnea, fatigue, respiratory tract infection, pneumonia, olfactory disorder, taste disorder and their complications.

[0109] More specifically, these compounds can be used for the above-mentioned diseases or symptoms caused by SARS-CoV-2 infection.

[0110] Combination therapy

[0111] The 3C-like protease inhibitors described in the present invention can be combined with other drugs to treat cancer, including at least one target drug / viral activity modulator, including Remdesivir (Remdesivir or GS-5734), Lopinavir (Lopinavir), Molnupiravir (Molnupiravir), Ritonavir (Ritonavir), Chloroquine (Chloroquine or Sigma-C6628), Hydroxychloroquine and / or α-interferon, etc.

[0112] Example

[0113] The compounds and preparation methods disclosed herein will be described in further detail below with reference to specific examples. It should be understood that the following examples are intended only to illustrate and explain the present invention and should not be construed as limiting the scope of protection of the present invention. All technical solutions implemented based on the contents of the present invention are encompassed within the scope of protection intended by the present invention.

[0114] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art; the reagents, raw materials, instruments, equipment, etc. used in the following examples are all available from commercial channels.

[0115] Example 1

[0116] Synthesis of compound 1

[0117]

[0118] Synthesis of intermediate 1-2

[0119] Under ice-cooling, 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) and mixed. The mixture was stirred at room temperature for 6 hours, then the reaction mixture was concentrated and diluted with ethyl acetate (200 mL). The mixture was washed with saturated sodium bicarbonate solution (100 mL x 2). The organic phase was separated, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain intermediate 1-2 (10 g) as a brown solid, which was used directly in the next reaction.

[0120] LCMS (ESI) m / z: 198.0 [M+H] + .

[0121] Synthesis of intermediates 1-3

[0122] 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 mixture was stirred overnight at room temperature. The reaction mixture was washed with water (200 mL) and saturated sodium chloride solution (200 mL). The organic phase was separated, 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 afford Intermediate 1-3 as a yellow solid (4.93 g, 46.1% yield).

[0123] LCMS (ESI) m / z: 212.0 [M+H] + .

[0124] Synthesis of intermediates 1-4

[0125] At room temperature, saturated ammonium chloride solution (100 mL) and reduced iron powder (3.91 g, 0.07 mol) were added to a solution of intermediate 1-3 (4.93 g, 0.02 mol) in anhydrous ethanol (100 mL). Stir at 80°C overnight. After cooling, the mixture was filtered, the filtrate was concentrated under reduced pressure, and diluted with ethyl acetate (100 mL). The mixture was washed with water (100 mL x 2), the organic phase was separated, 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 obtain intermediate 1-4 as a brown solid (3.4 g, 80.9% yield).

[0126] LCMS (ESI) m / z: 182.2 [M+H] + .

[0127] Synthesis of intermediates 1-6

[0128] To a solution of Intermediate 1-5 (10.0 g, 0.054 mol) in N,N-dimethylformamide (60 mL) at 0°C were 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). The mixture was stirred at room temperature for 5 hours. 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 then added at 0°C. The mixture was stirred at room temperature for 24 hours. The pH was adjusted to 3-4 with 1N HCl and the mixture was extracted with ethyl acetate (200 mL x 3). The organic phases were combined, washed with saturated sodium chloride solution (100 mL), separated, 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, dichloromethane:ethyl acetate=5:1) to give intermediate 1-6 (5.23 g, yield 45.1%) as a white solid.

[0129] LCMS (ESI) m / z: 252.0 [M+Na] + .

[0130] Synthesis of intermediates 1-7

[0131] 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 85°C for 16 hours. After cooling, water (100 mL) was added, and the mixture was extracted with ethyl acetate (200 mL x 3). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, petroleum ether:ethyl acetate = 2:1) to obtain intermediate 1-7 as a white solid (8.6 g, yield: 89%).

[0132] LCMS (ESI) m / z: 318.0 [M+H-56] + .

[0133] Synthesis of intermediates 1-8

[0134] 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. The mixture was concentrated under reduced pressure to obtain a white solid 1-8 (7.8 g), which was used directly in the next reaction.

[0135] LCMS (ESI) m / z: 318.0 [M+H] + .

[0136] Synthesis of intermediates 1-9

[0137] 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 at 100°C under a balloon of oxygen for 16 hours. After cooling, water (30 mL) was added, and the mixture was 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. The crude product was purified by column chromatography (silica gel, dichloromethane:methanol = 20:1) to afford Intermediate 1-9 as a white solid (120 mg, 40% yield).

[0138] LCMS (ESI) m / z: 395.0 [M+H] + .

