Benzodiazepine compounds as n-protein inhibitors

Abstract

The present disclosure relates to benzodiazepine compounds and pharmaceutically acceptable salts thereof, their uses in medicine, pharmaceutical compositions containing them, processes for their preparation, and intermediates used in such processes. The benzodiazepine compounds can be useful in the treatment, prevention, inhibition, and amelioration of RSV infection.

Description

Technical Field

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

[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 surfaces or hand-to-hand contact. Unlike influenza, it is not transmitted via small aerosol particles. Following successful vaccination, the incubation period is between four and six days, during which the virus spreads from the nasopharynx to the lower respiratory tract through the fusion of infected and uninfected cells and the shedding of necrotic epithelial cells. In infants, combined with increased mucus secretion and edema, this can lead to mucus blockage, resulting in overinflation and atrophy of distal lung tissue, indicative of bronchiolitis. Hypoxia is common, and feeding ability is often impaired due to respiratory distress. In RSV pneumonia, the inflammatory infiltration of the airways is composed of mononuclear cells and is more extensive, involving the bronchioles, bronchi, and alveoli. The duration and extent of viral shedding have been found to correlate with clinical signs and disease severity.

[0003] RSV is a leading cause of severe respiratory infections in infants and young children worldwide. While the highest morbidity and mortality rates occur in premature infants and those with chronic lung or heart disease, many infants hospitalized for RSV infection were originally healthy. Severe RSV infection in infancy can lead to years of recurrent wheezing and is associated with the development of asthma later in life.

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

[0005] Current anti-RSV treatments include ribavirin, but its use is fraught with concerns due to its toxicity, potential teratogenicity, and limited efficacy; and palizumab, a monoclonal antibody against RSV. Palizumab is intended for prophylactic rather than therapeutic treatment of RSV. Although this antibody is generally effective, its use is limited to premature and high-risk infants. In fact, its limited utility means it cannot be used for many people who require anti-RSV treatment. Therefore, there is an urgent need for effective alternatives to existing anti-RSV treatments.

[0006] Small molecules have also been proposed as RSV inhibitors. These small molecules include benzimidazole 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 preliminary development of the benzodiazepine compound RSV604, which has submicromolar anti-RSV activity, is described in Antimicrobial Agents and Chemotherapy, September 2007, 3346-3353 (Chapman et al.). Benzodiazepine inhibitors of RSV are also disclosed in publications including WO 2004 / 026843 and WO 2005 / 089770 (Arrow Therapeutics Limited); WO 2016 / 166546 and WO 2018 / 033714 (Durham University); WO 2017 / 015449, WO2018 / 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 (ReViralLimited).

[0007] The novel N-protein inhibitor RV299, which was in Phase 1 trials, has recently been discontinued, and to date, there are no approved N-protein inhibitors on the market for the treatment of RSV.

[0008] Therefore, improved treatment for RSV infection remains necessary. The compounds and methods described in this invention have one or more advantages, such as improved antiviral efficacy compared to RV299 and other known N-protein inhibitors, and unexpected and significant improvements in myelotoxicity and pharmacokinetic properties. Invention Overview This invention provides compounds of formula (A) and pharmaceutically acceptable salts thereof. Compounds of formula (A) inhibit the activity of viral N-proteins such as those in RSV and are suitable for treating, preventing, inhibiting, and improving viral infections, including RSV. Pharmaceutical compositions and medicaments comprising, alone or in combination with other RSV therapeutic agents, compounds of the present invention or pharmaceutically acceptable salts thereof. This invention also provides methods for preparing the compounds, pharmaceutically acceptable salts, and compositions of the present invention, and methods for using the foregoing. This “Summary of the Invention” is provided to introduce a series of concepts in a simplified form, which will be further described in the “Detailed Description of the Invention” below. This “Summary of the Invention” is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used solely as an aid in determining the scope of the claimed subject matter.

[0010] According to an embodiment of the present invention, formula (A) is provided: Where R 1 Choose from the group consisting of -CH3, -CD3, -CH2OH and -OCH3; or pharmaceutically acceptable salts thereof.

[0011] The following describes embodiments of the invention, wherein, for convenience, E1 is the same as the embodiment of formula (A) provided above or a pharmaceutically acceptable salt thereof.

[0012] It should be understood that the foregoing general description and the following detailed description are merely illustrative and explanatory and do not limit the invention for which protection is claimed. Invention Details The invention will 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 invention is not limited to specific synthetic preparation methods, which can, of course, be varied. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0014] E1 is a compound of formula (A) as defined above.

[0015] E2 is a compound as described in embodiment E1 or a pharmaceutically acceptable salt thereof, wherein R 1 Choose the group consisting of -CH3, -CD3, and -CH2OH.

[0016] E3 is a compound as described in embodiment E1 or a pharmaceutically acceptable salt thereof, wherein R 1 It is -CH3 or -CH2OH.

[0017] E4 is a compound or a pharmaceutically acceptable salt thereof as described in any of embodiments E1 to E3, wherein the compound is an S enantiomer.

[0018] E5 is the compound 2-(4-(ethanesulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide or a pharmaceutically acceptable salt thereof.

[0019] E6 is the compound (S)-2-(4-(ethanesulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide or a pharmaceutically acceptable salt thereof.

[0020] E7 is the compound as described in embodiment E6, which is (S)-2-(4-(ethanesulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0021] E8 is a pharmaceutically acceptable salt as described in implementation scheme E6.

[0022] E9 is a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof as described in any one of embodiments E1 to E7, and one or more pharmaceutically acceptable excipients.

[0023] E10 is a pharmaceutical composition comprising a compound according to any one of embodiments E1 to E7, and one or more pharmaceutically acceptable excipients.

[0024] E11 is a pharmaceutical composition comprising a pharmaceutically acceptable salt of any one of embodiments E1 to E6 or E8, and one or more pharmaceutically acceptable excipients.

[0025] E12 is a method for treating RSV infection, 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 E8.

[0026] E13 is the method of implementation scheme E12, which further includes administering a therapeutically effective amount of an additional RSV therapeutic agent.

[0027] E14 is the method of implementation scheme E13, wherein the additional RSV treatment 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, obeldesivir, and ribavirin.

[0028] E15 is the method of implementation scheme E14, wherein the additional RSV therapeutic agent is selected from the group consisting of: cesumatovir, ziresoxir, EDP-938, EDP-323, JNJ-64417184, PC786, S-337395, MRK-1, JNJ-8003, BI-D, AVG-158, AVG-233, and AZ-27.

[0029] E16 is the method of implementation scheme E15, wherein the additional RSV therapeutic agent is cisunatovir.

[0030] E17 is a method of any one of embodiments E12 to E16, wherein the compound is (S)-2-(4-(ethanesulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide.

[0031] E18 is a compound or a pharmaceutically acceptable salt thereof as described in any of embodiments E1 to E8, used as a medicine.

[0032] E19 is a compound or a pharmaceutically acceptable salt thereof as described in any of embodiments E1 to E8, used to treat RSV infection.

[0033] E20 is a compound or a pharmaceutically acceptable salt thereof as described in any one of embodiments E1 to E8, used to prepare a medicament for treating RSV infection.

[0034] The embodiments described above can be combined with any other embodiments described herein, and such other embodiments are not inconsistent with the combined embodiments. Furthermore, for any embodiment described herein, any compound or pharmaceutically acceptable salt thereof described in the examples may be claimed alone or in combination with one or more other compounds or pharmaceutically acceptable salts thereof from the examples.

[0035] definition Unless otherwise defined herein, scientific and technical terms used in connection with this invention shall have the meanings commonly understood by one of ordinary skill in the art.

[0036] The invention described herein may be suitably practiced in the absence of any elements not specifically disclosed herein.

[0037] "Compounds of the present invention" includes compounds of formula (A) and novel intermediates used in their preparation. Those skilled in the art will understand that compounds of the present invention include their possible conformational isomers (e.g., cis and trans isomers) and all optical isomers (e.g., enantiomers and diastereomers), racemic mixtures, diastereomers and other mixtures, and tautomers, in addition to such isomers. Those skilled in the art will also understand that compounds of the present invention include their solvates, hydrates, isomorphs, polymorphs, esters, salts, prodrugs, and isotopically labeled forms.

[0038] Unless otherwise indicated, as used herein, the singular forms “a / an” and “the” include plural references. For example, an “a” substituent includes one or more substituents.

[0039] As used herein, the term “about” when used to modify a numerically defined parameter (e.g., a dose of 100 mg) means that the parameter may vary by up to 10% less or more than its specified value. For example, a dose of about 100 mg means 100 mg ± 10%, that is, it may vary between 90 mg and 110 mg.

[0040] The term “pharmaceutically acceptable” means that the substances of the present invention (such as the compounds described herein) and any solvates or hydrates thereof, or compositions containing said substances or solvates or hydrates, are suitable for administration to an individual or patient.

[0041] Salt The term "pharmaceutically acceptable salt" refers to the following compounds of the present invention, which are generally prepared by reacting a free base or free acid with a suitable organic or inorganic acid, or a suitable organic or inorganic base, to provide a salt of the compounds of the present invention suitable for administration to an individual or patient.

[0042] In addition, compounds of formula I may also include other salts of such compounds, which are not necessarily pharmaceutically acceptable salts and may be used as intermediates for one or more of the following: 1) preparing compounds of formula I; 2) purifying compounds of formula I; 3) isolating enantiomers of compounds of formula I; or 4) isolating diastereomers of compounds of formula I.

[0043] Suitable acid addition salts are formed from acids that form non-toxic salts. Examples include (but are not limited to) acetates, adipates, aspartates, benzoates, benzenesulfonates, bicarbonates / carbonates, bisulfates / sulfates, borates, camphor sulfonates, citrates, cyclamates, ethanedisulfonates, ethanesulfonates, formates, fumarates, glucohepanoates, gluconates, glucurons, hexafluorophosphates, hibenzates, hydrochlorides / chlorides, hydrobromates / bromines, and hydroiodates / iodides. Hydroxyethanesulfonate, lactate, malate, maleate, malonate, methanesulfonate, methyl sulfate, naphthalene dicarboxylate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, dihydroxynaphthalate, phosphate / hydrogen phosphate / dihydrogen phosphate, pyroglutamate, gluconate, stearate, succinate, tannate, tartrate, toluenesulfonate, trifluoroacetate, 1,5-naphthalenedisulfonic acid, and xinofoate.

[0044] Suitable base salts are formed from bases that form non-toxic salts. Examples include (but are not limited to) aluminum, arginine, benzathine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, thiazoline, and zinc salts.

[0045] It can also form half-salts of acids and bases, such as half-sulfates and half-calcium salts.

[0046] For a review of suitable salts, see PAULEKUHN, GS et al., “Trends in Active Pharmaceutical Ingredient Salt Selection Based on Analysis of the Orange Book Database”, Journal of Medicinal Chemistry, 2007, 50(26):6665-6672.

