Benzodiazepine pyrazolo carboxamides as n-protein inhibitors

Abstract

This invention relates to benzodiazepine pyrazole carboxamides as defined in the specification and their pharmaceutically acceptable salts; to their use in medicine; to compositions containing them; to methods of their preparation; and to intermediates used in these methods. The compounds of this invention inhibit the activity of the RSV N-protein and can be used to treat, prevent, inhibit, and improve infections caused by viruses such as RSV.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Publication Number (43) Publication Date (21) Application Number 202580009930.X (22) Application Date 2025.01.13 (30) Priority Data 63 / 621,331 2024.01.16 US (85) PCT International Application Entering National Phase Date 2026.07.14 (86) PCT International Application Application Data PCT / IB2025 / 050374 2025.01.13 (87) PCT International Application Publication Data WO2025 / 153942 EN 2025.07.24 (71) Applicant: Pfizer Pharmaceuticals, USA (72) Inventors: M.J. Barrett, A.F. Bedenjak, S.K. Batachaya, G.S. Cockerell, J.A.C.R. Adams, J.A.D. Goode, A. Kagutka, R. Sharma, J. Tiloson III (74) Patent Agency: China Council for the Promotion of International Trade Patent & Trademark Office Co., Ltd., 11038 Patent Attorney: Chen Yingying (51) Int.Cl. C07D 498 / 04 (2006.01) A61P 31 / 14 (2006.01) A61K 31 / 5365 (2006.01) (54) Invention Title: Benzodiazepinel carboxamides as N-protein inhibitors (57) Abstract: This invention relates to benzodiazepinel carboxamides as defined in the specification and pharmaceutically acceptable salts thereof; to their use in medicine; to compositions containing them; to methods of their preparation; and to intermediates used in these methods. The compounds of the present invention inhibit the activity of RSV N-protein and can be used to treat, prevent, inhibit, and improve infections caused by viruses such as RSV. Claims: 3 pages Specification: 34 pages CN 122580320 A 2026.08.14 CN 1 22 58 03 20 A 1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R1 or R2 is independently selected from the group consisting of -CH3, -CD3, and -CH2OH. 2. A compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R1 is -CH3 or -CD3, and R2 is -CH3, -CD3, or -CH2OH. 3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R1 is -CH3 and R2 is -CH3 or -CH2OH. 4. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R1 is -CH3 and R2 is -CH2OH. 5. The compound of claim 4 or a pharmaceutically acceptable salt thereof, wherein the compound is a diastereomer of the following formula:6. Compound 2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide: or a pharmaceutically acceptable salt thereof. 7. The compound (S)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide: claims 1 / 3 page 2 CN 122580320 A or a pharmaceutically acceptable salt thereof. 8. A compound that is (S)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide. 9. A pharmaceutically acceptable salt of (S)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide. 10. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients. 11. A pharmaceutical composition comprising a pharmaceutically acceptable salt according to any one of claims 1 to 9, and one or more pharmaceutically acceptable excipients. 12. A method of treating RSV infection, the method comprising administering to a subject in need a therapeutically effective amount of a compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof. 13. The method of claim 12, further comprising administering a therapeutically effective amount of an additional RSV therapeutic agent. 14. The method of claim 13, wherein the additional RSV treatment agent is selected from the group consisting of: sisunatovir, zirexovir, EDP-938, EDP-323, JNJ-64417184, PC786, S-337395, MRK-1, JNJ-8003, BI-D, AVG-158, AVG-233, AZ-27, monorapvir, remdesivir, obedivir, and ribavirin.15. The method of claim 14, wherein the additional RSV therapeutic agent is selected from the group consisting of: sisunatovir, zirasoxorvir, EDP-938, EDP-323, JNJ-64417184, PC786, S-337395, MRK-1, JNJ-8003, BI-D, AVG-158, AVG-233, and AZ-27. 16. The method of claim 15, wherein the additional RSV therapeutic agent is sisunatovir. 17. The method according to any one of claims 12 to 16, wherein the compound is (S)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazin-3-carboxamide. Claims 2 / 3 Page 3 CN 122580320 A 18. The compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, used as a medicament. 19. The compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, used for treating RSV infection. 20. The compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, used to manufacture a medicament for treating RSV infection. Claims 3 / 3 Page 4 CN 122580320 A Benzodiazepines Carboxamides as N-Protein Inhibitors Technical Field

[0001] This invention relates to benzodiazepines carboxamide compounds and their use in the treatment or prevention of respiratory syncytial virus (RSV) infection. Background Art

[0002] RSV is an antisense single-stranded RNA virus belonging to the Paramyxoviridae family. RSV is easily transmitted through the secretions of infected individuals via surface contact or hand-to-hand transfer. Unlike influenza viruses, RSV is not transmitted through small aerosol particles. After successful vaccination, the incubation period is 4 to 6 days, during which time the virus spreads from the nasopharynx to the lower respiratory tract through the fusion of infected and uninfected cells and the shedding of necrotic epithelium. In infants, this spread can lead to mucus blockage, accompanied by increased mucus secretion and edema, which in turn causes overinflation and collapse of distal lung tissues, phenomena characteristic of bronchiolitis. Hypoxia is common and the ability to eat is usually impaired due to respiratory distress. In RSV pneumonia, inflammatory infiltration of the airways is composed of mononuclear cells and more extensively involves the bronchioles, bronchi, and alveoli. The timing and extent of viral shedding have been found to be associated with the clinical symptoms and severity of the disease.

[0003] RSV is a leading cause of severe respiratory infections in infants and young children worldwide.Even though many infants hospitalized for RSV infection are healthy, the highest morbidity and mortality rates still occur in preterm infants and infants with chronic lung or heart disease. Severe RSV infection in infancy can lead to recurrent wheezing over many years and is associated with the development of subsequent asthma.

[0004] RSV is also a leading cause of morbidity and mortality in the elderly, immunocompromised children and adults, and patients with chronic obstructive pulmonary disease (COPD) and congestive heart failure (CHF).

[0005] Current anti-RSV treatments include ribavirin, but its use is concerning due to its toxicity, potential teratogenicity, and limited efficacy; and palizumab, a monoclonal antibody against RSV. Such palizumab is used for prophylactic rather than therapeutic treatment of RSV. Although palizumab is often effective, its use is limited to preterm infants and high-risk infants. In fact, this limited use means that it cannot be provided to many people who need anti-RSV treatment. Therefore, there is an urgent need for an effective alternative to existing anti-RSV treatments.

[0006] Small molecules have also been proposed as RSV inhibitors. Such small molecules include benzimidazoles and benzodiazepines. For example, benzimidazole inhibitors of RSV are disclosed in WO 02 / 062290 and WO 03 / 053344 (Squibb Bristol Myers Co), WO 2010 / 103306 (AstraZeneca UK Ltd), and WO 2013 / 068769, WO 2016 / 055780, WO 2019 / 016566 and WO 2019 / 122928 (ReViral Limited). The discovery and initial development of RSV604 (i.e., a benzodiazepine compound with submicromolar anti-RSV activity) is described in Chapman et al., Antimicrobial Agents and Chemotherapy, September 2007, pp. 3346-3353.Benzodiazepine inhibitors of RSV have also been disclosed in patent publications, including WO 2004 / 026843 and WO 2005 / 089770 (Arrow Therapeutics Limited), WO 2016 / 166546 and WO 2018 / 033714 (Durham University), WO 2017 / 015449, WO 2018 / 129287 and WO 2018 / 226801 (Enanta Pharmaceuticals, Inc.), and WO 2021 / 079121, WO 2021 / 084280, WO 2021 / 032992, WO 2022 / 008911 and WO 2022 / 008912 (ReViral Limited).

[0007] RV299 (a novel N-protein inhibitor) has recently been terminated in Phase 1 and has not yet been approved for marketing as an N-protein inhibitor for the treatment of RSV.

[0008] Therefore, there remains a need for improved treatment of RSV viral infection. The compounds and methods of the present invention have one or more advantages, including: unexpectedly low predicted human dose-enhanced efficacy, improved safety, and unexpectedly improved metabolic stability compared to other known N-protein inhibitors. Summary of the Invention

[0009] The present invention provides in part compounds of formula (I) and pharmaceutically acceptable salts thereof. The compounds of formula (I) inhibit the activity of viral N-proteins such as those in RSV and can be used to treat, prevent, inhibit, and improve infections caused by viruses including RSV. The present invention also provides pharmaceutical compositions and medicaments comprising the compounds of the present invention or pharmaceutically acceptable salts thereof, alone or in combination with additional RSV therapeutic agents. This invention also provides in part a method for preparing the compounds of the invention, their pharmaceutically acceptable salts, and compositions, as well as a method for using the methods described above. This summary provides selected concepts introduced in a brief form, which will be further elaborated in the detailed description of the invention below. This summary is not intended to identify key or essential features of the claimed subject matter, nor should it be used solely as an auxiliary means of determining the scope of the claimed subject matter.

[0010] According to an embodiment of the invention, formula (I) or a pharmaceutically acceptable salt thereof is provided:

[0011]

[0012] wherein R1 or R2 is independently selected from the group consisting of -CH3, -CD3, and -CH2OH.

[0013] Embodiments of the invention are described below, wherein for convenience, E1 is the same as the embodiment of formula (I) or a pharmaceutically acceptable salt thereof provided above.

[0014] It should be understood that the above general description and the following detailed description of the invention are merely exemplary and explanatory illustrations and do not limit the scope of protection claimed by the present invention.

[0015] Detailed Description of the Invention

[0016] The present invention can be more readily understood by referring to the following detailed description of embodiments of the invention and examples included herein. It should be understood that the present invention is not limited to specific synthetic methods, which may of course vary. It should also be understood that the terminology used herein is only for describing particular embodiments and is not intended to limit the invention.

[0017] E1 is a compound of formula (I) as defined above.

[0018] E2 is a compound of E1 or a pharmaceutically acceptable salt thereof, wherein R1 is -CH3 or -CD3, and R2 is -CH3, -CD3, or -CH2OH.

[0019] E3 is a compound of E1 or a pharmaceutically acceptable salt thereof, wherein R1 is -CH3 and R2 is -CH3 or -CH2OH.

[0020] E4 is a compound or a pharmaceutically acceptable salt thereof, wherein R1 is -CH3 and R2 is -CH2OH.

[0021] E5 is the compound of claim 4 or a pharmaceutically acceptable salt thereof, said compound being a diastereomer of the following formula: Specification 2 / 34 page 6 CN 122580320 A

[0022] .

[0023] E6 is the compound 2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide or a pharmaceutically acceptable salt thereof, as shown in the following formula:

[0024] .

[0025] E7 is a compound of the following formula: (S)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide or a pharmaceutically acceptable salt thereof:

[0026]

[0027] E8 is the compound (S)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide as shown in the specification 3 / 34 page 7 CN 122580320 A

[0028] .

[0029] E9 is a pharmaceutically acceptable salt of (S)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide.

[0030] E10 is a pharmaceutical composition comprising a compound according to any one of embodiments E1 to E8 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients.

[0031] E11 is a pharmaceutical composition comprising a pharmaceutically acceptable salt according to any one of embodiments E1 to E9 and one or more pharmaceutically acceptable excipients.

[0032] E12 is a method for treating RSV infection, the method comprising administering to an individual in need a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof according to any one of embodiments E1 to E9.

[0033] E13 is a method according to embodiment E12, further comprising administering a therapeutically effective amount of an additional RSV therapeutic agent.

[0034] E14 is a method according to embodiment E13, wherein the additional RSV therapeutic agent is selected from the group consisting of: sisunatovir, ziresovir, EDP-938, EDP-323, JNJ-64417184, PC786, S-337395, MRK-1, JNJ-8003, BI-D, AVG-158, AVG-233, AZ-27, molnupiravir, remdesivir, obedivir, and ribavirin.

[0035] E15 is the method according to embodiment E14, wherein the additional RSV therapeutic agent is selected from the group consisting of: sisunatovir, zirasoxorvir, EDP-938, EDP-323, JNJ-64417184, PC786, S-337395, MRK-1, JNJ-8003, BI-D, AVG-158, AVG-233, and AZ-27.

[0036] E16 is the method according to embodiment E15, wherein the additional RSV therapeutic agent is sisunatovir.

[0037] E17 is a method according to any one of embodiments E12 to E16, wherein the compound is (S)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide.

[0038] E18 is a compound or a pharmaceutically acceptable salt thereof according to any one of embodiments E1 to E9, used as a medicament.

[0039] E19 is a compound or a pharmaceutically acceptable salt thereof according to any one of embodiments E1 to E9, used to treat RSV infection.

[0040] E20 is a compound or a pharmaceutically acceptable salt thereof according to any one of embodiments E1 to E9, used to manufacture a medicament for treating RSV infection.

[0041] Each of the above embodiments may be combined with any other embodiment described herein, provided that the other embodiment does not conflict with the combined embodiment. Furthermore, any compound or pharmaceutically acceptable salt thereof described in the examples may be claimed individually or in combination with one or more other compounds or pharmaceutically acceptable salts thereof in the examples, for use in any embodiment described herein. Specification 4 / 34 pages 8 CN 122580320 A

[0042] Definitions

[0043] Unless otherwise defined herein, the scientific and technical terms used in this invention have the meanings commonly understood by one of ordinary skill in the art.

[0044] The present invention can be suitably practiced even in the absence of any element not explicitly disclosed herein.

[0045] “Compounds of the present invention” includes compounds of formula (I) and novel intermediates used in their preparation. Those skilled in the art will understand that compounds of the present invention include conformational isomers (e.g., cis and trans isomers) and all optical isomers (e.g., enantiomers and diastereomers), as well as racemic mixtures, diastereomer mixtures and other mixtures of these isomers, and their tautomers (if present). Those skilled in the art will also understand that compounds of the present invention include solvates, hydrates, isomorphs, polymorphs, esters, salts, prodrugs and their isotopically labeled forms (if formed).

[0046] Unless otherwise stated, the singular forms “a / an” and “the” used herein include the plural forms. For example, a substituent includes one or more substituents.

[0047] As used herein, the term “about” when used to modify a numerically defined parameter (e.g., a dose of 100 mg) indicates that the value of the parameter may fluctuate by 10%. For example, a dose of about 100 mg means 100 mg ± 10%, i.e., the dose may vary between 90 mg and 110 mg.