[0139] 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)

[0140] To a solution of Intermediate 1-9 (120 mg, 0.3 mmol) in tetrahydrofuran (20 mL) was added lithium bis(trimethylsilyl)amide (0.6 mmol, 0.6 mL, 1.0 M in THF) and Intermediate 1-4 (67 mg, 0.37 mmol) sequentially at 0°C. The mixture was stirred at 0°C for 2 hours. The mixture was quenched with saturated ammonium chloride solution (30 mL) and 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 the filtrate was concentrated under reduced pressure. The crude product was purified by preparative high pressure chromatography (Gemini-C18 150 x 21.2 mm, 5 μm, acetonitrile-water (0.1% formic acid), gradient: 30%-60%) to afford 1 as a white solid (14.5 mg, 9% yield).

[0141] LCMS (ESI) m / z: 513.9 [M+H] + .

[0142] 1 H NMR(400MHz,DMSO-d6,DCl in D2O)δ9.17(d,J=2.0Hz,1H),9.05(d,J=5.5Hz,1H),8.77–8.74(m,1H),8.54(s,1H), 8.32-8.27(m,1H),7.88-7.82(m,2H),7.64-7.56(m,2H),5.34(s,2H),4.21(s,3H).

[0143] Synthesis of comparative compound 1

[0144]

[0145] Intermediates 1-8 and 1-4 were synthesized by referring to the synthesis method of compound 1.

[0146] Synthesis of intermediate 2-2

[0147] To a solution of 1-8 (250 mg, 0.79 mmol) in N,N-dimethylformamide (5 mL) were added triethylamine (398 mg, 3.94 mmol), 2-1 (324 mg, 2.37 mmol), copper acetate (242 mg, 1.35 mmol), and molecular sieves (300 mg) at room temperature. The mixed solution was stirred at 60°C under an oxygen flow for 16 hours. After completion of the reaction, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, dichloromethane:methanol = 30:1) to obtain compound 2-2 as a yellow oil (145.5 mg, yield: 45.1%).

[0148] LCMS (ESI) m / z: 409.1 [M+H]+ .

[0149] Synthesis of comparative compound 1

[0150] 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 ice-cooling. Under nitrogen protection, lithium bis(trimethylsilyl)amide (0.65 mL) was added at 0°C and stirred for 2 hours. After completion of the reaction, the mixed solution was quenched by addition of water (10 mL), extracted with ethyl acetate (10 mL*3), and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure. The crude product was purified by high-pressure preparative chromatography (column: Gemini-C18 150 x 21.2 mm, 5 μm. Mobile phase: ACN-H2O (0.05% NH3.H2O). Gradient: 35-40°C) to afford comparative compound 1 (49.5 mg, yield: 36.1%) as a white solid.

[0151] LCMS (ESI) m / z: 528.1 [M+H] + .

[0152] 1 H NMR(400MHz,CD3OD)δ8.43-8.32(m,2H),8.16(s,1H),7.70(s,2H),7.60(dd,J =18.4,8.2Hz,1H),7.55-7.05(m,2H),5.34(s,2H),4.17(s,3H),2.39(s,3H).

[0153] Biological Examples

[0154] In the following biological examples, compound 1 and comparative compound 1 among the test compounds were prepared by Example 1, and comparative compound 2 was prepared according to the method described in Y Unoh et al. (J. Med. Chem. 2022, 65, 9, 6499–6512, DOI: 10.1021 / acs.jmedchem.2c00117).

[0155] 1. Caco-2 cell model to evaluate the bidirectional permeability of compounds

[0156] 1.1 Instruments, Equipment and Materials

[0157] 1) The control drugs propranolol and Digoxin were purchased from MCE, and minoxidil was purchased from the China Food and Drug Administration.

[0158] 2) Caco-2 cells were purchased from the American Type Culture Collection (ATCC).

[0159] 3) FBS medium was purchased from Sigma, DMEM from Corning (Cambridge, MA), non-essential amino acids (NEAA), Hank's balanced salt solution (HBSS), and trypsin / EDTA from Thermo Fisher Scientific. Penicillin and streptomycin were purchased from Solebro.

[0160] 4) HTS-96-well Transwell plates and other sterile consumables were purchased from Corning.

[0161] 5) Millicell resistance measurement system was purchased from Millipore. Vision was purchased from Nexcelom Bioscience, Infinite 200PRO microplate reader was purchased from Tecan, and MTS2 / 4 orbital shaker was purchased from IKA Labortechnik.

[0162] 1.2 Experimental design

[0163] 1.2.1 Cell culture and plating

[0164] 1) High-glucose (4.5 g / L) DMEM medium containing L-glutamine, supplemented with 10% fetal bovine serum, 0.1 mg / mL streptomycin, and 100 units of penicillin was used for cell culture.

[0165] 2) Cultivate Caco-2 cells in cell culture flasks in an incubator set at 37°C, 5% CO2, and 95% relative humidity. Cells can be seeded in Transwell plates when they reach 70-90% confluence.

[0166] 3) Before seeding cells, add 50 μL of cell culture medium to each well of the upper Transwell chamber and 25 mL of cell culture medium to the lower culture plate. Incubate the culture plate in a 37°C, 5% CO2 incubator for 1 hour before seeding cells.