[0047] Pharmaceutically acceptable salts of the compounds of this invention can be prepared by methods well known to those skilled in the art, including (but not limited to) the following procedures: (i) By reacting the compound of the present invention with a desired acid or base; (ii) Using a desired acid or base, remove the acid- or base-unstable protecting group from a suitable precursor of the compound of the present invention, or open the ring of a suitable cyclic precursor (e.g., a lactone or lactam); or (iii) By converting one salt of the compound of the present invention into another salt. This can be achieved by reacting with a suitable acid or base or by means of a suitable ion exchange procedure.

[0048] These procedures are typically carried out in solution. The resulting salt can precipitate and be collected by filtration, or it can be recovered by evaporating the solvent.

[0049] solvates The compounds of this invention and their pharmaceutically acceptable salts may exist in both solvated and solvent-free forms. The term "solvent" is used herein to describe a molecular complex comprising a compound of this invention or its pharmaceutically acceptable salt and one or more pharmaceutically acceptable solvent molecules, such as ethanol. When the solvent is water, the term "hydrate" is used.

[0050] The currently accepted classification system for organic hydrates is based on the definition of segregated site hydrates, channel hydrates, or metal ion-coordinated hydrates – see KR Morris, *Polymorphism in Pharmaceutical Solids* (edited by HGBrittain, Marcel Dekker, 1995). Segregated site hydrates are those in which water molecules are separated from each other and do not directly contact each other by intercalation into organic molecules. In channel hydrates, water molecules reside in lattice channels, where they are adjacent to other water molecules. In metal ion-coordinated hydrates, water molecules are bound to metal ions.

[0051] When the solvent or water is tightly bound, the complex can have a well-defined stoichiometry independent of humidity. However, when the solvent or water is weakly bound, such as in channel solvates and hygroscopic compounds, the water / solvent ratio can depend on humidity and drying conditions. In these cases, non-stoichiometry becomes the standard.

[0052] complex The scope of this invention also includes multicomponent complexes (other than salts and solvates) in which the drug and at least one other component are present in stoichiometric or non-stoichiometric amounts. Complexes of this type include inclusion complexes (drug-host inclusion complexes) and cocrystals. The latter is generally defined as a crystalline complex of neutral molecular components bound together by non-covalent interactions, but can also be a complex of a neutral molecule and a salt. Cocrystals can be prepared by melt crystallization, by recrystallization from an autosolvent, or by physically grinding the components together – see O. Almarsson and MJ Zaworotko, ChemCommun, 17, 1889-1896 (2004). For a review of multicomponent complexes, see Haleblian, J PharmSci, 64 (8), 1269-1288 (August 1975).

[0053] solid form The compounds of this invention can exist in a continuous solid form ranging from completely amorphous to completely crystalline. The term "amorphous" refers to a state in which the material lacks long-range order at the molecular level and exhibits physical properties of a solid or liquid depending on temperature. Such materials typically do not produce distinctive X-ray diffraction patterns and, although exhibiting solid properties, are more formally described as liquids. Upon heating, solid properties transform into liquid properties, characterized by a state change, typically second-order ("glass transition"). The term "crystalline" refers to a solid phase in which the material has a regularly ordered internal structure at the molecular level and produces a distinctive X-ray diffraction pattern with defined peaks. Such materials will also exhibit liquid properties upon sufficient heating, but the transformation from solid to liquid is characterized by a phase transition, typically first-order ("melting point").

[0054] The compounds of this invention can also exist in a mesocrystalline state (intermediate phase or liquid crystal) under suitable conditions. The mesocrystalline state lies between a truly crystalline state and a truly liquid state (melt or solution). Mesocrystalline phenomena arising from temperature changes are described as "thermotropic," while mesocrystalline phenomena arising from the addition of a second component (such as water or another solvent) are described as "lyotropic." Compounds with the potential to form lyotropic interphases are described as "amphiphilic" and are characterized by ionicly polar head groups (such as -COO). - Na + -COO - K + or -SO3 - Na + ) or nonionic polar head groups (such as -N) - N + The molecular composition of (CH3)3). For more information, see NH Hartshorne and A. Stuart. Crystals and the Polarizing Microscope, 4th edition (Edward Arnold, 1970).

[0055] The compounds of this invention may exhibit polymorphism and / or one or more isomers (e.g., optical isomers, geometric isomers, or tautomers). The compounds of this invention may also be isotopically labeled. Such variations are implicit for the compounds of this invention as defined with reference to their structural characteristics, and are therefore within the scope of this invention. tautomer In the compounds of this invention, tautomerism (“tautomerism”) can occur when structural isomers can interconvert via a low-energy barrier. This can manifest as proton tautomerism in compounds of this invention containing, for example, imine, ketone, or oxime groups, or as so-called valence tautomerism in compounds containing aromatic moieties. Thus, a single compound can exhibit more than one type of isomerism.

[0057] It must be emphasized that, although for the sake of brevity the compounds of the present invention have been drawn in a single tautomer form herein, all possible tautomer forms are included within the scope of the present invention.

[0058] Stereoisomers Cis / trans isomers can be separated using techniques well known to those skilled in the art, such as chromatography and fractional crystallization.

[0059] Common techniques for preparing / separating individual 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 stepwise crystallization, and one or both of the diastereomeric isomers can be converted to the 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 enantiomerically enriched using chromatography (typically HPLC). The eluent is concentrated to obtain an enriched mixture. Chiral chromatography using subcritical and supercritical fluids can be employed. Methods for chiral chromatography used in some 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 references cited therein). When any racemate crystallizes, two different types of crystals may occur. The first type is the racemic compound mentioned above (true racemate), in which a homogeneous form of crystal containing equimolar amounts of two enantiomers is produced. The second type is a racemic mixture or aggregate, in which two crystalline forms, each containing a single enantiomer, are produced in equimolar amounts. Although the two crystalline forms present in a racemic mixture may have the same physical properties, their physical properties may differ from those of a true racemate. Racemic mixtures can be separated by techniques known to those skilled in the art—see, for example, E.L. Eliel and S.H. Wilen, Stereochemistry of Organic Compounds (Wiley, 1994).

[0060] isotope This invention includes all pharmaceutically acceptable isotopically labeled compounds of this invention, wherein one or more atoms are replaced by atoms having the same number of atoms, but with an atomic mass or mass number different from the atomic mass or mass number that constitutes the vast majority of atoms in nature.

[0061] Examples of suitable isotopes included in the compounds of the present invention may include isotopes of hydrogen, such as... 2 H and 3 H; carbon isotopes, such as 11 C 13 C and 14 C; isotopes of chlorine, such as36 Cl; isotopes of fluorine, such as 18 F; Iodine isotopes, such as 123 I and 125 I; Nitrogen isotopes, such as 13 N and 15 N; isotopes of oxygen, such as 15 O、 17 O and 18 O; isotopes of phosphorus, such as 32 P; and isotopes of sulfur, such as 35 S.

[0062] Certain isotopically labeled compounds of this invention (e.g., compounds doped with radioactive isotopes) are suitable for drug and / or substrate tissue distribution studies. Radioactive isotope tritium (i.e., 3 H) and carbon-14 (i.e. 14 C) It is particularly suitable for this purpose due to the ease of its incorporation and the readily available means of inspection.

[0063] Using heavier isotopes (such as deuterium, i.e.) 2 H) substitution can provide certain therapeutic advantages resulting from higher metabolic stability, such as prolonged in vivo half-life or reduced dose requirements.

[0064] Positron-emitting isotopes (such as 11 C 18 F, 15 O and 13 The N) replacement can be applied to positron emission tomography (PET) studies to examine substrate receptor occupancy.

[0065] The isotopically labeled compounds of the present invention can generally be prepared by techniques known to those skilled in the art, or by using appropriate isotopically labeled reagents instead of previously used unlabeled reagents, by methods similar to those described in the accompanying examples and preparations.

[0066] Pharmaceutically acceptable solvates according to the present invention include solvates in which the solvent for crystallization may be isotopically substituted, for example, D2O, d6-acetone, d6-DMSO.

[0067] Metabolites The scope of this invention also includes the active metabolites of the compounds of this invention, that is, compounds that are typically formed in vivo after drug administration via oxidation or dealkylation. Some examples of metabolites according to the invention include (but are not limited to): (i) In the case where the compounds of the present invention contain alkyl groups, their hydroxyalkyl derivatives (-CH ->-COH ->-CHO ->-CO2H): (ii) In the case where the compound of the present invention contains an alkoxy group, its hydroxyl derivative (-OR ->-OH).

[0068] Pharmaceutical Composition In another embodiment, the present invention comprises a pharmaceutical composition. For the purposes of pharmaceutical composition, the compound itself or a pharmaceutically acceptable salt thereof is simply referred to as the compound of the present invention.

[0069] "Pharmaceutical composition" refers to a mixture of one or more of the compounds of the present invention, or pharmaceutically acceptable salts, solvates, hydrates or prodrugs thereof, as active ingredients, and one or more pharmaceutically acceptable excipients.

[0070] The term "excipient" is used herein to describe any component other than the compounds of this invention. The selection of excipients will depend to a great extent on factors such as the mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[0071] As used herein, "excipient" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonics and absorption delay agents, carriers, diluents, etc. Examples of excipients include one or more of water, saline, phosphate-buffered saline, dextran, glycerol, ethanol, etc., and combinations thereof, and isotonics such as sugars, sodium chloride, or polyols such as mannitol or sorbitol may be included in said compositions. Examples of excipients also include various organic solvents (such as hydrates and solvates). Where necessary, pharmaceutical compositions may contain additional excipients such as flavorings, binders / adhesives, lubricants, disintegrants, sweeteners or flavoring agents, colorants or dyes, etc. For example, for oral administration, tablets containing various excipients (such as citric acid) can be used with various disintegrants (such as starch, alginate, and certain complex silicates) and binders (such as sucrose, gelatin, and acacia). Examples of excipients include (but are not limited to) calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycol. Additionally, lubricants (such as magnesium stearate, sodium lauryl sulfate, and talc) are generally suitable for tableting purposes. Similar types of solid compositions can also be used in the form of soft and hard filled gelatin capsules. Therefore, non-limiting examples of excipients also include lactose or milk candy and high molecular weight polyethylene glycol. When oral administration of aqueous suspensions or elixirs is required, the active compounds therein can be combined with various sweeteners or flavorings, colorants or dyes, and, if necessary, emulsifiers or suspending agents, as well as with other excipients (such as water, ethanol, propylene glycol, glycerin, or combinations thereof).

[0072] Examples of excipients also include pharmaceutically acceptable substances (such as humectants) or small amounts of auxiliary substances (such as humectants or emulsifiers, preservatives or buffers) that promote the shelf life or effectiveness of the compound.

[0073] The compositions of the present invention can be in various forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), dispersions or suspensions, tablets, capsules, pills, powders, and liposomes. The form depends on the intended mode of administration and therapeutic application.

[0074] Typical compositions are in the form of injectable or infusionable solutions, such as those commonly used to induce passive immunization in humans with antibodies. One mode 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.

[0075] Oral administration of solid dosage forms may be, for example, in the form of discrete units such as hard or soft capsules, pills, sachets, lozenges, or tablets, each containing a predetermined amount of at least one compound of the present invention. In another embodiment, oral administration may be in powder or granule form. In another embodiment, oral administration may be in the form of a spray-dried dispersion. In another embodiment, the oral dosage form is sublingual, such as a lozenge. In such solid dosage forms, the compound of the present invention is typically combined with one or more excipients. Such capsules or tablets may contain controlled-release formulations. In the case of capsules, tablets, and pills, the dosage form may also contain buffers or may be prepared using enteric coating.