[0048] The term “pharmaceutically acceptable” means that a substance (e.g., a compound described herein) and any solvate or hydrate thereof, or a composition containing the substance or solvate or hydrate described herein, is suitable for administration to a subject or patient.

[0049] Salt

[0050] The term "pharmaceutically acceptable salt" refers to the compounds of the present invention, which are typically prepared by reacting a free base or free acid with a suitable organic or inorganic acid, or a suitable organic or inorganic base, to obtain a salt of the compounds of the present invention suitable for administration to a subject or patient.

[0051] Furthermore, compounds of formula I may also include other salts of such compounds, which are not necessarily pharmaceutically acceptable salts, but may be used as intermediates for one or more of the following: 1) preparation of compounds of formula I; 2) purification of compounds of formula I; 3) isolation of enantiomers of compounds of formula I; or 4) isolation of diastereomers of compounds of formula I.

[0052] Suitable acid addition salts are formed from acids that form non-toxic salts. Examples include, but are not limited to: acetates, adipic acid salts, aspartate salts, benzoates, benzenesulfonates, bicarbonates / carbonates, bisulfates / sulfates, borates, camphorsulfonates, citrates, cyclohexylaminosulfonates, ethanedisulfonates, ethanesulfonates, formates, fumarates, glucohepanoates, glucuronates, glucuronides, hexafluorophosphates, phenoxylates, hydrochlorides / chlorides, hydrobromates / bromines, hydroiodates / iodides, hydroxyethylsulfonates, lactates, malates, maleates, malonates, methanesulfonates, methyl sulfates, naphthates, 2-naphthalenesulfonates, nicotinates, nitrates, orotates, oxalates, palmitates, dihydroxynaphthalates, phosphates / hydrogen phosphates / dihydrogen phosphates, pyroglutamates, glycosides, stearates, succinates, tannates, tartrates, toluenesulfonates, trifluoroacetates, 1,5-naphthalenedisulfonates, and sinetes.

[0053] Suitable base salts are formed from bases that form non-toxic salts. Examples include, but are not limited to, aluminum salts, arginine salts, benzyl sulfoxide salts, calcium salts, choline salts, diethylamine salts, diethanolamine salts, glycine salts, lysine salts, magnesium salts, meglumine salts, ethanolamine salts, potassium salts, sodium salts, tromethamine salts, and zinc salts.

[0054] Hemi-salts of acids and bases can also be formed, such as hemi-sulfates and hemi-calcium salts.

[0055] For a review of suitable salts, see PAULEKUHN, G.S. et al., “Trends in Active Pharmaceutical Ingredient Salt Selection Based on Analysis of the Orange Book, 5 / 34 pages, 9 CN 122580320 A Database,” Journal of Medicinal Chemistry, 2007, 50(26):6665-6672.

[0056] Pharmaceutically acceptable salts of the compounds of the present invention can be prepared by methods well known to those skilled in the art, including but not limited to the following procedures:

[0057] (i) reacting the compounds of the present invention with a desired acid or base;

[0058] (ii) removing an acid- or base-instable protecting group from a suitable precursor of the compounds of the present invention, or ring-opening a suitable cyclic precursor (e.g., a lactone or lactam) with a desired acid or base; or

[0059] (iii) converting one salt of the compounds of the present invention into another salt. This can be achieved by reacting with a suitable acid or base or by a suitable ion exchange method.

[0060] These methods are generally carried out in solution. The resulting salt can be collected by filtration precipitation or by evaporation of the solvent.

[0061] Solvates

[0062] The compounds of the present invention and their pharmaceutically acceptable salts can exist in both non-solvated and solvated forms. Hereinafter, the term “solvate” is used to describe a molecular complex comprising the compounds of the present invention or their pharmaceutically acceptable salts and one or more pharmaceutically acceptable solvent molecules (e.g., ethanol). When the solvent is water, the term “hydrate” is used.

[0063] The currently accepted classification system for organic hydrates divides them into isolated site hydrates, channel hydrates, or metal ion coordination hydrates—see KR Morris, Polymorphism in Pharmaceutical Solids (Ed. HG Brittain, Marcel Dekker, 1995). Isolated site hydrates are those in which water molecules are separated from each other by intermediate organic molecules and cannot directly contact each other. In channel hydrates, water molecules are located within lattice channels and are adjacent to other water molecules. In metal ion coordination hydrates, water molecules are bonded to metal ions.

[0064] When solvent or water molecules are tightly bound, the complex may have a well-defined stoichiometry and be independent of humidity. However, when solvent or water molecules are weakly bound, such as in channel solvates and hygroscopic compounds, the water / solvent ratio may depend on humidity and drying conditions. In this case, a non-stoichiometric ratio is the norm.

[0065] Complexes

[0066] The scope of the invention also includes multi-component complexes (other than salts and solvates) wherein the drug and at least one other component are present in stoichiometric or non-stoichiometric amounts. Such complexes include cage-like inclusion complexes (drug-host inclusion complexes) and cocrystals. The latter is generally defined as a crystalline complex in which neutral molecular components are bound together by non-covalent interactions, but may also be a complex of neutral molecules and salts.Eutectic can be prepared by melt crystallization, recrystallization from a solvent, or by physically grinding the components together—see O. Almarsson and MJ Zaworotko, Chem Commun, 17, 1889-1896. For a general review of multicomponent complexes, see Haleblian, J Pharm Sci, 64(8), 1269-1288 (August 1975).

[0067] Solid Form

[0068] The compounds of the present invention can exist in a continuous range of solid states, from completely amorphous to completely crystalline. The term “amorphous” refers to a material that lacks long-range order at the molecular level and may exhibit solid or liquid physical properties depending on the temperature. Typically, such materials do not produce obvious X-ray diffraction patterns and, although exhibiting solid properties, are more formally described as liquid. Upon heating, a transition from solid to liquid occurs, characterized by a change in state, typically a second-order phase transition (“glass transition”). The term "crystallization" refers to a solid phase of a material with a regular and ordered internal structure at the molecular level, producing a characteristic X-ray diffraction pattern with distinct peaks. Such materials also exhibit liquid properties when fully heated, but the transition from solid to liquid is characterized by a phase transition, usually a first-order phase transition ("melting point"). Specification 6 / 34 pages 10 CN 122580320 A

[0069] The compounds of the present invention can also exist in a mesocrystalline state (mesocrystalline or liquid crystal) under suitable conditions. The mesocrystalline state is between a true crystalline state and a true liquid state (melt or solution). The mesocrystalline phenomenon caused by temperature change is called a "thermally induced phase transition", while the mesocrystalline phenomenon caused by the addition of a second component (e.g., water or other solvent) is called a "lyotropic phase transition". Compounds with the potential to form a lyotropic mesophase are called "amphiphilic" compounds, whose molecules have ionic (e.g., -COO-Na+, -COO-K+ or -SO3-Na+) or nonionic (e.g., -N-N+(CH3)3) polar head groups. For more information, see N.H. Hartshorne and A. Stuart, Crystals and the Polarizing Microscope, 4th Edition (Edward Arnold, 1970).

[0070] The compounds of the present invention may exhibit polymorphism and / or one or more isomerisms (e.g., optical isomerism, geometric isomerism, or tautomerism). The compounds of the present invention may also be isotopically labeled. This variation is an inherent characteristic of the compounds of the present invention, as these compounds are defined by their structural features and are therefore within the scope of the present invention.

[0071] Tautomers

[0072] In the compounds of the present invention, structural isomers can interconvert through a low energy barrier, resulting in tautomerism (hereinafter referred to as "tautomerism"). For example, proton tautomerism may occur when the compounds of the present invention contain an imino, ketone, or oxime group; so-called valence tautomerism may occur in compounds containing aromatic moieties. Therefore, a single compound may exhibit more than one type of tautomerism.

[0073] It must be emphasized that, for the sake of brevity, the compounds of the present invention are represented herein only as single tautomers, but all possible tautomers are included within the scope of the present invention.

[0074] Stereoisomers

[0075] Cis-trans isomers can be separated by conventional techniques well known to those skilled in the art, such as chromatography and fractional crystallization.

[0076] Conventional techniques for preparing / separating single enantiomers include: chiral synthesis from suitable optically pure precursors, or resolution of racemic mixtures (or racemic mixtures of salts or derivatives) using, for example, chiral high-performance liquid chromatography (HPLC). The resulting diastereomeric mixture can be separated by chromatography and / or fractional crystallization, and one or both diastereomeric compounds can be converted into their corresponding pure enantiomers by methods well known to those skilled in the art. The chiral compounds (and their chiral precursors) of the present invention can be obtained by chromatography (typically HPLC) in an enantiomerically enriched form. The enriched mixture can be obtained by concentrating the eluent. Chiral chromatography using subcritical and supercritical fluids can be employed. The chiral chromatographic methods used in certain embodiments of the present invention are known in the art (see, for example, Smith, Roger M., Loughborough University, Loughborough, UK; Chromatographic Science Series (1998), 75 (Supercritical Fluid Chromatography with Packed Columns), pp. 223-249 and its references). When any racemate is crystallized, two different types of crystals may form. The first type is the racemic compound described above (the true racemate), in which a homogeneous crystal is formed containing equimolar amounts of the two enantiomers. The second type is a racemic mixture or aggregate in which two equimolar amounts of crystals are formed, each containing only one enantiomer. Although the two crystalline forms present in a racemic mixture have the same physical properties, they may have different physical properties compared to a true racemate.Racemic mixtures can be separated using conventional techniques known to those skilled in the art—for example, see E. L. Eliel and S. H. Wilen, Stereochemistry of Organic Compounds (Wiley, 1994).

[0077] Isotopes

[0078] This invention includes all pharmaceutically acceptable isotopically labeled compounds in which one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature. Specification 7 / 34 pages 11 CN 122580320 A

[0079] Examples of isotopes suitable for inclusion in compounds of the present invention include isotopes of hydrogen, such as 2H and 3H; isotopes of carbon, such as 11C, 13C and 14C; isotopes of chlorine, such as 36Cl; isotopes of fluorine, such as 18F; isotopes of iodine, such as 123I and 125I; isotopes of nitrogen, such as 13N and 15N; isotopes of oxygen, such as 15O, 17O and 18O; isotopes of phosphorus, such as 32P; and isotopes of sulfur, such as 35S.

[0080] Certain isotope-labeled compounds of the present invention, such as those containing radioactive isotopes, can be used for drug and / or substrate tissue distribution studies. Radioactive isotopes tritium (i.e., ³H) and carbon-14 (i.e., ¹⁴C) are particularly suitable for this purpose due to their ease of incorporation and detection.

[0081] Substitution with a heavier isotope (e.g., deuterium, i.e., ²H) can provide certain therapeutic advantages, such as greater metabolic stability, thereby extending the in vivo half-life or reducing dosage requirements.

[0082] Substitution with positron-emitting isotopes (e.g., ¹¹C, ¹⁸F, ¹⁵O, and ¹³N) can be used in positron emission tomography (PET) studies to detect substrate receptor occupancy.

[0083] The isotopically labeled compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art, or by processes similar to those described in the accompanying examples and preparation methods, wherein a suitable isotopically labeled reagent is used instead of a previously used unlabeled reagent.

[0084] Pharmaceutically acceptable solvates according to the invention include solvates in which the crystallization solvent can be isotopically substituted, such as D₂O, d₆-acetone, d₆-DMSO.

[0085] Metabolites

[0086] The scope of the present invention also includes the active metabolites of the compounds of the present invention, i.e. compounds formed in the body after administration of the drug, which are usually formed by oxidation or dealkylation reactions.Examples of metabolites according to the invention include, but are not limited to:

[0087] (i) compounds of the invention contain an alkyl group or a hydroxyalkyl derivative thereof (-CH -> -COH):

[0088] .

[0089] Pharmaceutical Compositions

[0090] In another embodiment, the invention includes pharmaceutical compositions. For the purposes of pharmaceutical compositions, compounds themselves or pharmaceutically acceptable salts thereof are collectively referred to as compounds of the invention.

[0091] “Pharmaceutical composition” means a mixture of one or more compounds of the invention or pharmaceutically acceptable salts, solvates, hydrates or prodrugs thereof as active ingredients, and one or more pharmaceutically acceptable excipients.

[0092] Hereinafter, the term “excipient” is used to describe any component other than compounds of the invention. The selection of excipients depends largely on factors such as the route of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[0093] As used herein, “excipient” includes any and all physiologically compatible solvents, dispersion media, coating agents, antimicrobial and antifungal agents, isotonic agents and absorption retardants, carriers, diluents, etc. Examples of excipients include one or more of water, physiological saline, phosphate-buffered saline, dextran, glycerol, ethanol, etc., and combinations thereof, and may include isotonic agents such as sugars, sodium chloride, or polyols (such as mannitol or sorbitol). Examples of excipients also include various organic solvents (e.g., hydrates and solvates). If desired, pharmaceutical compositions may contain other excipients such as flavoring agents, binders / bonding agents, lubricants, disintegrants, sweeteners or flavoring agents, colorants or dyes, etc. For example, for oral administration, tablets containing various excipients (e.g., citric acid) may be used with various disintegrants (e.g., starch, alginate, and certain complex silicates) and binders (e.g., sucrose, gelatin, and gum arabic). Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycol. In addition, lubricants such as magnesium stearate, sodium dodecyl sulfate, and talc are commonly used in tableting. Similar solid compositions can also be used in soft and hard-filled gelatin capsules. Therefore, non-limiting examples of excipients also include lactose or milk sugar and high molecular weight polyethylene glycol. When an aqueous suspension or elixir is required for oral administration, the active compound therein may be used in combination with various sweeteners or flavorings, colorants or dyes, and (if desired) emulsifiers or suspending agents, as well as other excipients (e.g., water, ethanol, propylene glycol, glycerin, or combinations thereof).

[0094] Examples of excipients also include pharmaceutically acceptable substances, such as wetting agents, or small amounts of adjuvants, such as wetting agents or emulsifiers, preservatives, or buffers, which can extend the shelf life of the compound or enhance its effectiveness.

[0095] The compositions of the present invention can be in various forms. For example, these forms include liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injections and infusions), dispersions or suspensions, tablets, capsules, pills, powders, and liposomes. The dosage form depends on the intended route of administration and therapeutic use.

