CN113214334A

CN113214334A - Compound for treating virus infection and preparation method and application thereof

Info

Publication number: CN113214334A
Application number: CN202010083301.0A
Authority: CN
Inventors: 杨成; 张起愿; 陆华龙
Original assignee: Huachuang Synthetic Pharmaceutical Co ltd
Current assignee: Huachuang Synthetic Pharmaceutical Co ltd
Priority date: 2020-02-05
Filing date: 2020-02-05
Publication date: 2021-08-06

Abstract

Provided herein are formulations, methods and compounds of formula (i) for treating viral infections, pneumovirinae virus infections, as well as processes and intermediates for synthesizing compounds of formula (i).

Description

Compound for treating virus infection and preparation method and application thereof

Technical Field

Provided herein are compounds, methods and pharmaceutical formulations for the treatment of pneumovirinae (pneumovlrlenae) viral infections, in particular, including respiratory tract viral infections, as well as processes and intermediates useful for preparing the compounds.

Background

Viruses of the Pneumovirinae subfamily are negative-sense, single-stranded RNA viruses, which are responsible for many epidemic human and animal diseases. The pneumovirinae subfamily of viruses is part of the family paramyxoviridae and includes Human Respiratory Syncytial Virus (HRSV). Almost all children experience HRSV infection by the time of their second birthday. HRSV is the leading cause of lower respiratory tract infections in infants and children, with 0.5% to 2% of these infected persons requiring hospitalization. Elderly and adults with chronic heart, lung disease, or those who are immunosuppressed also have a high risk of developing severe HRSV disease. No vaccine is currently available to prevent HRSV infection. The monoclonal antibody palivizumab is useful for immunoprophylaxis, but its use is limited to high-risk infants, e.g., premature infants or those with congenital heart or lung disease, and for general use, the cost is often prohibitive. Additionally, the nucleoside analog ribavirin has been approved as the only antiviral agent for the treatment of HRSV infections but has limited efficacy. Thus, there is a need for anti-pneumovirinae therapies.

Examples of pyrrolo [2, 3-d ] pyrimidine compounds useful for the treatment of viral infections are described in U.S.2012/0009147 a1(Cho et al), U.S.2012/0020921 AI (Cho et al), W02008/089105 a2(Babu et al), WO 2008/141079 AI (Babu et al), WO 2009/132135 a1(Butler et al), WO 2010/002877 a2(Francom), WO 2011/035231 AI (Cho et al), WO 2011/035250 a1(Butler et al), WO 2011/150288 a1(Cho et al), WO 2012/012465(Cho et al), WO 2012/012776 a1(Mackman et al), W02012/037038 (Clarke et al), WO 2012/087596 a1(Delaney et al) and WO 2012/142075 a1(Girijavallabhan et al).

There remains a need for new antiviral agents that can be used for the treatment of paramyxoviridae virus infections, including pneumovirinae virus infections such as HRSV infections, with a fertile efficacy and with acceptable toxicity profiles.

Disclosure of Invention

Provided herein are compounds, methods and pharmaceutical formulations for treating infections caused by the pneumovirinae virus family, including treating infections caused by human respiratory viruses.

There is provided a compound of formula (i) or a pharmaceutically acceptable salt thereof:

wherein:

R₁is composed of

R₂Is OH, OD or halogen;

R₃is OH or OD;

R₄is H, D, CN, C₁-C₄Alkyl radical, C₂-C₄Alkenyl radical, C₂-C₄Alkynyl, C₃-C₄Cycloalkyl, azido, halogen, guanidino or C₁-C₂A haloalkyl group;

R₅is H, D, CN, C₁-C₄Alkyl radical, C₂-C₄Alkenyl radical, C₂-C₄Alkynyl, C₃-C₄Cycloalkyl, azido, halogen, guanidino or C₁-C₂A haloalkyl group;

R₆selected from H,

R₅Is H or R₆Is not H, R₆Is H or R₅Is not H;

wherein:

n is selected from 1,2, 3 and 4;

R₇is selected from C₁-C₈Alkyl, -O-C₁-C₈Alkyl, benzyl, -O-benzyl, -CH₂-C₃-C₆Cycloalkyl, -O-CH₂-C₃-C₆Cycloalkyl and CF₃；

R₈Selected from phenyl, 1-naphthyl, 2-naphthyl,

R₉Is selected from H and CH₃；

R₁₀Is selected from H or C₁-C₆An alkyl group;

R₁₁selected from H, C₁-C₈Alkyl, benzyl, C₃-C₆Cycloalkyl and-CH₂-C₃-C₆A cycloalkyl group.

In another embodiment, an effective amount of a compound selected from formula i consisting of or a pharmaceutically acceptable salt, isomer, deutero, solvate or ester thereof is administered.

Detailed Description

Definition of

The terms halo and halogen refer to a halogen atom selected from F, CI, Br and I.

"azido" refers to an azido group, i.e., the group-N3. The term "n" as used herein refers to an integer, such as an integer selected from 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20, i.e., from 2 to 20 or from 2-20. In some instances, "n" refers to an integer group such as 1 to 3, 1 to 4, 1 to 6, 1 to 8, 2 to 4, 2 to 6, 2 to 8, etc., as used herein the term "haloalkyl" refers to an alkyl group as defined herein in which one or more hydrogen atoms are each replaced by a halogen substituent. For example, a (C1-C6) haloalkyl is a (C1-C6) alkyl in which one or more hydrogen atoms are replaced by a halogen substituent. Such ranges include from one halogen substituent on the alkyl group to complete halogenation of the alkyl group.

The term "(C) as used herein_1-n) Haloalkyl ", wherein n is an integer, alone or in combination with another group, is intended to mean an alkyl group having 1 to n carbon atoms as defined above wherein one or more hydrogen atoms are each replaced by a halogen substituent. Wherein n is 2 (C)_1-n) Examples of haloalkyl groups include, but are not limited to, chloromethyl, chloroethyl, dichloroethyl, bromomethyl, bromoethyl, dibromoethyl, fluoromethyl, difluoromethyl, trifluoromethyl, fluoroethyl, and difluoroethyl.

The term "(Ci-.) alkyl" as used herein, wherein n is an integer, alone or in combination with another group, is intended to refer to a non-cyclic, straight-chain or branched alkyl group containing from 1 to n carbon atoms. "(C1-4) alkyl" includes, but is not limited to, methyl, ethyl, propyl (n-propyl), butyl (n-butyl), 1-mesoethyl (isopropyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (isobutyl), and 1, 1-dimethylethyl (tert-butyl). The abbreviation Me denotes a methyl group, Et denotes an ethyl group, Pr denotes a propyl group, iPr denotes a 1-methylethyl group, Bu denotes a butyl group, and tBu denotes a1, 1-dimethylethyl group.

The term "alkyl" refers to a hydrocarbon containing primary, secondary, or tertiary atoms. For example, an alkyl group can have 1 to 4 carbon atoms (i.e., (C1-C4) alkyl), 1 to 3 carbon atoms (i.e., (C1-C3) alkyl), or 1 or 2 carbon atoms (i.e., (C1-C2) alkyl). Examples of suitable alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, isopropyl, -CH (CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, isobutyl, -CH2CH (CH3)2), 2-butyl (s-Bu, sec-butyl, -CH (CH3) CH2CH3), and 2-methyl-9-propyl (t-Bu, tert-butyl, -C (CH3) 3). "alkyl" also refers to a saturated, branched, or straight chain hydrocarbon group having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkyl groups include, but are not limited to, methylene (-CH2-), 1-ethyl (-CH (CH3) -), 1, 2-ethyl (-CH2CH2-), 1-propyl (-CH (CH2CH3) -), 1, 9-propyl (-CH2CH (CH3) -), 1, 3-propyl (-CH2CH2CH2-), 1, 4-butyl (-CH2CH2CH2-), and the like.

An "alkenyl group" is a straight or branched chain hydrocarbon containing a primary, secondary or tertiary carbon atom with at least one site of unsaturation, i.e., a carbon-carbon sp-double bond. By way of example, an alkenyl group can have 2 to 4 carbon atoms (i.e., C2-C4 alkenyl) or 2 to 3 carbon atoms (i.e., C2-C3 alkenyl). Examples of suitable alkenyl groups include, but are not limited to, vinyl (-CH ═ CH2) and allyl (-CH2CH ═ CH 2).