[0076] In another embodiment, oral administration may be carried out in 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 aroma agents.

[0077] In another embodiment, the invention includes parenteral dosage forms. "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) can be formulated using suitable dispersants, wetting agents, and / or suspending agents according to known techniques.

[0078] In another embodiment, the invention includes topical dosage forms. "Topical application" includes, for example, transdermal application (such as via a transdermal patch or iontophoresis device), intraocular application, or intranasal or inhalation application. Compositions for topical application also include, for example, topical gels, sprays, ointments, and creams. Topical formulations may include compounds that enhance the absorption or penetration of the active ingredient through the skin or other affected areas. When the compounds of the invention are applied via a transdermal device, application is achieved using a reservoir and porous membrane type patch or a solid matrix type patch. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibers, bandages, and microemulsions. Liposomes may also be used. Typical excipients include alcohols, water, mineral oils, liquid paraffin, white petrolatum, glycerin, polyethylene glycol, and propylene glycol. A penetration enhancer may be added – see, for example, BC Finnin and TMMorgan, J. Pharm. Sci., Vol. 88, pp. 955-958, 1999.

[0079] 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 in the form of micronized suspensions or solutions in pH-adjusted isotonic sterile saline. Other formulations suitable for ocular and ear application include ointments, biodegradable implants (i.e., absorbable gel sponges, collagen) and non-biodegradable implants (i.e., silicone), rice paper, lenses, and microparticle or vesicle systems, such as nonionic surfactant vesicles (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., gellan gum), may be incorporated with preservatives such as benzalkonium chloride. Such formulations may also be delivered by iontophoresis.

[0080] For intranasal or inhalation administration, the compounds of the present invention are preferably delivered in solution or suspension form using a pump-jet container squeezed or pumped by a patient, or in aerosol form using a pressurized container or nebulizer with a suitable propellant. Formulations suitable for intranasal administration are typically administered in dry powder form (alone, as a mixture, such as a dry blend with lactose, or as mixed component particles, such as mixed with phospholipids (e.g., phosphatidylcholine)) using a dry powder inhaler, or in aerosol form using a pressurized container, pump, nebulizer, nebulizer (preferably using a nebulizer that generates a fine mist using electrohydrodynamics) 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.

[0081] In another embodiment, the invention includes a rectal dosage form. Such a rectal dosage form may be in the form of, for example, suppositories. Cocoa butter is a conventional suppository base, but various alternatives may be used where appropriate.

[0082] Other excipients and administration methods known in pharmaceutical technology may also be used. The pharmaceutical compositions of the present invention can be prepared using any known pharmaceutical technique, such as efficient dispensing and administration procedures. Considerations above regarding efficient dispensing and administration procedures are well known in the art and described in standard textbooks. Discussions of pharmaceutical dispensing can be found, for example, in Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing, Easton, Pennsylvania, 1975; Liberman et al., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Kibbe et al., eds., Handbook of Pharmaceutical Excipients (3rd edition), American Pharmaceutical Association, Washington, 1999.

[0083] Acceptable excipients are non-toxic to recipients at the dosage and concentration used and may include: buffers, such as phosphates, citrates and other organic acids; salts, such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzyl chloride). 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 Ig; 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 EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN. TM PLURONICS TM Or polyethylene glycol (PEG).

[0084] For oral administration, the composition may be provided to the patient in tablet or capsule form 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 for symptomatic dose adjustment. The drug typically contains about 0.01 mg to about 500 mg of active ingredient, or in another embodiment, about 1 mg to about 100 mg or 50 to 500 mg of active ingredient. In the case of intravenous administration, the dose range may be about 0.01 to about 10 mg / kg / min during a constant rate infusion.

[0085] 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. Liposomes with extended cycle times are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be produced by a reverse-phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter having a defined pore size to produce liposomes with the desired diameter.

[0086] The compounds of this invention can also be encapsulated in microcapsules, for example, prepared by coagulation techniques or interfacial polymerization, wherein the microcapsules are, for example, hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules respectively in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing (2000).

[0087] Sustained-release formulations can be used. Suitable examples of sustained-release formulations include semi-permeable matrices containing solid hydrophobic polymers of the compounds of the present invention, said matrices being in the form of molded articles, such as membranes or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactic acid lactide (US Patent No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamic acid ester, non-degradable ethylene-vinyl acetate, and degradable lactic acid-glycolic acid copolymers, such as those used in LUPRON DEPOT. TM The injectable microspheres (containing lactic acid-glycolic acid copolymer and leuprolide acetate) contain lactic acid-glycolic acid copolymer, sucrose isobutyrate acetate, and poly-D-(-)-3-hydroxybutyric acid.

[0088] Preparations intended for intravenous administration must be sterile. This is readily achieved, for example, by filtration through a sterile filter membrane. The compounds of the present invention are typically placed in containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be punctured by a hypodermic needle.

[0089] Suitable emulsions can be commercially available fat emulsions, such as Intralipid. TM Liposyn TM Infonutrol TM Lipofundin TM and Lipiphysan TM Preparation. The active ingredient may be soluble in the premixed emulsion composition, or it may be soluble in oils (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and in an emulsion formed when mixed with phospholipids (e.g., lecithin, soybean lecithin, or soybean lecithin) and water. It should be understood that other ingredients, such as glycerol or glucose, may be added to adjust the emulsion tension. Suitable emulsions typically contain up to 20% oil, for example, 5% to 20% oil. Fat emulsions may contain fat droplets of 0.1 to 1.0 μm, especially 0.1 to 0.5 μm, and have a pH of 5.5 to 8.0.

[0090] The emulsion composition can be formed by mixing the compounds of the present invention with Intralipid. TM Emulsion compositions prepared from or their components (soybean oil, lecithin, glycerin and water).

[0091] 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 achieves local or systemic effects by oral or nasal inhalation. Compositions in preferably sterile, pharmaceutically acceptable solvents may be nebulized using a gas. Nebulized solutions may be inhaled directly from a nebulizer or the nebulizer may be attached to a face mask, hood, or intermittent positive pressure ventilation machine. Solution, suspension, or powder compositions may be administered from a device that delivers the formulation in a suitable manner (preferably orally or nasally).

[0092] Application and administration As used herein, the terms “treating,” “treat,” or “treatment” encompass both preventative (i.e., preventative) and remission (i.e., reducing, alleviating, or slowing the progression of a patient’s disease (or condition) or any tissue damage associated with the disease).

[0093] As used herein, the terms "subject," "individual," or "patient" are used interchangeably to refer to any animal, including mammals. Mammals according to the invention include dogs, cats, cattle, goats, horses, sheep, pigs, rodents, rabbits, primates, humans, and the like, and include mammals in the womb. In one embodiment, humans are suitable individuals. Human individuals can be of any sex and at any developmental stage.

[0094] As used herein, the phrase “therapeutic effective amount” refers to the amount of an active compound or agent that elicits a biological or medical response sought by a researcher, veterinarian, physician, or other clinician in a tissue, system, animal, individual, or human, including one or more of the following: (1) Prevention of disease; for example, prevention of disease, symptoms or disorders in individuals who may be susceptible to disease, symptoms or disorders but have not yet experienced or shown the pathology or symptomology of the disease. (2) Suppressing disease; for example, suppressing disease, symptoms, or disorders in an individual who is experiencing or exhibiting a pathology or symptomology of disease, condition, or disorder (i.e., halting or slowing the further development of pathology and / or symptomology); and (3) Improve disease; for example, improve the disease, condition or disorder in an individual who is experiencing or showing pathology or symptomology of disease, condition or disorder (i.e., reverse pathology and / or symptomology).

[0095] Typically, the compounds of the present invention are administered in amounts effective in treating the conditions described herein. The compounds of the present invention may be administered on their own or alternatively in the form of pharmaceutically acceptable salts. For purposes of administration and delivery, the compounds themselves or their pharmaceutically acceptable salts are simply referred to as the compounds of the present invention.

[0096] The compounds of the present invention are administered by any suitable route, in the form of a pharmaceutical composition suitable for such route, and at a dose that effectively achieves the desired therapeutic effect. The compounds of the present invention can be administered orally, parenterally, topically, intranasally, or by inhalation.

[0097] The compounds of this invention can be administered orally. Oral administration may include swallowing to allow the compound to enter the gastrointestinal tract, or administration via the buccal or sublingual route, thereby allowing the compound to enter the bloodstream directly from the oral cavity.

[0098] In another embodiment, the compounds of the present invention can also be administered parenterally, for example, directly into the bloodstream, muscles, or internal organs. Suitable methods of parenteral administration include intravenous, intra-arterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, and subcutaneous administration. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and infusion techniques.

[0099] In another embodiment, the compound of the present invention can also be applied topically to the skin or mucous membranes, i.e., transdermal or transepidermal. In another embodiment, the compound of the present invention can also be administered intranasally or by inhalation. In another embodiment, the compound of the present invention can also be administered rectally or vaginally. In another embodiment, the compound of the present invention can also be administered directly to the eyes or ears.

[0100] The dosing regimens of the compounds of the present invention and / or compositions containing said compounds are based on various factors, including: patient type, age, weight, sex, and medical condition; severity of condition; route of administration; and activity of the specific compound used. Therefore, dosing regimens can vary widely. 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., mg / kg body weight of the compounds of the present invention) for the treatment of the specified conditions discussed 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, and in yet another embodiment, from about 0.5 to about 30 mg / kg. It is not uncommon for the compounds of the present invention to be repeatedly administered multiple times a day (typically not exceeding four times). Where necessary, multiple doses may be used daily to increase the total daily dose.

[0101] Treatment methods and uses The compound is an inhibitor of the N-protein of RSV and is suitable for the treatment, prevention, inhibition and improvement of RSV virus infection.

[0102] Co-application The compounds of this invention can be used alone or in combination with one or more other RSV therapeutic agents. This invention provides any of the uses, methods, or compositions as defined herein, wherein the compounds of this invention or pharmaceutically acceptable salts thereof are used in combination with one or more other known therapeutic agents to treat RSV. Such combinations can provide greater clinical benefit than any single administration. Examples of greater clinical benefit may include significant reduction in RSV symptoms, faster time to symptom relief, reduced lung lesions, greater reduction in RSV load (viral burden) in patients, and lower mortality.

[0103] The term "in combination" administration of two or more compounds means that all compounds are administered close enough in time to affect an individual's treatment. Depending on the treatment regimen, two or more compounds may be administered simultaneously or sequentially via the same or different routes of administration, with or without specific time constraints. Alternatively, simultaneous administration may be achieved by mixing the compounds before administration or by administering the compounds in separate dosage forms at the same time but at the same or different sites of application.

[0104] The phrases “parallel application,” “joint application,” “simultaneous application,” “continuous application,” and “simultaneous application” refer to the combined application of compounds.