[0096] Typical compositions are injections or infusions, such as antibody compositions similar to those used for passive immunization in humans. One route of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In another embodiment, the compound is administered by intravenous infusion or injection. In yet another embodiment, the compound is administered by intramuscular or subcutaneous injection.

[0097] For example, oral administration of solid dosage forms can take the form of discrete units, such as hard capsules or soft capsules, pills, pouches, lozenges, or tablets, each unit containing a predetermined amount of at least one compound of the present invention. In another embodiment, oral administration can be in powder or granule form. In another embodiment, oral administration can be in spray-dried dispersion form. In another embodiment, oral administration can be in sublingual form, such as lozenges. In these solid dosage forms, the compounds of the present invention are typically combined with one or more excipients. Such capsules or tablets may contain controlled-release formulations. For capsules, tablets, and pills, these dosage forms may also contain buffers or be formulated with an enteric coating.

[0098] In another embodiment, oral administration may be in the form of a liquid dosage form. Liquid dosage forms for oral administration include, for example, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents (e.g., water) commonly used in the art. Such compositions may also contain excipients such as wetting agents, emulsifiers, suspending agents, flavoring agents (e.g., sweeteners), and / or flavorings.

[0099] In another embodiment, the present invention includes a parenteral dosage form. “Parenteral administration” includes, for example, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, and infusion. Injectable formulations (i.e., sterile injectable aqueous or oily suspensions) may be formulated according to the prior art using suitable dispersants, wetting agents, and / or suspending agents.

[0100] In another embodiment, the present invention comprises a topical dosage form. “Topical application” includes, for example, transdermal application (e.g., via a transdermal patch or iontophoresis device), intraocular application, intranasal application, or inhalation application. Compositions for topical application also include, for example, topical gels, sprays, ointments, and creams. Topical formulations may contain compounds capable of enhancing the absorption or penetration of the active ingredient through the skin or other affected areas. When the compounds of the present invention are applied via a transdermal device, the application will use a reservoir-type porous membrane patch or a solid matrix patch.Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, powders, dressings, foams, films, skin patches, sheets, implants, sponges, fibers, bandages, and microemulsions. Liposomes can also be used for this purpose. Common excipients include alcohols, water, mineral oils, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol, and propylene glycol. Permeation enhancers may be added—for example, see BC Finnin and TM Morgan, J. Pharm. Sci., vol. 88, pp. 955-958, 1999.

[0101] Formulations suitable for topical application to the eye include, for example, eye drops, wherein the compounds of the present invention are dissolved or suspended in suitable excipients. Typical formulations suitable for ocular or ear application may be micronized suspensions or solutions in isotonic, pH-adjusted sterile saline drops. Other formulations suitable for ocular and ear application include ointments, biodegradable (i.e., absorbable gel sponges, collagen) and non-biodegradable (e.g., silicone) implants, wafers, lenses, and particulate or vesicle systems, such as niosomes or liposomes. Polymers, such as cross-linked polyacrylic acid, polyvinyl alcohol, hyaluronic acid, cellulose polymers (e.g., hydroxypropyl methylcellulose, hydroxyethyl cellulose, or methylcellulose) or heteropolysaccharide polymers (e.g., gelatin), may be incorporated into the formulation along with a preservative (e.g., benzalkonium chloride). Such formulations may also be delivered via iontophoresis.

[0102] For intranasal and inhalation administration, the compounds of the present invention can be conveniently delivered as a solution or suspension by squeezing or pumping by a patient through a pump nebulizer; or as an aerosol via a pressurized container or nebulizer, in conjunction with a suitable propellant. Formulations suitable for intranasal administration are typically administered as a dry powder (either alone or mixed with other substances, such as lactose, or as a mixed component particle, such as with phospholipids, like phosphatidylcholine), via a dry powder inhaler; or as an aerosol via a pressurized container, pump, sprayer, nebulizer (preferably using an electrohydraulic nebulizer to generate a fine mist) or nebulizer, with or without a suitable propellant, such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. For intranasal use, the powder may contain a bioadhesive, such as chitosan or cyclodextrin.

[0103] In another embodiment, the invention comprises a rectal dosage form. The rectal dosage form may be, for example, a suppository. Cocoa butter is a conventional suppository base, but other alternatives may be used as needed.

[0104] Other excipients and routes of administration known in the pharmaceutical field may also be used. The pharmaceutical compositions of the present invention can be prepared using any known pharmaceutical techniques, such as efficient formulation and administration procedures.The considerations mentioned above regarding effective formulation and administration procedures are well-known in the field and described in standard textbooks. For example, Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, 1975; Liberman et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Kibbe et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington, 1999 all discuss drug formulation.

[0105] Acceptable excipients that are non-toxic to the recipient at the doses and concentrations used may include buffers such as phosphates, citrates, and other organic acids; salts such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, ... Asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as TWEENT™, PLURONIC™, or polyethylene glycol (PEG). Instructions for use, page 10 / 34, CN 122580320 A

[0106] For oral administration, the composition may be provided in the form of tablets or capsules containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 75.0, 100, 125, 150, 175, 200, 250, 500, 600, 750 or 1000 mg of active ingredient, in order to adjust the dosage according to the patient's symptoms.The drug typically contains about 0.01 mg to about 500 mg of the active ingredient, or in another embodiment, about 1 mg to about 100 mg of the active ingredient, or 50 mg to 500 mg. When administered intravenously, the dose range can be about 0.01 mg / kg / min to about 10 mg / kg / min during a constant-rate infusion.

[0107] Liposomes containing the compounds of the present invention can be prepared by methods known in the art, such as those described in U.S. Patent Nos. 4,485,045 and 4,544,545. U.S. Patent No. 5,013,556 discloses liposomes with improved circulation time. Particularly useful liposomes can be prepared by reverse-phase evaporation, which uses a lipid composition comprising phosphatidylcholine, cholesterol, and polyethylene glycol-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter with a specific pore size to obtain liposomes of the desired diameter.

[0108] The compounds of the present invention can also be encapsulated in microcapsules, such as microcapsules prepared by coagulation or interfacial polymerization, such as hydroxymethyl cellulose or gelatin microcapsules and polymethyl methacrylate microcapsules, which can be used in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. These techniques are disclosed in Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing (2000).

[0109] Sustained-release formulations can be used. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing the compounds of the present invention, which is in the form of a molded article, such as a film or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or polyvinyl alcohol), polylactic acid (US Patent No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamic acid ester, non-degradable ethylene-vinyl acetate copolymers, degradable lactic acid-glycolic acid copolymers (e.g., copolymers used for LUPRONDEPOT™, which are injectable microspheres composed of lactic acid-glycolic acid copolymers and leuprolide acetate), sucrose isobutyrate acetate, and poly-D-(-)-3-hydroxybutyrate.

[0110] Formulations intended for intravenous injection must be sterile. This can be easily achieved, for example, by filtration through a sterile filter membrane. The compounds of the present invention are typically placed in containers with sterile inlets, such as intravenous infusion bags or vials with stoppers that can be punctured by a hypodermic needle.

[0111] Suitable emulsions can be prepared using commercially available fat emulsions (e.g., Intralipid™, Liposyn™, Infonutrol™, Lipofundin™, and Lipiphysan™). The active ingredient can be dissolved in a premixed emulsion composition or dissolved in an emulsion formed by mixing oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and phospholipids (e.g., lecithin, soybean lecithin, or soybean lecithin) with water. It should be understood that other ingredients, such as glycerol or glucose, can be added to adjust the emulsion's viscosity. Suitable emulsions typically contain up to 20% oil, for example, 5% to 20%. Fat emulsions can contain fat droplets with a particle size of 0.1 to 1.0 μm, particularly 0.1 to 0.5 μm, and a pH range of 5.5 to 8.0.

[0112] The emulsion composition may be prepared by mixing the compounds of the present invention with Intralipid™ or its components (soybean oil, lecithin, glycerin, and water).

[0113] Compositions for inhalation or inhalation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the composition is administered orally or via nasal inhalation to produce local or systemic effects. Preferably, compositions in sterile pharmaceutically acceptable solvents may be nebulized. The nebulized solution may be inhaled directly from a nebulizer (page 11 / 34 of the specification, CN 122580320 A) or the nebulizer may be connected to a face mask, breathing tent, or intermittent positive pressure ventilator. Solution, suspension, or powder compositions may be administered by a device that delivers the formulation in a suitable manner, preferably orally or nasally.

[0114] Administration and Dosage

[0115] As used herein, the term “treatment” includes both preventative and palliative treatment, namely, relief, reduction, or delay of the progression of a patient’s disease (or condition) or any tissue damage associated with the disease.

[0116] In this document, the terms “subject,” “individual,” or “patient” are used interchangeably to refer to any animal, including mammals. According to the invention, mammals include canines, felines, bovines, capillaries, equines, sheep, suidae, rodents, lagomorphs, primates, humans, etc., and encompass mammals in utero. In one embodiment, humans are suitable subjects. Human subjects can be of any sex and at any developmental stage.

[0117] In this document, “therapeutic effective amount” means an amount of an active compound or agent capable of evoking a biological or medical response in a tissue, system, animal, individual, or human sought by a researcher, veterinarian, physician, or other clinician, which may include one or more of the following:

[0118] (1) disease prevention; for example, preventing an individual who may be susceptible to a disease, condition, or disorder but has not yet developed or exhibited the pathology or symptoms of that disease, condition, or disorder from developing such disease, condition, or disorder;

[0119] (2) disease inhibition; for example, inhibiting the disease, condition, or disorder in an individual who is experiencing or exhibiting the pathology or symptoms of such disease, condition, or disorder (i.e., preventing or slowing the further development of the pathology and / or symptoms); and

[0120] (3) disease improvement; for example, improving the disease, condition, or disorder in an individual who is experiencing or exhibiting the pathology or symptoms of such disease, condition, or disorder (i.e., reversing the pathology and / or symptoms).

[0121] Generally, the compounds of the present invention are administered in an amount effective in treating the conditions described herein. The compounds of the present invention may be administered in the form of the compound itself or in the form of a pharmaceutically acceptable salt. For administration purposes, the compounds themselves or their pharmaceutically acceptable salts are collectively referred to as compounds of the present invention.

[0122] The compounds of the present invention may be administered via any suitable route in the form of a pharmaceutical composition suitable for that route, and the dosage should achieve the intended therapeutic effect. The compounds of the present invention may be administered orally, parenterally, topically, intranasally, or by inhalation.

[0123] The compounds of the present invention may be administered orally. Oral administration may allow the compound to enter the gastrointestinal tract by swallowing, or may allow the compound to enter the bloodstream directly from the mouth by buccal or sublingual administration.

[0124] In another embodiment, the compounds of the present invention may also be administered via a parenteral route, for example, by direct injection into the blood, muscle, or visceral organs. Suitable parenteral administration routes include intravenous, intra-arterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, and subcutaneous. Suitable parenteral administration devices include needle (including microneedle) injectors, needle-free injectors, and infusion techniques.

[0125] In another embodiment, the compounds of the present invention may also be administered topically to the skin or mucous membranes, i.e., percutaneously or transdermally. In another embodiment, the compounds of the present invention may also be administered intranasally or by inhalation. In another embodiment, the compounds of the present invention may be administered rectally or vaginally. In another embodiment, the compounds of the present invention may also be administered directly to the eyes or ears.

[0126] The administration regimen of the compounds of the present invention and / or compositions containing said compounds depends on a variety of factors, including the patient's type, age, weight, sex, and medical condition; the severity of the condition; the route of administration; and the activity of the specific compound used. Therefore, administration regimens may vary considerably.In one embodiment, the total daily dose of the compounds of the present invention is typically from about 0.01 to about 100 mg / kg (i.e., milligrams of the compounds of the present invention per kg of body weight) for the treatment of the condition described herein. In another embodiment, the total daily dose of the compounds of the present invention is from about 0.1 to about 50 mg / kg, while in another embodiment, it is from about 0.5 to about 30 mg / kg. The administration of the compounds of the present invention can typically be repeated multiple times a day (usually not exceeding 4 times). If necessary, multiple daily administrations can be given to increase the total daily dose.

[0127] Treatment Methods and Uses

[0128] These compounds are inhibitors of RSV N-protein and can be used to treat, prevent, inhibit, and alleviate RSV viral infection.

[0129] Co-administration

[0130] The compounds of the present invention can be used alone or in combination with one or more other RSV therapeutic agents. The present invention provides any of the uses, methods, or compositions described herein, wherein the compounds of the present invention or pharmaceutically acceptable salts thereof are used in combination with one or more other known therapeutic agents to treat RSV. Such combinations may provide greater clinical benefit than any single drug used alone. Examples of greater clinical benefit include: significant reduction in RSV symptoms, faster symptom relief, reduced lung pathological damage, significant reduction in the amount of RSV (viral load) in the patient's body, and reduced mortality.

[0131] "Combination" administration of two or more compounds means that all compounds are administered close enough to produce a therapeutic effect on the subject. Two or more compounds may be administered simultaneously or sequentially, via the same or different routes, in the same or different administration regimens, and with or without specific time limits, depending on the treatment regimen. Furthermore, simultaneous administration can be achieved by mixing the compounds before administration, or by administering the compounds at the same or different administration sites in different dosage forms at the same time.

[0132] The terms "simultaneous administration," "co-administration," "synchronous administration," "sequential administration," and "simultaneous administration" all refer to the combined administration of compounds.

[0133] The compounds of the present invention and one or more other RSV therapeutic agents may be administered in fixed or non-fixed combinations of active ingredients. "Fixed combination" refers to the compound of the present invention or a pharmaceutically acceptable salt thereof, and one or more RSV therapeutic agents, administered simultaneously to a subject in a single composition or dose. "Non-fixed combination" refers to the compound of the present invention or a pharmaceutically acceptable salt thereof formulated with one or more RSV therapeutic agents in different compositions or doses so that it can be administered simultaneously or sequentially to a subject in need, with variable dosing intervals, wherein such administration can provide effective concentrations of two or more compounds in the subject.