[0167] 4) After cell digestion, transfer the cell suspension to a round-bottom centrifuge tube and centrifuge at 120g for 5 minutes.

[0168] 5) Resuspend the cells in culture medium to a final concentration of 6.86×105 cells / mL. Add 50 μL of the cell suspension to each well of a 96-well Transwell culture plate, with a final seeding density of 2.4×105 cells / cm 2 .

[0169] 6) Start changing the medium 48 hours after inoculation and culture for 14-18 days, changing the medium every other day.

[0170] 7) Change the culture medium as follows: Separate the Transwell chamber from the receiving plate, discard the culture medium in the receiving plate first, then discard the culture medium in the Transwell chamber. Finally, add 75 μL of fresh culture medium to each chamber and 25 mL of fresh culture medium to the receiving plate.

[0171] 1.2.2 Evaluation of cell monolayer integrity

[0172] 1) After 14-18 days of culture, Caco-2 cells should be fully confluent and differentiated. At this point, they can be used for penetration assays.

[0173] 2) The resistance of the monolayer membrane was measured using a resistance meter (Millipore, USA), and the resistance of each pore was recorded.

[0174] 3) After the assay is completed, place the Transwell culture plate back into the incubator.

[0175] 4) Calculation of resistance: Measure resistance (ohms) × membrane area (cm 2 ) = TEER value (ohms·cm 2 ), if the TEER value is <230ohms·cm 2 , then the hole cannot be used for penetration test.

[0176] 1.2.3 Solution preparation

[0177] Prepare 1 L of buffer (HBSS, 10 mM HEPES, pH 7.4) by weighing 2.38 g HEPES and 0.35 g sodium bicarbonate, dissolving each in 900 mL of water. Then, add 100 mL of 10× HBSS, stir, adjust the pH to 7.4, and filter.

[0178] 2) Prepare a DMSO stock solution of the test substance. Prepare a 1 mM DMSO stock solution of the control drug. Dilute with buffer to a 5 μM working solution. Dilute the test substance with buffer to a 5 μM working solution. The final DMSO content of the system is 0.5%.

[0179] 3) Prepare the dosing solution:

[0180]

[0181] 4) Prepare the receiving end solution:

[0182]

[0183] 1.2.4 Drug penetration test

[0184] 1) Remove the Caco-2 Transwell plate from the incubator. Rinse the cell monolayer twice with buffer and incubate at 37°C for 30 minutes.

[0185] 2) To measure the apical-to-basolateral transport rate of compounds, 125 μL of test compound and control solution was added to each well (apical) of the insert. 235 μL of HBSS (10 mM HEPES, pH 7.4) buffer was added to each well of the test compound receiving plate (basolateral), and 235 μL of HBSS (10 mM HEPES, pH 7.4) buffer was added to each well of the control receiving plate (basolateral). A 50 μL sample was transferred from the apical solution and added to 200 μL of acetonitrile containing internal standards (100 nM alprazolam, 200 nM labetalol, 200 nM caffeine, and 2 μM ketoprofen) as the 0-minute apical administration sample for measurement.

[0186] 3) To measure the basolateral to apical transport rate of compounds, 285 μL of test compound and control drug solutions were added to each well of the receiver plate (basolateral side). 75 μL of HBSS (10 mM HEPES, pH 7.4) buffer was added to each well of the test compound insert (apical side). 75 μL of HBSS (10 mM HEPES, pH 7.4) buffer was added to each well of the control drug insert (apical side). A 50 μL sample was transferred from the basolateral solution and added to 200 μL of acetonitrile containing internal standards (100 nM alprazolam, 200 nM labetalol, 200 nM caffeine, and 2 μM ketoprofen) as the 0-minute basolateral dosing sample for measurement.

[0187] 4) Incubate in a 37°C CO2 incubator for 2 hours.

[0188] 5) After the transport experiment, transfer 50 μL of sample from the dosing port (apical port for Ap→Bl direction, basolateral port for Bl→Ap direction) to 200 μL of acetonitrile containing internal standards (100 nM alprazolam, 200 nM labetalol, 200 nM caffeine, and 2 μM ketoprofen). Transfer 50 μL of sample from the receiving port (basolateral port for Ap→Bl direction, apical port for Bl→Ap direction) to 200 μL of acetonitrile containing internal standards (100 nM alprazolam, 200 nM labetalol, 200 nM caffeine, and 2 μM ketoprofen). Vortex for 5 minutes. Centrifuge the quenched samples at each time point at 3220 g for 30 minutes. Transfer 100 μL of the supernatant from each sample to a 96-well plate, and add 100 μL of purified water to the corresponding well. Vortex the sample analysis plate and perform LC / MS / MS analysis. All incubations were performed in duplicate.

