WO2023125535A1 - 氘代拟肽类化合物及其用途 - Google Patents

氘代拟肽类化合物及其用途 Download PDF

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WO2023125535A1
WO2023125535A1 PCT/CN2022/142333 CN2022142333W WO2023125535A1 WO 2023125535 A1 WO2023125535 A1 WO 2023125535A1 CN 2022142333 W CN2022142333 W CN 2022142333W WO 2023125535 A1 WO2023125535 A1 WO 2023125535A1
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compound
pharmaceutically acceptable
deuterium atoms
deuterium
acceptable salt
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PCT/CN2022/142333
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English (en)
French (fr)
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孙晓伟
梁敏
郭小丰
张晓琳
张豪豪
高娜
赵杰
淡墨
何影
高园
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石药集团中奇制药技术（石家庄）有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0819—Tripeptides with the first amino acid being acidic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the technical field of medicine, in particular, to deuterated peptidomimetic compounds and applications of the compounds.
  • Severe acute respiratory syndrome coronavirus 2 causes the coronavirus disease (COVID-19) global pandemic.
  • antiviral therapies are an important part of the healthcare response to the ongoing threat of COVID-19.
  • Pfizer announced that the anti-new coronavirus oral drug PAXLOVID can reduce the chance of hospitalization or death by 89% in adults at risk of severe disease, and has been approved by the FDA for emergency use authorization (EUA) for the treatment of mild SARS-CoV-2 To moderate adults and children and adult patients ⁇ 12 years old, weighing ⁇ 40kg, and patient groups with higher risk of severe disease.
  • EUA emergency use authorization
  • PF-07321332 (Namatevir), which limits viral replication by inhibiting 3CL protease.
  • the current clinical trial dose of PAXLOVID is 300 mg PF-07321332 and 100 mg ritonavir twice daily.
  • WHO updated the COVID-19 treatment guidelines, strongly recommending PAXLOVID for the treatment of mild to moderate COVID-19 patients with high risk of hospitalization.
  • Naimatevir has defects in PK druggability: 1) Poor metabolic stability, poor oral absorption, need to be taken together with strong CYP3A4 inhibitors, which limits the use of a variety of CYP enzyme metabolic substrate drugs, and affects the liver Kidney function, which increases the risk of medication for the elderly and people with underlying diseases; 2) P-glycoprotein substrate, poor absorption, higher dosage.
  • Deuterium modification A potentially attractive strategy to improve the metabolic properties of drugs is deuterium modification.
  • Deuterium technology is through the conversion between isotopes, some hydrogen atoms are replaced by deuterium atoms, so that the physical and chemical properties of drug molecules are changed. This effect is called isotope effect.
  • isotope effect In this approach, one attempts to slow down CYP-mediated drug metabolism or reduce the formation of undesirable metabolites by replacing one or more hydrogen atoms with deuterium atoms.
  • Deuterium is a safe and stable non-radioactive isotope of hydrogen. Compared with drug molecules not modified with deuterium atoms, the chemical properties are the same, and the effectiveness and safety of existing drugs have been verified. Based on the fact that hydrogen and deuterium have little effect on the entire molecule, they will not affect the biochemical efficacy and safety of drugs. Selectivity, retaining its effectiveness to the greatest extent.
  • the deuterated modification of drugs is one of the technical means to increase the exposure of drugs in vivo, reduce the impact of adverse drug metabolites, and improve drug efficacy.
  • the hydrogen atom at a specific position in the drug molecule is replaced by a deuterium atom, it not only maintains the original biological activity and selectivity of the drug, but the carbon-deuterium bond can also significantly improve the metabolic stability and prolong the half-life.
  • the dosage of the drug can be reduced, and the safety of the drug can be improved.
  • PF-07321332 can try to improve metabolic stability and pharmacokinetic characteristics through deuterium modification.
  • the pharmacokinetic properties of drugs in vivo are affected by many factors, which also show corresponding complexity.
  • the changes in the pharmacokinetic properties of deuterated drugs show great chance and unpredictability.
  • Deuterium at certain sites not only cannot prolong the half-life, but may shorten it; on the other hand, it is extremely difficult to replace hydrogen at certain positions on the drug molecule with deuterium.
  • the sites at which a drug is suitable for deuteration are not obvious, and the effects of deuteration are also unpredictable. Therefore, the selection of deuterium sites is very important for improving the metabolic stability and efficacy of drugs.
  • the increased binding strength conferred by deuterium can positively affect the metabolic properties of the drug, with the potential to improve drug efficacy, safety and/or tolerability.
  • the object of the present invention is to provide a compound with a deuterated peptidomimetic structure and its use for preventing and/or treating diseases caused by RNA virus infection sensitive to 3CL protease inhibitors, and related diseases to which PAXLOVID is applicable.
  • the first aspect of the present invention provides a deuterium-substituted compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof, which has the following structure
  • R 1 to R 15 are each independently hydrogen or deuterium
  • Y 1 to Y 14 are each independently hydrogen or deuterium
  • At least one of R 1 to R 15 and Y 1 to Y 14 is a deuterium atom.
  • 3-9 of R 1 to R 9 are deuterium atoms
  • R 1 to R 9 are all deuterium atoms
  • R 1 to R 9 are deuterium atoms
  • R 1 to R 9 are deuterium atoms
  • R 1 to R 3 or R 4 to R 6 or R 7 to R 9 are deuterium atoms
  • 3-6 of R 10 to R 15 are deuterium atoms
  • R 10 to R 15 are all deuterium atoms
  • R 10 to R 15 are deuterium atoms
  • R 10 to R 12 or R 13 to R 15 are deuterium atoms
  • 2-14 of Y 1 to Y 14 are deuterium atoms
  • Y 13 to Y 14 are deuterium atoms.
  • the second aspect of the present invention provides intermediate compounds or salts thereof represented by the following formulas (IV), (VII) and (VIII):
  • R 1 to R 15 and Y 1 to Y 6 are as described in the compound of formula (I);
  • R 1 to R 15 and Y 1 to Y 14 are as described in the compound of formula (I);
  • the salt of the compound of formula (IV) is hydrochloride, acetate or trifluoroacetate
  • the salt of the compound of formula (VII) is a potassium, sodium or lithium salt.
  • the present invention also provides a method for preparing the compound represented by formula (I) or a pharmaceutically acceptable salt thereof.
  • the present invention also provides a pharmaceutical composition, which comprises the compound of the present invention or a pharmaceutically acceptable salt thereof.
  • the present invention also provides a pharmaceutical composition, which comprises the compound shown in the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the present invention also provides a pharmaceutical composition, which comprises the compound shown in the present invention or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and other drugs, and the other drugs are CYP inhibitors.
  • the CYP inhibitor is preferably ritonavir.
  • the present invention also provides the compound shown in the present invention or a pharmaceutically acceptable salt thereof in the preparation of preventing and/or treating diseases or diseases caused by RNA virus infection sensitive to 3CL protease inhibitors, and PAXLOVID Use in medicines for applicable related diseases.
  • the present invention also provides a method for preventing and/or treating diseases caused by RNA virus infection sensitive to 3CL protease inhibitors, and related diseases for which PAXLOVID is applicable, which includes administering a therapeutically effective dose of The compound represented by general formula (I) or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the present invention.
  • the compound represented by the general formula (I) of the present invention or a pharmaceutically acceptable salt thereof can be administered in combination with other related drugs.
  • the other related drug is a CYP inhibitor
  • the CYP inhibitor is preferably ritonavir.
  • the present invention also provides a medicament for the prevention and/or treatment of diseases caused by RNA virus infection sensitive to 3CL protease inhibitors, and related diseases for which PAXLOVID is applicable, which includes a therapeutically effective dose of general A compound represented by formula (I) or a pharmaceutically acceptable salt thereof.
  • the drug can be administered in combination with other related drugs.
  • the other related drug is a CYP inhibitor
  • the CYP inhibitor is preferably ritonavir.
  • the drug is preferably used alone.
  • the RNA virus is a coronavirus, preferably a betacoronavirus, such as SARS-CoV, SARS-CoV-2, MERS-CoV.
  • the disease or condition associated with RNA virus infection is COVID-19.
  • the disease or disease associated with RNA virus infection is novel coronavirus infection or novel coronavirus pneumonia.
  • the disease or disease is a disease or disease caused by a new coronavirus (SARS-CoV-2) infection; preferably, the new coronavirus is a new coronavirus wild strain, a new coronavirus Delta variant , New coronavirus Omicron mutant strain.
  • SARS-CoV-2 coronavirus
  • the new coronavirus is a new coronavirus wild strain, a new coronavirus Delta variant , New coronavirus Omicron mutant strain.
  • the novel coronavirus Omicron variant is selected from Omicron BA.1, Omicron BA.2, Omicron BA.4 or Omicron BA.5 variants.
  • the term "pharmaceutically acceptable salt” or “pharmaceutically acceptable salt” refers to any salt suitable for use within the scope of sound medical judgment without undue toxicity, irritation, allergic reaction, or in contact with the tissues of mammals, especially humans. Salts commensurate with a reasonable benefit/risk ratio, such as the pharmaceutically acceptable salts of amines, carboxylic acids, and other types of compounds are well known in the art. The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or alone by reacting the free base or acid with a suitable reagent.