[0134] In one embodiment, the compounds of the present invention are administered in combination with additional RSV therapeutic agents that can be used to treat RSV infection, the therapeutic agents comprising a pharmaceutically acceptable salt of a drug of a specific name and a pharmaceutically acceptable solvate of said drug and salt. The additional RSV therapeutic agents include drugs that are inhibitors of F-protein, N-protein, L-protein, and nucleoside. Examples of additional RSV therapeutic agents include F-protein inhibitors cezavir and ziresorvir (Ark Bio); N-protein inhibitor EDP-938 (Enanta); non-nucleoside RSV polymerase inhibitors EDP-323 (Enanta), JNJ-64417184 (Johnson & Johnson / Janssen), PC786 (Pulmocide), S-337395 (Shionogi), MRK-1 (Merck), JNJ-8003 (Johnson & Johnson / Janssen), BI-D (Boehringer Ingelheim), AVG-158 (Aviragen), AVG-233 (Aviragen), AZ-27 (AstraZeneca); and nucleoside inhibitors monorapvir, remdesivir, obedivir, and ribavirin.

[0135] Synthetic Methods

[0136] The compounds of the present invention can be synthesized via synthetic routes that include processes similar to those well known in the field of chemistry, particularly in the cases described herein. Starting materials are generally available from commercial sources such as Sigma-Aldrich (located in St. Louis, Missouri), or can be easily prepared using methods well known to those skilled in the art (e.g., Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v. 1-19, Wiley, New York (1967-1999 ed.), or Beilsteins Handbuch der organischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin, including the supplement (also available through the Beilstein online database). The compounds used herein are related to or derived from compounds of significant scientific and commercial value, as described on page 13 / 34 of CN 122580320 A. Therefore, many such compounds are commercially available, or have been reported in the literature, or can be easily prepared from other commonly used substances using methods reported in the literature.

[0137] For a more detailed description of each reaction step, please refer to the "Examples" section below. Those skilled in the art will understand that other synthetic routes can be used to synthesize the compounds of this invention.Although specific starting materials and reagents are discussed below, other starting materials and reagents can be substituted to provide various derivatives and / or reaction conditions. Furthermore, many compounds prepared by the methods described below can be further modified using conventional chemical methods well known to those skilled in the art, according to this disclosure.

[0138] Those skilled in the art should understand that the experimental conditions listed in the following schemes are merely examples of suitable conditions for achieving the transformations shown, and that specific conditions for preparing the compounds of the present invention may need to be or are suitable to be changed. Furthermore, it should be understood that in order to provide the compounds required by the present invention, it may be necessary or desirable to perform transformations in a different order as described in the schemes, or to modify one or more transformations.

[0139] Examples

[0140] The following examples are provided to better understand the present invention. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way.

[0141] Experimental Procedures

[0142] The synthesis of various compounds of the present invention is described below. All starting materials in the intermediates and examples are commercially available or can be prepared by methods known in the art or as described herein.

[0143] Unless otherwise stated, all reactions were carried out using continuous stirring under a nitrogen or argon atmosphere. When appropriate, use a hot air gun under dynamic vacuum to dry the reaction apparatus and use anhydrous solvents (Sure-Seal™ from Sigma-Aldrich (St. Louis, Missouri) or DriSolv™ from EMD Chemicals (Gibston, New Jersey). In some cases, pass commercially available solvents through a column packed with 4 Å molecular sieves until the following water quality control standards are met: a) <100 ppm for dichloromethane, toluene, N,N-dimethylformamide, and tetrahydrofuran; and b) <180 ppm for methanol, ethanol, 1,4-dioxane, and diisopropylamine. For highly sensitive reactions, the solvent is further treated with metallic sodium, calcium hydride, or molecular sieves and distilled immediately before use. Other commercially available solvents and reagents without further purification are used. Reaction conditions (reaction time and temperature) may be varied when referring to synthesis procedures in other examples or methods. Products are typically dried under vacuum before proceeding with further reactions or submitting for bioassays.

[0144] When instructed, the reaction is heated by microwave radiation using a Biotage Initiator or Personal Chemistry Emrys Optimizer. Thin-layer chromatography (TLC), liquid chromatography-mass spectrometry (LCMS), high-performance liquid chromatography (HPLC), and / or gas chromatography-mass spectrometry (GCMS) are used to monitor reaction progress.TLC was performed on pre-coated silica gel plates containing a fluorescent indicator (254 nm excitation wavelength) and visually observed under UV light and / or after staining with I2, KMnO4, CoCl2, phosphomolybdic acid, and / or ammonium cerium molybdate. LCMS data were acquired on an Agilent 1100 series instrument equipped with a Leap Technologies autosampler, Gemini C18 column, acetonitrile / water gradient, and trifluoroacetic acid, formic acid, or ammonium hydroxide modifiers. Column eluent was analyzed using a Waters ZQ mass spectrometer with scans from 100 to 1200 Da in both positive and negative ion modes. Other similar instruments may also be used. HPLC data were typically obtained on an Agilent 1100 series instrument using the specified column, acetonitrile / water gradient, and trifluoroacetic acid or ammonium hydroxide modifier. GCMS data were obtained using a Hewlett Packard 6890 column oven equipped with an HP 6890 syringe, HP-1 column (12 m × 0.2 mm × 0.33 µm), and helium carrier gas. Samples were analyzed using electron ionization scans from 50 to 550 Da on an HP 5973 mass-selective analyzer. Purification was performed using a medium-performance liquid chromatography (MPLC) instrument with an Isco CombiFlash Companion, AnaLogix IntelliFlash 280, Biotage SP1, or Biotage Isolera One and pre-packed Isco RediSep or Biotage Snap silica cartridges. Chiral purification was performed by chiral supercritical fluid chromatography (SFC), typically using Waters, Berger, or Thar instruments; columns such as Chiral Technologies AD-H, ChiralPAK-AD, -AS, -IC, Chiralcel-OD, or -OJ columns; and mixtures of carbon dioxide with methanol, ethanol, 2-propanol, or acetonitrile (alone or modified with trifluoroacetic acid or propan-2-amine). UV detection was used to trigger fraction collection. For synthesis processes referring to other embodiments or methods, purification conditions may be modified: typically, the solvent and solvent ratio used for the eluent / gradient are selected to provide an appropriate Rf value or retention time.

[0145] Mass spectrometry data were obtained from LCMS analysis. Mass spectrometry (MS) was performed via atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), electron impact ionization (EI), or electron scattering (ES) ionization sources.Proton nuclear magnetic resonance (¹H NMR) chemical shifts are given in parts per million (ppm) relative to the lower field of tetramethylsilane (TMS) and recorded on Varian, Bruker, or Jeol spectrometers at 300, 400, 500, or 600 MHz. Chemical shifts are expressed in ppm (d) of the residual peaks in the reference deuterated solvent (CDCl₃, 7.26 ppm; CD₃OD, 3.31 ppm; CD₃CN, 1.94 ppm; (CD₃)₂SO, 2.50 ppm; D₂O, 4.79 ppm). Peak shapes are described as follows: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m, multiplet; br; s, broad singlet; app, apparent. Fluorine NMR (19F NMR) chemical shifts are expressed in parts per million (ppm) and recorded on Varian, Bruker, or Jeol spectrometers at 300, 400, 500, or 600 MHz. Analytical SFC data are typically obtained on Agilent or Berger analyzers as described above. Optical rotation data are obtained on a PerkinElmer Model 343 polarimeter using a 1 dm cell. Trace analysis was performed by Quantitative Technologies Inc. and within 0.4% of the calculated values.

[0146] Unless otherwise stated, chemical reactions were carried out at room temperature (approximately 23°C).

[0147] Unless otherwise stated, all reactants were commercially available and prepared using methods known in the literature without further purification.

[0148] The terms “concentration,” “evaporation,” and “concentration under vacuum” refer to the removal of solvent using a rotary evaporator at a bath temperature below 60°C under reduced pressure. The abbreviations “min” and “h” stand for “minutes” and “hours,” respectively. The terms “TLC” refer to thin-layer chromatography, “room temperature or ambient temperature” refers to a temperature between 18 and 25°C, “GCMS” refers to gas chromatography-mass spectrometry, “LCMS” refers to liquid chromatography-mass spectrometry, “UPLC” refers to ultra-high performance liquid chromatography, “HPLC” refers to high performance liquid chromatography, and “SFC” refers to supercritical fluid chromatography.

[0149] The retention times of HPLC, UPLC, LCMS, GCMS, and SFC were measured using the methods mentioned in the procedure.

[0150] The following compounds and intermediates were named using the naming rules provided by ChemDraw (version 20.1.1.125, PerkinElmer Informatics, Inc., Sheldon, Connecticut, USA).The nomenclature rules provided in ChemDraw 20.1.1.125 are well known to those skilled in the art, and are believed to generally conform to the IUPAC (International Union for Pure and Applied Chemistry) recommendations for organic chemistry nomenclature and the CAS index rules. Unless otherwise stated, all reactants are commercially available and have not been further purified or prepared using methods known in the literature.

[0151] Example 1: 2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide (1) Specification 15 / 34 pages 19 CN 122580320 A

[0152]

[0153] Intermediate 1: 3-amino-9-fluoro-5-phenyl-1,3-dihydro-2H-benzo[e][1,4]diazazon-2-one (C1)

[0154]

[0155] Step 1: Synthesis of (2-amino-3-fluorophenyl)(phenyl) methyl ketone

[0156]

[0157] 2-MeTHF (1000 mL, 10 times the volume (V)) was loaded into the reactor. 2-Amino-3-fluorobenzonitrile (100 g, 735 mmol, 1.0 equivalent) was added. The reactor was purged three times with nitrogen and the temperature was adjusted to between -5 and 5 °C (internal temperature 0 °C). At this temperature and under nitrogen, a 2-MeTHF solution of PhMgBr (2.8 M, 577 mL, 1616 mmol, 2.2 equivalent) was added dropwise over 30 minutes, and exothermic reaction was observed. The reactor was adjusted to between 20 and 30 °C (internal temperature 25 °C), and the reaction mixture was stirred at this temperature for 16 hours (reaction progress was monitored by HPLC analysis of aliquots quenched with 6 M HCl solution). The reactor was cooled to between -5 and 5 °C, and then 6 M HCl (735 mL, 6.0 equivalent) was added dropwise (exothermic reaction was observed). The reaction mixture was heated to 20–30 °C and stirred for 16 hours. 6 M NaOH (300 mL) was slowly added to the reactants until the pH reached between 8 and 9. The phases were separated, and the aqueous layer was extracted with 2-MeTHF (5 volumes x 2). The combined organic layers were washed with water (500 mL) and allowed to stand to combine with another batch for further post-processing and purification.The same reaction was repeated on a larger scale using 2-amino-3-fluorobenzonitrile (240 g, 735 mmol, 1 equivalent) and a 2-MeTHF solution of PhMgBr (2.8 M, 2390 mL, 3880 mmol, 2.2 equivalent), followed by the same procedure. The post-treated crude was combined with the previous batch in 2-MeTHF and concentrated at a temperature below 40 °C, with the solvent converted to MTBE (5 times the volume). Heptane (2.5 times the volume) was added dropwise to the mixture, and the mixture was filtered to collect the solid. The filter cake was washed with a larger volume of heptane (0.5 times the volume). The filter cake was dried between 35 and 45 °C for 16 hours to give the title compound as a solid (450.4 g, 77%). The calculated LCMS value was C13H11FNO + [M+H]+ 216.08, and the measured value was 216.10. Instructions for Use, Page 16 / 34, 20 CN 122580320 A

[0158] Step 2: Synthesis of 2-(1H-benzo[d][1,2,3]triazol-1-yl)-2-(((benzoxy)carbonyl)amino)acetic acid

[0159]

[0160] MeCN (500 mL, 5 times the volume) was added to the reactor and the temperature was adjusted to between 15 and 25 °C. Benzotriazole (100 g, 839 mmol, 1.0 equivalent), glyoxylic acid monohydrate (77.3 g, 839 mmol, 1.0 equivalent), benzoyl carbamate (127 g, 839 mmol, 1.0 equivalent) and the final p-toluenesulfonic acid monohydrate (TsOH H2O, 2.89 g, 16.8 mmol, 0.02 equivalent) were added with stirring. After the reaction mixture became a homogeneous solution, nitrogen gas was bubbled through for 5 minutes and the mixture was heated to between 35 and 45°C. After stirring at this temperature for 17 hours, the reaction mixture was heated to between 55 and 65°C over 1 hour and stirred at this temperature for 0.5 hours. The temperature was then slowly decreased to between 35 and 45°C over another 1 hour and stirred for 0.5 hours. The reaction mixture was further cooled to between 20 and 30°C and the solid was collected by filtration. After HPLC analysis of the solid and filtrate, the two components were recombined and concentrated to between 1 and 2 times their original volume. The mixture was stirred in the previous reactor at between 35 and 45°C for 18 hours and then cooled to between 20 and 30°C. The precipitate was collected by filtration and further dried by heating to give 196.6 g of the title compound as a solid (72%, 99.1% LCAP) for use without further purification.

[0161] Step 3: Synthesis of methyl carbamate (1-(1H-benzo[d][1,2,3]triazol-1-yl)-2-((2-benzoyl-6-fluorophenyl)amino)-2-oxoethyl)carbamate

[0162]

[0163] 2-(1H-benzo[d][1,2,3]triazol-1-yl)-2-(((benzyloxy)carbonyl)amino)acetic acid (182 g, 558 mmol, 1.2 equivalents) was loaded into the reactor and then THF (1000 L, 10 times the volume) was added. (2-amino-3-fluorophenyl)(phenyl)methyl ketone (100 g, 465 mmol, 1.0 equivalents) was added and the reactor was purged with nitrogen three times. The reaction mixture was cooled to between -35 and -25 °C and POCl3 (51.0 mL, 85.5 g, 558 mmol, 1.2 equivalents) was added. After stirring at the same temperature for 30 minutes, pyridine (74.8 mL, 73.5 g, 929 mmol, 2.0 equivalents) was added dropwise over 2 hours. The reaction mixture was stirred at between -35 and -25 °C for 16 hours. Water (2000 mL, 20 times the volume) was added to a second reactor, followed by NaHCO3 (390 g, 4650 mmol, 10.0 equivalents). The reaction mixture from the first reactor was added to the NaHCO3 solution with stirring, maintaining the temperature between 20 and 30 °C. The quenched mixture was stirred at the same temperature for 16 hours and EtOAc (1000 mL, 20 times the volume) was added. The separated aqueous layer was extracted with EtOAc (500 mL, 10 times the volume), and the combined organic layers were washed with water (10 times the volume). The organic phase was concentrated to approximately 500 mL under vacuum and below 40°C, and MeOH (5 times the volume) was added. The mixture was further concentrated to 280 mL under vacuum and below 40°C, and a larger amount of MeOH (5 times the volume) was added. The mixture was then concentrated to dryness under vacuum and below 40°C to obtain a gel-like title compound (243 g, quantitative, 93% LCAP) which was used unpurified for the next step.

