WO2022021841A1 - Inhibiteur principal de protéase du nouveau coronavirus, son procédé de préparation et son utilisation - Google Patents

Inhibiteur principal de protéase du nouveau coronavirus, son procédé de préparation et son utilisation Download PDF

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WO2022021841A1
WO2022021841A1 PCT/CN2021/076400 CN2021076400W WO2022021841A1 WO 2022021841 A1 WO2022021841 A1 WO 2022021841A1 CN 2021076400 W CN2021076400 W CN 2021076400W WO 2022021841 A1 WO2022021841 A1 WO 2022021841A1
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alkyl
alkylene
substituted
none
halogenated
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PCT/CN2021/076400
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Chinese (zh)
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杨胜勇
李琳丽
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四川大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4025—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil not condensed and containing further heterocyclic rings, e.g. cromakalim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/422—Oxazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/16—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the invention belongs to the technical field of organic synthetic medicines, and in particular relates to an inhibitor of novel coronavirus main protease, a preparation method and pharmaceutical use thereof.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • alpha-interferon and anti-HIV drugs lopinavir/ritonavir have been used clinically combination, but still has very limited efficacy and may have toxic side effects.
  • Remdesivir a broad-spectrum antiviral drug developed by Gilead Sciences, Inc., is also being explored to treat COVID-19, but more data is needed to prove its efficacy. Therefore, there is still an urgent need to develop safe and effective anti-SARS-CoV-2 drugs.
  • the genomic RNA of coronavirus is about 30knt long, has a 5' cap structure and a 3'-poly-a tail, and contains at least 6 open reading frames (ORFs).
  • ORF1a/b occupies approximately two-thirds of the genome length and directly translates two polyproteins: pp1a and pp1ab, with an a-1 frameshift between ORF1a and ORF1b.
  • Mpro major protease
  • 3CLpro 3C-like protease
  • PEPs papain-like proteases
  • Nonstructural proteins are involved in the production of subgenomic RNAs, encoding the four major structural proteins (envelope (E), membrane (M), spine (S), and nucleocapsid (N) proteins) and other auxiliary proteins to complete the Virus replication and invasion process.
  • E envelope
  • M membrane
  • S spine
  • N nucleocapsid
  • M pro is conserved among coronaviruses, and the substrates of M pro in different coronaviruses have some common features: amino acids from N-terminal to C-terminal are numbered in a paired form (-P4-P3-P2-P1 ⁇ P1 '-P2'-P3'), the cleavage site is between P1 and P1'.
  • Mpro has a unique substrate preference for glutamine at the P1 site (Leu-Gln ⁇ (Ser, Ala, Gly)), which is absent in host proteases, suggesting that by targeting the virus It is feasible to achieve high selectivity with M pro .
  • the absolute dependence of the virus on the correct function of this protease, coupled with the lack of a homologous human protease makes Mpro an ideal antiviral target.
  • the purpose of the present invention is to provide the inhibitor of novel coronavirus main protease and its preparation method and pharmaceutical use.
  • the present invention provides the compound shown in formula I, or its pharmaceutically acceptable salt, or its stereoisomer, or its optical isomer, or its isotopic substitution form:
  • X is O or S
  • Ring A is selected from the following groups unsubstituted or substituted by one or more R 6 : 5-6 membered saturated heterocyclyl, 5-6 membered unsaturated heterocyclyl, saturated heterofused ring, unsaturated heterofused ring group; R 6 is independently selected from C 1-6 alkyl, C 1-6 alkoxy, halogen, hydroxyl, cyano, amino, and carboxyl;
  • R 3 is L 3 M 0 L 4 R 3a ; wherein L 3 is selected from none, C 1-4 alkylene, halogenated C 1-4 alkylene, C 2-4 alkenylene, halogenated C 2- 4 alkenylene, L 4 is selected from none, C 1-4 alkylene, halogenated C 1-4 alkylene, M 0 is selected from none, O, S, NH, CO, CONH, NHCO, R 3a is The following groups are unsubstituted or substituted by one or more R 3b : 5-6 membered aryl, 5-6 membered heteroaryl, unsaturated heterofused ring group, unsaturated fused ring alkyl; R 3b are each independently Selected from C 1-5 alkyl substituted or unsubstituted by R 3c , C 1-5 alkoxy substituted or unsubstituted by R 3c , halogen, phenyl substituted or unsubstituted by R 3c , NR 14 R
  • R 5 is selected from COR 8 or WCOOR 7 ; wherein, R 8 is selected from hydrogen or W is selected from none, C 1-4 alkylene, C 2-4 alkenylene, C 2-4 alkynylene, R 7 is selected from C 1-6 alkyl; M is selected from none, CO, NH, CONH , NHCO, COO or OCO, L 0 is selected from none, C 1-4 alkylene, C 2-4 alkenylene, L 1 is selected from none, C 1-4 alkylene, C 2-4 alkenylene , R 8a is selected from C 1-5 alkyl, halogenated C 1-5 alkyl, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic group, 5-6 membered aryl or 5-6 membered aryl Yuan Heteroaryl.