  • the compounds of the present invention also include their "crystalline forms", the term "crystalline form” referring to a certain crystal lattice configuration of a crystalline substance. It is known in the art that crystal form is related to stability, dissolution and mechanical properties in pharmaceuticals. Different crystal forms of the same substance generally have different crystal lattices (eg, unit cells) with different physical properties characteristic of them. Different crystalline forms can be characterized by methods known in the art. For example, it can be identified by solid state characterization methods such as by X-ray powder diffraction (XRPD). Other characterization methods include Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA), Dynamic Vapor Sorption (DVS), Solid State NMR, etc. The crystalline form can be characterized by any one of the above methods, or by combining two or more methods.
  • XRPD X-ray powder diffraction
  • Other characterization methods include Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (
  • the compound of the present invention also includes its " solvate ", and term “ solvate “, “ solvate” means the physical combination of compound of the present invention and one or more solvent molecules (whether organic or inorganic). association. This physical association includes hydrogen bonding. In some cases, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, solvates will be able to be isolated. Solvent molecules in solvates may exist in regular and/or disordered arrangements. Solvates may contain stoichiometric or non-stoichiometric solvent molecules. "Solvate” encompasses both solution-phase and isolatable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.
  • the compound of the present invention also includes its "hydrate".
  • the term “therapeutically effective amount” refers to an amount of a compound sufficient to effectively treat a disease or condition described herein when administered to a patient in need thereof.
  • a “therapeutically effective amount” will vary depending on the compound, the condition and its severity, and the age of the patient to be treated, but can be adjusted as necessary by those skilled in the art.
  • the object of the present invention is to provide an antiviral drug with better metabolic stability and pharmacokinetic properties, higher drug efficacy and safety, lower dosage than existing drugs, and can reduce strong CYP such as ritonavir. Combined doses of effective inhibitors, or administered alone. Thereby improving the effectiveness of the drug, reducing the patient's medication risk, and improving medication compliance.
  • Said compound can be prepared by the following steps:
  • R 1 to R 15 and Y 1 to Y 14 are the same as those mentioned above.
  • the first step Amino-protected tertiary leucine compound (II) and azabicyclic compound (III) undergo amide condensation reaction to obtain compound (V);
  • compound (VI) removes the Boc protecting group under acidic conditions, and then reacts with trifluoroacetic acid or trifluoroacetic acid derivatives to obtain trifluoroacetic acid amide compound (VII);
  • Step 4 compound (VII) is condensed with compound (IV) to obtain compound (VIII);
  • Step 5 Compound (VIII) undergoes dehydration reaction to obtain the target product deuterium-substituted compound (I).
  • the condensation reaction of the first step and the fourth step selects solvents or mixed solvents with better solubility and stable properties, including but not limited to N,N-dimethylformamide, tetrahydrofuran, acetonitrile, acetone, methyl ethyl ketone, dioxygen Hexacyclic, N,N-dimethylacetamide, dimethyl sulfoxide, ethyl acetate, dichloromethane, chloroform, 1,2-dichloroethane, methanol, ethanol, isopropanol, purified water, etc.;
  • the selected condensing agents include but are not limited to 1-hydroxybenzotriazole, thionyl chloride, phosphorus oxychloride, 2-hydroxypyridine-N-oxide, dicyclohexylcarbodiimide, EDCI, HATU, etc.;
  • the acid-binding agents selected for the reaction include but are not limited to potassium carbonate, sodium carbonate, trie
  • the second step of the hydrolysis reaction uses water as a solvent or a mixed solvent with better compatibility with water.
  • Solvents with better compatibility with water include but are not limited to ethanol, methanol, isopropanol, acetone, N,N-dimethylformamide , tetrahydrofuran, N,N-dimethylacetamide, butanone, dioxane, sulfolane, dimethyl sulfoxide, acetonitrile, etc.;
  • the catalyst uses alkali or acid, including but not limited to sodium hydroxide, potassium hydroxide, hydrogen Lithium oxide, magnesium hydroxide, potassium carbonate, sodium carbonate, cesium carbonate, hydrochloric acid, sulfuric acid, phosphoric acid, trifluoroacetic acid, etc.
  • the reaction temperature range is 0°C to 60°C, preferably 15°C to 35°C.
  • the third step is to use acidic conditions to react and deprotect the group.
  • the solvent is a single or mixed solvent, including but not limited to dichloromethane, dioxane, water, N,N-dimethylformamide, tetrahydrofuran, N,N-di Methyl acetamide, dimethyl sulfoxide, ethyl acetate, acetonitrile, ethanol, methanol, isopropanol, acetone, methyl ethyl ketone, etc.; selected acids include but not limited to hydrochloric acid, sulfuric acid, phosphoric acid, trifluoroacetic acid, periodic acid , hydrobromic acid, etc.; selected trifluoroacetylation reagents include but not limited to trifluoroacetic acid, sodium trifluoroacetate, potassium trifluoroacetate, magnesium trifluoroacetate, methyl trifluoroacetate, ethyl trifluoroacetate, trifluor
  • the solvent used for the dehydration reaction in the fifth step includes but is not limited to dichloromethane, dioxane, N,N-dimethylformamide, tetrahydrofuran, N,N-dimethylacetamide, dimethyl sulfoxide, ethyl acetate , acetonitrile, methyl tert-butyl ether, anisole, n-hexane, cyclohexane, n-heptane, chloroform, one or more of 1,2-dichloroethane; selected dehydrating agents include but are not limited to Thionyl chloride, phosphorus oxychloride, phosphorus trichloride, phosphorus pentachloride, phosphorus pentoxide, acetic anhydride, trifluoroacetic anhydride, Burgess reagent, benzenesulfonic anhydride, methanesulfonic anhydride, trifluoromethanesulfon
  • the deuterated products at different sites described in this paper can be prepared using different compounds as starting reactants, as shown in the following structure:
  • the amino-protected tertiary leucine compound (II) can be the following compound A, compound D, compound G or compound J;
  • the azabicyclic compound (III) can be the following compound B, compound E, compound H or compound K;
  • Compound (IV) can be the following compound C or compound F;
  • Deuterated drugs (2)-(31) can use compound A or compound D or compound G or compound J as amino-protected tertiary leucine compound (II), and use compound B or compound E or compound H or compound K as nitrogen
  • the heterobicyclic compound (III) is prepared by using compound C or compound F as compound (IV) by the above-mentioned preparation method.
  • the structures of the compounds of the present invention are determined by nuclear magnetic resonance (NMR) or/and liquid chromatography-mass chromatography (LC-MS).
  • the starting materials in the examples of the present invention are known and commercially available, or can be synthesized using or following methods known in the art.
  • the synthetic route is as follows:
  • the filter cake (1.3g), triethylamine (1.8g) and ethyl trifluoroacetate (1.3g) were added to methanol (7ml), stirred at room temperature for 20h, and concentrated to dryness under reduced pressure. Add water (20ml) to the concentrate, adjust the pH of the aqueous phase to 2-3 with 1M dilute hydrochloric acid aqueous solution, extract with ethyl acetate (30ml), wash the organic phase with saturated sodium chloride and dry over anhydrous magnesium sulfate , filtered, and concentrated under reduced pressure to obtain intermediate 3-(3) (1.2g).
  • the preparation of intermediate 4-(3) refers to the preparation method of intermediate 4-(7) in Example 1, and intermediate 3-(7) needs to be replaced by intermediate 3-(3) in the synthesis operation.
  • the preparation of the deuterated drug (3) refers to the preparation method of the deuterated drug (7) in Example 1, and the intermediate 4-(7) needs to be replaced by the intermediate 4-(3) in the synthesis operation.
  • the synthetic route is as follows:
  • intermediate 4-(5) refers to the preparation method of intermediate 4-(7) in Example 1.
  • intermediate 3-(7) needs to be replaced by intermediate 3-(3), and compound C is replaced by For compound F.
  • the preparation of the deuterated drug (5) refers to the preparation method of the deuterated drug (7) in Example 1, and the intermediate 4-(7) needs to be replaced by the intermediate 4-(5) in the synthesis operation.
  • deuterated drug (1) deuterated drug (2), deuterated drug (4) and deuterated drug (6) refers to the preparation method of the above examples, and the mass spectrum data of these compounds are as follows:
  • mice rats, mice, humans, dogs and monkey liver microsomes were used to evaluate the metabolic stability of the compounds of the present invention.
  • NADPH Reduced nicotinamide adenine dinucleotide phosphate
  • Acetonitrile (chromatographically pure) was purchased from Merck, Germany.
  • the total volume of each incubation system is 100 ⁇ L, and the medium is 100 mM phosphate buffer (PBS, pH7.4), including liver microsomal protein at a final concentration of 0.50 mg/mL, 3.00 ⁇ M of the test compound and 1.00 mM NADPH, using Incubate in a water bath at 37°C, and add the same volume of ice-cold acetonitrile after 0, 5, 15, 30, 45, and 60 min of reaction to terminate the reaction.
  • the negative control was incubated with heat-inactivated liver microsomes of the corresponding species, and the incubation time points were 0, 15, and 60 min, respectively.
  • the remaining content of the test compound was detected by LC/MS/MS method. All incubation samples were double samples.
  • the deuterated drug (7) has better metabolic stability in liver microsomes of five species, and the stability is significantly better than that of PF -07321332, the half-life was significantly prolonged and the clearance rate was significantly reduced.
  • Deuterated drugs (5) were also improved compared to PF-07321332. It shows that the deuterated drug of the present invention has the drug potential of lower drug dosage, reducing or avoiding the combination with ritonavir and only needing to take the drug once a day.