[0164] Step 4: Synthesis of (9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)carbamate Specification 17 / 34 pages 21 CN 122580320 A

[0165]

[0166] (1-(1H-benzo[d][1,2,3]triazol-1-yl)-2-((2-benzoyl-6-fluorophenyl)amino)-2-oxoethyl)carbamate (crude product 243 g, 464 mmol, 1.0 equivalent) was loaded into a reactor and MeOH (1215 mL, 5 times the volume) was added. The reactor was purged with nitrogen 3 times and cooled to between -5 and 5°C.Add a MeOH solution of NH3 (7M, 663 mL, 4640 mmol, 10 equivalents). Heat the reaction mixture to between 20 and 30 °C and stir for 1 hour. Concentrate the reaction mixture under reduced pressure and below 40 °C to give the intermediate (1-amino-2-((2-benzoyl-6-fluorophenyl)amino)-2-oxoethyl)carbamate (240 g). Add MeOH (1200 mL, 5 times the volume) and concentrate the mixture to 2.5 times the volume. Add additional MeOH (600 mL, 2.5 times the volume). Readjust the temperature to between 20 and 30 °C and add AcOH (960 mL, 4 times the volume). Stir the reaction mixture again at between 20 and 30 °C for 16 hours and then add water (240 mL, 1 times the volume). Filter the solid and wash with a 5:1 MeOH:water (1 times the volume) solution. The filter cake was dried between 40 and 50 °C to obtain the title compound as a solid (117 g, 63%, 99.6% LCAP).

[0167] Step 5: Synthesis of 3-amino-9-fluoro-5-phenyl-1,3-dihydro-2H-benzo[e][1,4]diazazon-2-one (C1)

[0168]

[0169] Benzyl (9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)carbamate (117 g, 290 mmol, 1.0 equivalent) was charged into a reactor and then CH2Cl2 (936 mL, 8 times the volume) was added. The reactor was purged with nitrogen three times and the temperature was adjusted to between 15 and 25 °C. TfOH (58.0 mL, 109 g, 725 mmol, 2.5 equivalences) was added dropwise, and the reaction mixture was stirred at 15–25 °C for 16 hours. Water (234 mL) was added to the reactor while maintaining the same temperature. Meanwhile, a NaHCO3 solution was prepared by adding solid NaHCO3 (73.1 g, 870 mmol, 3.0 equivalences) to a second reactor containing water (5 times the volume). This NaHCO3 solution was added dropwise to the first reactor containing the reaction mixture, and the pH was adjusted to between 7 and 8. The quenched reaction mixture was stirred at 15–25 °C for 2 hours. The resulting solid was filtered and washed with water (2 times the volume) and ethyl acetate (0.5 times the volume). The filter cake was dried at 35–45 °C for 16 hours to give the title compound as a solid (79.8 g, quantitative, 100% LCAP, but contaminated with trace amounts of NaOTf by 19F NMR). LCMS: The calculated value is C15H13FN3O + [M+H]+ 270.10, and the measured value is 270.1.¹H NMR (400 MHz, (CD₃)₂SO) δ 7.61–7.48 (m, 4H), 7.48–7.37 (m, 2H), 7.25 (ddd, 1H), 7.10 (d, 1H), 4.32 (s, 1H). ¹⁹F NMR (376 MHz, (CD₃)₂SO) δ -124.16 (another small peak at -77.74 ppm, indicating trace amounts of NaOTf).

[0170] The known methods described in WO / 2004 / 026843, WO / 2005 / 090319, WO / 2017 / 015449 and WO / 2021 / 032992 can also be used to prepare the title compound C1 (CAS #1584714-99-7).

[0171] Intermediate 2: Synthesis of 2-(4-(ethylsulfonyl)-2-fluorophenyl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxylic acid (C2) Specification 18 / 34 pages 22 CN 122580320 A

[0172]

[0173] Step 6: Synthesis of 5,5-dimethyl-1,3,2-dioxathiacyclohexane 2,2-dioxide

[0174]

[0175] 5,5-dimethyl-1,3,2-dioxathiacyclohexane 2-oxide (8.0 g, 53.3 mmol, 1.0 equivalent), acetonitrile (80 mL) and water (80 mL) The mixture was loaded into a three-necked round-bottom flask equipped with a top stirrer and internal temperature monitoring, and cooled to an internal temperature of 0.4 °C with stirring. Ruthenium(III) trichloride (0.32 mL, 48 mg, 0.21 mmol, 0.004 equivalents) was added, followed by the addition of sodium periodate (13.7 g, 63.9 mmol, 1.2 equivalents) in portions over 10 minutes while maintaining an internal temperature below 14 °C. The reaction mixture was stirred at an internal temperature of 12 °C for 15 minutes and then quenched with a saturated aqueous solution of NaHSO3 (300 mL) while maintaining an internal temperature below 30 °C. The reaction mixture was diluted with ethyl acetate (500 mL) and the phases were separated. The organic layer was washed with a saturated aqueous solution of NaHSO3 (4 times) and brine (3 times), dried over Na2SO4, concentrated under vacuum, and dried under high vacuum for 4 days to give the title compound (8.25 g, 93%) as a white solid. 1H NMR (600 MHz, CDCl3) δ 4.34(s, 4H), 1.15(s, 6H).

[0176] Step 7: Synthesis of ethyl 3,5-dibromo-1H-pyrazole-4-carboxylate

[0177]

[0178] Sodium acetate trihydrate (127.0 g, 922 mmol, 6.8 equivalents) was mixed with water (200 mL) until most of the solid dissolved. Ethyl 4-pyrazolecarboxylate (19.0 g, 135.58 mmol, 1.0 equivalents) was dissolved in MeCN (100 mL). The sodium acetate mixture was added to the MeCN solution of ethyl 4-pyrazolecarboxylate. The resulting mixture was cooled to 0°C and bromine (15.4 mL, 47.9 g, 298 mmol, 2.2 equivalents) was slowly added. After 10 minutes, another portion of bromine (2.8 mL, 8.7 g, 54.2 mmol, 0.4 equivalents) was added. The reaction mixture was warmed to room temperature and stirred for another 3 hours. Add 10% sodium thiosulfate aqueous solution until the mixture turns pale yellow. Dilute the mixture with ethyl acetate and 2-MeTHF and separate the layers. Concentrate the organic phase under vacuum to produce a grayish-white solid. Dry the crude product in a vacuum oven at 50°C overnight. Suspend the resulting solid in water (150 mL) and stir for 2 hours. After filtration, dry the solid in a vacuum oven at 50°C for 4 hours and further dry at room temperature and high vacuum to remove residual AcOH. The title compound (38.72 g, 96%) is given as a white solid. LCMS values ​​for C6H7Br79Br79N2O2+, C6H7Br79Br81N2O2+, and C6H7Br81Br81N2O2+[M+H]+ were calculated to be 296.89, 298.89, and 300.88, respectively, while measured values ​​were 299.1, 297.1, and 301.1, respectively. ¹H NMR (400 MHz, CDCl3) δ 4.37 (q, 2H), 1.40 (t, 3H). Instructions for Use, Page 19 / 34, 23 CN 122580320 A

[0179] Step 8: Synthesis of ethyl 3,5-dibromo-1-(3-hydroxy-2,2-dimethylpropyl)-1H-pyrazole-4-carboxylate

[0180]

[0181] ethyl 3,5-dibromo-1H-pyrazole-4-carboxylate (50.0 g, 168 mmol, 1.0 equivalent), 5,5-dimethyl-1,3,2-dioxane-2,2-dioxide (33.5 g, 201 mmol, 1.2 equivalent), potassium carbonate (46.4 g, 336 mmol, 2.0 equivalent) and 1,4-dioxane (500 mL, 10 V, 0.34 M) were added to a three-necked 2 L round-bottom flask equipped with a reflux condenser and a top stirrer. The heterogeneous white mixture was cleaned three times by alternating vacuum and nitrogen cycles and then stirred at an internal temperature of 93°C for 21 hours.LCMS showed complete conversion to the desired product, and the reactants were subsequently cooled to 0°C with the slow addition of concentrated hydrochloric acid (12 M, 55 mL, 839 mmol, 4.0 equivalence). The mixture was stirred at 0°C for 5 minutes, warmed to room temperature, and stirred for 1 hour. Additional concentrated hydrochloric acid (12 M, 14 mL, 1 equivalence) was added, and the mixture was stirred at 46°C (internal temperature) for 2 hours. The reactants were cooled to 10°C and slowly quenched with a saturated aqueous solution of NaHCO3 (400 mL) until pH = 9. The separated aqueous layer was extracted with ethyl acetate (500 mL × 3). The combined organic layers were dried over MgSO4, filtered, and concentrated to give the title compound (72.2 g, 84 wt% potency, containing trace amounts of reagents 5,5-dimethyl-1,3,2-dioxane-2,2-dioxide and ethyl acetate) as an orange oil, which was used in the next step without further purification. LCMS calculated values ​​for C11H17Br 79Br79N2O3+, C11H17Br 79Br81N2O3+, and C11H17Br 81Br81N2O3+[M+H]+ were 382.96, 384.96, and 386.96, respectively, while measured values ​​were 383.2, 385.2, and 387.2. ¹H NMR (400 MHz, CDCl3) δ 4.35 (q, 2H), 4.12 (s, 2H), 3.30 (d, 2H), 2.91 (t, 1H), 1.39 (t, 3H), 0.95 (s, 6H).

[0182] Step 9: Synthesis of ethyl 2-bromo-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxylate

[0183]

[0184] ethyl 3,5-dibromo-1-(3-hydroxy-2,2-dimethylpropyl)-1H-pyrazol-4-carboxylate (72.0 g, 84% wt% potency, 160 mmol, 1.0 equivalent) and THF (720 mL, 10 V, 0.22 M) were added to a three-necked 2 L round-bottom flask equipped with a top stirrer. The homogeneous yellow solution was washed three times with alternating vacuum / nitrogen circulation and then cooled to an internal temperature of 3°C with stirring. KHMDS solution (1 M THF solution, 161 mL, 160 mmol, 1.0 equivalent) was added over 10 minutes via a feeding funnel, and the solution was then warmed to room temperature. The mixture was stirred for 2 hours, and LCMS showed complete conversion to the desired product. The reaction was quenched by adding water (300 mL), the phases were separated, and the aqueous layer was extracted with ethyl acetate (200 mL × 3).The combined organic layers were dried over MgSO4, filtered, and the filtrate was concentrated to give the title compound (40.0 g) as an orange oil, which was used in the next step without further purification (quantitative, 87 wt% efficiency). LCMS calculated values ​​for C11H16Br 79N2O3+ and C11H16Br 81N2O3+ [M+H]+ were 303.03 and 305.03, respectively, and the measured values ​​were 303.3 and 305.3. 1H NMR (400 MHz, CDCl3) δ 4.31 (q, 2H), 4.03 (s, 2H), 3.79 (s, 2H), 1.36 (t, 3H), 1.15 (s, 6H).

[0185] Step 10: Synthesis of 1-bromo-4-(ethylsulfonyl)-2-fluorobenzene (Instructions 20 / 34, page 24, CN 122580320 A)

[0186]

[0187] CuI (4.75 g, 24.9 mmol, 0.05 equivalent), K3PO4 (106 g, 499 mmol, 1.0 equivalent), and sodium ethanesulfinate (75.2 g, 648 mmol, 1.3 equivalent) were added to a three-necked round-bottom flask equipped with a condenser, a top stirrer, and an internal temperature sensor. Subsequently, 1-bromo-2-fluoro-4-iodobenzene (150 g, 498.5 mmol, 1.0 equivalent) and (2S, 4R)-N-(2,6-dimethylphenyl)-4-hydroxypyrrolidone-2-carboxamide (5.84 g, 24.9 mmol, 0.05 equivalent) were added. A 0.05 equivalent DMSO solution (1.66 L, 0.3 M) was used. The reactants were bubbled with nitrogen for 20 min and then heated at an internal temperature of 50 °C for 16 h. The reactants were cooled to <10 °C (internal temperature) in an ice bath and then quenched with a 1:3 mixture of concentrated NH4OH and saturated NH4Cl (1 L) while maintaining the internal temperature below 30 °C. A solid precipitated during quenching. The solid was filtered and washed with MTBE (1 L). The phases were separated and the aqueous layer was extracted with MTBE (700 mL × 3). The combined organic layers were collected and washed with saturated NH4Cl (400 mL × 2) and brine (400 mL × 2), dried over Na2SO4, and concentrated under vacuum to give 1-bromo-4-(ethylsulfonyl)-2-fluorobenzene (128.4 g, 96%) as a grayish-white solid. This product was used in subsequent reactions without further purification. GCMS: Calculated values ​​are C8H8Br 79FO2S + and C8H8Br 81FO2S + [M]+ 265.94 and 267.94, respectively, while measured values ​​are 265.9 and 267.9.1H NMR (400 MHz, (CD3)2SO) δ 8.05 (dd, 1H), 7.90 (dd, 1H), 7.67 (dd, 1H), 3.39 (q, 2H), 1.11 (t, 3H).