  • X is O or S
  • n is selected from an integer of 0-3, preferably an integer of 0-2;
  • R 1 and R 2 are each independently selected from hydrogen, C 1-5 alkyl, C 1-5 alkoxy, halogen, hydroxyl, cyano, amino, and carboxyl;
  • R 3 is L 3 M 0 L 4 R 3a ; wherein L 3 is selected from none, C 1-4 alkylene, halogenated C 1-4 alkylene, C 2-3 alkenylene, and L 4 is selected from none , C 1-4 alkylene, halogenated C 1-4 alkylene, M 0 is selected from none, O, S, NH, CO, CONH, NHCO, R 3a is unsubstituted or by one or more R 3b Substituted the following groups: phenyl,
  • R 3b are each independently selected from C 1-4 alkyl, halogen-substituted C 1-4 alkyl, deuterated C 1-4 alkyl, cyano-substituted C 1-4 alkyl, C 1-4 alkyl Oxyl, halogen-substituted C 1-4 alkoxy, deuterated C 1-4 alkoxy, cyano-substituted C 1-4 alkoxy, halogen, phenyl, halogenated phenyl, NR 14 R 15 , Hydroxyl, R 14 and R 15 are each independently selected from hydrogen or C 1-4 alkyl;
  • R 8 is selected from hydrogen or M is selected from None, CO, NH, CONH, NHCO, COO or OCO, L 0 is selected from None, C 1-3 alkylene, C 2-4 alkenylene, L 1 is selected from None, C 1-3 alkylene Alkyl, C 2-4 alkenylene, R 8a is selected from C 1-4 alkyl, halogenated C 1-4 alkyl, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic group, A 5- to 6-membered aryl group or a 5- to 6-membered heteroaryl group.
  • R 1 and R 2 are each independently selected from hydrogen, C 1-4 alkyl, C 1-4 alkoxy, halogen, and hydroxyl;
  • R 3 is selected from L 3 M 0 L 4 R 3a ;
  • L 3 is selected from none, C 1-3 alkylene, halogenated C 1-3 alkylene, C 2-3 alkenylene,
  • L 4 is selected from none, C 1-3 3 alkylene, halogenated C 1-3 alkylene, M 0 is selected from none, O, NH, CO, CONH,
  • R 3a is phenyl, phenyl substituted by one or more R 3b , R 3b
  • R 3b Each independently selected from C 1-4 alkyl, halogen-substituted C 1-4 alkyl, deuterated C 1-4 alkyl, cyano-substituted C 1-4 alkyl, C 1-4 alkoxy , halogen-substituted C 1-4 alkoxy, deuterated C 1-4 alkoxy, cyano-substituted C 1-4 alkoxy, halogen, phenyl, halogenated phenyl, NR
  • R 4 is selected from C 1-2 alkyl, COOR 10 , substituted or unsubstituted phenyl; the substituent is selected from hydroxyl, nitro; R a1 and R a2 are independently selected from hydrogen, C 1-3 alkyl, halogen; R 10 is C 1-3 alkyl;
  • R 8 is selected from hydrogen, CONHR 11 , L 2 COOR 12 , C 1-4 alkyl, halogenated C 1-4 alkyl; R 11 is selected from 3-6 membered saturated cycloalkyl, C 1-4 alkyl , benzyl, L 2 is a C 1-2 alkylene group, a C 2-3 alkenylene group, and R 12 is a C 1-3 alkyl group.
  • formula II is shown as formula II-1 or formula II-2:
  • X is O or S, preferably O;
  • R 1 and R 2 are each independently selected from hydrogen, C 1-3 alkyl, preferably methyl;
  • n is selected from an integer from 0 to 3
  • R 3b is independently selected from phenyl, halogenated phenyl, halogen, C 1-3 alkyl, halogenated or deuterated C 1-3 alkyl, C 1- 3 alkoxy, halogenated or deuterated C 1-3 alkoxy, hydroxyl;
  • R a1 and R a2 are independently selected from hydrogen, C 1-3 alkyl, and halogen;
  • R b is selected from hydrogen, C 1-3 alkyl, halogenated C 1-3 alkyl;
  • L 3 is selected from none, C 1-2 alkylene, halogenated C 1-2 alkylene, C 2 alkenylene, L 4 is selected from none, C 1-3 alkylene, halogenated C 1-3 Alkylene, M 0 is selected from none, O, NH, CO, CONH;
  • the halogen is preferably chlorine or fluorine.