[0189] 6) After the two-hour transport experiment, measure the fluorescence value. Prepare a 10mM Lucifer Yellow stock solution with water and dilute it to 100μM with transport buffer. Add 100μL of Lucifer Yellow solution to the top of the Transwell chamber and 300μL of transport buffer to the base. Incubate at 37°C in a CO2 incubator for 30 minutes. Remove 80μL of solution directly from the top and base (using the basolateral wells) and transfer to a new 96-well plate. Measure the cell fluorescence value (to detect membrane integrity) using a microplate reader with an excitation wavelength of 485nM and an emission wavelength of 530nM.

[0190] 1.3 Data Analysis

[0191] Data calculations were performed using Excel. Peak areas were calculated from ion chromatography results. The apparent permeability coefficient (P app , unit: cm / s×10 -6 ) is calculated using the following formula:

[0192]

[0193] In the formula: V A is the volume of the receiving end solution (Ap→Bl is 0.235 mL, Bl→Ap is 0.075 mL), Area is the Transwell-96 well plate membrane area (0.143 cm 2 ); time is the incubation time (unit: s).

[0194] P app(B-A) is the apparent permeability coefficient from the base to the top; P app(A-B) is the apparent permeability from the top to the base.

[0195] 2. In vitro evaluation of PXR activation potential in DPX2 cells

[0196] 2.1 Materials and Reagents

[0197] 1) DPX2 (a HepG2 cell line stably transfected with PXR and luciferase) cells were obtained from Puracyp Inc (Carlsbad, CA).

[0198] 2) The positive control drug rifampicin was purchased from Sigma (St. Louis, MO).

[0199] 3) CellTiter-Fluor TM Cell viability assay kit and One-Glo luciferase assay system were purchased from Promega (Madison, WI). FBS culture medium was purchased from Avantor, and DPX2 cell culture medium and drug administration medium were purchased from Puacyp.

[0200] 4) Reagent and consumables information

[0201] name Supplier Item No. batch number Puracyp Dosing Medium Puracyp D-500-100 - Puracyp Culture Medium Puracyp C-500-100 - FBS (fetal bovine serum) Avantor 76294-180 234D19 <![CDATA[CellTiter-Fluor TM Cell Viability Assay kit]]> Promega G6081 - One-Glo Luciferase Assay System Promega E6120 -

[0202] 2.2 Experimental design

[0203] 2.2.1 Cell culture and plating

[0204] 1) Preparation of DPX2 medium and dosing medium: add 45 ml of fetal bovine serum to 450 ml of DPX2 medium and 450 ml of dosing medium respectively.

[0205] 2) DPX2 cells were cultured in T-75 cell culture flasks in an incubator set at 37°C, 5% CO2, and 95% relative humidity. Cells were isolated when they reached 80-90% confluence.

[0206] 3) Rinse the cells cultured in the T-75 cell culture flask with 10 mL of PBS and aspirate. Add 3 mL of trypsin and incubate at 37°C for approximately 5 minutes or until the cells are completely detached and floating. Then, add excess serum-containing medium to inactivate the trypsin to terminate the digestion.

[0207] 4) After cell digestion, transfer the cell suspension to a conical-bottom centrifuge tube and centrifuge at 150g for 5 minutes. Resuspend the cells in culture medium to a final concentration of 4.5×10 5 cells / mL. 25 μL of cell suspension was added to each well of a 384-well culture plate. The plate was placed in an incubator and incubated for 24 hours. The cell plate can then be used for PXR experimental studies.

[0208] 2.2.2 Incubation of test compounds

[0209] 1) Solution Preparation: Prepare DMSO stock solutions of 1 and 10 mM test compound and 10 mM positive control rifampicin. The final concentrations of the test compound are 1 and 10 μM, respectively, and the final concentration of rifampicin is 10 μM. The DMSO content of the final system is 0.1%. A blank control is DMSO.

[0210] 2) Remove the cell plate from the incubator and directly add 25 nL of blank control solution, solution containing the test compound, and solution containing the positive control drug. Set up three replicates per group. After drug addition, transfer the cell plate to a cell culture incubator and continue incubation for 48 hours.

[0211] 3) Check the cell structure and monolayer integrity before testing to ensure it meets the study requirements.

[0212] 2.2.3 PXR activity testing

[0213] 1) After 48 hours of incubation, the cell plates can be used to test PXR activity.

[0214] 2) Make CellTiter-Fluor TM Allow the Cell Viability Assay Kit and ONE-Glo Luciferase Assay Reagent to reach room temperature. Add 10 μL of GF-AFC Substrate to a 10 mL Assay Buffer container to create a 2× reagent, then add 10 mL of PBS to dilute to a 1× reagent. Transfer the ONE-Glo Luciferase Assay Substrate to the ONE-Glo Luciferase Assay Buffer.

[0215] 3) Take out the DPX2-treated cell plate from the incubator and discard the culture medium in the cell plate. TM Pour the cell viability assay reagent into a sterile sample reservoir, use a multichannel pipette to add 25 μL of the reagent to each well, and place the cell plate in a 37°C CO2 incubator and incubate for 30 minutes.