[0188] Step 11: Synthesis of 2-(4-(ethylsulfonyl)-2-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborhexacyclopentane

[0189]

[0190] 1-bromo-4-(ethylsulfonyl)-2-fluorobenzene (128.4 g, 480 mmol, 1.0 equivalent), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborhexacyclopentane) (183 g, 721 mmol, 1.5 equivalent), KOAc (94.3 g, 961 mmol, 2.0 equivalent) and 1,4-dioxane (1.2 L, 0.4 M) were added to a three-necked round-bottom flask equipped with a condenser, a top stirrer and an internal temperature sensor. The reactants were bubbled under nitrogen for 15 minutes, followed by the addition of Pd(dppf)Cl2CH2Cl2 (13.4 g, 16.4 mmol, 0.025 equivalence). The reactants were then bubbled under nitrogen for another 15 minutes and subsequently heated at an internal temperature of 90°C for 16 hours. The reactants were cooled to room temperature, concentrated under vacuum, diluted with MTBE (1 L), filtered through a diatomaceous earth mat, and then washed with MTBE (0.5 L). The combined organic layers were concentrated under vacuum. The residue was dissolved in ethyl acetate (900 mL) and treated with SiliaMetS® mercaptan metal remover (60 g), followed by heating to reflux for 1.5 hours. The slurry was filtered. SiliaMetS® mercaptan metal remover (60 g) was added to the filtrate, and the mixture was heated to reflux for another 1.5 hours. After filtration, the filtrate was treated with DARCO activated carbon (55 g) and subsequently filtered through a diatomaceous earth mat to remove the DARCO activated carbon. The resulting filtrate was concentrated under vacuum and then diluted with heptane (1.1 L, 7.2 V). The slurry was heated to reflux for 2 hours to produce a homogeneous solution. The solution was slowly cooled to room temperature, at which point a solid precipitated from the solution. The solid was filtered and dried at 40 °C and under vacuum for 16 hours to give 2-(4-(ethylsulfonyl)-2-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentane (126 g, 95.6% efficiency, adjusted yield 120.5 g, 80%) as a grayish-white solid.LCMS: The calculated value was [C14H21BFO4S] + [M+H] + 315.2, but only the boric acid counterpart was observed, with a calculated value of [C8H11BFO4S] + [M+H] + 233.04 and an observed value of 233.2. ¹H NMR (600 MHz, (CD3)2SO) δ 7.91 (dd, 1H), 7.73 (dd, 1H), 7.67 (dd, 1H), 3.37 (q, 2H), 1.32 (s, 12H), 1.09 (t, 3H).

[0191] Step 12: Synthesis of ethyl 2-(4-(ethylsulfonyl)-2-fluorophenyl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxylate

[0192]

[0193] 2-bromo-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxylate (30.0 g, 85.8 mmol, 1.0 equivalent), 2-(4-(ethylsulfonyl)-2-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentane (39.6 g, 95% efficiency, 120 mmol, 1.4 equivalent) and XPhos were sequentially added to a three-necked round-bottom flask equipped with a condenser, top stirrer and internal temperature monitoring. Pd G3 (3.06 g, 3.43 mmol, 0.04 equivalence) in isopropanol (210 mL) and K2CO3 (35.6 g, 257 mmol, 3.0 equivalence) in water (42 mL) were slurries. The reactants were bubbled with nitrogen for 20 min and then stirred at an internal temperature of 75 °C for 1.5 h. The reactants were cooled to room temperature and quenched with water (390 mL). The resulting solids were filtered and the filtrate was left to stand overnight at room temperature with stirring. Additional solids were precipitated from the aqueous filtrate and collected by filtration. Two batches of solids were treated under reflux with ethyl acetate (1 L, 300 mL) and SiliCycle SiliaMetS® thiol 40–63 µm (loading: 1.47 mmol / g) (15 g, 5 g) for 2.5 h, respectively. The mixture was filtered and concentrated under vacuum to give a yellow solid. The first batch of solid was recrystallized in isopropanol (350 mL) to give the title compound (24.6 g, 70%) as a white solid. The second batch of solid was recrystallized in isopropanol (350 mL) and then further recrystallized in isopropanol (120 mL) to give the title compound (5.37 g, 15%) as a white solid. The overall yield was 30.0 g (85%). LCMS: calculated value C19H24FN2O5S + [M+H]+ 411.14, found value 411.4.1H NMR (600 MHz, (CD3)2SO) δ 7.80-7.74 (m, 2H) , 7.74-7.68 (m, 1H) , 4.16 (s, 2H) , 4.03 (q, 2H) , 3.94 (s, 2H) , 3.41 (q, 2H) , 1.13 (t, 3H), 1.08 (s, 6H), 1.04 (t, 3H).

[0194] Step 13: Synthesis of 2-(4-(ethylsulfonyl)-2-fluorophenyl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxylic acid (C2) Specification 22 / 34 pages 26 CN 122580320 A

[0195]

[0196] 2-(4-(ethylsulfonyl)-2-fluorophenyl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxylic acid ethyl ester (30.7 g, 74.8 mmol, 1.0 equivalent), ethanol (219 mL) and NaOH aqueous solution (1 M, 220 mL, 220 mmol, 3 equivalent) were added to a three-necked round-bottom flask equipped with a condenser, top stirrer and internal temperature monitoring to generate a white slurry. The mixture was stirred at an internal temperature of 65°C for 18 hours. The solution was cooled to room temperature and then acidified to pH 2 by dropwise addition of 12 M HCl aqueous solution. The resulting slurry was stirred at room temperature for 2 hours, then filtered and washed with water (300 mL × 2). The solid was suspended in THF (200 mL) and refluxed for 4 hours. The slurry was cooled to room temperature and filtered, then dried in a vacuum oven at 45°C to give the title compound (26.3 g, 92%) as a white solid. LCMS: Calculated value: C17H20FN2O5S + [M+H]+ 383.11, measured value: 383.3. 1H NMR (600 MHz, (CD3)2SO) δ 12.00 (s, 1H), 7.79–7.73 (m, 2H), 7.72–7.67 (m, 1H), 4.14 (s, 2H), 3.93 (s, 2H), 3.41 (q, 2H), 1.14 (t, 3H), 1.08 (s, 6H).

[0197] Step 14: 2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide (1)

[0198]

[0199] 2-hydroxypyridine-N-oxide (485.8 mg, 4.37 mmol, 1.2 equivalences) was added to 2-(4-(ethylsulfonyl)-2-fluorophenyl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxylic acid (1500.0 mg, 92.9 mmol) in a 100 mL round-bottom flask. An amber solution of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI HCl; 1.027 g, 5.35 mmol, 1.47 equivalence) was added over 6 minutes in 5 fractions. The reaction mixture formed a pale pink, slightly turbid solution. The resulting solution was stirred at room temperature for 50 minutes. LCMS indicated complete consumption of the carboxylic acid. 3-Amino-9-fluoro-5-phenyl-1,3-dihydro-2H-benzo[e][1,4]diazazepine-2-one (1.255 g, 94.6 wt% energy, 4.41 mmol, 1.21 equivalence) was added over 6 minutes in 5 fractions. The reaction mixture was stirred at room temperature for 2 hours, during which time the mixture transformed from a suspension to a clear solution. LCMS showed almost complete conversion to the desired product. Water (30 mL, 17 V) was added over 10 minutes via a feeding funnel at 15°C to quench the reaction, and the resulting free-flowing slurry was warmed to room temperature for 30 minutes. The precipitate was collected by filtration (see page 23 / 34 of the instruction manual, CN 122580320 A). The reaction flask was washed with water (30 mL × 5), and the filter cake was washed with water (30 mL × 3). The filter cake was dried overnight under nitrogen and vacuum to give the title compound as a solid (2410 mg, 99%). The calculated LCMS value was C32H30F2N5O5S + [M+H] + 634.19, and the measured value was 634.5.1H NMR (600 MHz, (CD3)2SO) δ 10.95 (brs , 1H) , 7.98 (d , 1H) , 7.74-7.65 (m, 3H) , 7.57 (ddd, 1H) , 7.54-7.51 (m, 1H) , 7.49-7.42 (m, 4H) , 7.30 (ddd , 1H) , 7.13 (d , 1H) , 5.37 (d , 1H) , 4.37 (s, 2H) , 4.01 (s, 2H) , 3.37 (q, 2H) , 1.16 (s, 3H) , 1.15 (s, 3H) , 1.11 (t, 3H). 19F NMR (564 MHz, (CD3)2SO) δ -109.78, -123.35.

[0200] Examples 2 and 3: (S)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide (2) and (R)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide (3)

[0201]

[0202] Racemic 2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide (2.41 g) was milled for 2 hours in a 6:1 water:MeCN (33 mL, 14 V) mixture at room temperature, filtered, and dried over a high vacuum.The solid containing two enantiomers of 2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide (2.41 g, 3.80 mmol) was purified by a preparative chiral SFC (instrument: Waters Prep 80, column: Regis Whelk-O(S,S) 250 mm × 30.0 mm 5u, mobile phase 60% CO2 / 40% (1:1 MeCN:MeOH), flow rate 150 mL / min, temperature 40 °C, back pressure 100 bar). The chiral purity of the isomers was assessed using an analytical chiral SFC (instrument: Agilent 1260 SFC-MS; column: Regis Whelk-O(S,S) 250 mm × 4.6 mm 5u; mobile phase: CO2 / (1:1 MeCN:MeOH), 95:5 to 0:100, flow rate 3.0 mL / min, back pressure 120 Bar, detection at 210 nm). The title compound (S)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide was isolated as a solid (using the analytical chiral SFC method described above; retention time 4.63 min, 1.160 g, 96%, 99.5% ee). Data for the (S) enantiomer: LCMS calculated value: C32H30F2N5O5S + [M+H]+ 634.19, measured value: 634.5. ¹H NMR (600 MHz, (CD3)2SO) δ 10.96 (brs, 1H), 7.98 (d, 1H), 7.75–7.65 (m, 3H), 7.57 (ddd, 1H), 7.55–7.50 (m, 1H), 7.50–7.40 (m, 4H), 7.30 (ddd, 1H), 7.13 (dd, 1H), 5.37 (d, 1H), 4.37 (s, 2H), 4.01 (s, 2H) , 3.37 (q, 2H), 1.16 (s, 3H), 1.15 (s, 3H), 1.11 (t, 3H). 19F NMR (564 MHz, (CD3)2SO) δ -109.77, -123.35.Another enantiomer, which was also isolated as a solid, was (R)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide (using the analytical chiral SFC method described above; retention time 4.74 min, 1.20 g, 99%, 98.4% ee). Data for the (R) enantiomer: LCMS calculated value: C32H30F2N5O5S + [M+H] + 634.19, measured value: 634.5. ¹H NMR (600 MHz, (CD3)2SO) δ 10.96 (brs, 1H), 7.99 (d, 1H), 7.73–7.64 (m, 3H), 7.57 (dd, 1H), 7.54–7.50 (m, 1H), 7.50–7.41 (m, 4H), 7.29 (ddd, 1H), 7.13 (d, 1H), 5.37 (d, 1H), 4.37 (s, 2H), 4.01 (s, 2H) , 3.37 (q , 2H) , 1.16 (s, 3H) , 1.15 (s, 3H) , 1.11 (t, 3H). 19F NMR (564 MHz, (CD3)2SO) δ -109.76, -123.38.

[0203] Examples 4 and 5: (R)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-((S)-9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6-(hydroxymethyl)-6-methyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide (4) and (S)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-((S)-9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1] ,4]diaza-3-yl)-6-(hydroxymethyl)-6-methyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide (5)

[0204] Step 1: Synthesis of ethyl 3,5-dibromo-1-((2,2,5-trimethyl-1,3-dioxane-5-yl)methyl)-1H-pyrazol-4-carboxylate

[0205]

[0206] 5-(bromomethyl)-2,2,5-trimethyl-1,3-dioxane can be prepared using the literature method (ACS Appl. Mater. Interfaces 2023, 15, 2246-2255). Ethyl 3,5-dibromo-1H-pyrazole-4-carboxylate (1.000 g, 3.356 mmol, 1.0 equivalence), 5-(bromomethyl)-2,2,5-trimethyl-1,3-dioxane (1.498 g, 6.713 mmol, 2.0 equivalence), K₂CO₃ (928 mg, 6.713 mmol, 2.0 equivalence), and DMSO (5 mL) were added to a vial. The reaction mixture was heated at 100 °C for 8 hours. The reaction mixture was cooled to room temperature and subsequently diluted with ethyl acetate and water. The organic layer was concentrated under vacuum, and the residue was dissolved in dichloromethane (DCM). Rapid chromatography (0-25% ethyl acetate in heptane) yielded ethyl 3,5-dibromo-1-((2,2,5-trimethyl-1,3-dioxane-5-yl)methyl)-1H-pyrazole-4-carboxylate (1.404 g, 95%) as a white, waxy solid. ¹H NMR (400 MHz, (CD₃)₂SO) δ 4.34 (s, 2H), 4.26 (q, 2H), 3.63-3.51 (m, 4H), 1.37 (s, 3H), 1.35 (s, 3H), 1.30 (t, 3H), 0.79 (s, 3H).

[0207] Step 2: Ethyl 3,5-dibromo-1-(3-hydroxy-2-(hydroxymethyl)-2-methylpropyl)-1H-pyrazole-4-carboxylate

[0208]

[0209] Ethyl 3,5-dibromo-1-((2,2,5-trimethyl-1,3-dioxane-5-yl)methyl)-1H-pyrazole-4-carboxylate (1.40 g, 3.181 mmol, 1.0 equivalent), MeCN (5 mL) and water (0.5 mL) were added to the flask. HCl (12.1 M, 53 µL, 0.64 mmol, 0.2 equivalent) was added to the reaction mixture. After 1 hour at room temperature, the reaction mixture was concentrated under no-blank conditions on page 25 / 34 of the true specification, CN 122580320 A, and the residue was subsequently dissolved in ethyl acetate. The organic layer was washed with a saturated aqueous solution of NaHCO3, dried (MgSO4), and then concentrated under vacuum to give ethyl 3,5-dibromo-1-(3-hydroxy-2-(hydroxymethyl)-2-methylpropyl)-1H-pyrazole-4-carboxylate (1.273 g, 100%) as a yellow oil. LCMS: Calculated values ​​for C11H17Br 79Br79N2O4+, C11H17Br 79Br81N2O4+, and C11H17Br 81Br81N2O4+ [M+H]+ were 398.96, 400.95, and 402.95, respectively; measured values ​​were 399.1, 401.1, and 403.0, respectively. ¹H NMR (600 MHz, (CD3)2SO) δ 4.62 (s, 2H), 4.26 (q, 2H), 4.18 (s, 2H), 3.33 (d, 2H), 3.28 (d, 2H), 1.30 (t, 3H), 0.69 (s, 3H).