  • This experiment evaluates the metabolic stability of the compound of the present invention in rats and cynomolgus monkeys, as well as the evaluation of the in vivo pharmacokinetics after oral administration.
  • Phenix Winnolin pharmacokinetic software (V8.0, American Certara company);
  • Tween 80 (Tween 80), purchased from Sigma company;
  • Methyl cellulose purchased from Sigma company;
  • Cynomolgus monkeys were purchased from Hainan Xinzhengyuan Biotechnology Co., Ltd.
  • T max * is represented by the median (minimum value, maximum value)
  • the deuterated drug (7) has higher plasma peak concentration and higher plasma exposure after intragastric administration, indicating that the deuterated drug (7) ) has more excellent pharmacokinetic behavior in vivo.
  • it has the application potential of taking a lower dosage than PF-07321332, or reducing the dosage of ritonavir in combination so that it does not need to be used in combination with ritonavir, so that it can expand the clinical use population and alleviate or reduce adverse reactions.
  • the exposure of the deuterated drug (7) alone group was significantly higher than that of the PF-07321332 single use group, and the C max and AUC last were 7.32 and 3.31 times that of PF-07321332, respectively; the deuterated drug (7) alone
  • the exposure of the treatment group was higher than that of the PF-07321332+ritonavir combination group, and the C max and AUC last were 1.40 and 1.76 times, respectively.
  • the exposure of deuterated drug (7) was significantly increased, which was 4.47 times of that of the single use group (AUC last ).
  • the human recombinant CYP3A4 isoenzyme incubation method was used to detect the metabolic stability of the compound of the present invention in the human CYP3A4 incubation system.
  • NADPH Reduced nicotinamide adenine dinucleotide phosphate
  • Acetonitrile (chromatographically pure), purchased from Germany Merck company;
  • Human CYP3A4 recombinase was purchased from BD Gentest, USA.
  • each incubation system is 100 ⁇ L
  • the medium is 100 mM phosphate buffer (PBS, pH 7.4), including the test compound at a final concentration of 3.0 ⁇ M and 1.0 mM NADPH, and incubated in a 37° C. water bath.
  • CYP3A4 recombinase protein was added to the buffer-substrate-cofactor mixture to initiate the reaction at a concentration of 50 pmol/mL.
  • the same volume of ice-cold acetonitrile was added to terminate the reaction. All incubation samples were double samples.
  • Test example 4 SARS-CoV-2 virus Mpro enzymatic inhibition test
  • Proteins and substrates SARS-CoV-2 Mpro protease wild type and P132H mutant were cloned and expressed by Shanghai WuXi Pharmatech New Drug Development Co., Ltd. Proteins were stored at -80°C. The protease substrate was synthesized by GenScrip Company, and the substrate sequence was KTSAVLQSGFRKM. Substrates were stored at -20°C.
  • Tris-HCl pH 7.3
  • 100mM NaCl 100mM EDTA
  • 5mM TCEP 0.1% BSA.
  • GraphPad Prism software was used to analyze and calculate the half maximal inhibitory concentration (IC 50 ) of the compound on Mpro protease.
  • the deuterated drug (7) has good inhibitory activity on the wild-type Mpro protease of SARS-CoV-2 and the common mutant P132H protease of the mutant strain Omicron, which are better than the positive control compound PF-07321332.
  • CPE cytopathic assay
  • test compound was diluted 1:3 with DMSO, the initial concentration was 100 ⁇ M, and each concentration was added to a 96-well culture plate in triplicate; at the same time, 2 ⁇ M CP100356 was added to each well.
  • the Vero cells were treated with the compound for 3 days, and the effect of the test compound on the proliferation of the Vero cells was evaluated. Cells were seeded in 96-well culture plates at a density of 4000/well. Cells were incubated at 37°C, 5% CO 2 , saturated humidity for 3 days.
  • Cell proliferation was detected using an ATP-based cell proliferation detection kit (Cell Titer Glo, Promega Corporation). Cells were treated with Cell Titer Glo reagent after equilibrating at room temperature for 30 minutes. The dish was then covered with aluminum foil and shaken for 15 minutes to allow for thorough mixing and lysis. Chemiluminescent detection was performed using a multifunctional microplate reader (Tecan Infinite M200). Set blank wells (blank, no cells) and DMSO control wells.
  • test compound was diluted 1:2 with DMSO, 12 concentration points, the initial concentration was 10 ⁇ M, each concentration was duplicated, and 96 well culture plate; at the same time, add 2 ⁇ M CP100356 to each well;
  • Omicron BA.2 strain the test compound was diluted 1:2 with DMSO, 6 concentration points, the initial concentration was 1.25 ⁇ M, and each concentration was added to 96-well culture plate in 4 replicate wells; at the same time, each well was added 2 ⁇ M CP100356;
  • Vero cells were added to the plate, and cultured in a 5% CO 2 , 37° C. incubator for 3 to 4 days.
  • Cell controls (cells, no compound treatment or virus infection) and virus controls (cells infected with virus, no compound treatment) were set up.
  • CPE cytopathic effect
  • IR (%) [1-(RLU compound -RLU blank control )/(RLU vehicle control -RLU blank control )] ⁇ 100%.
  • GraphPad Prism software was used for graphing, data analysis and IC50 calculation.
  • RLU stands for relative light unit.
  • Deuterated drug (7) has good inhibitory activity against SARS-CoV-2 wild type and mutant strains Omicron BA.1, Omicron BA.2, Omicron BA.4 and Omicron BA.5, which is better than the positive control compound PF-07321332 ;
  • the deuterated drug (5) has good inhibitory activity on SARS-CoV-2 wild type and mutant strain Omicron BA.1, which is better than the positive control compound PF-07321332.
  • Test example 6 in vivo anti-SARS-CoV-2 virus activity research
  • the anti-SARS-CoV-2 virus activity of the test compound in mice was evaluated by hACE2 transgenic mouse challenge test.
  • mice in the infection group were inoculated with 1 ⁇ 10 4 PFU of SARS-CoV-2 virus (Pubmed No: MT627325) by intranasal drops, and then divided into vehicle group (Vehicle) and deuterated drug (7) treatment group (300mg/kg, BID) and positive control compound PF-07321332 (300mg/kg, BID) treatment group. Simultaneously, a sham-infected control group was set up.
  • mice were administered twice a day for 7 consecutive administrations. Animal status and body weight were monitored daily. 2 hours after the last administration at 72 hours after exposure, all mice were euthanized after recording the body weight of the mice, and the lungs were taken. After extracting RNA from lung tissue, qPCR was performed to evaluate the lung viral load.
  • mice Record the weight and abnormal conditions of the mice, once a day, and record in time any abnormal conditions such as the mouse's state of malaise, hypothermia, disheveled coat, and arched back.
  • Results are expressed as mean ⁇ standard error (MEAN ⁇ SEM). Data analysis was performed using Prism. When P ⁇ 0.05, there is a significant difference.