[0216] 4) Remove the 384-well cell plate from the cell culture incubator, cool briefly to ambient temperature, and measure the cell fluorescence value using a microplate reader with an excitation wavelength of 400 nM and an emission wavelength of 505 nM.

[0217] 5) Pour ONE-Glo assay reagent into a sterile sample reservoir and add 25 μL directly to each well. Gently shake the plate to mix the solution. After incubating at room temperature for 5 minutes, read the chemiluminescence value of each well using a microplate reader.

[0218] 2.2.4 Data Analysis

[0219] 1) All data calculations were performed using Excel.

[0220] 2) Luciferase activity was determined by RLU / RFU, where RLU represents the average relative luminescence value of three parallel samples at each concentration of the test compound, and RFU represents the average relative fluorescence value of three parallel samples at each concentration of the test compound.

[0221] 3) The activation multiple is calculated by the following formula:

[0222]

[0223] The activation folds of the compounds of the present invention are reported in Table 2.

[0224] 4) The calculation formula for the positive control percentage is:

[0225] %Positive control=(Fold activation test compound / Foldactivationpositive control compound )*100%

[0226] 3. Intravenous and Oral Pharmacokinetics in Mice

[0227] 1. Dosage information

[0228]

[0229] Animal feeding control: fasting overnight before administration, free access to water, and feed 4 hours after administration. Dosing frequency: single administration on the day of the experiment.

[0230] The actual dosage volume was calculated based on the animal's body weight.

[0231] 2. Collection time

[0232]

[0233] Acceptable timeframe for blood sample collection

[0234]

[0235] 3. Blood sample collection and processing process

[0236]

[0237] 4. In vivo experimental evaluation

[0238]

[0239] 5. Experimental Animal Information

[0240]

[0241] 6. Sample analysis

[0242] The LC-MS / MS method was used to determine the blood drug concentration in plasma samples using the accompanying standard curve.

[0243] The plasma test results were analyzed using WinNonlin 8.3 (Phoenix TM ) or other similar software to calculate pharmacokinetic parameters. If applicable plasma drug concentration-time data are available, pharmacokinetic parameters will be calculated.

[0244] Pharmacokinetic data were presented using descriptive statistics such as mean, standard deviation, and sample size. Calculations were performed using Microsoft Excel 2013. Other pharmacokinetic parameters and statistical analyses were also performed and documented in the data summary.

[0245] 4. Enzyme Activity Assay

[0246] The biochemical experiments used the fluorescence resonance energy transfer (FRET) test method.

[0247] The assay system (120 μL) contained 108 μL of the main protease (WT or P132H) at a final concentration of 150 nM. The protein-free small molecule control group was added with 108 μL of protein diluent, which consisted of 50 mM Tris-HCl pH 7.4 and 1 mM EDTA.

[0248] Contains 10 μL substrate, final concentration of substrate is 20 μM; contains 2 μL of different concentrations of small molecules to be tested (3-fold serial dilution, dilution solvent is 100% DMSO), and the negative control NC here is added with 2 μL 100% DMSO.

[0249] Substrate: The fluorescent substrate is MCA-AVLQSGFR-LYS(DNP)-Lys-NH2.

[0250] The other components of the reaction system were reaction buffer 50 mM Tris-HCL pH 7.4, 1 mM EDTA.

[0251] The reaction process is as follows: First, mix 108 μL of the main protease or protein dilution with 2 μL of the small molecule and add it to a 96-well all-black microtiter plate (Corning Costar, #3916) and incubate for 30 minutes. After the incubation is completed, the fluorescent substrate is added and the detection is quickly started on the microtiter plate reader.

[0252] The enzyme-labeled detection method is:

[0253] During the kinetic monitoring process, the excitation light wavelength was 320 nm, the emission light wavelength was 405 nm, the detection interval was approximately 15 s, and the total detection time was 20 min. The fluorescence values of each reaction well at different times under these detection conditions were recorded, and the fluorescence values recorded in the first 200 s (generally 7-10 data points) were used for calculation.

[0254] The data processing and calculation process of this experiment is as follows:

[0255] First, the slope of the fluorescence value at each time point versus time for different reaction wells was calculated [Slope (fluorescence value RFU: time s)], which was recorded as the initial reaction rate V0. Initial velocity V0 = Slope (RFU in 200 s: time s).

[0256] The inhibition rate of the representative compound on the main protease was determined by the ratio of the initial reaction rate V0 of the enzyme to the initial reaction rate of the control group using different drug concentrations (Formula 1, Formula 2, and Formula 2 optimization), and then the IC was calculated using GraphPad Prism nonlinear fitting curve. 50 value.

[0257] Formula 1: Inhibition rate (%) = (RFU100％酶活性对照 -RFU 样品 ) / (RFU 100％酶活性对照 -RFU 空白对照 )×100%

[0258] Formula 2: Inhibition rate (%) = (NC initial velocity V0 - sample initial velocity V0) / NC initial velocity V0 × 100%;

[0259] NC enzyme activity is 100%, and the inhibition rate is 0%

[0260] Formula 2 optimization: Inhibition rate (%) = (NC initial velocity V0 - (sample initial velocity V0 - protein-free small molecule control group V0) / NC initial velocity V0 × 100%) is used to eliminate the problem of negative V0 (slope).