[0210] Step 3: 2-Bromo-6-(hydroxymethyl)-6-methyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxylic acid ethyl ester

[0211]

[0212] Add 3,5-dibromo-1-(3-hydroxy-2-(hydroxymethyl)-2-methylpropyl)-1H-pyrazol-4-carboxylic acid ethyl ester (1.10 g, 2.75 mmol, 1.0 equivalent) and THF (10 mL) to a flask. Slowly add KHMDS (1 M solution in THF, 2.75 mL, 2.75 mmol, 1.0 equivalent) to the reaction mixture. After 20 minutes, quench the reaction mixture with water, concentrate under vacuum, and dissolve the residue in dichloromethane.Rapid chromatography (50-100% ethyl acetate in heptane solution) yielded ethyl 2-bromo-6-(hydroxymethyl)-6-methyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxylate (940 mg, containing 9.5 wt% ethyl acetate, calculated yield 851 mg, 97%) as a clear oil / white foam mixture. LCMS: Calculated values ​​for C11H16Br 79N2O4+ and C11H16Br 81N2O4+ [M+H]+ were 319.03 and 321.0, respectively; measured values ​​were 319.2 and 321.1. ¹H NMR (600 MHz, (CD3)2SO) δ 5.08 (t, 1H), 4.26 (d, 1H), 4.17 (ddd, 2H), 4.14 (d, 1H), 3.97 (d, 1H), 3.76 (d, 1H), 3.32 (d, 2H), 1.23 (t, 3H), 0.98 (s, 3H).

[0213] Step 4: 2-(4-(ethylsulfonyl)-2-fluorophenyl)-6-(hydroxymethyl)-6-methyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxylate ethyl ester

[0214]

[0215] Add 2-bromo-6-(hydroxymethyl)-6-methyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxylate ethyl ester (800 mg, 2.51 mmol, 1.0 equivalent), 2-(4-(ethylsulfonyl)-2-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentane (1.02 g, 3.26 mmol, 1.5 equivalent), K2CO3 (1.04 g) to the vial. g (7.52 mmol, 3.0 equivalent) and XPhos Pd G3 (170 mg, 0.201 mmol). The vial was purged with nitrogen and then a mixture of 1,4-dioxane (10 mL) and water (2 mL) was added under nitrogen bubbling. The reaction mixture was heated at 95 °C for 2 hours and then cooled to room temperature. The reaction mixture was extracted with ethyl acetate, the organic layer was concentrated under vacuum and the residue was dissolved in dichloromethane, as per the instructions on page 26 / 34, 30 CN 122580320 A. Rapid chromatography (40-100% ethyl acetate in heptane solution) yielded 2-(4-(ethylsulfonyl)-2-fluorophenyl)-6-(hydroxymethyl)-6-methyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxylic acid ethyl ester (836 mg, 78%) as a brown foamy solid.LCMS: Calculated value: C19H24FN2O6S + [M+H]+ 427.13, measured value: 427.3. ¹H NMR (600 MHz, (CD3)2SO) δ 7.81–7.74 (m, 2H), 7.74–7.70 (m, 1H), 5.11 (t, 1H), 4.31 (d, 1H), 4.19 (d, 1H), 4.08 (d, 1H), 4.03 (q, 2H), 3.88 (d, 1H), 3.41 (q, 2H), 3.37 (d, 2H), 1.13 (t, 3H), 1.04 (t , 3H), 1.03 (s, 3H).

[0216] Step 5: Synthesis of (S)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-6-(hydroxymethyl)-6-methyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxylic acid ethyl ester and (R)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-6-(hydroxymethyl)-6-methyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxylic acid ethyl ester

[0217]

[0218] By preparative chiral SFC (instrument: Waters Prep 80, column: Chiral Technologies OX-H 250 mm × 30.0 mm 5u, mobile phase 80% CO2 / 20% (2-propanol + 0.2% 7N NH3 in MeOH solution), flow rate 100 Two enantiomers of 2-(4-(ethylsulfonyl)-2-fluorophenyl)-6-(hydroxymethyl)-6-methyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxylic acid ethyl ester were separated using an analytical chiral SFC (instrument: Agilent 1260 SFC-MS; column: Chiral Technologies OX-H 250 mm × 4.6 mm 5u; mobile phase CO2 / (2-propanol + 0.2% 7N NH3 in MeOH solution) 95:5 to 40:60, flow rate 3.00 mL / min, back pressure 100 bar, detection at 210 nm). The chiral purity of the isomers was assessed using an analytical chiral SFC (instrument: Agilent 1260 SFC-MS; column: Chiral Technologies OX-H 250 mm × 4.6 mm 5u; mobile phase CO2 / (2-propanol + 0.2% 7N NH3 in MeOH solution) 95:5 to 40:60, flow rate 3.00 mL / min, back pressure 100 bar, detection at 210 nm).Each isomer of 2-(4-(ethylsulfonyl)-2-fluorophenyl)-6-(hydroxymethyl)-6-methyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxylic acid ethyl ester [peak 1 (enantiomer 1): (400 mg, 48%, 100% ee, retention time 4.85 min); peak 2 (enantiomer 2): (344 mg, 41%, 98.2% ee, retention time 5.01 min)] was separated as a solid. The absolute stereochemistry of enantiomers 1 and 2 was not determined. Data for enantiomer 1: Using the analytical chiral SFC method described above, retention time 4.85 min; ¹H NMR (600 MHz, (CD₃)₂SO) δ 7.85–7.74 (m, 2H), 7.74–7.69 (m, 1H), 5.11 (t, 1H), 4.31 (d, 1H), 4.19 (d, 1H), 4.08 (d, 1H), 4.03 (q, 2H), 3.88 (d, 1H), 3.41 (q, 2H), 3.37 (d, 2H), 1.13 (t, 3H), 1.04 (t, 3H), 1.03 (s, 3H). Data for enantiomer 2: Using the analytical chiral SFC method described above, retention time was 5.01 minutes; ¹H NMR (600 MHz, (CD₃)₂SO) δ 7.82–7.75 (m, 2H), 7.75–7.67 (m, 1H), 5.12 (t, 1H), 4.32 (d, 1H), 4.20 (d, 1H), 4.09 (d, 1H), 4.04 (q, 2H), 3.89 (d, 1H), 3.42 (q, 2H), 3.38 (d, 2H), 1.14 (t, 3H), 1.05 (t, 3H), 1.04 (s, 3H).

[0219] Step 6: Synthesis of (R)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-((S)-9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6-(hydroxymethyl)-6-methyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide and (S)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-((S)-9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6-(hydroxymethyl)-6-methyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide and (S)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-((S)-9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6-(hydroxymethyl)-6-methyl-6,7-dihydro- 5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide

[0220]

[0221] Starting from the isolated enantiomers of ethyl 2-(4-(ethylsulfonyl)-2-fluorophenyl)-6-(hydroxymethyl)-6-methyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxylate, such as the preparation of (S)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide and (R)-2-(4-(ethylsulfonyl)- These two title compounds were prepared via a similar route to (2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide (Examples 2 and 3). The absolute stereochemistry of the right-hand side molecule is arbitrarily designated. The absolute stereochemistry of this 9-fluorobenzodiazepine is designated based on a comparison with a real sample of the metabolite produced from hepatocyte culture of the original Example 2. Data for the first metabolite, Example 4: 100% ee; LCMS: calculated value C32H30F2N5O6S + [M+H]+ 650.19, measured value 650.5.1H NMR (600 MHz, (CD3)2SO) δ 7.97 (d, 1H) , 7.76-7.64 (m, 3H) , 7.57 (t, 1H) , 7.52 (ddd , 1H) , 7.50-7.42 (m, 4H) , 7.32-7.26 (m, 1H) , 7.13 (d , 1H) , 5.37 (d , 1H) , 5.18 (t, 1H) , 4.52 (d , 1H) , 4.39 (d , 1H) , 4.15 (d , 1H) , 3.95 (d , 1H) , 3.45 (d , 2H) , 3.37 (q, 2H) , 1.11 (t, 3H) , 1.09 (s, 3H). 19F NMR (564 MHz, (CD3)2SO) δ -109.75, -123.37. Data from Example 5 of the second metabolite: 100% ee; LCMS: calculated value C32H30F2N5O6S + [M+H]+ 650.19, measured value 650.5. ¹H NMR (600 MHz, (CD3)2SO) δ 10.95 (brs, 1H), 7.95 (d, 1H), 7.77–7.66 (m, 3H), 7.57 (ddd, 1H), 7.53 (ddd, 1H), 7.50–7.39 (m, 4H), 7.30 (ddd, 1H), 7.13 (d, 1H), 5.37 (d, 1H), 5.17 (t, 1H). 4.51 (d, 1H), 4.40 (d, 1H), 4.15 (d, 1H), 3.94 (d, 1H), 3.45 (d, 2H), 3.37 (q, 2H), 1.11 (t, 3H), 1.10 (s, 3H). 19F NMR (564 MHz, (CD3)2SO) δ -109.75, -123.36.

[0222] Predictive Deuterated Analogs (PDAs) of Example 2

[0223] The compounds provided in Table 1 are predictive deuterated analogs (PDAs) of Example 2. Formula (A) is the general formula of Example 2 of deuteration, wherein Y1, Y2a, Y2b, Y3a, Y3b, Y4, Y5a, Y5b, Y5c, Y6a, Y6b, Y6c, Y7a, Y7b, Y7c, Y8, Y9 and Y10 are each independently H or D.Based on the metabolic profile of Example 1, the deuterated analogues of Example 2 in Table 1 were predicted by MetaSite (moldiscovery.com / software / metasite / ). Based on MetaSite predictions, Y1, Y2a, Y2b, Y3a, Y3b, Y4, Y5a, Y5b, Y5c, Y6a, Y6b, Y6c, Y7a, Y7b, Y7c, Y8, Y9, and Y10 are the most likely sites for metabolism. Specification 28 / 34 pages 32 CN 122580320 A

[0224]

[0225] Table 1

[0226] Specification 29 / 34 pages 33 CN 122580320 A

[0227] Examples A-1 to A-20 in Table 1 may provide certain therapeutic advantages due to better metabolic stability, such as increased in vivo half-life, reduced dose requirement, reduced CYP450 inhibition (competitive or time-dependent), or improved therapeutic index or tolerability.

[0228] Those skilled in the art can prepare additional deuterated analogs of Example 2, such deuterated analogs having different combinations of Y1 to Y10 provided in Table 1. Such additional deuterated analogs may provide therapeutic advantages similar to those achievable by the deuterated analog.

[0229] Determination of partition coefficient (logD)

[0230] Shake-flask method for determining logD - a miniaturized version of the logD determination described in the literature Stopher, D. and McClean, S. J., Pharm. Pharmacol., 1990, Vol. 42, 144 pages

[0231] Nadolol, mexiletine, propranolol, quinidine, amitriptyline and chlorpromazine were dissolved in DMSO to a concentration of 10 mM and used as control in each dish.

[0232] D-neo-inositol, (5-[[(2E)-3-[4-[[(5E)-5-[[(3-chloro-2,6-dichlorophenyl)methoxy]imino]-5,6-dideoxy-β-D-arabinose-hexafuranosyl]oxy]-3-hydroxyphenyl]-2-methyl-1-oxo-2-propenyl]amino]-5-deoxy-1,2-O-methylene-(9CI) (CAS 247598-17) was used as an internal standard (IS).

[0233] All liquid processing steps were performed using a Biomek FX. Buffer-saturated 1-octanol (149 µL / well) was added to replicate 1 mL capacity 96-well plates.Test compounds (10 mM solution in DMSO; 2 µL / well) and controls (10 mM solution in DMSO; 2 µL / well) were transferred from the Matrix 2D barcode storage tube to the 96-well plate.

[0234] After adding 1-octanol-saturated phosphate buffer (149 µL / well), the plate was sealed with a silicone well cap. The plate was laterally vigorously mixed at 10 rpm on a disc shaker for 1 hour at room temperature or laterally vigorously mixed at 2500 rpm on a plate mode shaker for 15 minutes, followed by centrifugation at 2500 rpm (1006×g) for 15 minutes. After removing the cap from the plate, aliquots from the 1-octanol phase (4 µL / well) were transferred to a new 1 mL volumetric dish containing 1-octanol 1S solution (796 µL / well). Aliquots (10 µL / well) from the buffer phase were transferred to another 1 mL volumetric plate containing buffer 1S solution (190 µL / well).

[0235] LC-MS Conditions

[0236] High-throughput microfluidic gradient LC-MS / MS (µfLC-MS / MS)

[0237] Samples were analyzed by microfluidic reverse-phase liquid chromatography combined with tandem mass spectrometry (µfLC-MS / MS). The LS-1 sample delivery system was controlled by LeadScape software (Sound Analytics, Niantic CT) and connected to the interface of a SCIEX 6500 / 6500+ triple quadrupole / Qtrap mass spectrometer operated by Analyst 1.7 HF3 acquisition software (SCIEX, Framingham MA). LC solvent was delivered using a Prolab Zirconium microfluidic LC pump (Reinach, Switzerland). Mobile phase A consisted of a 0.1% aqueous formic acid solution and mobile phase B consisted of a 0.1% formic acid solution in acetonitrile (Fisher Scientific). A 30 × 0.3 mm microfluidic column was obtained from Waters (Milford, MA).

[0238] A mobile phase with an initial composition of 97% A / 3% B was delivered to the system at a rate of 21 µL / min. After sample injection, the composition of mobile phase B was increased to 85% over 30 seconds, maintained at 85% for 5 seconds, and immediately returned to the initial conditions for column equilibration for 25 seconds (total cycle time 60 seconds). After sample analysis, multiple injections of LC-MS / MS chromatography were integrated and processed in LeadScape (Review Analyze mode).

[0239] The results (logD value) were calculated by adjusting the peak area by the dilution factor and incorporating the internal standard correction, and using the ratio of the corrected peak area.