[0105] The compounds of this invention and one or more other RSV therapeutic agents can be administered in fixed or non-fixed combinations of active ingredients. The term "fixed combination" means that the compounds of this invention or their pharmaceutically acceptable salts and one or more RSV therapeutic agents are administered simultaneously to an individual in a single composition or dose. The term "non-fixed combination" means that the compounds of this invention or their pharmaceutically acceptable salts and one or more RSV therapeutic agents are formulated into individual compositions or dosage forms such that they can be administered simultaneously or continuously to an individual in need at variable intervals, wherein such administration provides an effective amount of two or more compounds in the individual.

[0106] In one embodiment, the compound of the present invention is administered in combination with an additional RSV therapeutic agent suitable for treating RSV infection, said additional RSV therapeutic agent comprising a pharmaceutically acceptable salt of a specifically named agent and a pharmaceutically acceptable solvate of said agent and salt. Other examples of therapeutic RSV agents include F-protein inhibitors cisunatovir and ziresoxir (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 monorapir, remdesivir, obedivir, and ribavirin.

[0107] Synthesis method The compounds of this invention can be synthesized by synthetic routes (which include methods similar to those known in the field of chemistry), particularly according to the description contained herein. Starting materials are generally available from commercial sources, such as Sigma-Aldrich (St. Louis, Missouri), or readily prepared using methods well known to those skilled in the art (e.g., by methods commonly described below: Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, Volumes 1-19, Wiley, New York (1967-1999), or Beilsteins Handbuch der organischen Chemie, 4, Aufl. edition, Springer-Verlag, Berlin, including supplements (also available through the Beilstein online database)). The compounds used herein are related to or derived from compounds of significant scientific and commercial interest, and therefore many such compounds are commercially available or reported in the literature or readily prepared from other generally available materials by methods reported in that literature.

[0108] For a more detailed description of individual reaction steps, 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. While 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 according to this disclosure using conventional chemical methods well known to those skilled in the art.

[0109] Those skilled in the art will understand that the experimental conditions described in the following scheme are illustrative of conditions applicable to achieving the demonstrated transformations, and that it may be necessary or required to modify the precise conditions used to prepare the compounds of the present invention. Furthermore, it should be understood that it may be necessary or required to perform the transformations in a different order than described in the scheme, or to adjust one or more of the transformations, to obtain the desired compounds of the present invention.

[0110] Example To better understand the present invention, the following embodiments are described. These embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0111] Experimental Procedure The synthesis of various compounds of the present invention is described below. All of these intermediates and starting materials in the examples are commercially available or can be prepared by methods known in the art or as described herein.

[0112] Unless otherwise specified, all reactions were carried out under a nitrogen or argon atmosphere with continuous stirring. Where appropriate, the reaction equipment was dried under a dynamic vacuum using a hot air gun, and anhydrous solvent (Sure-Seal from Sigma-Aldrich (St. Louis, Missouri)) was used. TM The product may be from DriSolv, EMD Chemicals, Gibbstown, NJ. TM (Product). In some cases, commercially available solvents are passed through a column packed with 4Å molecular sieves until the following QC standards for water are met: a) for dichloromethane, toluene, N,N - Dimethylformamide and tetrahydrofuran, <100 ppm; b) For methanol, ethanol, 1,4-dioxane and diisopropylamine, <180 ppm. For extremely 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 are used without further purification. For synthesis following procedures in other examples or methods, reaction conditions (reaction time and temperature) may be varied. Products are typically dried under vacuum before further reactions or submission for bioassays.

[0113] If indicated, the reactants are heated by microwave irradiation using a Biotage Initiator or a Personal Chemistry EmrysOptimizer. Reaction progress is monitored using thin-layer chromatography (TLC), liquid chromatography-mass spectrometry (LCMS), high-performance liquid chromatography (HPLC), and / or gas chromatography-mass spectrometry (GCMS). TLC is performed on pre-coated silica gel plates with a fluorescent indicator (254 nm excitation wavelength) and observed under UV light and / or with staining agents such as I₂, KMnO₄, CoCl₂, phosphomolybdic acid, and / or cerium ammonium molybdate. LCMS data are 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 is analyzed using a Waters ZQ mass spectrometer in positive and negative ion modes from 100 to 1200 Da. Other similar instruments are also used. HPLC data were acquired on Agilent 1100 series instruments using specified columns, acetonitrile / water gradients, and trifluoroacetic acid or ammonium hydroxide modifiers. GCMS data were acquired using a Hewlett Packard 6890 oven equipped with an HP 6890 syringe, an HP-1 column (12 m × 0.2 mm × 0.33 µm), and helium carrier gas. Samples were analyzed using an HP 5973 mass-selective detector with scans from 50 to 550 Da of electron ionization. Purification was performed by medium-performance liquid chromatography (MPLC) using Isco CombiFlashCompanion, AnaLogix IntelliFlash 280, Biotage SP1, or Biotage Isolera One instruments and pre-packed Isco RediSep or Biotage Snap silica columns. Chiral purification is typically performed using Waters, Berger, or Thar instruments; columns such as Chiral Technologies AD-H, ChiralPAK-AD, ChiralPAK-AS, ChiralPAK-IC, Chiralcel-OD, or Chiralcel-OJ columns; and a CO2 mixture containing methanol, ethanol, 2-propanol, or acetonitrile (adjusted alone or with trifluoroacetic acid or propan-2-amine) by chiral supercritical fluid chromatography (SFC). UV detection is used to trigger fraction collection. For synthesis following procedures in other examples or methods, the purification method may vary: generally, the solvent and solvent ratio used for the eluent / gradient are selected to provide appropriate R... f Or the retention period.

[0114] Mass spectrometry data are reported from LCMS analysis. Mass spectrometry (MS) analysis is performed using atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), electron impact ionization (EI), or electron scattering (ES) ionization sources. Proton NMR spectra are presented at low field parts per million relative to tetramethylsilane. 1 Chemical shifts were recorded on 300, 400, 500, or 600 MHz Varian, Bruker, or Jeol spectrometers using 1H NMR. Chemical shifts were referenced to residual peaks of deuterated solvents (CDCl3, 7.26 ppm; CD3OD, 3.31 ppm; CD3CN, 1.94 ppm; (CD3)2SO, 2.50 ppm; D2O, 4.79 ppm) expressed in parts per million (ppm, d). Peak shapes were described as follows: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m, multiplet; br; s, broad singlet; app, apparent. Fluorine NMR (… 19 Chemical shifts (F NMR) are given in parts per million and recorded on Varian, Bruker, or Jeol spectrometers at 300, 400, 500, or 600 MHz. Analytical SFC data are typically acquired on Agilent or Berger analytical instruments as described above. Optical rotation data are acquired using a 1 dm unit on a PerkinElmer Model 343 polarimeter. Microanalysis is performed by Quantitative Technologies and is within 0.4% of the calculated values.

[0115] Unless otherwise specified, chemical reactions are carried out at room temperature (approximately 23 degrees Celsius).

[0116] Unless otherwise noted, all reactants were commercially available and used without further purification, or prepared using methods known in the literature.

[0117] The terms "concentration," "evaporation," and "concentration in vacuum" refer to the removal of solvent under reduced pressure in a rotary evaporator at a bath temperature below 60°C. The abbreviations "min" and "h" represent "minutes" and "hours," respectively. The terms "TLC" refer to thin-layer chromatography, "room temperature or ambient temperature" means a temperature between 18°C ​​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, and "HPLC" refers to high performance liquid chromatography. "SFC" refers to supercritical fluid chromatography.

[0118] Use the methods specified in the procedure to measure HPLC, UPLC, LCMS, GCMS, and SFC retention times.

[0119] The compounds and intermediates described below are named using the naming conventions provided by ChemDraw (version 20.1.1.125, PerkinElmer Informatics, Shelton, Connecticut, USA). The naming conventions provided by ChemDraw 20.1.1.125 are well known to those skilled in the art and are believed to generally conform to the recommendations of the International Union for Pure and Applied Chemistry (IUPAC) regarding the Nomenclature of Organic Chemistry and the CAS Index rules. Unless otherwise stated, all reactants were commercially available without further purification or prepared using methods known in the literature.

[0120] Example 1: 2-(4-(ethanesulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (1) Intermediate 1: 3-Amino-9-fluoro-5-phenyl-1,3-dihydro-2H-benzo[e][1,4]diaza-2-one (C1): Step 1: Synthesis of (2-amino-3-fluorophenyl)(phenyl) methyl ketone 1000 mL of 2-MeTHF (1000 V) was added to 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°C and 5°C (internal temperature at 0°C). A solution of PhMgBr in 2-MeTHF (2.8 M, 577 mL, 1616 mmol, 2.2 equivalent) was added dropwise to the reactor at -5°C and 5°C over 30 minutes under nitrogen, and exothermic reaction was observed. The reactor was adjusted to between 20°C and 30°C (internal temperature at 25°C), and the reaction mixture was stirred at this temperature for 16 hours (the reaction progress was monitored by HPLC of aliquots quenched with 6 M HCl solution). The reactor was cooled to between -5°C and 5°C, and then 735 mL of 6 M HCl (6.0 equivalent) was added dropwise (exothermic reaction was observed). The reaction mixture was heated to between 20°C and 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 V × 2). The combined organic layers were washed with water (500 mL) and set aside to be combined with another batch for further processing and purification. Following the same procedure, the same reaction was repeated on a larger scale with 2-amino-3-fluorobenzonitrile (240 g, 735 mmol, 1 equivalent) and 2-MeTHF containing PhMgBr (2.8 M, 2390 mL, 3880 mmol, 2.2 equivalents). The crude product treated in 2-MeTHF was combined with the previous batch and concentrated, kept below 40°C, and the solvent was switched to MTBE (5 V). Heptane (2.5 V) was added dropwise to the mixture, and the solid was collected by filtration and the filter cake was washed with more heptane (0.5 V). The filter cake was dried at 35–45 °C for 16 hours to give the title compound (450.4 g, 77%) as a solid. LCMS C 13 H 11 FNO + [M+H] + The calculated value is 216.08, and the measured value is 216.10.

[0121] Step 2: Synthesize 2-(1 H -benzo[ d [1,2,3]triazol-1-yl)-2-(((benzoxy)carbonyl)amino)acetic acid MeCN (500 mL, 5 V) was charged into the reactor, and the temperature was adjusted between 15°C and 25°C. Benzotriazole (100 g, 839 mmol, 1.0 equivalent), glyoxylic acid monohydrate (77.3 g, 839 mmol, 1.0 equivalent), and methyl carbamate (127 g, 839 mmol, 1.0 equivalent) were added with stirring, followed by p-toluenesulfonic acid monohydrate (TsOH•H₂O, 2.89 g, 16.8 mmol, 0.02 equivalent). After the reaction mixture became a homogeneous solution, nitrogen gas was bubbled through it for 5 minutes, and the mixture was heated to between 35°C and 45°C. After stirring at this temperature for 17 hours, the reaction mixture was heated to between 55°C and 65°C over 1 hour and stirred at this temperature for 0.5 hours. The temperature was then slowly decreased again to between 35°C and 45°C over 1 hour and stirred for 0.5 hours. The reaction mixture was further cooled to 20°C and 30°C, and the solids were collected by filtration. After HPLC analysis of the solids and filtrate, the two components were recombined and concentrated to between 1 and 2 V. The mixture was stirred in the previous reactor between 35°C and 45°C for 18 hours and cooled to between 20°C and 30°C. The precipitate was collected by filtration and further heated to dry, yielding 196.6 g of the title product as a solid (72%, 99.1% LCAP), which was used without further purification.