  • the structure of the compound is one of the following structures:
  • the present invention also provides a pharmaceutical composition, wherein the pharmaceutical composition is the above compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer, or an isotopic substitution form thereof. Active ingredient, plus pharmaceutically acceptable excipients.
  • the present invention also provides the use of the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer, or an isotopic substituted form thereof, in the preparation of a coronavirus proteolytic enzyme inhibitor; preferably
  • the coronavirus proteolytic enzyme is a coronavirus main protease; more preferably, the coronavirus proteolytic enzyme is SARS-COV-2M pro .
  • the present invention also provides the use of the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer, or an isotopic substituted form thereof, in the preparation of an anti-coronavirus drug, preferably,
  • the coronavirus is a novel coronavirus SARS-CoV-2.
  • the present invention also provides the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopic substituted form thereof in the preparation of prevention and/or treatment of SARS-COV-2M pro
  • the disease related to SARS-COV-2M pro is novel coronavirus pneumonia COVID-19.
  • the medicine of described coronavirus proteolytic enzyme inhibitor, anti-coronavirus or the medicine of prevention and/or treatment of viral pneumonia can suppress the activity of SARS-COV-2M pro and/or can suppress SARS-COV-2 infection cell.
  • C a-b alkyl denotes any alkyl group containing "a" to "b” carbon atoms.
  • C 1-6 alkyl refers to a straight or branched chain alkyl group containing 1 to 6 carbon atoms.
  • Substituted herein refers to the replacement of 1, 2 or more hydrogen atoms in a molecule by a different atom or molecule, including 1, 2 or more substitutions on isotopic or isotopic atoms in the molecule .
  • Isotopic substitution form refers to a compound obtained by replacing one or more than two atoms in a compound with its corresponding isotope, for example, hydrogen in the compound is replaced by protium, deuterium or tritium.
  • “Pharmaceutically acceptable” means that a carrier, vehicle, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients that make up a pharmaceutical dosage form and physiologically compatible with receptor compatible.
  • Salt is an acid and/or base salt of a compound or its stereoisomer with inorganic and/or organic acids and/or bases, including zwitterionic salts (inner salts), and quaternary ammonium salts , such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. It can also be obtained by mixing a compound, or a stereoisomer thereof, with a certain amount of acid or base as appropriate (eg, equivalent). These salts may form a precipitate in solution and be collected by filtration, or recovered after evaporation of the solvent, or obtained by lyophilization after reaction in an aqueous medium.
  • “Pharmaceutically acceptable salts” may be hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, Succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate.
  • Halogen is fluorine, chlorine, bromine or iodine.
  • Aryl refers to an all-carbon monocyclic or fused polycyclic (ie, rings sharing adjacent pairs of carbon atoms) groups having a conjugated pi-electron system, such as phenyl.
  • the aryl group contains no heteroatoms, such as nitrogen, oxygen, or sulfur, while the point of attachment to the parent must be on a carbon atom on the ring with a conjugated pi-electron system.
  • Aryl groups can be substituted or unsubstituted.
  • the "5- to 6-membered aryl group” refers to an aryl group having 5 or 6 ring carbon atoms.
  • Heteroaryl refers to a heteroaromatic group containing one to more heteroatoms.
  • the heteroatoms referred to herein include oxygen, sulfur and nitrogen.
  • the heteroaryl group can be optionally substituted or unsubstituted.
  • the "5- to 6-membered heteroaryl group” refers to a heteroaryl group having 5 or 6 ring atoms.
  • Cycloalkyl refers to a saturated or unsaturated cyclic hydrocarbon substituent; the cyclic hydrocarbon may be monocyclic or polycyclic.
  • a 3- to 6-membered saturated cycloalkyl group refers to a saturated cycloalkyl group having 3 to 6 ring carbon atoms.
  • Heterocyclyl refers to a saturated or unsaturated cyclic hydrocarbon substituent; cyclic hydrocarbons may be monocyclic or polycyclic and carry at least one ring heteroatom (including but not limited to O, S or N).
  • the "3- to 6-membered saturated heterocyclic group” refers to a saturated heterocyclic group having 3 to 6 ring atoms.
  • Fused cycloalkyl refers to a polycyclic cycloalkyl in which two rings share two adjacent carbon atoms.