[0240] The logD value of each compound was calculated using the following equation: Specification 30 / 34 Page 34 CN 122580320 A

[0241]

[0242] Evaluation of antiviral activity against RSV

[0243] Plaque reduction assay

[0244] The RSV plaque reduction assay is an infectious assay that allows for the quantification of the number of infectious units at different sites of RSV infection. Since each plaque originates from a single infectious viral particle, antiviral efficacy can be accurately calculated by counting plaques in the presence and absence of antiviral compounds. HepG2 cells (ECACC: 85011430) were passaged in flasks and seeded in 24-well plates in DMEM containing antibiotics and supplemented with 10% fetal bovine serum (FBS). During seeding and subsequent incubation, the cells were cultured in DMEM containing 2% FBS. 100 plaque-forming units (PFU) / well of RSV (RSV A2 VR-1540) were mixed with serially diluted compounds. Subsequently, 100 µL of the virus / compound mixture was added to the confluent HepG2 cell monolayer. The plate was incubated in a humidified incubator at 37°C and 5% CO2 for 2 hours. The inoculum was then removed, and 1 ml of covering medium (4% CMC in 2% DMEM solution) was added to each well. The cells were incubated in a humidified incubator at 37°C and 5% CO2 for 48 hours and then fixed with 75% acetone-25% methanol solution. The plate was washed under running water, and blocking medium (2% skim milk in PBS-Tween solution) was added to each well. The plate was incubated on a shaker at 37°C for 1 hour. The blocking medium was removed, and 200 µl / well of primary antibody (anti-RSV multi-strain antibody) was added to the blocking medium. The plate was incubated at 37°C for 90 minutes. The plate was washed twice under running water, and then 200 µl / well of secondary antibody (rabbit anti-goat HRP conjugate) was added to the blocking medium. The plate was incubated at 37°C for 1 hour. The plate was washed twice under running water and then 200 µl / well of immunostaining reagent was added at 37°C for 10 minutes. After removing the immunostaining reagent, the plate was washed twice under running water and allowed to stand and air dry before scanning on a CTL BioSpot® S6 Macro analyzer. Plaque counts were used to calculate the infection percentage relative to the mean plaque count of the RSV virus control wells. The mean plaque count for each diluted compound in the replicate wells was plotted to construct a dose-response curve and obtain EC50 and EC90 values. EC90 values ​​are provided in Table 2.

[0245] Human hepatocyte clearance assay in vitro

[0246] Human hepatocyte clearance assay - literature Di, L., et al., Eur. J. Med. Chem., 2012, 57, 441-448

[0247] High-throughput human hepatocyte stability assays were performed using 384-well plates.Ten enriched cryopreserved human hepatocytes from donors were purchased from Celsis IVT (Baltimore, MD). The cryopreserved hepatocytes were thawed and resuspended in Williams E medium (WEM GIBCO-BRL, lot number C1984, customer formulation #91-5233EC) supplemented with HEPES and Na2CO3. Cell counts were performed using the Trypan Blue exclusion method. The hepatocyte suspension was added to the 384-well plate using a Multidrop® liquid dispenser (Multidrop DW, Thermo Scientific, Waltham, MA). The plate was then covered and transferred to a Sciclone® ALH 3000 workstation (Caliper Life Sciences, Hopkinton, MA) equipped with two 6-position Mecour heat exchangers. Test compounds were diluted with buffer and added to the hepatocytes on the Sciclone® workstation. The final incubation medium contained 500,000 cells / mL and 1 μM of the test compound, in a total volume of 15 μL, and contained 0.1% DMSO. Incubation was performed at 37°C. At different time points (0, 3, 10, 30, 60, and 120 min), the reaction was quenched with cold acetonitrile containing an internal standard (IS, CP-628374). The sample was centrifuged at 4°C and 3000 rpm for 10 min (Eppendorf, Hauppauge, NY). The supernatant was transferred to a new, sealed dish using a BioMek® liquid processor (Beckman Coulter, Inc., Danvers MA) before LC-MS / MS analysis. Detailed LC-MS / MS analysis conditions are as described previously (Reference: Di, L., et al., J. Pharm. Sci. 2011, 100, 4974-4985). Propranolol (2D6, 1A2 and 2C19 substrates), midazolam (CYP3A4), triazolam (CYP3A4, low clearance), and naloxone (UGT2B7) were used as positive controls.

[0248] Relay method using human hepatocytes – literature Di, L., et al., Drug Metabolism and Disposition, 2012, 40, 1860-1865

[0249] Enriched cryopreserved human hepatocytes from 10 donors were purchased from Celsis IVT (Baltimore, MD). This batch of enriched hepatocytes was used in all studies. When selecting new batches of hepatocytes, labeled compounds were used to verify enzyme activity.After thawing, hepatocytes were resuspended in Williams E medium supplemented with HEPES and Na2CO3 (customer formulation #91-5233EC; Invitrogen, Grand Island, NY). Cells were counted using the Trypan Blue exclusion method, and 1 μM of the compound (dimethyl sulfoxide, 0.025% final concentration; methanol, 0.125% final concentration) was added to a 24-well plate containing 500,000 cells / ml, for a final incubation volume of 0.50 ml. The plate was covered with a Breathe-Easy breathable membrane (Diversified Biotech, Dedham, MA) and incubated for 4 hours in a humidified incubator at 37°C, 95% O2 / 5% CO2, 75% relative humidity, and 150 rpm. At times 0 and 4 hours, 25 μl of the hepatocyte suspension was removed from the incubator and added to 50 μl of ice-cold acetonitrile containing internal standards to quench the reaction. Samples were centrifuged at 3000 rpm (Eppendorf, Hauppauge, NY) for 10 min at 4 °C, and 50 μl of supernatant was transferred to a clean plate, completely dried, and reconstructed before LC-MS / MS analysis. The residual hepatocyte suspension in the incubation plate was centrifuged (3000 rpm, 10 min, 4 °C). 300 μl of supernatant was transferred to a clean 24-well plate and stored at -80 °C until the next relay experiment. For the second relay experiment, the supernatant plate was warmed to 37 °C for 20 min, and hepatocytes were added to the sample to generate a final cell density of 500,000 cells / ml. The plate was incubated at 37 °C for 4 h, and samples were taken and processed as described above. Five relays were performed for a total incubation time of 20 h. If more relays were needed for compounds with significantly low clearance rates, the supernatant from the last relay was retained. A standard curve was prepared under the same conditions.

[0250] LC-MS / MS Quantification

[0251] The LC mobile phase was as follows: (A) HPLC-grade water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid. Compounds were eluted from the column (Kinetex C18, 30 × 2 mm, 2.6 μm; Phenomenex, Torrance, CA) at a flow rate of 0.4 ml / min for 2.0 min using a solvent gradient from 5% (A) to 95% (B). The cycle time was 3 min / injection. 5 μl aliquots of the sample were injected for analysis using a CTC PAL autosampler (LEAP Technology, Carrboro, NC). A full scan mode from m / z 150 to 600 was applied to detect each compound.Data collection, processing, and analysis were performed using LCquan software (version 2.5; Thermo Fisher Scientific). Terfenadine was used as an internal standard for LC-MS / MS quantification in positive ion multiple reaction monitoring mode. All tested compounds showed good linearity (R² > 0.99) and the limits of quantitation for all compounds were 1 nM.

[0252] The in vitro clearance rate in humans was calculated using the methods described in the above literature.

[0253] In vitro bone marrow assay - Chen, W., et al., J. Pharmacokinetics and Pharmacodynamics, 2020, 47, 163-182

[0254] Initial human bone marrow mononuclear cells (Lonza) were cultured in stem line II hematopoietic stem cell expansion medium (Sigma Aldrich) supplemented with 5% FBS and induced by the following cytokines (R&D systems): 25 ng / mL stem cell factor (SCF), 10 ng / mL G-CSF, 10 ng / mL granulocyte-macrophage colony-stimulating factor, 3 U / mL erythropoietin (EPO), 15 ng / mL thrombopoietin (TPO), 10 ng / mL IL3, 10 ng / mL IL6 and 25 ng / mL Flt3 ligand. Cell culture was maintained in an incubator at 37°C, 5% CO2 and 98% humidity (see reference for further details). Cells were pre-incubated for 1 day and then exposed to DMSO or a compound for up to 5 days. Cell counts were manually determined using a hemocytometer in 10 µL aliquots and converted to total cell counts by adjusting the volume of cell culture medium in each well. Although total cell counts included myeloid, erythroid, and megakaryocyte lines, neutrophil precursors comprised the majority of the entire cell population under the stimulation conditions of cell culture. Therefore, efficacy in reducing total cell counts was used as an indicator of antiproliferative activity (see Hu W, Sung T, Jessen BA, Thibault S, Finkelstein MB, Khan NK, Sacaan AI (2016), Mechanistic investigation of bone marrow suppression associated with palbociclib and its differentiation from cytotoxic chemotherapies, Clin. Cancer Res., 22(8): 2000-2008). Results are shown in Table 2.

[0255] Comparison of antiviral activity, metabolic stability, and toxicity

[0256] Table 2 shows the biological data for the compounds of Example 2 and Comparative Compounds 1 to 3. The comparative compounds are compounds of Examples 25, 41, and 197 of WO 2022 / 008911 and can be prepared as described therein.

[0257] Table 2

[0258]

[0259] Apparent intrinsic clearance (CLint,app) values ​​produced in Example 2 during incubation with human hepatocytes under relay conditions

[0260] In drug development projects, a high apparent intrinsic clearance (CLint,app) measured in in vitro metabolic stability assays using liver microsomes and / or hepatocytes is an undesirable characteristic of new chemical entities, indicating poor pharmacokinetics and resulting in the need for unacceptable total dose regimens to produce pharmacological effects. Optimization of metabolic clearance (CLint,app) is an important consideration for potential clinical drug candidates, given its role in controlling half-life (t1 / 2) and oral absorption. The regulation of lipophilicity, as represented by the partition coefficient (logD), remains a key design principle in drug development. Higher lipophilicity is positively correlated with a logD value <3, indicating a higher likelihood of maintaining favorable metabolic stability, as measured by human hepatocyte (HHEP) assays. As shown in Table 2, removing the methyl group and substituting it with a dimethyl group, such as in Comparative Compound 1 and Comparative Compound 2, increases logD, resulting in similar moderate metabolic clearance values ​​assessed in in vitro HHEP assays, which measure the consumption of the parent molecule over time. Furthermore, introducing an ethyl sulfone group reduces the logD value by up to half a log unit, but this reduction in logD does not translate into a significant reduction in CLint,app (Comparative Compound 1 and Comparative Compound 3). When the 6,6-genomic-dimethyl modification is combined with the introduction of an ethyl sulfone as shown in Example 2 of this application, we observed a significant and unexpected improvement in the metabolic stability of HHEP. The determination of low CLint,app values ​​indicated in Table 2 needs to be evaluated in HHEP assays under relay conditions to capture slow metabolic processes.

[0261] The introduction of an ethyl sulfone group also results in enhanced RSV EC90 potency of compounds such as Example 2 of the present invention (compared to Comparative Compound 1 or 2). When evaluating the compounds of the present invention in in vitro human bone marrow assays, the introduction of an ethyl sulfone group and modification of the core having a 6,6-genomic-dimethyl motif resulted in a concomitant improvement in toxicity as observed by the applicant. In vitro bone marrow assays were used to assess the effect of the compounds on the proliferation of bone marrow progenitor cells, with a reduction in total cell count as a measure of antiproliferative activity.In vitro results of the bone marrow toxicity test have shown predictability of neutropenia in clinical trials of palbociclib, as reported in the aforementioned literature. In Table 2, the compounds of Example 2 and Comparative Compound 3 demonstrate beneficial human bone marrow toxicity IC50 values ​​compared to Comparative Compound 1 and Comparative Compound 2, indicating improved safety of the ethyl sulfone analog.

[0262] The specific combination of the gem-methyl and ethyl sulfone substitution provides an unexpected combination of simultaneously improved properties: (1) significantly enhanced potency against RSV N-protein; (2) significantly improved metabolic stability as assessed by human hepatocytes; and (3) improved safety as assessed by bone marrow toxicity testing. The compounds of Example 2 are unique in this series of compounds due to the combination of such improvements relative to Comparative Compound 1, 2, or 3. The effects of these changes result in an improved antiviral agent that exhibits an unexpectedly low predictable human dose, improved safety, and unexpectedly improved metabolic stability compared to Comparative Compounds 1, 2, and 3, which translates into a longer pharmacokinetic half-life.

[0263] Several references are cited in this application. The entire contents of these references are incorporated herein by reference for all purposes.

[0264] Those skilled in the art will understand that various modifications and variations can be made to this invention without departing from its scope or spirit. Those skilled in the art should be able to understand other embodiments of the invention by reading this specification and practicing the invention disclosed herein. This specification and embodiments are for reference only; the true scope and spirit of the invention are indicated by the following claims. Specification 34 / 34 pages 38 CN 122580320 A.

Claims

1. Compound of formula (I) Or a pharmaceutically acceptable salt thereof, wherein R 1 Or R 2 Choose independently from the following groups: -CH3, -CD3, and -CH2OH.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 1 It is -CH3 or -CD3, and R 2 It can be -CH3, -CD3, or -CH2OH.

3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 1 It is -CH3, and R 2 It is -CH3 or -CH2OH.

4. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 1 It is -CH3, and R 2 It is -CH2OH.

5. The compound according to claim 4, or a pharmaceutically acceptable salt thereof, wherein the compound is a diastereomer of the following formula: 。 6. Compound 2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide: Or its pharmaceutically acceptable salt.

7. Compound (S)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide: Or its pharmaceutically acceptable salt.

8. A compound, which is (S)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide: 。 9. A pharmaceutically acceptable salt of (S)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide.

10. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

11. A pharmaceutical composition comprising a pharmaceutically acceptable salt according to any one of claims 1 to 9, and one or more pharmaceutically acceptable excipients.

12. A method for treating RSV infection, the method comprising administering to a subject in need a therapeutically effective amount of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9.

13. The method of claim 12, further comprising administering an additional therapeutically effective amount of RSV therapeutic agent.

14. The method of claim 13, wherein the additional RSV treatment agent is selected from the group consisting of: sisunatovir, zirexovir, EDP-938, EDP-323, JNJ-64417184, PC786, S-337395, MRK-1, JNJ-8003, BI-D, AVG-158, AVG-233, AZ-27, monorapvir, remdesivir, obedivir, and ribavirin.

15. The method of claim 14, wherein the additional RSV therapeutic agent is selected from the group consisting of: sisunatovir, zirasoxorvir, EDP-938, EDP-323, JNJ-64417184, PC786, S-337395, MRK-1, JNJ-8003, BI-D, AVG-158, AVG-233, and AZ-27.

16. The method of claim 15, wherein the additional RSV therapeutic agent is sisunatovir.

17. The method according to any one of claims 12 to 16, wherein the compound is (S)-2-(4-(ethylsulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazazon-3-yl)-6,6-dimethyl-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-carboxamide.

18. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, used as a medicine.

19. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, for the treatment of RSV infection.

20. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, used in the manufacture of a medicament for treating RSV infection.