[0122] 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 2-(1) H -benzo[ d[1,2,3]triazol-1-yl)-2-(((benzyloxy)carbonyl)amino)acetic acid (182 g, 558 mmol, 1.2 equivalents) was charged into the reactor, followed by THF (1000 L, 10 V). (2-amino-3-fluorophenyl)(phenyl)methyl ketone (100 g, 465 mmol, 1.0 equivalents) was added, and the reactor was purged three times with nitrogen. The reaction mixture was cooled to between -35°C 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 between -35°C and -25°C for 16 hours. Water (2000 mL, 20 V) was added to the second reactor, followed by NaHCO3 (390 g, 4650 mmol, 10.0 equivalent). The reaction mixture from the first reactor was added to this NaHCO3 solution with stirring, and the temperature was maintained between 20°C and 30°C. The quenched mixture was stirred at the same temperature for 16 hours, and EtOAc (1000 mL, 20 V) was added. The separated aqueous layer was extracted with EtOAc (500 mL, 10 V), and the combined organic layers were washed with water (10 V). The organic phase was concentrated to approximately 500 mL under vacuum at below 40°C, and MeOH (5 V) was added. The mixture was further concentrated to 280 mL under vacuum at below 40°C, and MeOH (5 V) was added again. The mixture was concentrated to dryness under vacuum at below 40°C to give the title compound (243 g, quantitative, 93% LCAP) in a gel form, which was used in the next step without purification.

[0123] Step 4: Synthesis of methyl benzo(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)carbamate (1-(1) H -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 charged into a reactor and MeOH (1215 mL, 5 V) was added. The reactor was purged three times with nitrogen and cooled to between -5°C and 5°C. MeOH containing NH3 (7 M, 663 mL, 4640 mmol, 10 equivalent) was added. The reaction mixture was heated to between 20°C and 30°C and stirred for 1 hour. The reaction mixture was concentrated under reduced pressure below 40°C to give intermediate (1-amino-2-((2-benzoyl-6-fluorophenyl)amino)-2-oxoethyl)carbamate (240 g). MeOH (1200 mL, 5 V) was added and the mixture was concentrated to 2.5 V. Add additional MeOH (600 mL, 2.5 V). Adjust the temperature back to between 20°C and 30°C and add AcOH (960 mL, 4 V). Stir the reaction mixture between 20°C and 30°C for another 16 hours, then add water (240 mL, 1 V). Filter the solid and wash with a 5:1 MeOH:water mixture (1 V). Dry the filter cake between 40°C and 50°C to give the title compound as a solid (117 g, 63%, 99.6% LCAP).

[0124] Step 5: Synthesis of 3-amino-9-fluoro-5-phenyl-1,3-dihydro-2H-benzo[e][1,4]diaza-2-one (C1) (9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1-phenyl) was added to the reactor. H -benzo[ e[1,4]-Diazin-3-yl)benzoyl carbamate (117 g, 290 mmol, 1.0 equivalent) was added, followed by CH2Cl2 (936 mL, 8 V). The reactor was purged three times with nitrogen and the temperature was maintained between 15°C and 25°C. TfOH (58.0 mL, 109 g, 725 mmol, 2.5 equivalent) was added dropwise while the reaction mixture was stirred between 15°C and 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 equivalent) to a second reactor containing water (5 V). This NaHCO3 solution was added dropwise to the first reactor containing the reaction mixture, and the pH was adjusted between 7 and 8. The quenched reaction mixture was stirred between 15°C and 25°C for 2 hours. The obtained solid was filtered and washed with water (2 V) and EtOAc (0.5 V). The filter cake was dried between 35 °C and 45 °C for 16 hours to give the title compound (79.8 g, quantitative, 100% LCAP, but contaminated with trace amounts of NaOTf, based on...) in solid form. 19 F NMR). LCMS: C 15 H 13 FN3O + [M+H] + The calculated value is 270.10, and the measured value is 270.1. 1 H NMR (400 MHz, (CD3)2SO) δ7.61-7.48 (m, 4H), 7.48-7.37 (m, 2H), 7.25 (ddd, 1H), 7.10 (d, 1H), 4.32 (s,1H). 19 F NMR (376 MHz, (CD3)2SO) δ -124.16 (another small peak at -77.74 ppm indicates trace NaOTf).

[0125] The title compound C1 (CAS No. 1584714-99-7) can also be prepared using known procedures described in WO / 2004 / 026843, WO / 2005 / 090319, WO / 2017 / 015449 and WO / 2021 / 032992.

[0126] Intermediate 2: 2-(4-(ethanesulfonyl)-2-fluorophenyl)-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxylic acid (C2) Step 6: Synthesis of ethyl 5-amino-3-bromo-1H-pyrazole-4-carboxylate hydrochloride A 1.740 L, 0.13 M solution of MeCN containing 35.0 g (230 mmol, 1.0 equivalent) of ethyl 5-amino-1H-pyrazole-4-carboxylate was placed in a 3 L three-necked round-bottom flask to obtain a pale brown solution. The mixture was bubbled with nitrogen and cooled to -10 °C (internal temperature). Carefully added in portions (6 g × 8) over 30 minutes... N -Brominated succinimide (NBS, 48.2 g, 271 mmol, 1.2 equivalents) was reacted while maintaining a temperature between -10 °C and 0 °C. The reaction mixture became a pale orange homogeneous solution. The mixture was heated to room temperature and stirred at 25 °C for 1 hour. The crude reactant was cooled to -15 °C, and concentrated HCl (12 M, 18.8 mL, 226 mmol, 1.0 equivalents) was added via syringe. A grayish-white precipitate formed, and the mixture was stirred at -15 °C for 1 hour. The solid was collected by filtration and washed with low-temperature MeCN (200 mL). The grayish-white solid was dried under vacuum overnight to give a solid. This reaction was repeated three more times on the same scale, and the solids from all four batches were combined and dried in a vacuum oven at 50 °C for 19 hours to give the title compound (164.74 g, 66%) as a grayish-white solid. LCMS: C6H9 79 BrN3O2 + and C6H9 81 BrN3O2 + [M+H] + The calculated values ​​are 234.00 and 236.00, and the measured values ​​are 234.0 and 236.1. 1 HNMR (600 MHz, (CD3)2SO) δ 4.17 (q, 2H), 1.25 (t, 3H).

[0127] Step 7: Synthesis of ethyl 2-bromo-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxylate PhMe (600 mL) and 5-amino-3-bromo-1 H1,1,3,3-Tetraethoxy-2-methylpropane hydrochloride (100 g, 369.7 mmol, 1.0 equivalent) was loaded into a 3 L jacketed reactor. The reactor was flushed with additional PhMe (200 mL). Liquid form of 1,1,3,3-tetraethoxy-2-methylpropane (95.3 g, 407 mmol, 1.1 equivalent) was added, followed by additional PhMe (124 mL) to the reagent container. The reactor was set to an internal temperature of 65 °C, and the stirring was set to 300 rpm. The reaction mixture gradually became homogenized after heating. After 1 h, the stirring was increased to 350 rpm, and solids began to form on the sides of the reaction vessel. The reactor was set to 47 °C, with a cooling rate of 0.2 K / min. The precipitation rate increased when the internal temperature reached 45 °C. The reaction mixture was maintained at 45 °C for 2 h, and then cooled to 20 °C. The mixture was stirred at this temperature for 19 h, and the solids were collected by filtration. The filter cake was washed with PhMe (100 mL) and further dried by vacuum drying through a funnel. The solid was then transferred to a vacuum oven at 50°C and purged with nitrogen for four days to provide the title compound (85.3 g, 81%) as a white solid. LCMS: C 10 H 11 79 BrN3O2 + and C 10 H 11 81 BrN3O2 + [M+H] + The calculated values ​​are 284.00 and 286.00, while the measured values ​​are 284.2 and 286.1. 1 H NMR (400 MHz, (CD3)2SO) δ 9.10 (dd, 1H), 8.76 (d, 1H), 4.32 (q, 2H), 2.37 (s, 3H), 1.32 (t, 3H).

[0128] Step 8: Synthesis of 1-bromo-4-(ethanesulfonyl)-2-fluorobenzene 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 probe. Then, 1-bromo-2-fluoro-4-iodobenzene (150 g, 498.5 mmol, 1.0 equivalent) and (2... S 4 R )- NA solution of 2,6-dimethylphenyl)-4-hydroxypyrrolidine-2-carboxamide (5.84 g, 24.9 mmol, 0.05 equivalents) in DMSO (1.66 L, 0.3 M) was prepared. 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 (1 L) of concentrated NH4OH: saturated NH4Cl while maintaining the internal temperature below 30 °C. A solid precipitated during quenching. The solid was filtered off 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 obtain 1-bromo-4-(ethanesulfonyl)-2-fluorobenzene (128.4 g, 96%) as a grayish-white solid. This substance was used as is in the following reactions without further purification. GCMS: C8H8 79 BrFO2S + and C8H8 81 BrFO2S + [M] + The calculated values ​​are 265.94 and 267.94. The measured values ​​are 265.9 and 267.9. 1 H 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).

[0129] Step 9: Synthesis of 2-(4-(ethanesulfonyl)-2-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane Add 1-bromo-4-(ethanesulfonyl)-2-fluorobenzene (128.4 g, 480 mmol, 1.0 equivalent), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-di(1,3,2-dioxaborane) (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) to a three-necked round-bottom flask equipped with a condenser, a top stirrer, and an internal temperature probe. Bubble the reactants under nitrogen for 15 minutes, then add Pd(dppf)Cl2•CH2Cl2 (13.4 g, 16.4 mmol, 0.025 equivalent). Bubble the reactants again under nitrogen for 15 minutes, then heat 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 0.5 L MTBE (0.5 L). The combined organic layers were concentrated under vacuum. The residue was dissolved in EtOAc (900 mL) and filtrated with Silicia. MetS ® Treat with 60 g of Thiol metal remover, then heat to reflux for 1.5 hours. Filter the slurry. Add Silicia to the filtrate. MetS ® Thiol metal scavenging agent (60 g) was added and the mixture was reheated to reflux for 1.5 hours. After filtration, the filtrate was treated with DARCO activated carbon (55 g) and then passed through Celite. ® The solid was removed by filtration. 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 obtain a homogeneous solution. The solution was slowly cooled to room temperature, at which point the solid precipitated from the solution. The solid was filtered off and dried under vacuum at 40 °C for 16 hours to obtain a grayish-white solid of 2-(4-(ethanesulfonyl)-2-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane (126 g, 95.6% efficiency, adjusted yield 120.5 g, 80%). LCMS: [C 14 H 21 BFO4S] + [M+H] + The calculated value is 315.2, but this only applies to the [C8H] observed for boric acid. 11 BFO4S] + [M+H] + The calculated value is 233.04, and the measured value is 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).