  • Hetero-fused ring group refers to a polycyclic heterocyclic group, which contains at least one heteroatom, and two rings in the polycyclic heterocyclic group share two adjacent carbon atoms or heteroatoms.
  • Alkylene refers to a group in which an alkyl group has lost one atom.
  • C 1 alkylene C 2 alkylene:
  • Alkenylene refers to a group in which an alkenyl group has lost one atom.
  • alkenyl group has lost one atom.
  • Alkynylene refers to a group in which an alkynyl group has lost one atom.
  • alkynyl group For example C 2 alkynyl:
  • the experimental results show that the present invention provides a compound that can effectively inhibit the activity of the new coronavirus main protease M pro , the compound can effectively inhibit the replication of SARS-COV-2 virus in cells, and inhibit the SARS-COV-2 virus in cells.
  • Infection resists in vivo SARS-COV-2 infection in transgenic mice; reduces viral load in the lungs of SARS-COV-2-infected transgenic mice, and reduces lung chemokine ligand 10 (CXCL10) and beta-type in mice
  • the gene expression level of interferon (IFN- ⁇ ) reduces the number of neutrophils (NEU) and macrophages (MAC) in the lungs of mice, and improves the pathological damage of the lungs of mice.
  • the compounds provided by the present invention also have good in vivo safety and pharmacokinetic properties.
  • the compounds of the present invention have very good application prospects in the preparation of SARS-CoV-2 M pro inhibitors, anti-SARS-CoV-2 drugs, and drugs for preventing and/or treating novel coronavirus pneumonia.
  • Fig. 1 is the inhibitory activity curve of compound 26 to SARS-COV-2M pro .
  • Fig. 2 is the inhibitory activity curve of compound 33 on SARS-COV-2M pro .
  • Figure 3 is the inhibitory activity curve of compound 37 against SARS-COV-2M pro .
  • Figure 4 Inhibition experiments of compounds on the replication of SARS-COV-2 in human alveolar epithelial cells.
  • FIG. 1 Pulmonary viral load in SARS-CoV-2-infected mice.
  • Figure 6 Lung histopathological sections (3dpi) of SARS-CoV-2-infected mice.
  • Figure 7 Representative cytokine expression levels (3dpi) in the lungs of SARS-CoV-2-infected mice.
  • Figure 8 Pulmonary neutrophil and macrophage counts (3 dpi) in SARS-CoV-2-infected mice.
  • the raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.
  • the compound 1 of the present invention is prepared according to the above-mentioned preparation route, and the reaction conditions of each step in the route are as follows:
  • i a, dimethyl 2-fluoromalonate, benzyl alcohol, toluene, p-toluenesulfonic acid, 110°C; b, isopropanol, n-hexane, -10°C;
  • Dibenzyl 2-fluoromalonate (18.4g, 60.9mmol, 1.0eq) was dissolved in 100mL of isopropanol, the temperature was raised to 45°C, sodium hydroxide (2.55g, 63.9mmol, 1.05eq) was dissolved in 60mL of water and then slowly Slowly drip, dripping time > 1 hour. After the dropwise addition, the reaction was continued for 30 minutes, the isopropanol was evaporated under reduced pressure, 50 mL of water was added, and the pH value was adjusted to about 9 with saturated sodium bicarbonate solution.
  • the aqueous phase was extracted twice with dichloromethane 20mL ⁇ 2 dichloromethane, the pH value of the aqueous phase was adjusted to 1-2 with 6mol/L hydrochloric acid, extracted three times with 40mL ⁇ 3 isopropyl ether, the organic phases were combined and washed with 30mL saturated brine once.
  • the organic phase was dried by adding anhydrous magnesium sulfate, filtered, and concentrated to obtain a viscous residue, which was added with 60 mL of n-hexane and stirred overnight. A white solid was precipitated, filtered, and the filter cake was dried under vacuum at 40 °C to obtain 6.5 g of the product with a yield of 50.3%.
  • Boc-L-aspartate 4-methyl ester (2.2g, 8.8mmol, 1.0eq) was dissolved in 50mL of anhydrous tetrahydrofuran, protected by argon replacement, cooled to 0°C, added CDI (1.5g, 9.3mmol, 1.05eq) ) and incubated for 1 hour.
  • the reaction solution was cooled to -20°C, 1.5 eq of intermediate 4 was slowly added, and the reaction was incubated for 1 hour, and then the temperature was raised to room temperature for 6 hours.
  • reaction solution was slowly poured into 300 mL of 2M dilute hydrochloric acid under an ice-water bath, extracted three times with 100 mL ⁇ 3 ethyl acetate, the organic phases were combined, washed with saturated sodium bicarbonate solution until weakly alkaline, and washed once with 50 mL of saturated brine. Anhydrous magnesium sulfate was added to dry, filtered and concentrated, and the obtained crude product was directly used in the next reaction.