The term "(C) as used herein₂-n) alkenyl ", wherein n is an integer, alone or in combination with another groupIs intended to mean an unsaturated, acyclic, linear or branched group containing two to n carbon atoms, at least two of which are bonded to each other by a double bond. Examples of such groups include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, and 1-butenyl. Unless otherwise indicated, the term "(C)_2-n) Alkenyl "is understood to encompass possible individual stereoisomers, including but not limited to (E) and (Z) isomers, and mixtures thereof. When (C)_2-n) When an alkenyl group is substituted, it is understood that, unless otherwise indicated, there is substitution on any of its carbon atoms that would otherwise carry a hydrogen atom such that the substitution would result in a chemically stable compound, as would be recognized by one of skill in the art.

"alkynyl" is a straight or branched chain hydrocarbon containing a primary, secondary or tertiary carbon atom having at least one site of unsaturation, i.e., a carbon-carbon sp-bond. For example, an alkynyl group can have 2 to 4 carbon atoms (i.e., C2-C4 alkynyl) or 2 to 3 carbon atoms (i.e., C2-C3 alkynyl). Examples of suitable alkynyl groups include, but are not limited to, ethynyl (-C-CH), propynyl (-CH2C-CH), and the like.

The term "(C) as used herein₂-n) alkynyl ", wherein n is an integer, alone or in combination with another group, is intended to mean an unsaturated, acyclic, linear or branched group containing two to n carbon atoms, at least two of which are bonded to each other by a triple bond. Examples of such groups where n is 4 include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and 1-butynyl. When (C)_2-n) When an alkynyl group is substituted, it is understood that, unless otherwise indicated, there is substitution on any of its carbon atoms which would otherwise carry a hydrogen atom such that the substitution would result in a chemically stable compound, as would be recognized by one of skill in the art.

The term cycloalkyl refers to a cyclic aliphatic group. Cycloalkyl groups herein may be referred to by the number of carbon atoms in the ring, e.g., "C3-C4 cycloalkyl" refers to cycloalkyl having 3 or 4 carbon ring atoms or "C3-C6 cycloalkyl" refers to cycloalkyl having 3, 4, 5 or 6 carbon ring atoms, i.e., a cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl ring.

The term "carbocycle" or "carbocyclyl" refers to a saturated (i.e., cycloalkyl) or partially unsaturated (e.g., cycloalkenyl, cycloalkadienyl, etc.) ring having the indicated number of carbon atoms, e.g., 3 to 4 carbon atoms or 3 to 6 carbon atoms as a monocyclic ring system. In one embodiment, a carbocycle is a monocyclic ring containing 3-6 ring carbons (i.e., (C3-C6) carbocycle). Non-limiting examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-9-enyl, 1-cyclohex-3-enyl and cyclohex-1, 3-dienyl rings.

Each carbocyclyl group may be substituted with 0, 1,2 or 3 substituents independently selected from halogen, -OH, -CN, -NO2, -NH2, -NH (C1-C6 alkyl), -N (C1-C6 alkyl) 2, C1-C6 alkyl, C1-C6 alkoxy and-CF 3.

Pharmaceutical preparation

Also provided herein is a pharmaceutical formulation comprising a pharmaceutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, solvate and/or ester thereof, and a pharmaceutically acceptable carrier or excipient. Also provided are separate pharmaceutical formulations, each comprising a pharmaceutically effective amount of a compound of formula (i) or one of the specific compounds of the examples herein, or a pharmaceutically acceptable salt, solvate and/or ester thereof, and a pharmaceutically acceptable carrier or excipient.

The compounds herein are formulated with conventional carriers and excipients, which are selected according to common practice. Tablets will contain excipients, glidants, fillers, binders and the like. Aqueous formulations are prepared in sterile form and will generally be isotonic when intended for non-oral delivery. All formulations optionally contain Excipients such as those listed in the Handbook of Pharmaceutical Excipients (1986). Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextran, hydroxyalkyl cellulose, hydroxyalkyl methyl cellulose, stearic acid, and the like. The pH of the formulation ranges from about 3 to about 11, but is typically about 7 to 10.

While the active ingredients may be administered separately, they are preferably provided in the form of a pharmaceutical formulation. Both veterinary and human formulations comprise at least one active ingredient as defined above, together with one or more acceptable carriers and optionally other therapeutic ingredients, particularly those as discussed herein. The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not physiologically deleterious to the recipient thereof.

Formulations include those suitable for the aforementioned routes of administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations are commonly found in Remington's Pharmaceutical Sciences (Mack Publishing co., Easton, PA). Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations suitable for oral administration may be provided in the following dosage forms: discrete units such as capsules, cachets or tablets each containing a predetermined amount of active ingredient; a powder or granules; solutions or suspensions in aqueous or non-aqueous liquids; or an oil-in-water or water-in-oil emulsion. The active ingredient may also be administered as a bolus, electuary or paste.

Tablets are made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersing agent. Molded tablets may be prepared by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may optionally be formulated so as to provide slow or controlled release of the active ingredient therefrom.

For infections of the eye or other external tissues such as the mouth and skin, the formulations are preferably applied as a topical ointment or cream containing one or more active ingredients in an amount of, for example, 0.075 to 20% w/w (including active ingredients in 0.1 w/w increments between 0.1% and 20%, such as 0.6 w/w%, 0.7 w/w%, etc.), preferably 0.2 to 15 w/w%, most preferably 0.5 to 10 w/w). When formulating ointments, the active ingredient may be used with either paraffin or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base.

If desired, the aqueous phase of the cream base may comprise, for example, at least 30% w/w of a polyhydric alcohol, i.e. an alcohol having two or more hydroxyl groups such as propylene glycol, 1, 3-butylene glycol, mannitol, sorbitol, glycerol and polyethylene glycols (including PEG400) and mixtures thereof. Topical formulations may desirably contain compounds that enhance the absorption or permeation winding of the active ingredient through the skin or other affected areas. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogs.

The oil phase of the emulsion can be composed of known ingredients in a known manner. Although this phase may comprise only emulsifiers, it desirably comprises a mixture of at least one emulsifier with a fat or oil or with both a fat and an oil. Preferably, the hydrophilic emulsifier is introduced together with the lipophilic emulsifier which acts as a stabilizer. It also preferably comprises both oil and fat. Emulsifiers, with or without stabilizers, constitute the so-called emulsifying waxes, which, together with oils and fats, constitute the so-called emulsifying ointment bases, which form the oily disperse phase of cream formulations.

Emulsifiers and emulsion stabilizers suitable for use in the formulation include cetyl alcohol, stearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.

Suitable oils or fats for the formulation are selected based on achieving the desired cosmetic properties. The cream should preferably be a non-greasy, non-staining and washable product with a suitable consistency to avoid leakage from tubes or other containers. Blends of straight or branched chain, mono-or dibasic alkyl esters such as diisoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acid, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a branched chain ester known as Crodamol CAP may be used, the latter three being preferred esters. These may be used alone or in combination, depending on the desired properties. Alternatively, high melting point lipids such as white soft paraffin and/or rolling body paraffin or other mineral oils are used.

The pharmaceutical formulations herein comprise a combination with one or more pharmaceutically acceptable carriers or excipients and optionally other therapeutic agents. The pharmaceutical formulations containing the active ingredient may be in any form suitable for the intended method of administration. When used, for example, orally, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, solutions, syrups or elixirs may be prepared. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents, including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide palatable preparations. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets are acceptable. These excipients may be, for example: inert diluents, such as calcium or sodium carbonate, lactose, calcium or sodium phosphate; granulating and disintegrating agents, such as corn starch or alginic acid; binding agents, such as starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques, including microencapsulation, to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed alone or with a wax.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as naturally-occurring phosphatides (e.g., lecithin), condensation products of an alkylene oxide with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents and one or more sweetening agents, such as sucrose or saccharin.

Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. Oral suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those disclosed above. Other excipients, for example sweetening, flavoring and coloring agents, may also be present.

The pharmaceutical composition may also be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil, for example olive oil or arachis oil, a mineral oil, for example liquid paraffin, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, for example gum acacia and gum tragacanth, naturally-occurring phosphatides, for example soy bean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate and condensation products of these partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsion may also contain sweeteners and flavoring agents. Syrups and elixirs may be formulated with sweetening agents, for example glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavouring or a colouring agent.

The pharmaceutical compositions may be in the form of a sterile injectable preparation or intravenous formulation such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation or intravenous preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol or as a lyophilisate. Among the acceptable carriers and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-and diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables.

The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, sustained release formulations for oral administration to humans may contain about 1 to 1000mg of the active material formulated with a suitable and convenient amount of carrier material, which may be about 5 to about 95% (weight: weight) of the total composition. The pharmaceutical composition may be prepared to provide an easily measurable amount for administration. For example, an aqueous solution intended for intravenous infusion may contain from about 3 to 500ug of active ingredient per mL of solution so that infusion of the appropriate volume at a rate of about 30mL/hr can occur.

Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient. The active ingredient is preferably present in such formulations at a concentration of from 0.5 to 20% w/w, advantageously from 0.5 to 10% w/w, in particular about 1.5% w/w.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth; lozenges comprising the active ingredient in an inert base such as gelatin and glycerol, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

Formulations for rectal administration may be presented as suppositories with a suitable base comprising, for example, cocoa butter or a salicylate.

Formulations suitable for intrapulmonary or nasal administration have a particle size, for example in the range of 0.1 to 500 microns, such as 0.5, 1, 30, 35 etc., which are administered by rapid inhalation through the nasal passage or by oral inhalation to reach the alveolar sacs. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol or dry powder administration may be prepared according to conventional methods and may be delivered with other therapeutic agents such as compounds previously used in the treatment or prevention of infections of the pneumovirinae sub-family as described below.

Another embodiment provides a novel, effective, safe, non-irritating and physiologically compatible inhalable composition suitable for treating pneumovirinae infections and potentially accompanying bronchiolitis, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof. Preferred pharmaceutically acceptable salts are inorganic acid salts, including hydrochloride, hydrobromide, sulphate or phosphate salts, as these may cause less lung irritation. Preferably, the inhalable formulation is delivered to the endobronchial space in an aerosol comprising particles having a Mass Median Aerodynamic Diameter (MMAD) between about 1 and about 5 μm. Preferably, the compounds of formula (I) are formulated for aerosol delivery using a nebulizer, a pressurized metered dose inhaler (pMDI) or a Dry Powder Inhaler (DPI).

Non-limiting examples of nebulizers include nebulizers, ultrasonic, pressurized, vibrating perforated plates, or equivalent nebulizers, including those employing adaptive aerosol Delivery technology (Denyer, j. aerosol medicine Drug Delivery 2010, 23Supp 1, S1-S10). Jet nebulizers use air pressure to break up liquid solutions into aerosol droplets. Ultrasonic nebulizers work with piezoelectric crystals, which shear a liquid into small aerosol droplets. Pressurized spray systems force solutions under pressure through small pores to generate aerosol droplets. Vibrating perforated plate apparatus utilizes rapid vibration to shear a stream of liquid into the appropriate droplet size.

In a preferred embodiment, the formulation for spraying uses a nebulizer capable of aerosolizing a formulation of a compound of formula (I) or formula (II) into particles of the desired MMAD to deliver an aerosol comprising particles of MMAD predominantly between about 1 μm and about 5 μm into the endobronchial space. For optimal therapeutic effectiveness and to avoid upper respiratory and systemic side effects, most aerosolized particles should not have an MMAD greater than about 5 μm. If the aerosol contains a large number of particles with an MMAD greater than 5 μm, the particles will deposit in the upper airway to reduce the amount of drug delivered to sites of inflammation and bronchoconstriction in the lower respiratory tract. If the MMAD of the aerosol is less than about 1 μm, the particles have a tendency to remain suspended in the inhaled air and subsequently exhale during exhalation.

When formulated and delivered according to the methods herein, an aerosol formulation for nebulization will deliver a therapeutically effective dose of a compound of formula (I) to a site of pneumovirinae infection sufficient to treat the pneumovirinae infection. The amount of drug administered must be adjusted to reflect the efficiency of delivery of the therapeutically effective dose of the compound of formula (I). In a preferred embodiment, the combination of an aqueous aerosol formulation with nebulization, spraying, pressurization, vibrating perforated plate, or ultrasonic nebulizer (depending on the nebulizer) allows about at least 20 to about 90%, typically about 70%, of the administered dose of the compound of formula (I) to be delivered into the airway. In a preferred embodiment, at least about 30 to about 50% of the active compound is delivered. More preferably, about 70 to about 90% of the active compound is delivered.

In another embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is delivered as an inhalable powder. The compounds are administered intrabronchially in dry powder formulations using dry powder or metered dose inhalers to effectively deliver fine particles of the compounds to the endobronchial space. For delivery by DPI, the compound of formula (I) is processed into particles with MMAD mainly between about 1 μm and about 5 μm by milling spray drying, critical fluid processing, or precipitation from solution. Media milling, jet milling and spray drying equipment and procedures capable of producing particle sizes with MMAD between about 1 μm and about 5 μm are well known in the art.

In one embodiment, excipients are added to the compound of formula (I) prior to processing into particles of the desired size. In another embodiment, excipients are blended with particles of a desired size to aid in the dispersion of the drug particles, for example by using lactose as an excipient.

Particle size determination is performed using equipment well known in the art. For example, multi-stage Anderson cascade impactor or other suitable methods such as those specifically mentioned as characterizing devices for metered dose and aerosol in dry powder inhalers within united states pharmacopeia chapter 601.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents.

The formulations are presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, or suitable fraction thereof, of the active ingredient as hereinbefore described.

It will be appreciated that the formulations may contain, in addition to the ingredients particularly mentioned above, other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may contain flavouring agents.

Veterinary carriers are materials used for the purpose of administering the composition and can be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary arts and are compatible with the active ingredient. These veterinary compositions may be administered orally, parenterally or by any other desired route.

The compounds herein are used to provide controlled release pharmaceutical formulations containing one or more of the compounds as active ingredients ("controlled release formulations") wherein the release of the active ingredient is controlled and regulated to allow for less frequent dosing or to improve the pharmacokinetic or toxicity profile of a given active ingredient.

The effective dose of the active ingredient will depend at least on the nature of the condition being treated, the toxicity, whether the compound is used prophylactically (lower dose) or against an active viral infection, the method of delivery and the pharmaceutical formulation, and is determined by the clinician using conventional dose escalation studies. It can be expected to be from about 0.0001 to about 100mg/kg body weight/day, typically from about 0.01 to about 10mg/kg body weight/day, more typically from about 0.01 to about 5mg/kg body weight/day, and most typically from about 0.05 to about 0.5mg/kg body weight/day.

For example, a candidate daily dosage for an adult human weighing about 70kg will be in the range of 1mg to 1000mg, preferably between 5mg and 500mg, and may be in single or multiple doses.

Route of administration

One or more of the compounds (herein referred to as active ingredients) may be administered by any route suitable for the condition being treated. Suitable routes include oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), vaginal, and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural), and the like. It will be appreciated that the preferred route may vary, for example, with the recipient. The advantage of the compounds herein is that they are orally available as a host and can be administered orally.

Combination therapy

The compositions are also used in combination with other active ingredients. For the treatment of infections with viruses of the pneumovirinae sub-family, preferably, the further active therapeutic agent is active against infections with viruses of the pneumovirinae sub-family, in particular respiratory syncytial virus infections. Non-limiting examples of such other active therapeutic agents are ribavirin, palivizumab, mevizumab, RSV-IGIV (Respicam), MEDI-557, A-60444 (also known as RSV604), MDT-637, BMS-433771, ALN-RSVO, ALX-0171, and mixtures thereof.

Infection by many viruses of the pneumovirinae subfamily is respiratory infection. Thus, other active therapeutic agents used to treat respiratory symptoms and sequelae of infection may be used in combination with the compounds of formula (I). The other therapeutic agent is preferably administered orally or by direct inhalation. For example, other preferred additional therapeutic agents for combination with the compound of formula (I) to treat viral respiratory infections include, but are not limited to, bronchodilators and corticosteroids.

Glucocorticoids, originally introduced as asthma therapy in 1950 (Carryer, Journal of allergy, 21, 282-287, 1950), remain the most effective and consistently effective therapy for this disease, but their mechanism of action is not fully understood (Morris, j. Unfortunately, oral glucocorticoid therapy is associated with serious adverse side effects such as trunk obesity, hypertension, glaucoma, glucose intolerance, accelerated cataract formation, bone mineral loss, and psychological effects, all of which limit its use as a long-term therapeutic (Goodman and Gilman, 10th edition, 2001). One solution to systemic side effects is the delivery of steroid drugs directly to the site of inflammation. Inhaled Corticosteroids (ICS) have been developed to alleviate the serious side effects of oral steroids. Non-limiting examples of corticosteroids that may be used in combination with compounds of formula (I) are dexamethasone, dexamethasone sodium phosphate, fluoromethalone acetate, loteprednol etabonate, hydrocortisone, prednisolone, fludrocortisone, triamcinolone acetonide, betamethasone, beclomethasone dipropionate, methylprednisolone, fluocinolone acetonide, flunisolide, fluocortebutan-21-butylate, flumethasone pivalate, budesonide, halobetasol propionate, mometasone furoate, fluticasone propionate, ciclesonide; or a pharmaceutically acceptable salt thereof.