[0130] Step 10: Synthesis of ethyl 2-(4-(ethanesulfonyl)-2-fluorophenyl)-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxylate A mixture of 2-bromo-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxylate (29.9 g, 105 mmol, 1.0 equivalent), K₂CO₃ (43.6 g, 316 mmol, 3.0 equivalent), 2-(4-(ethanesulfonyl)-2-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane (45.0 g, 137 mmol, 96% efficiency, 1.3 equivalent), isopropanol (300 mL), and deionized water (60 mL) bubbled under nitrogen was added to a flask equipped with a top stirrer. The flask was purged with nitrogen, and then XPhos Pd G₃ (4.69 g, 5.26 mmol, 0.05 equivalent) was added. The flask was purged again with nitrogen, and the mixture was stirred at 75°C (internal temperature) for 2.5 hours. While still hot, add 240 mL of deionized water to the reaction mixture, and a solid precipitate is observed. Slowly cool the two-phase mixture to room temperature. Filter the mixture to separate the grayish-brown solid. Wash the solid with 1:1 iPrOH:water (100 mL). Transfer the solid to a flask and add SiliCycle Silia. MetS ® Thiol 40-63 µm (loading: 1.40 mmol / g) (20 g). MEK (300 mL) was added and the mixture was stirred at 75 °C for 16 hours. The mixture was filtered and the filter cake was washed once with CH2Cl2. Activated carbon (10 g) was added to the filtrate, and the mixture was stirred at 40 °C for 1 hour. The mixture was filtered and the filtrate was concentrated under vacuum to give a yellow solid. The solid was slurried in EtOH (150 mL) for 1 hour. The mixture was filtered and the filter cake was washed once with MTBE to give ethyl 2-(4-(ethanesulfonyl)-2-fluorophenyl)-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxylate (36.5 g, 89%) as a dark green solid. LCMS: C 18 H 19 FN3O4S + [M+H] +The calculated value is 392.11, and the measured value is 392.3. 1 H NMR (600 MHz, (CD3)2SO) δ 9.23-9.19 (m, 1H), 8.82 (d,1H), 7.91-7.84 (m, 3H), 4.17 (q, 2H), 3.46 (q, 2H), 2.41 (s, 3H), 1.16 (t,3H), 1.11 (t,3H).

[0131] Step 11: Synthesis of 2-(4-(ethanesulfonyl)-2-fluorophenyl)-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxylic acid (C2). To a flask equipped with a top stirrer, add ethyl 2-(4-(ethanesulfonyl)-2-fluorophenyl)-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxylate (36.4 g, 88.0 mmol, 1.0 equivalent), NaOH (8.85 g, 221 mmol, 2.5 equivalent), deionized water (111 mL), and EtOH (200 mL). Stir the mixture at room temperature for 17 hours. Raise the temperature to 45°C and stir the mixture for another 3 hours. Concentrate the solution under vacuum until a solid begins to precipitate. Adjust the pH to 2 with 3 M HCl aqueous solution, which causes a white solid to precipitate. Filter the mixture and wash the white solid once with water. Suspend the solid in MEK (150 mL) and stir for 1 hour, then filter. The filter cake was vacuum dried at 50°C for 16 hours to obtain 31 g (97%) of 2-(4-(ethanesulfonyl)-2-fluorophenyl)-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxylic acid as a white solid. LCMS: C 16 H 15 FN3O4S + [M+H] + The calculated value is 364.08, and the measured value is 364.3. 1 H NMR (600 MHz, (CD3)2SO) δ12.42 (s, 1H), 9.17 (dd, 1H), 8.77 (d, 1H), 7.90-7.82 (m, 3H), 3.45 (q, 2H), 2.40 (d, 3H), 1.17 (t, 3H).

[0132] Example 1: 2-(4-(ethanesulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (1) In a 100 mL round-bottom flask, 2-hydroxypyridine-N-oxide (HOPO, 749 mg, 6.60 mmol, 1.2 equivalents) was added to an amber solution of 2-(4-(ethanesulfonyl)-2-fluorophenyl)-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxylic acid (2000.0 mg, 5.50 mmol, 1.0 equivalent) in DMSO (22.0 mL, 11 V, 0.25 M). The reaction mixture formed a pale pink, slightly turbid solution. N -(3-Dimethylaminopropyl)- N ′-Ethylcarbodiimide hydrochloride (EDCI•HCl, 1560 mg, 8.15 mmol, 1.48 equivalents) was added and the resulting pale yellow solution was stirred at room temperature for 30 minutes. LCMS indicated complete depletion of formic acid. 3-Amino-9-fluoro-5-phenyl-1,3-dihydro-2-ethylcarbodiimide hydrochloride was added in a single batch. H -benzo[ e [1,4]diazine-2-one (1530 mg, 5.50 mmol, 1.0 equivalent). The reaction mixture was stirred at room temperature for 1 hour, during which time the mixture changed from a suspension to a clear solution. LCMS showed almost complete conversion to the desired product. The reaction was quenched with water (34 mL, 17 V) for 10 minutes via a separate funnel at 0 °C, and the resulting free-flowing slurry was warmed to room temperature for 0.5 h. The precipitate was collected by filtration and washed with water (30 mL × 5 for reaction flasks and 3 × 30 mL × 3 for filter cake). The filter cake was dried under vacuum under nitrogen overnight to give the title compound (3252.0 mg, 96%) as a solid. LCMS: C 31 H 25 F2N6O4S + [M+H] + The calculated value is 615.16, and the measured value is 615.4. 1H NMR(600 MHz, (CD3)2SO) δ 10.97 (s, 1H), 9.55 (d,1H), 9.32 (dd, 1H), 8.96 (d, 1H), 7.85-7.76 (m, 3H), 7.59 (ddd, 1H), 7.55-7.48 (m, 3H), 7.47-7.43 (m, 2H), 7.31 (ddd, 1H), 7.15 (d, 1H), 5.52 (d, 1H), 3.41 (q, 2H), 2.46 (s, 3H), 1.14 (t, 3H). 19 F NMR (564 MHz, (CD3)2SO) δ -109.69, -123.37.

[0133] Examples 2 and 3: (S)-2-(4-(ethanesulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazon-3-yl)-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (2) and (R)-2-(4-(ethanesulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazon-3-yl)-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (3) 2-(4-(ethanesulfonyl)-2-fluorophenyl)- N -(9-Fluoro-2-oxo-5-phenyl-2,3-dihydro-1 H -benzo[ e [1,4]diazan-3-yl)-6-methylpyrazolo[1,5- aTwo enantiomers of pyrimidine-3-carboxamide (600 mg, 0.976 mmol) were separated by a preparative chiral SFC (instrument: Waters Prep 80; column: Regis Whelk-O (S,S) 250 mm × 30.0 mm 5 μm; mobile phase: 60% CO2 / 40% (1:1 MeCN:MeOH); flow rate: 80 mL / min; temperature: 40 °C; back pressure: 120 bar). The chiral purity of the isomers was assessed by an analytical chiral SFC (instrument: Agilent 1260 SFC-MS; column: Regis Whelk-O (S,S) 250 mm × 4.6 mm 5 μm; mobile phase: CO2 / (1:1 MeCN:MeOH), 95:5 to 0:100; flow rate: 3.00 mL / min; back pressure: 100 bar; detection at 210 nm). Title compound ( S )-2-(4-(ethanesulfonyl)-2-fluorophenyl)- N -(9-Fluoro-2-oxo-5-phenyl-2,3-dihydro-1 H -benzo[ e [1,4]diazan-3-yl)-6-methylpyrazolo[1,5- a Pyrimidine-3-carboxamide (289.3 mg, 48%, 100% ee, retention time 5.14 min) was separated in solid form. LCMS C 31 H 25 F2N6O4S + [M+H] + The calculated value is 615.16, and the measured value is 615.6. 1 H NMR (400MHz, (CD3)2SO) δ 10.97 (s, 1H), 9.56 (d, 1H), 9.33-9.30 (m, 1H), 8.95 (d, 1H),7.85-7.77 (m, 3H), 7.59 (dd, 1H), 7.55-7.48 (m, 3H), 7.48-7.41 (m, 2H), 7.31(ddd, 1H), 7.15 (d, 1H), 5.53 (d, 1H), 3.41 (q, 2H), 2.46 (s, 3H), 1.14 (t,3H). 19 F NMR (564 MHz, (CD3)2SO) δ -109.70, -123.38. Another ( R )-2-(4-(ethanesulfonyl)-2-fluorophenyl)- N -(9-Fluoro-2-oxo-5-phenyl-2,3-dihydro-1H -benzo[ e [1,4]diazan-3-yl)-6-methylpyrazolo[1,5- a Pyrimidine-3-carboxamide (290 mg, 48%, 95.5% ee, retention time 5.29 min) was also separated in solid form. R Enantiomer data: LCMS C 31 H 25 F2N6O4S + [M+H] + The calculated value is 615.16, and the measured value is 615.5. 1 H NMR (600 MHz, (CD3)2SO) δ 10.97 (s, 1H), 9.55 (d, 1H), 9.32 (s, 1H), 8.96(d, 1H), 7.91-7.71 (m, 3H), 7.59 (dd, 1H), 7.56-7.48 (m, 3H), 7.48-7.41 (m,2H), 7.31 (ddd, 1H), 7.15 (d, 1H), 5.53 (d, 1H), 3.41 (q, 2H), 2.46 (s, 3H), 1.14 (t, 3H). 19 F NMR (564 MHz, (CD3)2SO) δ -109.71, -123.38.

[0134] Example 4: ( S )-2-(4-(ethanesulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6-(hydroxymethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide Intermediate 3: Ethyl 2-bromo-6-(hydroxymethyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate (C3) Step 12: Synthesis of ethyl 2-bromo-6-carboxypyrazolo[1,5-a]pyrimidine-3-carboxylate Add 5-amino-3-bromo-1-methylpropane to a solution of methanetriformaldehyde (0.579 g, 0.578 mmol, 1.03 equivalents) in ethanol (EtOH, 20.4 mL). H1,52 g (5.62 mmol, 1.0 equivalent) of pyrazole-4-carboxylate and acetic acid (HOAc, 7.60 mL) were used. The reaction mixture was heated to 70 °C for 1 hour, after which LCMS indicated complete depletion of the starting material. The reaction mixture was cooled to room temperature and concentrated to give a residue. The residue was wet-milled with methyl tert-butyl ether (MTBE, 10 mL) for 16 hours. The resulting suspension was filtered and the filter cake was dried to give 1.59 g (95%) of 2-bromo-6-carboxypyrazolo[1,5-a]pyrimidine-3-carboxylate as a white solid. The solid was used in the next reaction without further purification. LCMS: C 10 H8Br 79 N3O3+ and C 10 H8Br 81 N3O3 + [M+H] + The calculated values ​​are 297.99 and 299.98, and the measured values ​​are 297.9 and 299.8. 1 H NMR (400MHz, CDCl3) δ 10.11 (s, 1H), 9.20 (d, 1H), 9.10 (d, 1H), 4.50 (d, 2H), 1.46 (t, 3H).