  • the compound 3 of the present invention is prepared according to the above-mentioned preparation route, and the reaction conditions of each step in the route are as follows:
  • Boc-L-dimethyl glutamate (12 g, 43.6 mmol, 1.0 eq) was dissolved in 100 mL of anhydrous tetrahydrofuran, protected by argon replacement, cooled to -78°C, and slowly added dropwise 94 mL of LiHMDS tetrahydrofuran solution (1M tetrahydrofuran solution, 94mmol, 2.2eq), the reaction was incubated for 1 hour after the dropwise addition. 3.24 mL of bromoacetonitrile (46.6 mmol, 1.1 eq) was slowly added dropwise to the reaction solution, and the reaction was incubated for 6 hours and then quenched with 50 mL of saturated ammonium chloride solution.
  • the pH value of saturated citric acid aqueous solution was adjusted to neutral, the tetrahydrofuran was evaporated under reduced pressure, extracted once with 10 mL of ethyl acetate, the aqueous phase was adjusted to pH 3-4 with saturated aqueous citric acid solution, extracted three times with 20 mL ⁇ 3 ethyl acetate, and the organic The phase was washed with 20 mL of saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain 0.78 g of an off-white solid with a yield of 93.2%.
  • reaction solution was slowly poured into 300 mL of 2M dilute hydrochloric acid under an ice-water bath, extracted three times with 100 mL ⁇ 3 ethyl acetate, the organic phases were combined, washed with saturated sodium bicarbonate solution until weakly alkaline, and washed once with 50 mL of saturated brine. Anhydrous magnesium sulfate was added to dry, filtered and concentrated, and the obtained crude product was directly used in the next reaction.
  • intermediate 18 500 mg was dissolved in 5 mL of dichloromethane, and then 5 mL of dioxane hydrochloride was added. After the reaction was completed, the mixture was spin-dried to obtain intermediate 19 with a yield of 85%.
  • Ethyl (1S,3aR,6aS)-2-(2-(2,4-dichlorophenoxy)acetyl)octahydrocyclopenteno[c]pyrrole-1-carboxylate (Intermediate 22, 200 mg, 0.52 mmol) was dissolved in 20 mL of methanol, then 2M sodium hydroxide solution (10 mL) was added, and the reaction was stirred at 25 ° C for 4 hours. After the reaction was monitored by TLC, the methanol was spin-dried, and the pH was adjusted to weakly acidic with hydrochloric acid. Methyl chloride was extracted three times, and the organic phases were combined and dried over anhydrous sodium sulfate, and then the crude product was selected and dried, and the crude product was directly carried out to the next step.
  • the compound 9 of the present invention is prepared according to the above-mentioned preparation route, and the reaction conditions of each step in the route are as follows:
  • the raw material 23 (quinoline 2-carboxylic acid 1.0 g, 11.6 mmol), 1-hydroxybenzotriazole (2.03 g, 15.08 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbon two Imine hydrochloride (4.43g, 23.3mmol), 30mL of N,N-dimethylformamide was placed in a round-bottomed flask and stirred at room temperature for 0.5 hours, 2.5mL of N,N-diisopropylethylamine was added, and 0.59 mL of N,N-diisopropylethylamine was added.
  • the intermediate 28 (((1R,5S)-6,6-dimethyl-3-(quinoline-2-carbonyl)-3-azabicyclo[3.1.0]hexane-2-carbonyl)-L -Phenylalanine) 500 mg, dissolved in 30 mL of dry methanol, sodium borohydride was added at room temperature, stirred for 3 hours, quenched by adding water, spin-dried methanol, and the aqueous phase was extracted with ethyl acetate (50 mL ⁇ 3), The collected organic phase was dried with sodium sulfate, and after suction filtration, the organic phase was spin-dried to obtain intermediate 29 with a yield of 80%.
  • the compound 14 of the present invention is prepared according to the above-mentioned preparation route, and the reaction conditions of each step in the route are as follows:
  • reaction was carried out under argon protection at room temperature for 12 hours.
  • the compound 15 of the present invention is prepared according to the above-mentioned preparation route, and the reaction conditions of each step in the route are as follows:
  • TLC detected the end of the reaction, quenched by adding water, spin-dried methanol, and extracted the remaining aqueous phase with ethyl acetate (50 mL ⁇ 3). The organic phases were combined, dried with anhydrous sodium sulfate, filtered and spin-dried to obtain a white solid as a crude product, which was directly used in the next reaction.