Abstract

一种具有氘代拟肽类结构的化合物和含有其的药物组合物，以及使用该组合物用于预防和/或治疗对3CL蛋白酶抑制剂敏感的RNA病毒感染引起的病症，以及PAXLOVID适用的相关病症的用途。所述的化合物与PF-07321332相比具有更高的血浆峰浓度和更高的血浆中暴露，有更优异的体内药代动力学行为，更高的抗病毒活性。

Description

氘代拟肽类化合物及其用途
本申请要求申请日为2021年12月28日的中国专利申请CN202111629214.1、申请日为2022年05月30日的中国专利申请202210600995.X、申请日为2022年7月6日的中国专利申请CN 202210788379.1和申请日为2022年11月28日的中国专利申请CN202211508394.2的优先权。所述在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明涉及医药技术领域，具体而言，涉及氘代拟肽类化合物以及所述化合物的用途。
背景技术
严重急性呼吸综合征冠状病毒2(SARS-CoV-2、新型冠状病毒、2019-nCoV)引起冠状病毒病(COVID-19)全球大流行病。除了疫苗，抗病毒疗法也是应对COVID-19持续威胁的医疗保健应对措施的重要组成部分。2021年11月辉瑞公司宣布抗新冠病毒口服药PAXLOVID可将患严重疾病风险的成年人住院或死亡的几率降低89％，获得FDA紧急使用授权(EUA)批准，用于治疗SARS-CoV-2轻至中度成人和≥12岁、体重≥40kg的儿童和成人患者，及具有较高重症风险的患者人群。其主要活性成分为PF-07321332(奈玛特韦)，通过抑制3CL蛋白酶来限制病毒复制。PAXLOVID目前临床试验剂量为每日两次，每次300毫克PF-07321332与100毫克利托那韦同时服用。2022年4月22日，WHO更新了COVID-19治疗指南，强烈推荐PAXLOVID用于治疗具有高住院风险的轻中度新冠肺炎患者。
奈玛特韦存在PK成药性的缺陷：1)代谢稳定性较差，口服吸收不佳，需要与强效CYP3A4抑制剂同时服用，限制多种CYP酶代谢底物类药物的使用，并影响肝肾功能，增加老年人、患有基础疾病人群用药风险；2)P-糖蛋白底物，吸收较差，给药剂量较高。
改善药物代谢特性的一个潜在的有吸引力的策略是氘代修饰。氘代技术是通过同位素之间的转换，部分氢原子替换为氘原子，使药物分子理化性质得到改变，该效应被称为同位素效应。在这种方法中，人们试图减慢CYP介导的药物代谢，或者通过用氘原子取代一个或多个氢原子来减少不良代谢产物的形成。氘是氢的一种安全稳定的非放射性同位素。和未用氘原子修饰的药物分子相比，化学性质相同，现有药物的有效性和安全性都已经过验证，基于氢和氘对整个分子的影响微乎其微，不会影响药物的生物化学效力和选择性，最大程度的保留其有效性。
药物的氘代修饰是提高药物体内暴露量、降低药物不良代谢产物影响、提高药效的技术手段之一。药物分子中特定位置的氢原子被氘原子取代后，不仅保持药物的原有生物活性和选择性，碳氘键还会明显提高代谢稳定性，延长半衰期。与氘代前的药物相比可以降低用药剂量，提高用药安全性。PF-07321332可尝试通过氘代修饰改善代谢稳定性和药代动力学特征。
但是，由于代谢过程复杂，药物在生物体内的药代动力学性质受到多方面因素影响，也表现出相应的复杂性。与相应的非氘代药物相比，氘代药物药代动力学性质的变化表现出极大的偶然性和不可预测性。某些位点的氘代非但不能延长半衰期，反而可能会使其缩短；另一方面，药物分子上某些位置的氢被氘取代也有极大难度。药物适合氘代的位点并非显而易见的，氘代效果也是不可预期的。因此氘代位点的选择对于改善药物的代谢稳定性以及药效至关重要。合理选择特定位点氘代修饰的情况下，氘赋予的结合强度增加可以积极影响药物的代谢特性，提高药物疗效、安全性和/或耐受性的潜力。
发明内容
本发明的目的在于提供一种具有氘代拟肽类结构的化合物及其预防和/或治疗对3CL蛋白酶抑制剂敏感的RNA病毒感染引起的病症，以及PAXLOVID适用的相关病症的用途。
本发明的第一方面，提供了一种如下式(I)所示的氘取代化合物或其药学上可接受的盐，其具有如下结构
Figure PCTCN2022142333-appb-000001
其中，
R 1～R 15各自独立地为氢或氘；
Y 1～Y 14各自独立地为氢或氘；
且，R 1～R 15和Y 1～Y 14中至少有一个为氘原子。
在本发明的一个优选实施方案中，R 1～R 9中3-9个为氘原子；
优选的，R 1～R 9均为氘原子；
优选的，R 1～R 9中6个为氘原子；
优选的，R 1～R 9中3个为氘原子；
优选的，R 1～R 3或R 4～R 6或R 7～R 9为氘原子；
在本发明的一个优选实施方案中，R 10～R 15中3-6个为氘原子；
优选的，R 10～R 15均为氘原子；
优选的，R 10～R 15中3个为氘原子；
优选的，R 10～R 12或R 13～R 15为氘原子；
在本发明的一个优选实施方案中，Y 1～Y 14中2-14个为氘原子；
进一步优选的，Y 13～Y 14为氘原子。
以下为举例结构，包括但不限于下列结构式化合物或其药学上可接受的盐：
Figure PCTCN2022142333-appb-000002
Figure PCTCN2022142333-appb-000003
Figure PCTCN2022142333-appb-000004
Figure PCTCN2022142333-appb-000005
本发明的第二方面，提供了如下式(IV),(VII)和(VIII)所示的中间体化合物或其盐：
Figure PCTCN2022142333-appb-000006
其中Y 7～Y 14的定义如式(I)化合物所述；
Figure PCTCN2022142333-appb-000007
其中R 1～R 15、Y 1～Y 6的定义如式(I)化合物所述；
Figure PCTCN2022142333-appb-000008
其中R 1～R 15、Y 1～Y 14的定义如式(I)化合物所述；
在本发明的一个优选实施方案中，式(IV)化合物为
Figure PCTCN2022142333-appb-000009
在本发明的一个优选实施方案中，式(IV)化合物的盐为盐酸盐、乙酸盐或三氟乙酸盐；
在本发明的一个优选实施方案中，式(VII)化合物为
Figure PCTCN2022142333-appb-000010
Figure PCTCN2022142333-appb-000011
在本发明的一个优选实施方案中，式(VII)化合物的盐为钾盐、钠盐或锂盐。
在本发明的一个优选实施方案中，式(VIII)化合物为
Figure PCTCN2022142333-appb-000012
Figure PCTCN2022142333-appb-000013
Figure PCTCN2022142333-appb-000014
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明的第三方面，本发明还提供一种制备式(I)所示的化合物或其药学上可接受的盐的方法。
本发明的第四方面，本发明还提供一种药用组合物，其包含本发明所述的化合物或其药学上可接受的盐。
本发明还提供一种药用组合物，其包含本发明所示的化合物或其药学上可接受的盐，以及药学上可接受的载体。
本发明还提供一种药用组合物，其包含本发明所示的化合物或其药学上可接受的盐，以及药学上可接受的载体，还包括其他药物，所述其他药物为CYP抑制剂。所述CYP抑制剂优选为利托那韦。
本发明的第五方面，本发明还提供本发明所示的化合物或其药学上可接受的盐在制备预防和/或治疗对3CL蛋白酶抑制剂敏感的RNA病毒感染引起的疾病或病症，以及PAXLOVID适用的相关病症的药物中的用途。
本发明的第六方面，本发明还提供一种预防和/或治疗对3CL蛋白酶抑制剂敏感的RNA病毒感染引起的病症，以及PAXLOVID适用的相关病症的方法，其包括向患者施用治疗有效剂量的通式(I)所示的化合物或其药学上可接受的盐，或本发明所述药物组合物。
本发明的通式(I)所示的化合物或其药学上可接受的盐，可联合其他相关药物组合施用。
在一些实施例中，所述其他相关药物为CYP抑制剂，所述CYP抑制剂优选为利托那韦。
本发明的第七方面，本发明还提供一种药物，用于预防和/或治疗对3CL蛋白酶抑制剂敏感的RNA病毒感染引起的病症，以及PAXLOVID适用的相关病症，其包括治疗有效剂量的通式(I)所示的化合物或其药学上可接受的盐。
在一些实施例中，所述的药物可联合其他相关药物组合施用。
在一些实施例中，所述其他相关药物为CYP抑制剂，所述CYP抑制剂优选为利托那韦。
在一些实施例中，所述药物优选单独使用。
在以上第五、六和七方面中：
在一些实施例中，所述RNA病毒为冠状病毒，优选为β冠状病毒，如SARS-CoV、SARS-CoV-2、MERS-CoV。
在一些实施例中，所述RNA病毒感染相关的疾病或病症为COVID-19。
在一些实施例中，所述RNA病毒感染相关的疾病或病症为新型冠状病毒感染或新型冠状病毒肺炎。
在一些实施例中，所述疾病或病症为新型冠状病毒(SARS-CoV-2)感染引起的疾病或病症；优选地，所 述新型冠状病毒为新型冠状病毒野生株、新型冠状病毒Delta变异株、新型冠状病毒Omicron变异株。
在一些实施例中，所述新型冠状病毒Omicron变异株选自Omicron BA.1、Omicron BA.2、Omicron BA.4或Omicron BA.5变异株。
定义
除另有规定外，术语“药学上可接受的盐”或“可药用盐”是指在合理医学判断范围内适用于与哺乳动物特别是人的组织接触而无过度毒性、刺激、过敏反应等并与合理的效益/风险比相称的盐，比如胺、羧酸和其他类型化合物的医学上可接受的盐在所属领域中是被熟知的。可以在本发明化合物的最终分离和纯化期间原位制备所述盐，或单独通过将游离碱或游离酸与合适的试剂反应制备所述盐。