Other anti-inflammatory agents that act through an anti-inflammatory cascade mechanism may also be useful as other therapeutic agents in combination with the compounds of formula (I) for the treatment of viral respiratory infections. Administration of "anti-inflammatory signal transduction modulators" (referred to herein as AISTM) women phosphodiesterase inhibitors (e.g., PDE-4, PDE-5 or PDE-7 specific inhibitors), transcription factor inhibitors (e.g., blocking NFKB by IKK inhibition) or kinase inhibitors (e.g., blocking P38MAP, JNK, PI3K, EGFR or Syk) are logical approaches to cut inflammation because these small molecules target a limited number of common intracellular pathways-those signal transduction pathways that are critical for anti-inflammatory therapeutic intervention (see review by p.j.barnes, 2006). These non-limiting additional therapeutic agents include: 5- (2, 4-difluoro-phenoxy) -1-isobutyl-1H-indazole-6-carboxylic acid (2-dimethylamino-ethyl) -amide (P38Map kinase inhibitor ARRY-797); 3-cyclopropylmethoxy-N- (3, 5-dichloro-pyridin-4-yl) -4-fluoromethoxy-benzamide (PDE-4 inhibitor roflumilast); 4- [2- (3-cyclopentyloxy-4-methoxyphenyl) -9-phenyl-ethyl ] -pyridine (PDE-4 inhibitor CDP-840); n- (3, 5-dichloro-4-pyridinyl) -4- (difluoromethoxy) -8- [ (methylsulfonyl) amino ] -1-dibenzofurancarboxamide (PDE-4 inhibitor Oglemilast); n- (3, 5-chloropyridin-4-yl) -2- [1- (4-fluorobenzyl) -5-hydroxy-IH-indol-3-yl ] -2-oxo-acetamide (PDE-4 inhibitor AWD 12-281); 8-methoxy-9-trifluoromethyl-quinoline-5-carboxylic acid (3, 5-dichloro-1-oxo-pyridine-4-coffin) -amide (PDE-4 inhibitor Sch 351591); 4- [5- (4-fluorophenyl) -2- (4-methanesulfinyl-phenyl) -1H-imidazol-4-yl ] monopyridine (P38 inhibitor SB-203850); 4- [4- (4-fluoro-phenyl) -1- (3-phenyl-propyl) -5-pyridin-4-yl-1H-imidazol-9-yl ] -but-3-yn-1-ol (P38 inhibitor RWJ-67657); 4-cyano-4- (3-cyclopentyloxy-4-methoxyphenylyl) -cyclohexanecarboxylic acid 2-diethylamino-ethyl ester (9-diethyl-ethyl ester prodrug of cilomilast, PDE-4 inhibitor); (3-chloro-4-fluorophenyl) - [ 7-methoxy-6- (3-morpholin-4-yl-propoxy) -quinazolin-4-yl ] -amine (gefitinib, EGFR inhibitor); and 4- (4-methyl-piperazin-1-ylmethyl) -N- [ 4-methyl-3- (4-pyridin-3-yl-pyrimidin-2-ylamino) -phenyl ] -benzamide (imatinib, EGFR inhibitor).

Combinations comprising inhaled β 2-adrenoceptor agonist bronchodilators such as formoterol, salbutamol or salmeterol with a compound of formula (I) are also suitable but non-limiting combinations useful in the treatment of respiratory viral infections.

Inhaled β 2-adrenoceptor agonist bronchodilators such as formoterol or salmeterol in combination with ICS have also been used to treat both bronchoconstriction and inflammation (Sym bicort and Advair, respectively). Combinations comprising these combinations of ICS and β 2-adrenoreceptor agonists, as well as compounds of formula (I), are also suitable and non-limiting combinations useful for treating respiratory viral infections.

Anticholinergic agents have potential uses for the treatment or prevention of pulmonary bronchoconstriction and thus can be used as further therapeutic agents in combination with compounds of formula (I) or formula (II) for the treatment of viral respiratory infections. These anticholinergic agents include, but are not limited to, antagonists of muscarinic receptors (particularly the M3 subtype), which have shown therapeutic efficacy in the control of cholinergic tone in human COPD (Witek, 1999); 1- { 4-hydroxy-1- [3, 3, 3-tris- (4-fluoro-phenyl) -propionyl ] -pyrrolidine-9-carbonyl) -pyrrolidine-2-carboxylic acid (1-methyl-piperidin-4-ylmethyl) -amide; 3- [3- (2-diethylamino-acetoxy) -9-phenyl-propionyloxy ] -8-isopropyl-8-methyl-8-azonian-bicyclo [3.2.1] octane (ipratropium-N, N-glycine diethyl ester); 1-cyclohexyl-3, 4-dihydro-1H-isoquinoline-2-carboxylic acid 1-aza-bicyclo [2.2.2] oct-3-yl ester (solifenacin); 2-hydroxymethyl-4-methanesulfinyl-2-phenyl-butyric acid 1-aza-bicyclo [2.2.2] oct-3-yl ester (revatoxyl) the compounds of formula (I) may also be combined with mucolytic agents to treat infections and symptoms of respiratory infections. One non-limiting example of a mucolytic agent is ambroxol. Similarly, a compound of formula (I) or formula (II) may be combined with an expectorant to treat both the infection and symptoms of respiratory infections. One non-limiting example of an expectorant is guaifenesin. Nebulized hypertonic saline is used to improve the immediate and long-term clearance of the small airways in patients with lung disease (Kuzik, j. pediatrics 2007, 266). The compounds of formula (I) may also be combined with nebulized hypertonic saline, particularly when the pneumovirinae virus is infected with bronchiolitis. The combination of a compound of formula (I) with hypertonic saline may further comprise any of the other agents discussed above. In one embodiment, about 3% nebulized hypertonic saline is used. Any compound may also be combined with one or more other active therapeutic agents in a single dosage form for simultaneous or sequential administration to a patient. The combination therapy may be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations.

Co-administration of a compound with one or more other active therapeutic agents herein generally refers to the simultaneous or sequential administration of the compound and one or more other active therapeutic agents such that a therapeutically effective amount of both the compound and the one or more other active therapeutic agents are present in the body of the patient.

Co-administration includes administering a unit dose of the compound before or after administering a unit dose of one or more other active therapeutic agents, e.g., within seconds, minutes, or hours of administering one or more other active therapeutic agents. For example, a unit dose of the compound may be administered first, followed by a unit dose of one or more other active therapeutic agents within seconds or minutes. Alternatively, a unit dose of one or more other therapeutic agents may be administered first, followed by administration of the unit dose of the compound within seconds or minutes. In some cases, it may be desirable to first administer a unit dose of the compound and then administer a unit dose of one or more other active therapeutic agents after a period of several hours (e.g., 1-12 hours). In other cases, it may be desirable to first administer a unit dose of one or more other active therapeutic agents, and then administer a unit dose of a compound described herein after a period of several hours (e.g., 1-12 hours).

Combination therapy may provide a "synergistic effect" or "synergistic effect", i.e., the effect obtained when the active ingredients are used together is greater than the sum of the effects produced by the compounds when used alone. A synergistic effect is obtained when the active ingredients are applied in the following manner: (1) co-formulated in a combined preparation and administered or delivered simultaneously; (2) by alternating or parallel delivery as separate formulations; or (3) by some other scheme. When delivered in alternation therapy, a synergistic effect may be obtained when the compounds are administered or delivered sequentially, e.g. in separate tablets, pills or capsules, or by different injections in separate syringes.

Generally, during alternation therapy, an effective dose of each active ingredient is administered sequentially, i.e., consecutively, whereas in combination therapy, effective doses of two or more active ingredients are administered together. Synergistic antiviral effect refers to an antiviral effect that is greater than the expected purely additive effect of the individual compounds in combination.

In yet another embodiment, the present application provides a method of treating a pneumovirinae virus infection in a human, the method comprising administering to the human a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, solvate, and/or ester thereof. Also provided are separate methods of treating a pneumovirinae virus infection in a human, each comprising administering to the human a therapeutically effective amount of a compound of formula (i) or one of the specific compounds of the examples herein or a pharmaceutically acceptable salt, solvate and/or ester thereof and a pharmaceutically acceptable carrier or excipient.

In another embodiment, there is provided a method of treating pneumovirinae infection in a human by administering to the human a therapeutically effective amount of a racemate, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate or solvate of the compound of formula (I) or a pharmaceutically acceptable salt or ester thereof.