  • the compound 42 of the present invention was prepared according to the above-mentioned preparation route, and the reaction conditions of each step in the route were as follows:
  • reaction was further carried out for 2 hours, and then acetic acid and tetrahydrofuran were added under low temperature conditions for quenching, and the resulting black suspension was further stirred for 10 minutes while warming to room temperature.
  • the reaction was further diluted with ethyl acetate, washed with water, saturated sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated by column chromatography to obtain a pale yellow solid as intermediate 38.
  • the compound 50 of the present invention is prepared according to the above-mentioned preparation route, and the reaction conditions of each step in the route are as follows:
  • Recombinant SARS-CoV-2M pro (750 nM final concentration) was mixed with serial dilutions of each compound in 25 ⁇ L of assay buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 mM DTT) and incubated 10 minutes.
  • the reaction was initiated by adding 25 ⁇ L of fluorogenic substrate (MCA-AVLQ ⁇ SGFR-Lys(Dnp)-Lys-NH2) at a final concentration of 20 ⁇ M, and the fluorescence signal at 320nm (excitation)/405nm (emission) was measured with a microplate reader.
  • Vmax of the reaction to which different concentrations of compound were added and the Vmax of the reaction to which DMSO was added was calculated and used to generate an IC50 curve.
  • half inhibitory concentration (IC50 ) values against SARS-CoV-2M pro were measured at 9 concentrations and 3 independent replicates. All experimental data were analyzed using GraphPad Prism software.
  • Anti-M pro IC 50 ( ⁇ M) 1 1.1 2 0.11 3 0.018 4 0.012 5 0.011 6 0.009 7 0.015 8 0.017 9 24.24 10 0.585 11 0.960 12 16.93
  • the compounds of the present invention can effectively inhibit the activity of SARS-CoV-2M pro , and can be used to prepare SARS-CoV-2M pro inhibitors and anti-new coronavirus medicines, and the preparation of medicines for the prevention and/or treatment of novel coronavirus pneumonia.
  • Experimental Example 2 Inhibition experiment of the compounds of the present invention on cell death caused by SARS-COV-2 infection of Vero E6 cells
  • the antiviral activity of the compounds was preliminarily evaluated by detecting the inhibitory effect of the compounds on cell death caused by SARS-COV-2 infection of Vero E6 cells.
  • NT 33 5.57 34 1.64 35 NT 36 2.23 37 2.97 38 NT 39 NT 40 NT 41 NT 42 NT 43 0.53 44 0.66 45 0.83 46 NT 47 NT 48 NT 49 NT 50 NT 51 NT 52 NT 53 NT 55 NT 56 NT 57 NT 58 NT 59 NT 60 NT 61 NT 62 NT 63 NT 64 NT 65 NT 66 NT 67 NT 68 NT 69 NT 70 NT 71 NT 72 NT 73 NT
  • NT stands for untested cell viability
  • the compound of the present invention can effectively inhibit the cell death caused by SARS-COV-2 infection of Vero E6 cells, indicating that the compound of the present invention can effectively inhibit the replication of SARS-COV-2 virus in cells.
  • Cytotoxicity assessment of compounds was performed using Vero E6 cells.
  • the specific experimental protocol is as follows: Vero E6 cells were seeded in a 96-well plate at a cell density of 2 ⁇ 10 4 cells/well, 100 ⁇ L/well, and incubated overnight at 37° C. in a 5% CO2 incubator. The next day, 200 ⁇ L of drug-containing medium was added to each well. The compound was initially diluted at 200 ⁇ M, with a 5-fold gradient dilution. There were 6 gradients in total. Three replicate wells were set for each concentration. Each group of experiments set a negative control and blank without drug. control. After 72 hours of drug treatment, the cell viability was detected by using the CCK-8 kit, and the toxicity of the compound to Vero E6 cells and the cytotoxicity concentration (CC 50 ) value were calculated. All experimental data were analyzed using GraphPad Prism software.
  • the compounds of the present invention have very low toxicity to Vero E6 cells.
  • the anti-SARS-COV-2 activity of compounds 3 and 39 in Vero E6 cells was evaluated using the plaque assay.
  • Vero E6 was seeded in a 24-well cell culture plate at 1.0 ⁇ 10 5 cells per well, and incubated overnight at 37°C for use. After adding the serially diluted drug, SARS-CoV-2 infected cells were added, and the MOI was about 0.002. After culturing in a 37°C cell incubator for 1 hour, the drug-containing infection supernatant was removed, washed with PBS, and 0.5 mL of sodium carboxymethyl cellulose containing different concentrations of drugs was added, and the final concentration of sodium carboxymethyl cellulose was 0.9%. Placed in a 37°C cell incubator for 72 hours.