除另有规定外，本发明化合物还包括其“晶型”，术语“晶型”是指晶体物质的某种晶格构型。本领域已知的是，晶型在制药中和稳定性、溶出性和机械性有关。相同物质的不同晶型通常具有其特有的不同物理性质的不同的晶格(例如晶胞)。不同的晶型可通过本领域已知的方法进行表征。例如，可通过固态表征方法例如通过X射线粉末衍射(XRPD)来鉴定。其它表征方法包括示差扫描量热法(DSC)、热解重量分析(TGA)、动态蒸汽吸附(DVS)、固态NMR等。可以使用上述任一种方法对晶型进行表征，或者组合使用两种以上的方法进行表征。
除另有规定外，本发明化合物还包括其“溶剂化物”，术语“溶剂化物”、“溶剂合物”意指本发明化合物与一个或多个溶剂分子(无论有机的还是无机的)的物理缔合。该物理缔合包括氢键。在某些情形中，例如当一个或多个溶剂分子纳入结晶固体的晶格中时，溶剂化物将能够被分离。溶剂化物中的溶剂分子可按规则排列和/或无序排列存在。溶剂化物可包含化学计量或非化学计量的溶剂分子。“溶剂化物”涵盖溶液相和可分离的溶剂化物。示例性溶剂化物包括但不限于水合物、乙醇合物、甲醇合物和异丙醇合物。溶剂化方法是本领域公知的。
除另有规定外，本发明化合物还包括其“水合物”，术语“水合物”是指水分子以配位键或共价键与化合物中的阳离子或阴离子结合，或指水离子不直接与阳离子或阴离子结合而是以一定比例存在于固体晶格的确定位置而形成的物质。
除另有规定外，术语“治疗有效量”是指在给予有需要的患者时足以有效治疗本发明所述的疾病或病症的化合物的量。“治疗有效量”将根据化合物、病症及其严重度、以及欲治疗患者的年龄而变化，但可由本领域技术人员根据需要进行调整。
本发明的有益效果为：
本发明的目的是提供一种代谢稳定性和药代动力学性质更佳，药效和安全性更高的抗病毒药物，服用剂量比现有药物更低，可以降低利托那韦等CYP强效抑制剂的联用剂量，或者单独给药。从而提高药物的有效性、降低患者的用药风险、及改善用药的依从性。
所述化合物可通过下述步骤制备：
Figure PCTCN2022142333-appb-000015
其中R 1～R 15、Y 1～Y 14的定义同前所述。
第一步：氨基保护的叔亮氨酸化合物(Ⅱ)与氮杂双环化合物(Ⅲ)进行酰胺缩合反应制得化合物(Ⅴ)；
第二步：化合物(Ⅴ)经水解得到化合物(Ⅵ)；
第三步：化合物(Ⅵ)经酸性条件脱去Boc保护基，再与三氟乙酸或三氟乙酸衍生物反应得到三氟乙酸酰胺类化合物(Ⅶ)；
第四步：化合物(Ⅶ)与化合物(Ⅳ)进行缩合反应制备得到化合物(Ⅷ)；
第五步：化合物(Ⅷ)经脱水反应得到目标产物氘取代化合物(Ⅰ)。
其中，第一步和第四步的缩合反应选用溶解性较佳且性质稳定的溶剂或混合溶剂，包括不限于N,N-二甲基甲酰胺、四氢呋喃、乙腈、丙酮、丁酮、二氧六环、N,N-二甲基乙酰胺、二甲基亚砜、乙酸乙酯、二氯甲烷、氯仿、1,2-二氯乙烷、甲醇、乙醇、异丙醇、纯化水等；选用的缩合剂包括但不限于1-羟基苯并三氮唑、二氯亚砜、三氯氧磷、2-羟基吡啶-N-氧化物、二环己基碳二亚胺、EDCI、HATU等；反应选用的缚酸剂包括不限于碳酸钾、碳酸钠、三乙胺，N,N-二异丙基乙胺，碳酸铯等；反应温度范围0℃～60℃，优选15℃～35℃。
第二步水解反应采用水为溶剂或水与相溶性较好的混合溶剂，与水相溶性较好的溶剂包括不限于乙醇、甲醇、异丙醇、丙酮、N,N-二甲基甲酰胺、四氢呋喃、N,N-二甲基乙酰胺、丁酮、二氧六环、环丁砜、二甲基亚砜、乙腈等；催化剂采用碱或酸，包括不限于氢氧化钠、氢氧化钾、氢氧化锂、氢氧化镁、碳酸钾、碳酸钠、碳酸铯、盐酸、硫酸、磷酸、三氟乙酸等；反应温度范围0℃～60℃，优选15℃～35℃。
第三步采用酸性条件下反应脱保护基，溶剂为单一或混合溶剂，包括不限于二氯甲烷、二氧六环、水、N,N-二甲基甲酰胺、四氢呋喃、N,N-二甲基乙酰胺、二甲基亚砜、乙酸乙酯、乙腈、乙醇、甲醇、异丙醇、丙酮、丁酮等；选用的酸包括不限于盐酸，硫酸，磷酸，三氟乙酸，高碘酸，氢溴酸等；选用的三氟乙酰化试剂包括不限于三氟乙酸、三氟乙酸钠、三氟乙酸钾、三氟乙酸镁、三氟乙酸甲酯、三氟乙酸乙酯、三氟乙酸苯酯、三氟乙酸酐等；反应温度范围5℃～80℃，优选15℃～65℃。
第五步脱水反应选用溶剂包括不限于二氯甲烷、二氧六环、N,N-二甲基甲酰胺、四氢呋喃、N,N-二甲基乙酰胺、二甲基亚砜、乙酸乙酯、乙腈、甲基叔丁基醚、苯甲醚、正己烷、环己烷、正庚烷、氯仿、1,2-二氯乙烷中的一种或多种；选用的脱水剂包括不限于二氯亚砜、三氯氧磷、三氯化磷、五氯化磷、五氧化二磷、醋酸酐、三氟乙酸酐、伯吉斯试剂、苯磺酸酐、甲磺酸酐、三氟甲磺酸酐等；反应温度范围10℃～80℃，优选15℃～35℃。
文中描述的不同位点氘代产物可以采用不同的化合物作为起始反应物制备，如下所示结构：
氨基保护的叔亮氨酸化合物(Ⅱ)可以为下述化合物A、化合物D、化合物G或化合物J；
氮杂双环化合物(Ⅲ)可以为下述化合物B、化合物E、化合物H或化合物K；
化合物(Ⅳ)可以为下述化合物C或化合物F；
Figure PCTCN2022142333-appb-000016
氘代药物(1)的制备：
Figure PCTCN2022142333-appb-000017
氘代药物(2)～(31)可采用化合物A或化合物D或化合物G或化合物J作为氨基保护的叔亮氨酸化合物(Ⅱ)，采用化合物B或化合物E或化合物H或化合物K作为氮杂双环化合物(Ⅲ)，采用化合物C或化合物F作为化合物(Ⅳ)通过上述制备方法进行制备。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件或者按照制造厂商所建议的条件。除非另行定义，文中所使用的所有专业与科学用语与本领域专业人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法之中。文中所示的较佳实施方法与材料仅做示范之用。
本发明的化合物结构是通过核磁共振(NMR)或/和液质联用色谱(LC-MS)来确定的。
本发明实施例中的起始原料是已知的并且可以在市场上买到，或者可以采用或按照本领域已知的方法来合成。
实施例1
(1R,2S,5S)-N-{(S)-1-氰基-2-[(S)-2-氧代-3-吡咯烷基-5,5-二氘]乙基}-3-[(S)-3,3-二甲基-2-(2,2,2-三氟乙酰氨基)丁酰基]-6,6-二甲基-3-氮杂双环[3.1.0]己烷-2-甲酰胺，氘代药物(7)的制备
合成路线如下：
Figure PCTCN2022142333-appb-000018
化合物C-2的制备
将化合物C-3(9.0g，28.6mmol)和氘代甲醇(MeOD,72ml)加入反应瓶中，搅拌溶解后加入氯化钴(2.23g，17.2mmol)，降温至0℃，30min内分批加入硼氘化钠(4.79g，114.4mmol)，加毕后转移至室温反应24h。加入饱和氯化铵水溶液淬灭，减压蒸馏，使用乙酸乙酯萃取水相3次，合并有机相，用无水硫酸钠干燥，过滤，减压浓缩，硅胶柱层析分离(正庚烷/乙酸乙酯梯度洗脱)，减压浓缩后得化合物C-2(3.73g)。
1H NMR(600MHz,CDCl 3)δ1.44(9H,s),1.83-1.86(2H,m),2.11-2.15(1H,m),2.44-2.51(2H,m),3.74(3H,s),4.31-4.33(1H,m),5.47(1H,br s),5.94(1H,br s).LCMS m/z 311.0[M+Na] +.
化合物C-1的制备
将化合物C-2(3.7g，12.8mmol)与氨甲醇溶液(7M，18.5ml)加入反应瓶中，室温反应60h。减压浓缩得化合物C-1(3.5g)。
1H NMR(600MHz,CD 3OD)δ1.47(9H,s),1.73-1.78(1H,m),1.85-1.89(1H,m),2.02-2.07(1H,m),2.34-2.37(1H,m),2.48-2.50(1H,m),4.10-4.12(1H,m).LCMS m/z 296.0[M+Na] +.
化合物C的制备
将化合物C-1(3.4g，12.4mmol)与异丙醇(30ml)加入反应瓶中，然后滴加氯化氢乙酸乙酯溶液(5.5M，10ml)，50℃反应4h后，冷却至室温搅拌过夜，减压浓缩后得化合物C(2.12g)。
1H NMR(600MHz,CD 3OD)δ1.68-1.90(1H,m),2.00-2.11(2H,m),2.43-2.45(1H,m),2.75-2.80(1H,m),4.04-4.05(1H,dd).LCMS m/z 195.9[M+Na] +.
中间体4-(7)的制备
将2-羟基吡啶-N-氧化物(0.37g)加入到中间体3-(7)(6.00g)和化合物C(3.34g)的丁酮(60ml)溶液中，0℃下搅拌下，向其中加入N,N-二异丙基乙胺(7ml)，和EDCI(3.1g)。室温下搅拌20h，将反应液用乙酸乙酯/甲基叔丁基醚(1:1,60ml)稀释，然后用水(20ml)和饱和氯化钠溶液(20ml)进行洗涤后，将有机相再用1M稀盐酸水溶液(20ml)和饱和氯化钠溶液(20ml)洗涤，将有机相用无水硫酸镁干燥，过滤，减压浓缩，硅胶柱层析分离(二氯甲烷/甲醇梯度洗脱)，减压浓缩后得中间体4-(7)(6.0g)。
1H NMR(600MHz,DMSO-d 6)δ0.84(3H,s),0.98(9H,s),1.02(3H,s),1.37(1H,d),1.48-1.50(2H,m),1.60-1.64(1H,m),1.91-1.99(1H,m),2.10-2.14(1H,m),2.37-2.43(1H,m),3.66(1H,d),3.88-3.90(1H,m),4.28-4.32(2H,m),4.42(1H,d),7.03(1H,br s),7.31(1H,br s),7.53(1H,s),8.29(1H,d),9.41(1H,d).LCMS m/z 542.1[M+Na] +.