[0261] The GraphPad Prism software nonlinear fitting curve {Nonlinear regression (curve fit)-[inhibitor] vs. response-Variable slope (four parameters)} was used to fit the half-maximal inhibitory concentration IC50 value and the inhibition curve.

[0262] Note: Each test sample (sample and control) was replicated three times within the same group, and three biological replicates were performed. Standard deviation analysis was performed based on the biological replicates. The inhibition rate was generally calculated using Formula 2.

[0263] 5. Anti-SARS-CoV-2

[0264] (1) Experimental materials

[0265] Cell lines: African green monkey kidney cells Vero E6 (ATCC, CRL-1586), human colorectal adenocarcinoma cells Caco-2 (ATCC, HTB-37), and human lung adenocarcinoma cells Calu-3 (ATCC, HTB-55).

[0266] Virus strain: 2019-nCoV-WIV04 (IVCAS 6.7512)

[0267] Infectious dose: MOI = 0.01

[0268] (2) Experimental methods

[0269] 1) 100 μL containing 2×10 4 The cells were seeded into 96-well plates and placed in a 37°C constant temperature and humidity incubator for overnight culture;

[0270] 2) After cells have adhered for 20 hours, the culture medium was aspirated and 100 μL of culture medium containing the indicated concentration of test compound plus CP-100356 was added to each well. Eight dilutions of each test compound were prepared, with 3-4 replicates per dilution. A DMSO-treated group and a normal cell group were also established. Except for the normal cell group, all other wells were treated with complete culture medium containing 0.01 MOI of virus and incubated at 37°C in a constant temperature and humidity incubator for 72 hours.

[0271] 3) 72 hours after infection, the cytopathic rate was recorded using a full-field cell scanner. Inhibition rate = (1 - cytopathic rate of the test compound group) × 100%.

[0272] 4) Calculate EC using four-parameter fitting based on the inhibition rate results 50 .

[0273] 6. Anti-drug resistance mutation L50F+E166A+L167F SARS-COV-2

[0274] (1) Construction of prokaryotic expression vector

[0275] 1) Design and synthesis of mutation primers

[0276] Primer software was used to design the mutation primers for L50F and E166A+L167F. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primer sequences are shown in the table below:

[0277]

[0278] 2) Cloning and transformation of mutant plasmids

[0279] Using the wild-type main protease plasmid as a template, PCR amplification was performed using the Fast Mutagenesis System Single Point Mutation Kit (purchased from TRAN). The amplified product was digested with the DMT restriction endonuclease to methylate the plasmid template and then transformed into competent cells with degraded methylated plasmids. The specific steps are as follows:

[0280] PCR system and conditions

[0281]

[0282] a) PCR: 94℃5min, 94℃30s, 66℃20s, 72℃1min, 30cycles, 72℃10min.

[0283] b) Digestion of PCR products: Add 1 μL of DMT enzyme to the PCR product, mix well, and incubate at 37°C for 1 hour.

[0284] c) Transformation: Add 8 μL of digested product to 50 μL of competent cells, mix well, and incubate on ice for 30 minutes. Heat shock the culture in a 42°C water bath for 45 seconds, then immediately place on ice for 3 minutes. Add 250 μL of antibiotic-free LB medium equilibrated to room temperature and incubate at 200 rpm and 37°C for 1 hour. Spread 100 μL of the bacterial solution evenly on a plate containing antibiotics and incubate at 37°C overnight.

[0285] d) Monoclonal identification: Single colonies were selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. After correct sequencing, the plasmid was extracted and transformed into BL21 competent cells for protein expression.

[0286] (II) Expression and purification of mutant proteins

[0287] 1) Prokaryotic expression of proteins

[0288] The successfully constructed mutant plasmid was transformed into BL21 (DE3) competent cells, and a single colony was selected the next day and cultured at 37°C until the bacterial liquid D 600 When the nanometer value reached 0.6-0.8, IPTG with a final concentration of 0.5 mol / L was added to induce expression overnight.

[0289] Protein purification by Ni-NAT affinity chromatography and molecular sieve purification

[0290] Buffer:

[0291] a) Affinity chromatography basal buffer: 50 mM Tris, 500 mM NaCl, 10% glycerol, pH = 8.0;

[0292] b) Molecular sieve buffer: 25 mM Hepsin, 150 mM NaCl, pH = 7.4.

[0293] Collect the precipitate from the overnight expression culture, crush it under high pressure, and centrifuge it at 18,000 rpm for 40 minutes. Collect the supernatant and purify it by Ni-NAT affinity chromatography. For nickel affinity chromatography, wash the nickel column with ddH2O and then equilibrate it with equilibration buffer. Bind the filtered bacterial supernatant to the nickel affinity chromatography gel, wash it with wash buffer containing 20 mM imidazole, and elute it with elution buffer containing 500 mM imidazole.