Also provided are separate methods of treating a pneumovirinae infection in a human in need thereof, each method comprising administering to the human a therapeutically effective amount of a racemate, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate or solvate of the compound of formula (II) or one of the specific compounds of the examples herein or a pharmaceutically acceptable salt, solvate and/or ester thereof.

In yet another embodiment, the present application provides a method of treating a human respiratory viral infection in a human, comprising administering to the human a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, solvate, and/or ester thereof.

In yet another embodiment, the present application provides a method of treating a human respiratory viral infection in a human, comprising administering to the human a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, solvate, and/or ester thereof, and at least one additional active therapeutic agent.

Also provided are separate methods of treating a human respiratory viral infection in a human in need thereof, each method comprising administering to the human a therapeutically effective amount of a compound of formula (I) or one of the specific compounds of the examples herein or a pharmaceutically acceptable salt, solvate and/or ester thereof.

Also provided are separate methods of treating a human respiratory viral infection in need thereof, each method comprising administering to the human a therapeutically effective amount of a compound of formula (I) or one of the specific compounds of the examples herein or a pharmaceutically acceptable salt, solvate, and/or ester thereof and at least one additional active therapeutic agent.

Also provided are separate methods of treating a human respiratory viral infection in need thereof, wherein said human is also experiencing bronchitis, each method comprising administering to said human a therapeutically effective amount of a compound of formula (I) or one of the specific compounds of the examples herein or a pharmaceutically acceptable salt, solvate and/or ester thereof.

Also provided are separate methods of treating a human respiratory viral infection in need thereof, wherein the human is also experiencing pneumonia, each method comprising administering to the human a therapeutically effective amount of a compound of formula (I) or one of the specific compounds of the examples herein or a pharmaceutically acceptable salt, solvate, and/or ester thereof.

Also provided are separate methods of ameliorating respiratory symptoms in a human undergoing a respiratory viral infection in a human, each method comprising administering to the human a therapeutically effective amount of a compound of formula (I) or one of the specific compounds of the examples herein or a pharmaceutically acceptable salt, solvate, and/or ester thereof.

Respiratory symptoms in persons experiencing respiratory viral infections may include stuffy or runny nose, coughing, wheezing, sneezing, shortness or difficulty of breathing, asphyxia, bronchitis, and pneumonia.

Also provided is an embodiment comprising the use of a compound of formula (I), or a pharmaceutically acceptable salt, solvate and/or ester thereof, in the manufacture of a medicament for the treatment of a pneumovirinae virus infection or a respiratory virus infection.

Also provided is a pharmaceutical formulation comprising a pharmaceutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, solvate and/or ester thereof, and a pharmaceutically acceptable carrier or excipient. Also provided is a pharmaceutical formulation comprising a pharmaceutically effective amount of a compound of formula (I) or one of the specific compounds of the examples herein, or a pharmaceutically acceptable salt, solvate and/or ester thereof, and a pharmaceutically acceptable carrier or excipient.

Also provided is a pharmaceutical formulation comprising a pharmaceutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, solvate and/or ester thereof, and a pharmaceutically acceptable carrier or excipient and a pharmaceutically effective amount of at least one additional active therapeutic agent.

Also provided are separate embodiments that include a compound of formula (I) or one of the specific compounds of the examples herein, or a pharmaceutically acceptable salt, solvate, and/or ester thereof, for use in the treatment of a pneumovirinae virus infection or a respiratory virus infection in a human.

Also provided are separate embodiments that include a compound of formula (I) or one of the specific compounds of the examples herein, or a pharmaceutically acceptable salt, solvate, and/or ester thereof, for use as a medicament.

Also provided are separate embodiments, including methods of manufacturing a medicament intended for the treatment of a pneumovirinae virus infection or a respiratory virus infection in a human, the method characterized by using a compound of formula (I) or one of the specific compounds of the examples herein or a pharmaceutically acceptable salt, solvate and/or ester thereof.

Also provided is a compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or ester thereof, for use in treating a pneumovirinae virus infection or a respiratory virus infection in a human.

Also provided are compounds as described in the specification. Pharmaceutical compositions as described in the specification are also provided. Also provided are methods of using the compounds of formula (I) as described in the specification. Also provided are methods of making compounds of formula (I) as described in the specification.

Metabolites of compounds

In vivo metabolites of the compounds described herein also fall within the scope herein to the extent that such products are novel and unobvious over the prior art. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, esterification, etc. of the administered compound, primarily due to enzymatic processes. Accordingly, new and nonobvious compounds produced by a process comprising contacting a compound with a mammal for a period of time sufficient to produce a metabolite thereof are included in the invention. Such products are typically identified by preparing a radiolabeled (e.g., 14C or 3H) compound, parenterally administering to an animal such as rat, mouse, guinea pig, monkey at a detectable dose (e.g., greater than about 0.5mg/kg), allowing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours) and isolating its conversion products from urine, blood or other biological samples. These products are easy to isolate as they are labelled (others are isolated by using antibodies capable of binding to epitopes present in the metabolite). The metabolite structure is determined in a conventional manner, for example by MS or NMR analysis. In general, analysis of metabolites is performed in the same manner as conventional drug metabolism studies well known to those skilled in the art. The conversion products, so long as they are not otherwise found in vivo, can be used in diagnostic tests for therapeutic doses of the compounds, even if they do not have their own HSV antiviral activity.

Formulations and methods for determining the stability of compounds in alternative gastrointestinal secretions are known. A compound is defined herein as being stable in the gastrointestinal tract when less than about 50 mole% of the protected groups are deprotected in the displaced intestinal or gastric fluid upon incubation for 1 hour at 37 ℃. Simply because the compounds are stable to the gastrointestinal tract does not mean that they do not hydrolyze in vivo. The prodrug will generally be stable in the digestive system, but may be substantially hydrolyzed to the parent drug in the digestive lumen, liver, lung or other metabolic organs or generally within the cell. As used herein, a prodrug is understood to be a compound that is chemically designed to effectively release the parent drug after overcoming the biological barriers of oral delivery.

Preparation of the Compounds

Example 1: (2S) -ethyl 2- (chloro (phenoxy) phosphorylamino) propionate (chloride A1)

Ethyl alanine ester hydrochloride (2.54g, 16.5mmol) was dissolved in anhydrous dichloromethane (30ml) and the mixture was cooled to 0 ℃ under N2(g) with stirring. Phenyl dichlorophosphate (2.23mL, 25mmol) was added followed by triethylamine dropwise over 10 min. The reaction mixture was then slowly warmed to RT and stirred for 12 h. Anhydrous acetic anhydride (150mL) was added and the mixture was stirred for 30 min. The solid formed was removed by filtration and the filtrate was concentrated under reduced pressure. The residue was subjected to silica gel chromatography eluting with 0-50% EtOAc in hexanes to afford chloride a1(1.86g, 39%).

¹H NMR(300MHz,CDC13)δ7.39-7.27(m,5H),4.27(m,3H),1.52(m,3H),1.32(m,3H)。

³¹P NMR(121.4MHz,CDCl₃)δ8.2,7.8。

Example 2: (2S) -2- (chloro (phenoxy) phosphorylamino) propionic acid 9-ethylbutyl ester (chloride B1)

The same procedure as for chloride A1 was used to prepare 2-ethylbutylalanine chloroaminophosphate B1, except that 2-ethylbutylalanine ester was used instead of ethyl alanine ester. The crude material was used for the next reaction. Treatment with methanol or ethanol formed an alternative product with the desired LCMS signal.

Example 3: (2S) -isopropyl 2- (chloro (phenoxy) phosphorylamino) propionate (chloride C1)

The same procedure as for chloride A1 was used to prepare isopropylalanine chloroaminophosphate C1, except that isopropylalanine ester was used instead of ethyl alanine ester. The crude material was used for the next reaction. Treatment with methanol or ethanol formed an alternative product with the desired LCMS signal.

Example 4: s, S '-2, 2' - ((4-nitrophenoxy) phosphoryl) bis (ethane-2, 1-diyl) bis (2, 2-dimethylthiopropionate)

(intermediate D1)

Phosphorus oxychloride (280. mu.L, 30.8mmol) was dissolved in 50ml dichloromethane and the solution cooled to-78 ℃. The thioester (10.0g 61.7mmol) was dissolved in 50ml of dichloromethane and slowly added to the phosphorus oxychloride solution. TEA (890. mu.L, 61.6mmol) was then added dropwise and allowed to cool and stir for 30 minutes. Then warmed to room temperature and allowed to stir for 2 hours. P-nitrophenol (4.28g, 30.8mmol) was added in one portion, then TEA (4.5ml, 30.8mmol) was added slowly and stirred at room temperature for 30 min. TLC (70: 30 hexane/EtOAc) showed only one spot, but LC/MS had two peaks (product and bis-p-nitrophenol ester). The solution was diluted with ether and the solids were removed by filtration and discarded. The mother liquor was concentrated and purified by silica gel chromatography to give a mixture of the product and bis-p-nitrophenolate salt. The mixture was then repurified by HPLC to provide intermediate D1(5.89g 37.7%).