氘代药物(7)的制备
将中间体4-(7)(1.2g，2.3mmol)与二氯甲烷(6ml)加入反应瓶中，搅拌下加入N-甲基吗啉(0.94g)，然后加入三氟乙酸酐(0.97g)室温反应2h。加入纯化水淬灭反应，分相后用饱和氯化钠水溶液洗涤有机相，将有机相减压浓缩。加入甲基叔丁基醚(12ml)打浆1h，过滤。滤饼用醋酸异丙酯(3.5ml)溶解后，加入正己烷(30ml)，搅拌过夜，纯化，并在50℃真空干燥4h后得氘代药物(7)(0.7g)。
1H NMR(600MHz,DMSO-d 6)δ0.85(3H,s),0.98(9H,s),1.03(3H,s),1.31(1H,d),1.56-1.58(1H,dd),1.66-1.72(2H,m),2.05-2.09(1H,m),2.12-2.17(1H,m),2.37-2.43(1H,m),3.69(1H,d),3.90-3.92(1H,m),4.16(1H,s),4.41(1H,d),4.95-4.99(1H,m),7.65(1H,s),9.03(1H,d),9.41(1H,d).LCMS m/z 502.2[M+H] +。
实施例2
(1R,2S,5S)-3-[(S)-3,3-二(三氘代甲基)-2-(2,2,2-三氟乙酰氨基)丁酰基-4,4,4-三氘]-N-{(S)-1-氰基-2-[(S)-2-氧代-3-吡咯烷基-5,5-二氘]乙基}-6,6-二甲基-3-氮杂双环[3.1.0]己烷-2-甲酰胺，氘代药物(3)的制备合成路线如下：
Figure PCTCN2022142333-appb-000019
化合物A的制备
向1M氢氧化钠水溶液(18mL)中加入二氧六环(12mL)和化合物A-1(1.52g)，0℃下，向反应液中滴加Boc 2O(2.25g)，滴加完毕后0℃搅拌5min，升至室温搅拌13h。将反应液减压浓缩蒸除二氧六环，用1M稀盐酸水溶液将反应液pH调至2-3，用乙酸乙酯(30ml)萃取，将有机相用饱和氯化钠洗涤后用无水硫酸镁干燥，过滤，将滤液减压浓缩至干后得化合物A(1.8g)。
1H NMR(600MHz,DMSO-d 6)δ1.38(9H,s),3.73(1H,d),6.78(1H,d),12.11(1H,br s).
中间体1-(3)的制备
将化合物A(1.78g)和化合物E即(1R,2S,5S)-6,6-二甲基-3-氮杂双环[3.1.0]己烷-2-甲酸甲酯盐酸盐(1.58g)和HATU(3.21g)加入到乙腈(30ml)和DMF(3ml)中，向其中加入N,N-二异丙基乙胺(3.0g)。室温下搅拌20h，减压浓缩至无液体流出，向浓缩物中加入乙酸乙酯(10ml)，用纯化水(10ml)洗涤，用1M稀盐酸水溶液(20ml)和饱和氯化钠溶液(20ml)洗涤，将有机相用无水硫酸镁干燥，过滤，减压浓缩至干，硅胶柱层析分离(正己烷/乙酸乙酯梯度洗脱)，减压浓缩后得中间体1-(3)(2.3g)。
1H NMR(600MHz,DMSO-d 6)δ0.85(3H,s),1.01(3H,s),1.35(9H,s),1.41(1H,d),1.49-1.55(1H,m),3.65(3H,s),3.79(1H,dd),3.93(1H,d),4.05(1H,d),4.21(1H,s),6.73(1H,d).
中间体2-(3)的制备
将中间体1-(3)(2.2g)溶于四氢呋喃(10ml)，向其中加入氢氧化锂(0.56g)的水溶液(3ml)，室温搅拌4h。向反应液中加入水(50ml)后减压浓缩，用1M稀盐酸水溶液将反应液pH调至2-3，用乙酸乙酯(30ml)萃取，将有机相用饱和氯化钠洗涤后用无水硫酸镁干燥，过滤，将滤液减压浓缩至得中间体2-(3)(2.0g)。
1H NMR(600MHz,DMSO-d 6)δ0.84(3H,s),1.01(3H,s),1.35(9H,s),1.38-1.40(1H,d),1.46-1.52(1H,m),3.77(1H,dd),3.91(1H,d),4.04(1H,d),4.12(1H,s),6.67(1H,d),12.64(1H,s).
中间体3-(3)的制备
将中间体2-(3)(2.0g)加入氯化氢乙酸乙酯溶液(20ml)中，室温搅拌反应3h，减压浓缩至干。向浓缩物中加入乙酸乙酯(5ml)打浆1h，过滤，用乙酸乙酯(5ml)洗涤滤饼，将滤饼干燥得1.3g。
将该滤饼(1.3g)、三乙胺(1.8g)和三氟乙酸乙酯(1.3g)加入到甲醇(7ml)中，室温搅拌20h，减压浓缩至干。向浓缩物中加入水(20ml)，用1M稀盐酸水溶液将水相pH调至2-3，用乙酸乙酯(30ml)萃取，将有机相用饱和氯化钠洗涤后用无水硫酸镁干燥，过滤，减压浓缩得中间体3-(3)(1.2g)。
1HNMR(600MHz,DMSO-d 6)δ0.83(3H,s),1.01(3H,s),1.43(1H,d),1.53(1H,dd),3.72(1H,d),3.85(1H,dd),4.15(1H,s),4.43(1H,d),9.41(1H,d),12.73(1H,br s).
中间体4-(3)的制备
中间体4-(3)的制备参照实施例1中间体4-(7)的制备方法，合成操作中需将中间体3-(7)替换为中间体3-(3)。
1H NMR(600MHz,DMSO-d 6)δ0.84(3H,s),1.02(3H,s),1.39(1H,d),1.48-1.52(2H,m),1.61-1.67(1H,m),1.91-1.96(1H,m),2.12-2.16(1H,m),2.37-2.43(1H,m),3.68(1H,d),3.87-3.91(1H,m),4.29-4.31(2H,m),4.42(1H,d),7.03(1H,br s),7.31(1H,br s),7.53(1H,s),8.28-8.29(1H,d),9.40(1H,d).LCMS m/z 551.1[M+Na] +.
氘代药物(3)的制备
氘代药物(3)的制备参照实施例1氘代药物(7)的制备方法，合成操作中需将中间体4-(7)替换为中间体4-(3)。
1H NMR(600MHz,DMSO-d 6)δ0.85(3H,s),1.03(3H,s),1.31-1.32(1H,d),1.55-1.58(1H,dd),1.65-1.72(2H,m),2.04-2.09(1H,m),2.11-2.17(1H,m),2.36-2.43(1H,m),3.69(1H,d),3.90-3.92(1H,m),4.16(1H,s),4.41(1H,d),4.94-4.99(1H,m),7.65(1H,s),9.03(1H,d),9.41(1H,d).LCMS m/z 511.2[M+H] +.
实施例3
(1R,2S,5S)-3-[(S)-3,3-二(三氘代甲基)-2-(2,2,2-三氟乙酰氨基)丁酰基-4,4,4-三氘]-N-{(S)-1-氰基-2-[(S)-2-氧代-3-吡咯烷基]乙基}-6,6-二甲基-3-氮杂双环[3.1.0]己烷-2-甲酰胺，氘代药物(5)的制备
合成路线如下：
Figure PCTCN2022142333-appb-000020
中间体4-(5)的制备
中间体4-(5)的制备参照实施例1中间体4-(7)的制备方法，合成操作中需将中间体3-(7)替换为中间体3-(3)，将化合物C替换为化合物F。
1H NMR(600MHz,DMSO-d 6)δ0.84(3H,s),1.02(3H,s),1.39(1H,d),1.47-1.52(2H,m),1.60-1.67(1H,m),1.91-1.96(1H,m),2.13-2.16(1H,m),2.37-2.43(1H,m),3.00-3.05(1H,m),3.12-3.15(1H,m),3.68(1H,d),3.87-3.90(1H,m),4.28-4.31(2H,m),4.42(1H,d),7.03(1H,br s),7.31(1H,br s),7.53(1H,s),8.28(1H,d),9.40(1H,d).LCMS m/z 549.1[M+Na] +.
氘代药物(5)的制备
氘代药物(5)的制备参照实施例1氘代药物(7)的制备方法，合成操作中需将中间体4-(7)替换为中间体4-(5)。
1H NMR(600MHz,DMSO-d 6)δ0.87(3H,s),1.04(3H,s),1.31-1.34(1H,d),1.56-1.59(1H,dd),1.66-1.73(2H,m),2.05-2.09(1H,m),2.12-2.17(1H,m),2.37-2.42(1H,m),3.02-3.06(1H,m),3.11-3.17(1H,m),3.70(1H,d),3.90-3.92(1H,m),4.17(1H,s),4.40(1H,d),4.95-4.99(1H,m),7.68(1H,s),9.02(1H,d),9.40(1H,d).LCMS m/z 509.2[M+H] +.