[0294] The supernatant, precipitate, flow-through, and protein samples eluted from each concentration gradient were collected after disruption, boiled, and then subjected to SDS-PAGE electrophoresis to verify protein expression. The eluted protein was concentrated using an ultrafiltration tube with a molecular weight cutoff of 30 kD at 3500 rpm and 4°C.

[0295] The protein was concentrated to the standard loading volume of AKTA PURIFIER molecular sieves (approximately 0.5 ml) and further purified using GE Healthcare product Superdex 200 Increase. The protein sample after molecular sieve purification was collected and concentrated to 10 mg / ml, aliquoted and frozen at -80°C until use.

[0296] (3) Fluorescence resonance energy transfer assay (FRET) Ki value measurement

[0297] 1) Reaction buffer: 50 mM Tris pH 7.4, 1 mM EDTA, 0.01% triton X-100.

[0298] 2) Testing system

[0299] Protein (108uL): Main protease (L50F+E166A+L167F), final concentration 500nM, small molecule control group without protein, add 108uL reaction buffer;

[0300] Substrate (10uL): Fluorescent substrate MCA-AVLQ-SGFR-Lys(Dnp)-Lys-NH2 (stored at 24mM). Dilute the 24mM substrate to 0.06mM and 0.12mM with DMSO, so that the final concentrations in the reaction system are 5uM and 10uM, respectively.

[0301] Small molecules (2 μL): Compound 1, Comparative Compound 1, and Comparative Compound 2 (stocked at 50 mM). Dilute the 50 mM concentration of the small molecules to 0.06 mM with 100% DMSO, for a final concentration of 1 μM in the reaction system. Serial dilution of the small molecules: Starting at 0.06 mM (final concentration 1 μM), serially dilute the above small molecules 2-fold to well 8. The negative control (NC) is prepared by adding 2 μL of 100% DMSO.

[0302] 3) Reaction process

[0303] First, mix 108 μL of the main protease or reaction buffer with 2 μL of the small molecule in a 96-well black microtiter plate (Corning Costar, #3916) and incubate for 30 minutes. After the incubation is complete, add the fluorescent substrate and quickly start the assay on a microtiter plate reader.

[0304] 4) Board layout:

[0305]

[0306]

[0307] 5) ELISA detection method

[0308] During the kinetic assay, the excitation wavelength was 320 nM, the emission wavelength was 405 nM, the detection interval was approximately 15 s, and the total detection time was 20 min. The fluorescence values at different times in each reaction well under these test conditions were recorded, and the fluorescence values recorded in the first 200 s were used for calculation (usually 7-10 data points).

[0309] 6) Data Analysis

[0310] a) Slope calculation: The fluorescence value within the first 200 s was selected to calculate the slope. The slope of the blank group was subtracted to obtain the V at each small molecule concentration. 1 / V was calculated and Dixon plotting was performed using GraphPad Prism 8.0.1.

[0311] b) Dixon plotting to determine Ki: Plot the slope 1 / V of a cluster of straight lines drawn at different fixed I values against the corresponding I at two different substrate concentrations. The intersection of the fitted straight lines at different substrate concentrations is the -Ki.

[0312] The structures and data comparisons of the compounds of the present invention and comparative compounds 1-2 are shown in Tables 1 and 2 below.

[0313] Table 1

[0314]

[0315] Table 2

[0316]

[0317]

[0318] Note:

[0319] Caco2:P app(B-A) is the apparent permeability coefficient from the base to the top; P app(A-B) is the apparent permeability from the top to the base

[0320] IV: intravenous

[0321] PO: Oral

[0322] T 1 / 2 :half life

[0323] C max : Peak plasma concentration

[0324] AUC last : Area under the drug-time curve, used to evaluate the extent of drug absorption

[0325] F%: Bioavailability

[0326] PXR: receptor activation-induced

[0327] The above data show that the compounds of the present invention, while maintaining excellent inhibitory activity, also achieve better stability, lower drug side effects, better pharmacokinetic properties, and better biological activity than the control compounds against the drug-resistant mutation L50F+E166A+L167F main protease produced under pressure screening of the peptidomimetic anti-SARS-COV-2 drug ALG-097161.

[0328] Specifically, the Caco2 permeability test characterizes the permeability of the drug. app(B-A) Indicates the permeation effect from blood to small intestine, P app(A-B) The compound of the present invention has a better penetration effect from the small intestine to the blood than the other two comparative compounds, and is less likely to penetrate from the blood to the small intestine, that is, the drug penetration effect is excellent.

[0329] In terms of PK, the half-life of compound 1, whether intravenously injected or orally, is longer than that of comparative compound 1 and comparative compound 2, indicating that the drug is eliminated more slowly in the body and has better in vivo stability. max ) remains consistent, its AUC last The value is significantly higher than that of the other two compounds, indicating that compound 1 has excellent absorption and in vivo exposure. In addition, compound 1 has more excellent bioavailability and has better pharmacokinetic properties than the other two compounds.