  • inhibition rate (%) (number of plaques in virus control wells - number of plaques in sample wells)/number of plaques in virus control wells*100
  • the experimental results are shown in Table 5.
  • the compounds of the present invention can effectively inhibit SARS-COV-2 infection in Vero E6 cells; in particular, compound 3 has an EC 50 of 0.2373 ⁇ M, and its activity is better than that of the positive control Remdesivir (EC 50 of 0.692 ⁇ M).
  • test compounds 60 male Sprague-Dawley (SD) rats, weighing 200-230 g, were randomly divided into 3 groups with 3 rats in each group.
  • the series of test compounds were administered by gavage (p.o.), intravenous (i.v.) and intraperitoneal (i.p.), respectively, according to the protocol in Table 6 below. They were fasted for 12 h before the experiment and had free access to water. 2h after the administration of unified food.
  • Gavage, intravenous and intraperitoneal administration solutions were formulated in DMSO/HS15/NaCl (5/3/92, v/v/v).
  • Administer the drug according to the dosage shown in Table 6, record the administration time, and collect blood through the jugular vein at the time set above or other suitable methods. Each sample is collected about 0.20mL, heparin sodium is anticoagulated, and placed on ice after collection. superior. Plasma was centrifuged within 1 hour (centrifugation conditions: 6800g, 6 minutes, 2-8°C). Plasma samples were stored in a -80°C freezer prior to analysis. The grouping and blood collection time points are shown in Table 6, with 3 animals at each time point.
  • the oral exposure of compound 26 was 842 h*ng/mL, and the bioavailability was 7.2%.
  • the oral exposure of compound 39 was 14586 h*ng/mL, and the bioavailability was 14.7%.
  • the oral exposure of compound 40 was 2888h*ng/mL, and the bioavailability was 22.1%.
  • the oral exposure of compound 43 was 258 h*ng/mL, and the bioavailability was 4.8%.
  • the oral exposure of compound 44 was 381 h*ng/mL and the bioavailability was 4.1%.
  • the oral exposure of compound 45 was 968 h*ng/mL, and the bioavailability was 5.1%.
  • ACE2 Humanized angiotensin-converting enzyme 2
  • mice (age: 8-10 weeks) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (#T037659. Compounds were dissolved in 5% (v/v) DMSO ( Sigma-Aldrich), 3% (v/v) HS15 (GLPBIO) and 92% saline. SARS-CoV-2 (stain107) intranasal infection and administration were performed according to the experimental protocol in Table 9. All mice were observed and Body weights were monitored daily until sacrificed.
  • lung tissue was fixed with 4% paraformaldehyde for at least 7 days, embedded in paraffin and cut into 3 ⁇ m sections. Sections were stained with hematoxylin and eosin (H&E) and analyzed by light microscopy. Lung injury was assessed according to histological features (thickening of alveolar septa, hemorrhage, inflammatory cell infiltration, etc.).
  • a specific embodiment of a representative inflammatory cytokine and chemokine assay in the lung is: using the PrimeScript TM RT kit (Takara), RNA extracted from the lungs was reverse transcribed into cDNA and then analyzed by Ex Taq TM II (TliRNaseH Plus) (Takara) and ViiA TM quantified gene expression. Primer sequences used to quantify inflammatory gene expression are shown in Table 10.
  • a specific embodiment for the determination of inflammatory cells (neutrophils and macrophages) in the lungs is performed by fixing mouse lung tissue in 4% paraformaldehyde for at least 7 days, then paraffin-embedding according to standard procedures. Cut into 4 ⁇ m sections.
  • rat monoclonal antibody F4/80 Humanabio, 1:100
  • rabbit polyclonal antibody Ly6G Servicebio, 1:300
  • HRP horseradish peroxidase
  • TSA tyramide signal amplification
  • the above experiments used a placebo as a control.
  • the placebo is a formulation in the same dosage form as the test drug, but without the active pharmaceutical ingredient.
  • the experimental results show that the compounds of the present invention can effectively resist SARS-COV-2 infection in vivo in transgenic mice.
  • the present invention provides a novel coronavirus main protease inhibitor represented by formula I and a preparation method and application thereof.
  • the compound represented by formula I can effectively inhibit the activity of SARS-CoV-2M pro , and can be used to prepare a SARS-CoV-2M pro inhibitor to block the replication and transcription of SARS-CoV-2 virus in patients.
  • the compounds of the present invention have very good application prospects in the preparation of SARS-CoV-2 M pro inhibitors, anti-SARS-CoV-2 drugs, and drugs for preventing and/or treating novel coronavirus pneumonia.