1H NMR(400MHz,CDCl₃)δ8.29-8.21(m,2H),7.46-7.36(m,2H),4.23(br q,J＝7.7Hz,4H),3.16(br t,J＝6.7Hz,4H),1.23(s,18H).

³¹P NMR(162MHz,CDCl₃)δ-7.72(S).

Example 5: lactone Compound E1

Commercial lactitol (15g, 35.7mmol) was dissolved in anhydrous DMSO (30mL) under N2 (g). Addition of Ac₂O (30mL), and the obtained productThe resulting reaction mixture was stirred at RT for 48 h. The reaction mixture was poured onto ice H₂O (750mL) and the mixture was stirred for 20 min. The mixture was extracted with EtOAc (3x200mL) and the combined organic extracts were then washed with H20(3x200 mL). The organic extract was purified over anhydrous MgSO₄Dried, filtered and concentrated under reduced pressure. The residue was dissolved in dichloromethane and subjected to silica gel chromatography eluting with 25% EtOAc in hexanes to afford lactone E1(13.9g, 93%).

¹H NMR(400MHz,DMSO)δ7.30-7.34(m,13H),7.19-7.21(m,2H),4.55-4.72(m,6H),4.47(s,2H),4.28(d,J＝3.9Hz,IH),3.66(m,2H).

LCMS m/z436.1[M+H₂O],435.2[M+OH]-Tr＝2.82min。

HPLC Tr ═ 4.59[ 2-98% ACN in H2), at a flow rate of 2ml/min over 5 min.

Example 6: lactone compound F1

Commercial lactitol (15g, 35.7mmol) was dissolved in anhydrous DMSO (30mL) under N2 (g). Thionyl chloride (3mL) was added and the resulting reaction mixture was stirred at RT for 48 h. The reaction mixture was poured onto ice H₂O (750mL) and the mixture was stirred for 20 min. The mixture was extracted with EtOAc (3x200mL) and the combined organic extracts were then washed with H20(3x200 mL). The organic extract was purified over anhydrous MgSO₄Dried, filtered and concentrated under reduced pressure. The residue was dissolved in dichloromethane and subjected to silica gel chromatography eluting with 40% EtOAc in hexane to afford lactone F1(12.1g, 88%).

Example 7: lactone compound G1

The same procedure as for lactone compound E1 was used to prepare lactone compound G1, except that (3R,4R,5R) -4- (benzyloxy) -5- (benzyloxymethyl) -3-fluorodihydrofuran-2 (3H) -one was used instead of lactitol.

Example 8: lactone compound H1

The same procedure as for lactone compound F1 was used to prepare lactone compound H1, except that (3R,4R,5R) -4- (benzyloxy) -5- (benzyloxymethyl) -3-fluorodihydrofuran-2 (3H) -one was used instead of lactitol.

Example 9: preparation of Compound 1

ZT1 (prepared according to W02009/132135) (0.5g, 2.4mmol) was suspended in anhydrous THF (10mL) under N2 (g). The suspension was stirred and TMSCl (0.67ml,5.28mmol) was added. The mixture was stirred at RT for 20min and then cooled to-78 ℃ after which time N-BuLi solution (6mL, 1.6N in hexane, 9.6mmol) was added slowly. The reaction mixture was stirred at-78 ℃ for 10min and then lactone E1(1g, 2.4mmol) was added via syringe. When the reaction was complete as measured by LCMS, AcOH was added to quench the reaction. The mixture was concentrated under reduced pressure and the residue was dissolved in a mixture of dichloromethane and water (100mL, 1: 1). The organic layer was separated and washed with water (50 mL). The organic layer was then dried over anhydrous MgSO₄Dried, filtered and concentrated under reduced pressure. The residue was subjected to silica gel chromatography eluting with 0-50% EtOAc in hexanes to provide the product as a 1:1 mixture of anomers (432mg, 32.5% yield).

LCMS m/z 555[M+H]

Intermediate 1(2.2g, 4.0mmol) was dissolved in anhydrous dichloromethane (100ml) and the solution was cooled to 0 ℃ under N2(g) with stirring. TMSCN (1.9lmL, 14) was addedmmol) and the mixture stirred for another 10 min. TMSOTf (3.3mL, 18.0mmol) was added slowly to the reaction and the mixture was stirred for 1 h. The reaction mixture was then diluted with dichloromethane (120ml) and NaHCO was added₃Aqueous solution (120mL) to quench the reaction. The reaction mixture was stirred for another 10min and the organic layer was separated. The aqueous layer was extracted with dichloromethane (150ml) and the combined organic extracts were extracted over anhydrous MgSO₄Dried, filtered and concentrated under reduced pressure. The residue was dissolved in a minimum amount of dichloromethane and subjected to silica gel chromatography eluting with a gradient of 0-75% EtOAc and hexanes to provide intermediate 2 as a mixture of anomers. (1.6g, 70.7%).

LCMS m/z 564[M+H].

Intermediate 2(100mg,0.18mmol) was dissolved in anhydrous dichloromethane (5ml) under N2(g) and cooled to-78 deg.C and BCl was added₃Solution (1N in dichloromethane, 0.506mL, 0.506mmol) and the reaction mixture was stirred at-78 deg.C for 1 h. When the reaction was complete according to LC/MS, MeOH was added to quench the reaction. The reaction mixture was warmed to RT and the solvent was removed under reduced pressure. The residue was subjected to C18 reverse phase HPLC eluting with H20 (0.1% TFA) for 5min followed by a gradient of 0-70% MeCN in H20 (0.1% TFA) over 35min to elute levorotatory compound 1(26mg, 52%) and dextrorotatory compound 1(26mg, 52%).

LCMS m/z 294.2[M+H]，291.2[M-H]。

Example 10: preparation of Compound 2

Compound 2 was prepared using the same procedure as compound 1 except that lactone compound G1 was used instead of lactone compound E1.

Example 11: preparation of Compound 3

Intermediate 1(2.2g, 4.0mmol) was dissolved in anhydrous THF (100ml) and the solution was cooled to 0 ℃ under N2(g) with stirring. A solution of methyl magnesium chloride (4mL, 12mmol) was added and the mixture was stirred overnight. Acetic acid solution (10mL) was added to quench the reaction. Concentrating under reduced pressure. The residue was dissolved in a minimum amount of dichloromethane and subjected to silica gel chromatography eluting with a gradient of 0-75% EtOAc and hexanes to provide intermediate 3 as a mixture of anomers. (1.1g, 49.7%).

LCMS m/z 553.6[M+H].

Intermediate 3(100mg,0.18mmol), Degussa catalyst (0.3g) and acetic acid 30ml were mixed together under N2(g), stirred with hydrogen for 2 hours, filtered to remove the catalyst and the mixture concentrated under reduced pressure. The residue was dissolved in a minimum amount of water and subjected to reverse phase HPLC to give the levorotatory 323mg 46%) and dextrorotatory 323mg 46%).

LCMS m/z 283.6[M+H]，281.6[M-H]。

Example 12: preparation of Compound 4

Compound 4 was prepared using the same procedure as in compound 3, except intermediate 1 of compound 2 was used instead of intermediate 1 of compound 3.

Example 13: preparation of Compound 5

Compound 1(10mg, 0.034mmol) was dissolved in trimethyl phosphate (2mL) and cooled to 0 deg.C, the mixture was stirred under an atmosphere of N2(g), and 1-methylimidazole (0.320mL, 5mmol) was added followed by the chloride C1(0.240mL, 4.4 mmol). The reaction mixture was stirred at 0 ℃ for 2h and then allowed to warm slowly to RT. While monitoring by LC/MS. When LCMS indicated completion, the reaction mixture was treated with water (5mL) and then concentrated under reduced pressure. The residue was dissolved in dichloromethane and subjected to silica gel chromatography eluting with 0-100% EtOAc in hexane. The product fractions were collected and concentrated. The residue was subjected to preparative HPLC to give compound 5(5.8mg 29.4%).

LCMS m/z 563[M+H].