[0330] PXR represents a drug-drug interaction. Specifically, PXR activation induces metabolic enzymes (such as CYP3A4), which can affect the pharmacokinetics of exogenous and endogenous substances. Overactivation of CYP3A4 can accelerate the metabolism of CYP3A4-mediated anti-tumor drugs, analgesics, and other drugs, affecting the efficacy of combined medications. Compound 1 of the present invention exhibits significantly lower PXR values at various concentrations than comparative compounds 1 and 2, demonstrating a lower risk of drug interactions and toxic metabolism.

[0331] The biochemical activity of compound 1 against the main protease of drug-resistant mutation L50F+E166A+L167F is more advantageous than that of comparative compounds 1 and 2.

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof: (I)。 2. A pharmaceutical composition comprising a compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, and optionally comprising at least one physiologically / pharmaceutically acceptable excipient.

3. The pharmaceutical composition of claim 2, wherein The pharmaceutical composition may also contain other active ingredients.

4. The pharmaceutical composition of claim 3, wherein The other active ingredients are selected from: Remdesivir (Remdesivir or GS-5734), Lopinavir (Lopinavir), Molnupiravir (Molnupiravir), Ritonavir (Ritonavir), Chloroquine (Chloroquine or Sigma-C6628), Hydroxychloroquine or α-interferon.

5. The pharmaceutical composition of claim 2, wherein The pharmaceutical composition is an RNA-dependent RNA polymerase inhibitor, a 3CLpro protease inhibitor, a CYP3A4 inhibitor or a host-targeted antiviral drug.

6. A kit comprising the compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, and other active ingredients.

7. The kit according to claim 6, wherein The other active ingredients are selected from: remdesivir, lopinavir, monoprevir, ritonavir, chloroquine, hydroxychloroquine or α-interferon.

8. Use of a compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof, and optionally other active ingredients, in the preparation of a medicament for treating or preventing viral infection.

9. The method according to claim 8, wherein The medicament is used to treat or prevent diseases, conditions, syndromes and / or disorders caused by viral infection.

10. The method according to claim 8, wherein Other active ingredients are present in the medicament, and the compound and the other active ingredients are present in the same unit dosage form.

11. The method according to claim 8, wherein Other active ingredients are present in the pharmaceutical, and the compound and other active ingredients are present in separate unit dosage forms.

12. The method according to claim 8, wherein The other active ingredients are selected from: remdesivir, lopinavir, monoprevir, ritonavir, chloroquine, hydroxychloroquine or α-interferon.

13. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for treating or preventing viral infection in combination with other active ingredients.

14. The method according to claim 13, wherein The medicament is used to treat or prevent diseases, conditions, syndromes and / or disorders caused by viral infection.

15. The method according to claim 13, wherein The other active ingredients are selected from: remdesivir, lopinavir, monoprevir, ritonavir, chloroquine, hydroxychloroquine or α-interferon.

16. Use of other active ingredients in the preparation of a medicament for treating or preventing viral infection in combination with a compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof.

17. The method according to claim 16, wherein The medicament is used to treat or prevent diseases, conditions, syndromes and / or disorders caused by viral infection.

18. The method according to claim 16, wherein The other active ingredients are selected from: remdesivir, lopinavir, monoprevir, ritonavir, chloroquine, hydroxychloroquine or α-interferon.

19. Use of the compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, or the pharmaceutical composition according to any one of claims 2 to 5, or the kit according to any one of claims 6 to 7, in the preparation of a medicament for treating or preventing viral infection.

20. The use according to claim 19, wherein The medicament is used to treat or prevent diseases, conditions, syndromes and / or disorders caused by viral infection.

21. The method of claim 19, wherein The other active ingredients are selected from: remdesivir, lopinavir, monoprevir, ritonavir, chloroquine, hydroxychloroquine or α-interferon.

22. The method according to any one of claims 8 to 21, wherein The compound or pharmaceutical composition inhibits viral proliferation.

23. The use according to claim 22, wherein The compound or pharmaceutical composition inhibits the activity of viral 3CL protease.

24. The method according to claim 23, wherein The 3CL protease has a P132H mutation.

25. The use according to claim 22, wherein The virus is a coronavirus.

26. The use according to claim 22, wherein The virus is an alpha coronavirus and / or a beta coronavirus.

27. The method of claim 22, wherein The virus is SARS-CoV-2.

28. The use according to any one of claims 9, 14, 17 and 20, 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, fatigue, respiratory tract infection, pneumonia, olfactory disorder, taste disorder and complications thereof, or a combination thereof.

29. The method of claim 28, wherein The virus is a coronavirus.

30. The use of claim 28, wherein The virus is an alpha coronavirus and / or a beta coronavirus.

31. The method of claim 28, wherein The virus is SARS-CoV-2.

Images