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Abstract

La présente invention concerne un inhibiteur principal de protéase du nouveau coronavirus, son procédé de préparation et son utilisation. Plus particulièrement, l'invention concerne un composé représenté par la formule (I), un sel pharmaceutiquement acceptable, un stéréoisomère, un isomère optique, ou une forme isotopiquement substituée de celui-ci. Le composé peut inhiber de manière efficace l'activité du SARS-CoV-2 M pro, peut être utilisé pour préparer un inhibiteur de SARS-CoV-2 M pro, et bloquer la réplication et la transcription des virus de SARS-CoV-2 chez un patient. Le composé a de très bonnes perspectives d'application dans la préparation d'un inhibiteur de SARS-CoV-2 M pro, d'un médicament anti-SARS-CoV-2, et d'un médicament pour prévenir et/ou traiter une nouvelle pneumonie à coronavirus.
PCT/CN2021/076400 2020-07-31 2021-02-09 Inhibiteur principal de protéase du nouveau coronavirus, son procédé de préparation et son utilisation WO2022021841A1 (fr)

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WO2022218442A1 (fr) * 2021-04-16 2022-10-20 南京明德新药研发有限公司 Composé de peptide court de proline à cycle modifié et son utilisation
CN115490681A (zh) * 2022-07-08 2022-12-20 歌礼生物科技（杭州）有限公司 三嗪衍生物
WO2022266363A1 (fr) * 2021-06-16 2022-12-22 The Scripps Research Institute Inhibiteurs de protéase pour le traitement d'infections à coronavirus
WO2023165334A1 (fr) * 2022-03-01 2023-09-07 成都威斯克生物医药有限公司 Dérivés de ceto amide et leur utilisation pharmaceutique
WO2023168844A1 (fr) * 2022-03-07 2023-09-14 广州谷森制药有限公司 Composé de lactame deutéré, son procédé de préparation, composition et son utilisation
WO2023185763A1 (fr) * 2022-04-01 2023-10-05 中国科学院上海药物研究所 Composé peptidomimétique, procédé de préparation, composition pharmaceutique et utilisation associée
GB2619610A (en) * 2020-07-20 2023-12-13 Enanta Pharm Inc Functionalized Peptides as Antiviral Agents
WO2024081318A1 (fr) * 2022-10-13 2024-04-18 Aligos Therapeutics, Inc. Composés antiviraux
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GB2619610A (en) * 2020-07-20 2023-12-13 Enanta Pharm Inc Functionalized Peptides as Antiviral Agents
US11976084B2 (en) 2020-11-23 2024-05-07 Enanta Pharmaceuticals, Inc. Spiropyrrolidine derived antiviral agents
CN117003813A (zh) * 2021-04-16 2023-11-07 福建广生中霖生物科技有限公司 环修饰的脯氨酸短肽化合物及其应用
WO2022218442A1 (fr) * 2021-04-16 2022-10-20 南京明德新药研发有限公司 Composé de peptide court de proline à cycle modifié et son utilisation
US12116367B2 (en) 2021-04-16 2024-10-15 Fujian Akeylink Biotechnology Co., Ltd. Ring-modified proline short peptide compound and use thereof
CN117003813B (zh) * 2021-04-16 2024-03-08 福建广生中霖生物科技有限公司 环修饰的脯氨酸短肽化合物及其应用
WO2022266363A1 (fr) * 2021-06-16 2022-12-22 The Scripps Research Institute Inhibiteurs de protéase pour le traitement d'infections à coronavirus
WO2023165334A1 (fr) * 2022-03-01 2023-09-07 成都威斯克生物医药有限公司 Dérivés de ceto amide et leur utilisation pharmaceutique
WO2023168844A1 (fr) * 2022-03-07 2023-09-14 广州谷森制药有限公司 Composé de lactame deutéré, son procédé de préparation, composition et son utilisation
CN115043900A (zh) * 2022-03-31 2022-09-13 深圳博瑞医药科技有限公司 一种拟肽化合物及其制备2019-nCoV主蛋白酶抑制剂的用途
WO2023185763A1 (fr) * 2022-04-01 2023-10-05 中国科学院上海药物研究所 Composé peptidomimétique, procédé de préparation, composition pharmaceutique et utilisation associée
CN115490681B (zh) * 2022-07-08 2023-04-18 歌礼生物科技（杭州）有限公司 三嗪衍生物
CN115490681A (zh) * 2022-07-08 2022-12-20 歌礼生物科技（杭州）有限公司 三嗪衍生物
WO2024081318A1 (fr) * 2022-10-13 2024-04-18 Aligos Therapeutics, Inc. Composés antiviraux

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