Example 14: preparation of Compound 6

Compound 6 was prepared using the same procedure as compound 5, except that compound 2 was used instead of compound 1.

Example 15: preparation of Compound 7

Compound 7 was prepared using the same procedure as compound 5, except that compound 3 was used instead of compound 1.

Example 16: preparation of Compound 8

Compound 8 was prepared using the same procedure as compound 5, except that compound 4 was used instead of compound 1.

Example 17: preparation of Compound 9

Compound 9 was prepared using the same procedure as compound 5, except chloride B1 was used instead of chloride C1.

Example 18: preparation of Compound 10

Compound 10 was prepared using the same procedure as compound 5, except chloride a1 was used instead of chloride C1.

Example 19: preparation of Compound 11

Compound 1(0.028g,0.096mmol) was dissolved in NMP (1mL) and THF (0.5mL) was added. Tert-butylmagnesium chloride (1.0M solution in THF, 0.054ml, 0.054mmol) was then added at room temperature under an argon atmosphere for 10 minutes before a solution of intermediate D1(27.3mg, 0.054mmol) in THF (0.1ml) was added and the resulting mixture was warmed to 50 ℃ and after 24 hours the resulting residue was purified by preparative liquid phase. Compound 11(11mg 16%) was obtained.

LCMS m/z662[M+H]。

Example 19: preparation of Compound 12

Compound 12 was prepared using the same procedure as compound 11, except that compound 2 was used instead of compound 1.

Example 20: preparation of Compound 13

Compound 13 was prepared using the same procedure as compound 11, except that compound 3 was used instead of compound 1.

Example 21: preparation of Compound 14

Compound 14 was prepared using the same procedure as compound 11, except that compound 4 was used instead of compound 1.

Example 22: preparation of Compound 15

Compound 1(30mg, 0.10mmol) was dissolved in trimethyl phosphate (lmL) and stirred under N2 (g). Phosphorus oxychloride (0.067mL, 0.73mmol) was added and the mixture was stirred for 2 h. The time to > 80% monophosphate formation was determined by analytical ion exchange column monitoring. Tributylamine (0.44mL, 1.85mmol) and triethylammonium pyrophosphate (0.327g0.72mmol) dissolved in anhydrous DMF (1mL) were added as a solution. The reaction mixture was stirred for 20min and then quenched by the addition of a 1N solution of triethylammonium bicarbonate in water (5 mL). The mixture was concentrated under reduced pressure and the residue was redissolved in water. The solution was subjected to ion exchange chromatography to give compound 15(11mg, 20% yield).

LCMS m/z 532[M-H]。

Example 23: preparation of Compound 16

Compound 16 was prepared using the same procedure as compound 15, except that compound 2 was used instead of compound 1.

Example 24: preparation of Compound 17

Compound 17 was prepared using the same procedure as compound 15, except that compound 3 was used instead of compound 1.

Example 25: preparation of Compound 18

Compound 18 was prepared using the same procedure as compound 15, except that compound 4 was used instead of compound 1.

Example 26: antiviral activity

Another embodiment relates to a method of inhibiting a viral infection comprising the step of treating a sample or patient suspected of being in need of such inhibition with a composition herein.

Samples suspected of containing viruses contemplated herein include natural or artificial materials such as living organisms; tissue or cell culture; biological samples such as biological material samples (blood, serum, urine, cerebrospinal fluid, tears, sputum, saliva, tissue samples, etc.); a laboratory sample; food, water or air samples; biological product samples such as cell extracts, particularly recombinant cells that synthesize the desired glycoprotein, and the like. Typically, the sample is suspected of containing an organism that induces a viral infection, often a pathogenic organism such as a tumor virus. The sample may be contained in any medium, including water and organic solvent/water mixtures. Samples include living organisms such as humans, and man-made materials such as cell cultures.

If desired, the antiviral activity of the compound after administration of the composition can be observed by any method, including direct and indirect methods of detecting such activity. Quantitative, qualitative, and semi-quantitative methods of detecting such activity are contemplated. One of the above screening methods is generally used, however, any other method (e.g., observing a physiological property of a living organism) is also suitable.

The antiviral activity of the compounds can be measured using standard screening protocols known. For example, the following general protocol can be used to measure the antiviral activity of a compound.

Antiviral Activity and cytotoxicity assay for respiratory Virus (2019-nCoV)

Activity against 2019-nCoV

Antiviral activity against 2019-nCoV was determined in HEp-2 cells using an infectious cytopathic cytoprotective assay. In this assay, compounds that inhibit viral infection and/or replication produce a cytoprotective effect against virus-induced cell killing, which can be quantified using cell viability reagents. The technique used here is a new improvement of the method described in the published literature (Chapman et al, anti Agents chemither.2007, 51 (9): 3346-53).

HEp-2 cells were obtained from ATCC (Manassas, VI) and maintained in MEM medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells were passaged twice weekly and maintained in the sub-confluent stage. Titrations were performed on the compound test stock 2019-nCoV strain to determine the appropriate dilution of the viral stock to produce the desired cytopathic effect in HEp-2 cells.

For antiviral assays, HEp-2 cells were grown in large cell culture flasks to near confluency but not to complete confluency. Compounds to be tested were pre-diluted in DMSO in 384-well compound dilution plates in a standardized dose response format of 8 or 40 samples/plate. Samples of each test compound serially diluted in three-fold increments were prepared in plates and the test samples were transferred via an acoustic transfer device (Echo, labcell) at 100 nl/well into cell culture assay 384-well plates. Each compound dilution is transferred to a dry assay plate in single or quadruplicate samples, which are stored until the assay is ready to begin. The positive and negative controls were arranged in vertical blocks (1 column) at opposite ends of the plate.

Subsequently, an infectious mixture was prepared using a viral stock with a cell density of 50000/ml at the appropriate dilution previously determined by titration and added via an automated device (uFlow, Biotek) to the assay plate with the compound at 20 uL/well. Each plate contained negative and positive controls (16 replicates each) to generate 0% and 100% virus inhibition standards, respectively. After infection with 2019-nCoV, the assay plates were incubated for 4 days in a cell culture incubator at 37 ℃. After incubation, the Cell viability reagent Cell TiterGIo (Promega, Madison, WI) was added to the assay plates, which were briefly incubated and then the fluorescence readings were measured (Envision, Perkin Elmer) in all assay plates. Percent inhibition of 2019-nCoV-induced cytopathic effects was determined from residual cell viability levels. These numbers were calculated for each test concentration relative to 0% and 100% inhibition controls, and EC was determined by non-linear regression at a concentration that inhibited the 2019-nCoV induced cytopathic effect by 50%₅₀The value is obtained. Various potent anti-2019-nCoV tool compounds were used as positive controls for antiviral activity.

Cytotoxicity assays in HEp-2 cells

Cytotoxicity of test compounds was determined in uninfected HEp-2 cells using cell viability reagents in parallel with antiviral activity in a similar manner to that previously described for other cell types (cihler et al, antibodies Agents Chemother.2008, 52 (2): 655-65). For the measurement of compound cytotoxicity, the same protocol as the determination of antiviral activity was used, except that the cells were not infected with 2019-nCoV. In contrast, uninfected cell mixtures at the same density were added at 20 ul/well to plates containing the pre-diluted compounds, also at 100 nl/well. The assay plates were then incubated for 4 days, followed by cell viability assays using the same CeIITiter Glo reagent addition and measurement of fluorescence readings. Untreated cells and cells treated with 2uM puromycin (Sigma, st. louis, MO) served as 100% and 0% cell viability controls, respectively. Percent cell viability was calculated for each test compound concentration relative to 0% and 100% controls, and CC was determined by non-linear regression at a compound concentration that reduced cell viability by 50%₅₀The value is obtained.