[0135] Step 13: Synthesis of ethyl 2-bromo-6-(hydroxymethyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate Sodium borohydride (NaBH4, 0.014 g, 0.369 mmol, 1.1 equivalents) was added in a single stirring at 0 °C to a stirred solution of ethyl 2-bromo-6-carboxypyrazolo[1,5-a]pyrimidine-3-carboxylate (0.100 g, 0.334 mmol) and CeCl3•7H2O (0.137 g, 0.369 mmol, 1.1 equivalents) in a 1:1 methanol:dichloromethane (MeOH:DCM, 1.4 mL, 0.20 M). The reaction mixture was stirred at 0 °C for 30 min, after which LCMS indicated complete depletion of the starting material. The reaction mixture was quenched with 10 drops of 1M HCl, followed by quenching with water (10 mL). The resulting mixture was concentrated to give a residue. 10 mL of water and 10 mL of ethyl acetate were added to the residue. The layers were separated, and the organic layer was concentrated to give a crude intermediate as a yellow solid, which was used in the next reaction without further purification.

[0136] 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 0.074 g, 0.33 mmol, 0.98 equivalents) was added to a stirred solution of the crude intermediate from the previous step in dioxane (3.6 mL, 0.09 M) at room temperature. The reaction mixture was stirred for 5 min, after which TLC (1:1 petroleum ether: ethyl acetate) indicated complete depletion of the crude starting material and formation of the product. The reaction mixture was quenched with 1 mL of saturated sodium bisulfite solution and extracted with ethyl acetate (10 mL). The organic layer was separated and evaporated under reduced pressure to give the crude product. The residue was purified by silica gel column chromatography to give ethyl 2-bromo-6-carboxypyrazolo[1,5-a]pyrimidine-3-carboxylate (0.038 g, 35%) as a white solid. LCMS: C 10 H 11 Br 79 N3O3+ and C 10 H 11 Br 81 N3O3 + [M+H] + The calculated values ​​are 300.00 and 302.00, and the measured values ​​are 300.0 and 302.0. 1 H NMR (400 MHz, (CD3)2OD) δ 9.17-8.99 (m, 1H), 8.85 (d, 1H), 5.60 (t, 1H), 4.63 (d, 2H), 4.33(q, 2H), 1.32 (t, 3H).

[0137] Example 4: ( S )-2-(4-(ethanesulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6-(hydroxymethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (4) The title compound was synthesized using intermediate 3 as the starting material, following a procedure similar to that in Examples 2 and 3.

[0138] LCMS: C 31 H 25 F2N6O5S + [M+H] + The calculated value is 631.16, and the measured value is 631.2. 1H NMR (400 MHz, (CD3)2DO) δ 10.96 (brs, 1H), 9.57 (d, 1H), 9.40 - 9.20 (m,1H), 9.02 (d, 1H),7.93-7.71 (m, 3H), 7.59 (ddd, 1H), 7.55-7.42 (m, 5H), 7.31 (td, 1H), 7.15 (d,1H), 5.67 (t, 1H), 5.53 (d, 1H), 4.72 (d, 2H), 3.42 (q, 2H), 1.14 (t, 3H). 19 FNMR (376 MHz, (CD3)2DO) δ -109.69, -123.37.

[0139] Predictive deuterated analog (PDA) of Example 2 The compounds provided in Table 2 are predictive deuterated analogs (PDAs) of Example 2. Formula (D) is the general formula for deuterated Example 2, wherein Y 1a Y 1b Y 1c Y 2 Y 3 Y 4 Y 5 Y 6a Y 6b Y 6c Y 7 Y 8 Y 9 and Y 10 Each is independently H or D. The deuterated analogues of Example 2 in Table 2 were predicted using MetaSite (moldiscovery.com / software / metasite / ) based on the metabolomic profile of Example 2. Based on MetaSite predictions, Y... 1a Y 1b Y 1c Y 2 Y 3 Y 4 Y 5 Y 6a Y 6b Y 6c Y 7 Y 8 Y 9 and Y 10 The most likely location is where it will be metabolized.

[0140] Table 2 Examples D-1 to D-20 in Table 2 may provide certain therapeutic advantages resulting from greater 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.

[0141] Those skilled in the art can use the Y provided in Table 2 1 -Y 10 Different combinations were used to prepare additional deuterated analogs of Example 2. These additional deuterated analogs can provide similar therapeutic advantages that can be achieved by deuterated analogs.

[0142] Evaluation of antiviral activity against RSV Analysis of plaque reduction: RSV plaque reduction analysis is an infectiousness analysis that quantifies the number of infectious units in well-defined lesions of RSV infection. Since each plaque originates from a single infectious viral particle, accurate calculations of antiviral activity can be obtained by counting plaques in and out of the presence of antiviral compounds. HEp-2 cells (ATCC, CCL23) were passaged in culture flasks and seeded into 24-well plates in DMEM containing antibiotics and supplemented with 10% FBS. Cells were cultured in DMEM containing 2% FBS during seeding and subsequent culture. 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 HEp-2 cell monolayer. The plates were incubated at 37°C in a humidified 5% CO2 incubator for 2 hours. The inoculum was then removed, and 1 ml of covering material (4% CMC in 2% DMEM) was added to each well. Cells were cultured at 37°C in a humidified 5% CO2 incubator for 48 hours, and then fixed with 75% acetone-25% methanol solution. The plates were washed under running water, and blocking solution (PBS-Tween containing 2% skim milk powder) was added to each well. The plates were then incubated on a shaker at 37°C for 1 hour. The blocking solution was removed, and 200 μl / well of blocking solution containing primary antibody (anti-RSV polyclonal antibody) was added. The plates were then incubated at 37°C for 90 minutes. The plates were washed twice under running water, and then 200 μl / well of blocking solution containing secondary antibody (rabbit anti-goat HRP conjugate) was added. The plates were then incubated at 37°C for 1 hour. The plates were washed twice under running water, and then 200 μl / well of immunostaining reagent was added at 37°C for 10 minutes. The immunostaining reagent was then removed, the plates were washed twice under running water and air-dried, and then scanned on a CTL BioSpot® S6 Macro analyzer. Infection percentage (%) was calculated using plaque count, relative to the average plaque count in the RSV control wells. The average plaque count from replicate wells of each compound dilution was plotted to construct dose-response curves, and EC50 was obtained. 50 and EC 90 Value. EC 90 The values ​​are provided in Table 1.

[0143] Analysis of human hepatocyte clearance rate - Di, L. et al. Eur. J. Med. Chem 2012, 57, 441- 448. High-throughput stability analysis of human hepatocytes was performed in 384-well configurations. Ten donor-mixed, cryopreserved human hepatocytes were purchased from Celsis IVT (Baltimore, MD). The cryopreserved hepatocytes were thawed and resuspended in Williams E medium (WEM GIBCO-BRL, catalog number C1984, custom formulation number 91-5233EC) supplemented with HEPES and Na2CO3. Cell counts were performed using the Trypan Blue exclusion assay. Multidrop was used. ® A liquid dispenser (Multidrop DW, Thermo Scientific, Waltham, MA) was used to add hepatocyte suspension to 384-well discs. The discs were then capped and transferred to a Sciclone device equipped with two 6-position Mecour heat exchangers. ® ALH 3000 workstation (Caliper Life Sciences, Hopkinton, MA). The test compound will be used in Sciclone. ® The solution was diluted with buffer and added to hepatocytes. The final culture contained 500,000 cells / mL and 1 μM of the test compound, with a total volume of 15 μL and 0.1% DMSO. Cultures were performed at 37°C. The reaction mixture was quenched with acetonitrile containing an internal standard (IS, CP-628374) at low temperature at different time points (0, 3, 10, 30, 60, 120 min). The sample was centrifuged at 3000 rpm (Eppendorf, Hauppauge, NY) for 10 min at 4°C. BioMek was used for further analysis. ® The supernatant was transferred to a new tray using a liquid processor (Beckman Coulter, Danvers MA), which was then sealed before LC-MS / MS analysis. Detailed LC-MS / MS analytical conditions have been previously described (Di, L. et al.). J. Pharm. Sci. 2011, 100 , 4974-4985). Propranolol (2D6, 1A2 and 2C19 substrates), midazolam (CYP3A4) and triazolam (CYP3A4, low clearance) and naloxone (UGT2B7) were used as positive controls.

[0144] Using a relay method with human liver cells - Di, L. et al., Drug Metabolism and Disposition , 2012, 40, 1860-1865. Ten donor-mixed, cryopreserved human hepatocytes were purchased from Celsis IVT (Baltimore, MD). This batch of mixed hepatocytes was used in all studies. When selecting new batches of hepatocytes, enzyme activity was verified using a labeling compound. After thawing, the hepatocytes were resuspended in Williams medium E (custom formulation 91-5233EC; Invitrogen, Grand Island, NY) supplemented with HEPES and Na2CO3. Cells were counted using the trypan blue exclusion method, and a compound at a final concentration of 1 μM (dimethyl sulfoxide, final concentration 0.025%; methanol, final concentration 0.125%) was incorporated into 24-well hepatocyte discs containing 500,000 cells / ml, for a final culture volume of 0.50 ml. The culture dish was covered with a Breathe-Easy membrane (Diversified Biotech, Dedham, MA) and incubated for 4 hours at 150 rpm in a humidified incubator at 37°C, 95% O2 / 5% CO2, and 75% relative humidity. At times 0 and 4 hours, 25 μl of hepatocyte suspension was removed from the culture and added to 50 μl of ice-cold acetonitrile containing an internal standard to quench the reaction. The sample was centrifuged at 3000 rpm (Eppendorf, Hauppauge, NY) for 10 minutes at 4°C, and 50 μl of the supernatant was transferred to a clean dish, completely dried and reconstituted, and then analyzed by liquid chromatography / mass spectrometry (LC-MS / MS). The remaining hepatocyte suspension in the culture dish was centrifuged (3000 rpm, 10 minutes, 4°C). 300 μl of the supernatant was transferred to a clean 24-well dish and stored at -80°C until the next follow-up experiment. For the second relay experiment, the supernatant dish was heated to 37°C for 20 minutes, and hepatocytes were added to the sample to obtain a final cell density of 500,000 cells / ml. The dish was incubated at 37°C for 4 hours, and samples were taken and processed as described above. Five relay experiments were performed to reach a total incubation time of 20 hours. If more relay experiments were required for compounds with extremely low clearance rates, the supernatant from the last relay experiment was retained. A standard curve was prepared under the same conditions.

[0145] LC-MS / MS quantification:The LC mobile phases were as follows: (A) HPLC-grade water containing 0.1% formic acid, and (B) acetonitrile containing 0.1% formic acid. Compounds were eluted from a column (Kinetex C18, 30 × 2 mm, 2.6 μm; Phenomenex, Torrance, CA) at a flow rate of 0.4 ml / min using a solvent gradient from 5% (A) to 95% (B) over 2.0 min. Cycle times were 3 min per injection. 5 μl aliquots of sample were injected for analysis using a CTC PAL autosampler (LEAP Technology, Carrboro, NC). Full scan mode from m / z 150 to 600 was used 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 cation multiple reaction monitoring mode. All tested compounds exhibited good linearity, with R0... 2 >0.99, and the quantitative limit for all compounds is 1 nM.