氘代药物(1)、氘代药物(2)、氘代药物(4)及氘代药物(6)的制备均参考以上实施例的制备方法，这些化合物的质谱数据如下：
Figure PCTCN2022142333-appb-000021
Figure PCTCN2022142333-appb-000022
生物学测试评价
以下结合测试例进一步描述解释本发明，但这些实施例并非意味着限制本发明的范围。
试验例1、肝微粒体代谢稳定性试验
(1)目的：
本实验采用大鼠，小鼠，人，犬和猴肝微粒体评价本发明化合物的代谢稳定性。
(2)试剂：
混合人肝微粒体，购于美国Corning公司；
混合雄性SD大鼠肝微粒体，购于美国Corning公司；
混合雄性ICR小鼠肝微粒体，购于美国Corning公司；
混合Beagle犬肝微粒体，购于美国Xenotech公司；
混合食蟹猴肝微粒体，购于RILD公司；
还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)，购于德国罗氏公司；
乙腈(色谱纯)，购于德国Merck公司。
(3)肝微粒体孵育体系：
每个孵育体系总体积为100μL，介质为100mM磷酸缓冲液(PBS，pH7.4)，包括终浓度为0.50mg/mL的肝微粒体蛋白、3.00μM的待测化合物和1.00mM的NADPH，采用37℃水浴进行孵育，分别在反应0、5、15、30、45、60min后加入同体积冰冷乙腈终止反应。阴性对照采用相应种属的热失活肝微粒体进行孵育，孵育时间点分别为0、15、60min。采用LC/MS/MS方法检测待测化合物的剩余含量。所有孵育样本均为双样本。
(4)数据处理：
使用Excel软件，以孵育体系中药物的ln剩余率对孵育时间作图，进行线性回归得到斜率k，计算半衰期T 1/2(min)、固有清除率CL int(mL/min/kg)、肝清除率CL hb(mL/min/kg)和剩余率Remaining(T＝60min)值。
(5)结果
Figure PCTCN2022142333-appb-000023
注：-，无法计算。
从上表中可以看出与非氘代化合物PF-07321332相比，氘代药物(7)在五个种属的肝微粒体中均具有更好的代谢稳定性，稳定性均显著优于PF-07321332，半衰期显著延长、清除率显著降低。氘代药物(5)相较于PF-07321332也均有改善。表明本发明氘代药物具有更低的药物服用剂量、减少或避免与利托那韦联用及每日仅需服药一次的成药潜力。
试验例2、体内药代动力学实验
(1)目的：
本实验评价本发明化合物在大鼠和食蟹猴体内的代谢稳定性，以及经口服给药后的体内药代动力学的评估。
(2)试剂及试验动物：
Waters ACQUITY UPLC超高效液相系统(Waters公司)；
Xevo-TQ XS三重四级杆质谱仪(Waters公司)；
Phenix Winnolin药动学软件(V8.0，美国Certara公司)；
R320低速冷冻离心机(北京白洋医疗器械)；
TGL-16M高速台式冷冻离心机(湘仪仪器有限公司)；
MS105电子分析天平(梅特勒-托利多(上海)有限公司)；
吐温80(Tween 80)，购于Sigma公司；
甲基纤维素(MC)，购于Sigma公司；
SD大鼠购于北京维通利华试验动物技术有限公司；
食蟹猴购于海南新正源生物技术有限公司。
(3)大鼠体内药代动力学实验方法
(3.1)药液配制：
2％Tween 80:98％0.5％MC水溶液(V：V)。
(3.2)给药方案：
健康成年雄性SD大鼠6只(每组3只动物)，禁食过夜(自由饮水)后灌胃给予PF07321332及氘代药物(7)10mg/kg，给药体积为10mL/kg。于给药前及给药后0.5、1、2、4、6、8、12、24h由颈静脉采血0.2mL，4℃离心5min分离血浆，于-20℃保存待测。建立LC-MS/MS法测定血浆中的原形药物浓度，绘制血药浓度-时间曲线，采用WinNonlin 7.2软件计算主要药动学参数。
大鼠药代动力学参数(po)
Figure PCTCN2022142333-appb-000024
注：T max*用中位数(最小值，最大值)表示
从上表中可以看出与非氘代化合物PF-07321332相比，灌胃给药后氘代药物(7)具有更高的血浆峰浓度和更高的血浆中暴露，说明氘代药物(7)具有更优异的体内药代动力学行为。在临床上具备服用剂量比PF-07321332更低、或减少利托那韦联用量以至不需要与利托那韦联用的应用潜力，从而能够扩大临床使用人群，减轻或减少不良反应。
(4)食蟹猴体内药代动力学研究
(4.1)药液配制：
2％Tween 80:98％0.5％MC水溶液(V：V)。
(4.2)给药方案：
健康成年食蟹猴8只，雌雄各半，禁食过夜(自由饮水)，随机分为4组，分别进行单药灌胃和联合利托那韦(Ritonavir)灌胃给药，给药5mL/kg[溶媒为2％Tween 80:98％0.5％MC水溶液(V：V)]；于给药后0.25、0.5、1、2、4、8、10、24、32、48h，分别从猴四肢静脉取血1mL置于K2-EDTA抗凝管中，置于湿冰中，4℃离心分离血浆，血浆转移分装于2.0mL离心管中立即置于-80℃冰箱保存。采用LC-MS/MS法测定血浆中待测化合物浓度。
食蟹猴药代动力学参数(po)
Figure PCTCN2022142333-appb-000025
经口灌胃后，氘代药物(7)单用组暴露量显著高于PF-07321332单用组，C max和AUC last分别为PF-07321332的7.32和3.31倍；氘代药物(7)单用组比PF-07321332+利托那韦联用组暴露量高，C max和AUC last分别为1.40和1.76倍。与利托那韦联用后，氘代药物(7)暴露量显著提高，为单用组的4.47倍计(AUC last计)。
结果表明本发明化合物有良好的体内药代动力学，具有更低服药剂量或无需与利托那韦联合用药的潜力。
试验例3、对人CYP3A4代谢酶的代谢稳定性试验
(1)目的：
本实验采用人重组CYP3A4同工酶孵化法，检测本发明化合物在人CYP3A4孵育体系中的代谢稳定性。
(2)试剂：
还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)，购于德国罗氏公司；
乙腈(色谱纯)，购于德国Merck公司；
人CYP3A4重组酶购于美国BD Gentest公司。
(3)重组酶孵育实验：
每个孵化体系总体积为100μL，介质为100mM磷酸缓冲液(PBS，pH7.4)，包括终浓度为3.0μM的待测化合物和1.0mM的NADPH，采用37℃水浴进行孵化。预孵化3min后，向缓冲液-底物-辅助因子混合物中加入CYP3A4重组酶蛋白起始反应，浓度为50pmol/mL，反应60min后加入同体积冰冷乙腈终止反应。所有孵化样本均为双样本。
(4)数据处理：
采用试验例1同样的方式分析和处理数据。
氘代药物(7)、氘代药物(5)和PF07321332在人CYP3A4重组酶中的T 1/2、固有清除率C lint(CYP450)
Figure PCTCN2022142333-appb-000026
结果表明氘代药物(7)在人CYP3A4重组酶中稳定性显著优于阳性对照化合物PF-07321332；氘代药物(5)亦有改善。
试验例4、SARS-CoV-2病毒Mpro酶学抑制测试
(1)目的：
采用体外酶学试验，检测氘代药物对新型冠状病毒(SARS-CoV-2)野生型WT和P132H突变Mpro蛋白酶的抑制活性。选用PF-07321332作为阳性对照化合物。
(2)试剂：
蛋白和底物：SARS-CoV-2Mpro蛋白酶野生型和P132H突变型由上海药明康德新药开发有限公司克隆表达。蛋白保存在-80℃中。蛋白酶底物由GenScrip公司合成，底物序列为KTSAVLQSGFRKM。底物保存在-20℃中。
仪器：液体工作站(Labcyte，型号：Echo655)、Multidrop分液仪(Thermo，型号：Multidrop combi)、生化培养箱(Binder，型号：KT115)和酶标仪(Molecular Devices，型号：SpectraMax M4)。
试剂：Tris-HCl(pH 7.3)、100mM NaCl、1mM EDTA、5mM TCEP和0.1％BSA。
(3)实验步骤：
化合物用DMSO进行1:3系列稀释10个浓度点，每个浓度双复孔，加入实验板中。受试化合物起测浓度为5μM。阴性对照孔含有酶和底物，但不含化合物，作为无抑制作用对照。阳性对照孔含有底物、酶和高浓度的PF-07321332，作为100％抑制作用对照。将Mpro蛋白酶加入含化合物的实验板中，室温和化合物共培养30分钟；然后加入反应底物在30℃恒温培养箱共孵育60分钟。用多功能酶标仪读板检测荧光读数。
(4)数据处理：
采用GraphPad Prism软件分析计算化合物对Mpro蛋白酶的半数抑制浓度(IC 50)值。
(5)结果：
Figure PCTCN2022142333-appb-000027
由上述结果可知，氘代药物(7)对SARS-CoV-2野生型Mpro蛋白酶、变异株Omicron常见突变P132H蛋白酶均具有良好抑制活性，均优于阳性对照化合物PF-07321332。
试验例5、SARS-CoV-2病毒CPE活性评价
(1)目的：
通过细胞病变试验(CPE)评价受试化合物在Vero细胞中的抗SARS-CoV-2病毒活性。
(2)实验步骤：
a.CC 50测定：
受试化合物用DMSO进行1:3倍比稀释，起始浓度为100μM，每个浓度三复孔，加入96孔培养板中；同时每孔加入2μM CP100356。用化合物处理Vero细胞3Day，评估待测化合物对Vero细胞增殖的影响。细胞以4000/孔密度接种于96孔培养板。将细胞在37℃、5％CO 2、饱和湿度条件下孵育3天。