Cytotoxicity assays in MT-4 cells

MT-4 cell lines were obtained from NIH AIDS Research and Reference Reagent Program (Germantown, MD) and cultured in RPMI-1640 medium (Irvine Scientific, Santa Ana, Calif., Cat #9160) supplemented with 10% FBS, 100 units/mL penicillin, 100 units/mL streptomycin, and 2 mMl-glutamine. MT-4 cells were passaged twice weekly to maintain cell densities below 0.6X10⁶cells/mL. Complete RPMI-1640 medium containing 100x concentration of 3-fold serial dilutions of compounds (26nM to 530uM) was inoculated in quadruplicate into black 384-bundle plates. After compound addition, 2x10 was added to each well using a MicroFIo liquid dispenser (BioTek, Winooski, VT)³MT-4 cells and cells were incubated at 37 ℃ in 5% CO₂The cells were incubated in an incubator for 5 days. After incubation, cells were allowed to equilibrate to 25 ℃ and Cell viability was determined by adding 25uL of Cell-Titer Glo viability reagent. The mixture was incubated at 25 ℃ for 10 minutes and the fluorescence signal was quantified on a Victor fluorescence plate reader. CC (challenge collapsar)₅₀The value is defined by the Cell-timer Glo signalThe concentration of compound determined to reduce cell viability by 50%. Data were analyzed using the Pipeline Pilot Plate Data analysis Collection software (version 7.0, Accelrys, San Diego, Calif.). CC (challenge collapsar)₅₀Values were calculated by non-linear regression analysis using the 4-parameter sigmoidal dose-response equation: y ═ bottom + (top-bottom)/(1 +10[ (LogCC)₅₀-X) slope]) Where "top" and "bottom" are fixed at 100% and 0% cell viability, respectively. CC (challenge collapsar)₅₀Values were calculated as mean ± standard deviation of 3 independent experiments.

From the above results, it is understood that the compound EC of the present invention₅₀、HEp-2 CC₅₀、TM-4CC₅₀The concentrations were superior to the control compounds, indicating that the compounds of the invention have greater bacteriostatic ability than the control compounds and are less toxic to normal cells.

2019-nCoV RNP formulation

The 2019-nCoV Ribonucleoprotein (RNP) complex was prepared from a modified method by Mason et al (1). At 7.1 × 10⁴Individual cell/cm²HEp-2 cells were seeded in MEM + 10% Fetal Bovine Serum (FBS) and allowed to stick to the wall overnight at 37 deg.C (5% C02). After attachment, cells were infected with 2019-nCoV in 35mL MEM + 2% FBS. At 20 hours post infection, the medium was replaced with MEM + 2% FBS supplemented with 2ug/mL actinomycin D and returned to 37 ℃ for one hour. The cells were then washed once with PBS and treated with 35mL PBS +250ug/mL lysolecithin for one minute, after which all liquid was aspirated. By scraping them into 1.2mL buffer A [50mM TRIS acetate (pH8.0), 100mM potassium acetate, ImM DTT and 2ug/mL actinomycin D]To collect cells and lysis occurs by repeated passes through an 18 gauge needle (10 times). The cell lysate was placed in ice for 10 minutes and then centrifuged at 2400 for 10 minutes at 4 ℃. The supernatant was removed (S1) and supplemented with 1% Triton X-100 of 600uL buffer B [10mM TRIS acetate (pH8.0), 10mM potassium acetate and 1.5mM MgCl₂]In which the Pellet (PI) is broken by repeated passes through an 18-gauge needle (10 times). The resuspended pellet was placed in ice for 10 minutes and then centrifuged at 2400 for 10 minutes at 4 ℃. The supernatant was removed (S2) and the pellet was disrupted in 600uL of buffer B supplemented with 0.50/deoxycholate and 0.10/Tween 40 (P2). The resuspended pellet was placed in ice for 10 minutes and then centrifuged at 2400 for 10 minutes at 4 ℃. The supernatant (S3) fraction containing the enriched 2019-nCoV RNP complexes was collected and the protein concentration was determined by UV absorbance at 280 nm. Aliquots of 2019-nCoV RNP S3 were stored at-80 ℃.

2019-nCoV RNP test

The transcription reaction was performed in 30uL of reaction buffer [50mM TRIS-acetate (pH8.0), 120mM potassium acetate, 5% glycerol, 4.5mM MgCl₂3mM DTT, 2mM ethylene glycol-bis (2-aminoethyl ether) -tetraacetic acid (EGTA), 50ug/mL BSA, 2.5U Rnasin (Promega), ATP, GTP, UTP, CTP, and 1.5uCi [ a-³²p]NTP(3000Ci/mmol)]Contains 25ug of crude RSV RNP complex. The radiolabeled nucleotides used in the transcription assay were selected to match the nucleotide analogs used to assess 2019-nCoV RNP transcription inhibition. With it K_ⅢThe final concentration of half (ATP 20uM, GTP 12.5uM, UTP 6uM and CTP 2uM) was added with cold competitive NTP. The remaining three nucleotides were added at a final concentration of 100 uM.

To determine whether the nucleotide analogs inhibited the 2019-nCoV RNP transducion, compounds were added using 6-step serial dilutions in 5-fold increments. After incubation at 30 ℃ for 90 min, the RNP reaction was stopped with 350uL Qiagen RLT lysis buffer and RNA was purified using the Qiagen RNeasy 96 kit. Purified RNA was denatured in RNA sample-laden buffer (Sigma) at 65 ℃ for 10min and tested on a 1.20/agarose/MOPS gel containing 2M formaldehyde. The agarose gels were dried and exposed to a Storm phosphor imaging screen and developed using Storm phosphor imaging agent (GE Healthcare). The concentration of compound that reduced total radiolabeled transcript by 50% (IC) was calculated by non-linear regression analysis of two parallel samples₅₀)。

Reference documents: mason, S., Lawetz, C., Gaudette, Y., Do, F., Scuten, E., Lagace, L., Simonaau, B.and Liuzzi, M. (2004) polymerization-dependent scanning assay for respiratory synthetic RNA transaction activity and identification of an inorganic acid Research,32, 4758-.

Examples	IC₅₀/μM
		Compound 1	0.54
Compound 2	0.63
		Compound 3	1.11
Compound 4	1.21
		Compound 5	0.35
Compound 6	0.42
		Compound 7	1.03
Compound 8	1.16
		Compound 9	0.57
Compound 10	0.68
		Compound 11	1.07
Compound 12	0.97
		Compound 13	0.88
Compound 14	0.95
		Compound 15	0.63
Compound 16	0.70
		Compound 17	0.71
Compound 18	0.83
		Control Compounds	1.71

According to the results, the half inhibitory concentration of the compound is better than that of the control compound, which shows that the bacteriostatic ability of the compound is stronger than that of the control compound.

Claims

1. A compound of formula (I), including pharmaceutically acceptable salts, isomers, deuterons, solvates or esters thereof:

(Ⅰ)

wherein:

R₁is composed of

、

、

、

、

、

、

、

、

、

、

、

、

、

、

、

、、

、

、

、、

R₂Is OH, OD or halogen;

R₃is OH or OD;

R₆selected from H,

、

、

And;

R₅is H or R₆Is not H, R₆Is H or R₅Is not H;

wherein:

n is selected from 1,2, 3 and 4;

R₈Selected from phenyl, 1-naphthyl, 2-naphthyl,

And

；

R₉is selected from H and CH₃；

R₁₀Is selected from H or C₁-C₆An alkyl group;

2. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R₄H, CN, methyl, ethyl, ethenyl, ethynyl, azido, guanidino, F, CI, -CH₂CI、-CH₂F、-CHF₂or-CF₃。

3. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R₂Is F.

4. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R₂Is OH.

5. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R₃Is OH.

6. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R₅H, CN, methyl, ethyl, ethenyl, ethynyl, azido, guanidino, F, CI, -CH₂CI、-CH₂F、-CHF₂or-CF₃。

7. A compound according to claim 2, or a pharmaceutically acceptable salt thereof, wherein R₄Is CN.

8. A compound according to claim 2, or a pharmaceutically acceptable salt thereof, wherein R₄Is methyl, ethyl, ethenyl or ethynyl.

9. A compound according to claim 6, or a pharmaceutically acceptable salt thereof, wherein R₅Is CN.

10. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R₆Selected from:

、

、

and;

wherein:

R₇is selected from C₁-C₈Alkyl, -O-C₁-C₈Alkyl, benzyl and-CH₂-C₃-C₆A cycloalkyl group;

R₁₁is selected from C₁-C₈Alkyl, benzyl, C₃-C₆Cycloalkyl and-CH₂-C₃-C₆A cycloalkyl group.

11. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein:

(A) R₇is selected from C₁-C₈An alkyl group; or

(B) R₇Is selected from C₁-C₆An alkyl group; or

(C) R₇Is selected from C₁-C₅An alkyl group; or

(D) R₇Is selected from C₁-C₄An alkyl group; or

(E) R₁₁Is selected from C₁-C₈An alkyl group; or

(F) R₁₁Is selected from C₁-C₄An alkyl group.

12. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, further comprising when R is₂R is F₄Not a methyl group.

13. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R₆Selected from H and:

、

and

。

14. the compound of claim 1, comprising the structure:

。

15. a pharmaceutical formulation comprising a pharmaceutically effective amount of a compound according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or adjuvant.

16. Use of a compound according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a pneumovirinae virus infection or a coronavirus infection in a human.