[0146] The intrinsic clearance rate in the human body was calculated using the method previously described in the references above.

[0147] Screening of recombinant human aldehyde oxidase (rHAO) using recombinant human aldehyde oxidase (AOX) - Cronin, C. et al. J. Protein Expression and Purification , 2021, 177. At 37°C, recombinant human AOX was diluted to approximately 50 mU / mL with 100 mM KH₂PO₄-KOH buffer (pH 7.4) containing 3 mM MgCl₂. -1 1. The AOX concentrations used in the analysis were normalized to the AOX activity against zoniporide to maintain database consistency. Reactions in 384-well pans were initiated by mixing 40 μL of diluted AOX with 21 nL of 10 mM unknown or positive substrate control using an acoustic liquid dispenser (ATS, EDCBiosystems). After mixing, 3 μL aliquots were removed at different times (2, 5, 10, 15, 30, 45, 60, and 120 minutes) and added to 35 μL of acetonitrile. After quenching and centrifugation at 1200×g for 10 minutes, the supernatant was transferred to an injection pan, mixed with 3 times its volume of water, and then analyzed by LC / MS.

[0148] The LC / MS system consisted of two Shimadzu pumps, an ADDA dual-arm autosampler (ApricotDesigns), two 1.5 × 5 mm polymethacrylate columns (11-03455-DB; Optimize Technologies), and a Sciex QTRAP 5500 mass spectrometer (AB Sciex) equipped with an electrospray ionization source. The capture and elution buffers consisted of mobile phase A (2.5% methanol / 2.5% acetonitrile / 95% 2 mM ammonium acetate) and mobile phase B (45% methanol / 45% acetonitrile / 10% 2 mM ammonium acetate), diluted at 1 mL / min. -1 The flow rate was controlled. Initially, the column was desalted and equilibrated at 100% A for 8 seconds, then switched to 100% B for 16 seconds to elute the analyte. A dual-arm autosampler dispensed 12 injections of 14 μL each over a 3.5-minute run. The analyte to internal standard peak area ratio was calculated using Sound Review v1.1 (Sound Analytics) and imported into an IDBS E-Workbook Suite v9.2.0 (IDBS) template for final calculations. A graph showing the percentage of the natural logarithm of the remaining parent compound relative to time was used to determine the first-order rate constant (k) and t0. ½ Value. Use t according to the following formula. ½ The control score (FOC) value is calculated, where the control AOX substrate is zopoietin: FOC = t ½ Zopolide / t ½ Unknown object. For t ½ For compounds with a melting point >120 min (analytical limit), the FOC value is reported as FOC < 0.15 (i.e., FOC of zopolide). The results are provided in Table 1.

[0149] In vitro bone marrow analysis - Chen, W. et al., J. Pharmacokinetics and Pharmacodynamics, 2020, 47, 163-182. Primary human bone marrow mononuclear cells (Lonza) were cultured in stemline 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 cultures were maintained in an incubator at 37°C, 5% CO2, and 98% humidity (see references for further details). Cells were pre-cultured for 1 day and then exposed to DMSO or a compound for 5 days. Cell counts were manually determined using a hemocytometer in 10 μL aliquots, and the total cell count was converted by adjusting the volume of cell culture medium in each well. Although the total cell count includes bone marrow, erythrocyte, and megakaryocyte lines, neutrophil precursors constitute the majority of the total cell population due to the stimulation conditions of cell culture. Therefore, the effect of reducing the total cell count was used as a measure 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). The results are presented in Table 1.

[0150] Comparison of metabolic stability, toxicity and antiviral activity Table 1 provides biodata for the compounds of Example 2 and comparative compounds 1-3. The comparative compounds are compounds of Examples 3 and 34 of WO2021032992 and compounds of Example 23 of WO2022180397, and can be prepared as described therein.

[0151] Table 1

[0152] The apparent intrinsic clearance rate (CL) generated in human hepatocyte culture under relay conditions for Example 2 int,app )value High apparent intrinsic clearance (CL) measured in in vitro metabolic stability analysis using liver microsomes and / or hepatocytes int,app This refers to the undesirable characteristics of novel chemical entities in drug discovery processes, exhibiting suboptimal pharmacokinetics, characterized by plasma clearance values ​​close to hepatic blood flow and elimination half-life (t). 1 / 2 The short duration of action and poor oral bioavailability due to extensive first-pass metabolism in the gastrointestinal tract and liver. Ultimately, CL... int,app This would result in an unacceptable total dosing regimen for the pharmacological effects. Comparative compound 1 showed low metabolic CL after being cultured in cryopreserved human hepatocytes. int,app It is primarily produced via single oxidation at the C5 position of the fused pyrazolopyrimidine ring. The enzyme responsible for this biotransformation reaction has been identified as cytosolic aldehyde oxidase (AO). Those skilled in the art can identify structural motifs that may be susceptible to AO oxidation (e.g., electron-deficient imine substituents that are part of a heteroaromatic ring system (such as a pyridine or pyrimidine ring, etc.). For example, Comparative Compound 2 contains an unsubstituted C5 position on its pyrazolopyrimidine ring, and it is predicted that it can be metabolized by AO in a manner similar to that described in the case of Comparative Compound 1. Metabolic instability was observed after culturing Comparative Compound 2 in rHAO, where t 1 / 2 The values ​​and FOC are not significantly different from those mentioned in the case of Comparative Compound 1. The structure of the C5 oxidative metabolite of Comparative Compound 1 was also clearly confirmed by comparing its analytical properties with those of the chemically synthesized standard.

[0153] Novel pyrazolopyrimidine derivatives were screened for AO metabolic predisposition in human hepatocytes and rHAO cultures. Unexpectedly, the presence of a C6-methyl substituent on the pyrazolopyrimidine ring in Comparative Compound 1 resulted in a compound (Comparative Compound 3), which showed stable AO metabolism in rHAO analysis (t...). 1 / 2 >240 minutes). Similar structure-metabolism findings were also noted after comparing the AO metabolic potential of Comparative Compound 2 and Example 2. The sensitivity of Comparative Compound 2 to AO metabolism in rHAO culture disappeared in Example 2 (Comparative Compound 2 rHAO t 1 / 2 = 34.4 minutes, Example 2 rHAO t 1 / 2 >240 minutes). Therefore, based on these observations, it appears that placing a single methyl group near a site in the pyrazolopyrimidine skeleton that is readily metabolized by AO resolves the AO metabolic predisposition issue. In the case of Example 2, the structural change also caused metabolic CL in human hepatocytes. int,app Significantly reduced.

[0154] As mentioned above, given the role of metabolic clearance in controlling t 1 / 2And its effects on oral absorption are important considerations for optimization as a potential clinical candidate. As shown in Table 1, eliminating the AO metabolic tendency noted for Comparatives 2 and 1 and Example 2 also resulted in CL in human hepatocyte cultures at normal culture time (approximately 2 hours). int,app Significant reduction (<4 mL / min / 10) 6 ), and subsequently obtained CL in human hepatocyte culture under relay conditions (approximately 20 hours of culture). int,app The estimated value (0.38 mL / min / 10) 6 Furthermore, as in Example 2, the incorporation of C6-methyl and ethyl sulfone substitution resulted in a significantly lower molecule scavenging rate compared to that of Comparative Compound 1 or Comparative Compound 2. int,app The value predicted by SAR.

[0155] The inclusion of the ethyl sulfone portion also enables the RSV EC of compounds such as those in Example 2 of this invention. 90 Enhanced potency (compared to Comparatives 3 or 1). The toxicity resulting from the incorporation of the ethyl sulfone moiety was improved, as assessed in in vitro human bone marrow analysis. In vitro bone marrow analysis was used to assess the effect of the compound on bone marrow progenitor cell proliferation, with the effect on the reduction of total cell count used as a measure of antiproliferative activity. In the clinical trials of palbociclib reported in the aforementioned references, in vitro results from bone marrow toxicity analysis showed predictability for neutropenia. In Table 1, Examples 2 and Comparatives 2 showed unexpectedly good human bone marrow toxicity IC50 compared to Comparatives 3 and 1. 50 This indicates that the safety of ethyl sulfone analogs has been improved.

[0156] Overall, the specific combination of C6-methyl and ethyl sulfone substitution unexpectedly provides a combination of improved properties: (1) improved stability against oxidation by human aldehyde oxidase; (2) significantly improved metabolic stability as assessed in human hepatocytes; (3) significantly enhanced potency against RSV N-protein; and (4) improved safety as assessed in bone marrow toxicity analysis. Example 2 is unique in this series of compounds compared to RV299 in that it possesses this combination of improvements. The effects of these changes result in an improved antiviral agent with unexpectedly low predicted human dose, improved safety, and unexpectedly improved metabolic stability, which translates into a longer pharmacokinetic half-life compared to Comparatives 1, 2, and 3.

[0157] Throughout this application, references are made to various publications. For all purposes, the disclosures of these publications are incorporated herein by reference in their entirety.

[0158] It will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. Other embodiments of the invention will become apparent to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This specification and examples are intended to be illustrative only, wherein the true scope and spirit of the invention are indicated by the following claims.

Claims

1. Compounds of formula (A) Where R 1 Choose from the group consisting of -CH3, -CD3, -CH2OH and -OCH3; or pharmaceutically acceptable salts thereof.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 1 Choose the group consisting of -CH3, -CD3, and -CH2OH.

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

4. The compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein the compound is an S enantiomer.

5. Compound 2-(4-(ethanesulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide or a pharmaceutically acceptable salt thereof.

6. The compound (S)-2-(4-(ethanesulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide or a pharmaceutically acceptable salt thereof.

7. The compound of claim 6, wherein it is (S)-2-(4-(ethanesulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide.

8. A pharmaceutically acceptable salt of (S)-2-(4-(ethanesulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide.

9. A pharmaceutical composition comprising the compound of any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

10. A pharmaceutical composition comprising the compound as described in any one of claims 1 to 7, and one or more pharmaceutically acceptable excipients.

11. A pharmaceutical composition comprising a pharmaceutically acceptable salt as described in any one of claims 1 to 6 or claim 8, and one or more pharmaceutically acceptable excipients.

12. A method for treating RSV infection, the method comprising administering to an individual in need a therapeutically effective amount of the compound as described in any one of claims 1 to 8 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, ziresovir, EDP-938, EDP-323, JNJ-64417184, PC786, S-337395, MRK-1, JNJ-8003, BI-D, AVG-158, AVG-233, AZ-27, molnupiravir, remdesivir, obeldesivir, and ribavirin.

15. The method of claim 14, wherein the additional RSV therapeutic agent is selected from the group consisting of: cesumatovir, ziresoxir, 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 cezunatovir.

17. The method of any one of claims 12 to 16, wherein the compound is (S)-2-(4-(ethanesulfonyl)-2-fluorophenyl)-N-(9-fluoro-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-6-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide.

18. The compound of any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, used as a medicine.

19. The compound of any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, for the treatment of RSV infection.

20. The compound of any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, used to prepare a medicament for treating RSV infection.

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