使用基于ATP的细胞增殖检测试剂盒(Cell Titer Glo,Promega Corporation)检测细胞增殖情况。细胞在室温平衡30分钟后用Cell Titer Glo试剂处理。然后将培养皿用铝箔覆盖并振荡15分钟，使其充分混合和裂解。使用多功能酶标仪(Tecan Infinite M200)进行化学发光检测。设定空白孔(blank，无细胞)及DMSO对照孔。
b.EC 50测定：
WT、Omicron BA.1、BA.4、BA.5毒株：受试化合物用DMSO进行1:2倍比稀释，12个浓度点，起始浓度为10μM，每个浓度双复孔，加入96孔培养板中；同时每孔加入2μM CP100356；
Omicron BA.2毒株：受试化合物用DMSO进行1:2倍比稀释，6个浓度点，起始浓度为1.25μM，每个浓度4复孔，加入96孔培养板中；同时每孔加入2μM CP100356；
板中加入100CCID 50SARS-CoV-2病毒(WT、Omicron BA.1、BA.2、BA.4、BA.5)；
板中加入Vero细胞，于5％CO 2、37℃培养箱中培养3～4天。设置细胞对照(细胞，无化合物处理或病毒感染)和病毒对照(细胞感染病毒，无化合物处理)。显微镜下观察和记录细胞病变效应(CPE)，计算EC 50。
(3)数据处理：
用下列公式来计算检测化合物的抑制率(Inhibition rate，IR)：
IR(％)＝[1-(RLU 化合物-RLU 空白对照)/(RLU 溶媒对照-RLU 空白对照)]×100％。采用GraphPad Prism软件进行作图、数据分析及IC 50计算。
RLU即相对光单位(relative light unit)。
(4)结果：
结果显示，在加入2μM P-gp抑制剂CP100356情况下，0.04～100μM氘代药物(7)、氘代药物(5)对Vero 细胞增殖均无影响，两个化合物CC 50＞100μM。
在加入2μM P-gp抑制剂CP100356情况下，PF-07321332、氘代药物(5)和(7)对WT、Omicron BA.1等毒株的抗病毒活性如下：
Figure PCTCN2022142333-appb-000028
在加入2μM P-gp抑制剂CP100356情况下，PF-07321332、氘代药物(7)对Omicron BA.2、BA.4和BA.5等毒株的抗病毒活性如下：
Figure PCTCN2022142333-appb-000029
氘代药物(7)对SARS-CoV-2野生型及变异株Omicron BA.1、Omicron BA.2、Omicron BA.4和Omicron BA.5均有良好抑制活性，优于阳性对照化合物PF-07321332；氘代药物(5)对SARS-CoV-2野生型及变异株Omicron BA.1均有良好抑制活性，优于阳性对照化合物PF-07321332。
试验例6、体内抗SARS-CoV-2病毒活性研究
(1)目的：
通过hACE2转基因小鼠攻毒试验评价受试化合物在小鼠体内的抗SARS-CoV-2病毒活性。
(2)实验步骤：
采用雄性ACE2转基因人源化小鼠(CAG-hACE2-IRES-Luc-Tg小鼠，12周龄，NM-TG-200002，购自上海南方模式生物科技股份有限公司)进行攻毒试验。BSL-3实验室中，感染组小鼠经滴鼻接种1×10 4PFU SARS-CoV-2病毒(Pubmed No：MT627325)，后分为溶媒组(Vehicle)、氘代药物(7)治疗组(300mg/kg，BID)以及阳性对照化合物PF-07321332(300mg/kg，BID)治疗组。同时设假染毒对照组。动物每天给药两次，连续给药7次。每天监测动物状态、体重。染毒72h末次给药2h后，记录小鼠体重后全部小鼠实施安乐死，取肺脏。肺脏组织抽提RNA后进行qPCR评价肺部病毒载量。
(3)观察指标及数据处理：
a.记录小鼠体重及异常情况，每天1次，如出现小鼠状态萎靡、体温降低、毛色蓬乱、弓背静卧等异常状况及时记录。
b.检测每只小鼠肺部病毒载量。
结果以均值±标准误差(MEAN±SEM)表示。利用Prism进行数据分析。当P＜0.05认为有显著性差异。
(4)结果：
a.攻毒后Vehicle组小鼠体重从Day 1～Day 4呈下降趋势，Day 4体重变化为-13.4％；氘代药物(7)对攻毒后小鼠体重具有维持作用，Day4体重变化为-2.6％，优于阳性对照化合物PF-07321332(-8.2％)。
小鼠体重(g)
Figure PCTCN2022142333-appb-000030
b.攻毒后Vehicle组小鼠肺部病毒载量为1.85E+07copies/g，300mg/kg氘代药物(7)连续7次给药后显著降低为3.23E+06copies/g，较Vehicle组降低82.6％；同剂量PF-07321332连续7次给药后降低为 5.50E+06copies/g，较Vehicle组降低70.3％。
小鼠肺部病毒载量(copy/g lung)
Groups Mean SEM P
Vehicle 1.85E+07 5.26E+06 —
PF-07321332 5.50E+06 2.36E+06 0.0541
氘代药物(7) 3.23E+06 * 2.22E+06 0.0281
与Vehicle组相比，*P＜0.05
结果表明，氘代药物(7)对小鼠体重具有优于阳性对照化合物PF-07321332的体重维持以及降低肺部病毒载量的作用，提示氘代药物(7)体内抗SARS-CoV-2病毒活性要好于阳性对照化合物PF-07321332。

Claims (10)

  1. 一种如下式(I)所示的氘代化合物或其药学上可接受的盐，其具有如下结构
    Figure PCTCN2022142333-appb-100001
    其中，
    R 1～R 15各自独立地为氢或氘；
    Y 1～Y 14各自独立地为氢或氘；
    且，R 1～R 15和Y 1～Y 14中至少有一个为氘原子。
  2. 权利要求1所述化合物或其药学上可接受的盐，其中，R 1～R 9中3-9个为氘原子；优选的，R 1～R 9均为氘原子；优选的，R 1～R 9中6个为氘原子；优选的，R 1～R 9中3个为氘原子；优选的，R 1～R 3或R 4～R 6或R 7～R 9为氘原子。
  3. 权利要求1或2所述化合物或其药学上可接受的盐，其中，R 10～R 15中3-6个为氘原子；优选的，R 10～R 15均为氘原子；优选的，R 10～R 15中3个为氘原子；优选的，R 10～R 12或R 13～R 15为氘原子。
  4. 权利要求1～3中任一项所述化合物或其药学上可接受的盐，其中，Y 1～Y 14中2-14个为氘原子；进一步优选的，Y 13～Y 14为氘原子。
  5. 根据权利要求1～3中任一项所述化合物或其药学上可接受的盐，结构如下所示：
    Figure PCTCN2022142333-appb-100002
    Figure PCTCN2022142333-appb-100003
    Figure PCTCN2022142333-appb-100004
    Figure PCTCN2022142333-appb-100005
  6. 一种药用组合物，其包含权利要求1～5中任一项所述化合物，或其药学上可接受的盐。
  7. 一种药用组合物，其包含有效量的权利要求1～5中任一项所述化合物，或其药学上可接受的盐以及药学上可接受的载体，其特征在于该药物组合物还包括其他药物，所述其他药物为CYP抑制剂；优选的，CYP抑制剂为利托那韦。
  8. 权利要求1～5中任一项所述化合物或其药学上可接受的盐，或权利要求6-7的药用组合物在制备预防和/或治疗对3CL蛋白酶抑制剂敏感的RNA病毒感染引起的疾病或病症，以及PAXLOVID适用的相关病症的药物中的用途；
    优选的，所述RNA病毒为冠状病毒；优选为β冠状病毒；进一步优选为：SARS-CoV、SARS-CoV-2或MERS-CoV。
  9. 权利要求8所述的用途，其中，所述疾病或病症为新型冠状病毒SARS-CoV-2感染引起的疾病或病症；
    优选的，所述新型冠状病毒为新型冠状病毒野生株、新型冠状病毒Delta变异株、新型冠状病毒Omicron变异株；
    进一步优选的，所述新型冠状病毒Omicron变异株选自Omicron BA.1、Omicron BA.2、Omicron BA.4或Omicron BA.5变异株。
  10. 化合物或其盐，所述结构如下式(VII)和(VIII)所示：
    Figure PCTCN2022142333-appb-100006
    其中R 1～R 15、Y 1～Y 6的定义如权利要求1-5任一项式(I)化合物所述；
    Figure PCTCN2022142333-appb-100007
    其中R 1～R 15、Y 1～Y 14的定义如权利要求1-5任一项式(I)化合物所述；
    优选的，式(VII)化合物为
    Figure PCTCN2022142333-appb-100008
    Figure PCTCN2022142333-appb-100009
    Figure PCTCN2022142333-appb-100010
    优选的，式(VII)化合物的盐为钾盐、钠盐或锂盐；
    优选的，式(VIII)化合物为
    Figure PCTCN2022142333-appb-100011
    Figure PCTCN2022142333-appb-100012
    Figure PCTCN2022142333-appb-100013
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