A pharmaceutical composition for preventing and treating influenza virus and use thereof

Abstract

The present application discloses an anti-influenza virus pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof or a compound of formula (II) or a pharmaceutically acceptable salt thereof, wherein R 1 and R 2 are as defined in the specification. The present application also discloses the use of the pharmaceutical composition in the preparation of a medicament for preventing or treating influenza.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority and benefit to Chinese Patent Application No. 202110250982.X, filed with the State Intellectual Property Office of China on March 8, 2021, which is incorporated herein by reference in its entirety. Technical Field

[0003] This application relates to the field of pharmaceutical chemistry, specifically to a pharmaceutical composition of a class of anti-influenza virus peramivir derivatives and its pharmaceutical uses. Background Technology

[0004] Influenza viruses, due to their high variability and susceptibility to humans, have become a major source of emerging and novel viral infectious diseases, posing a significant challenge to public health systems. Meanwhile, reports of drug resistance to existing antiviral drugs have been increasing in recent years. Against this backdrop, highly effective, high-dose, and novel treatment routes and regimens for influenza virus infections are urgently needed.

[0005] Peramivir (Chinese chemical name: (1S,2S,3R,4R)-3-[(1S)-1-(acetylamino)-2-ethylbutyl]-4-guanidino-2-hydroxycyclopentanecarboxylic acid; English chemical name: (1S,2S,3R,4R)-3-[(1S)-1-(acetylamino)-2-ethylbutyl]-4-(carbamimidoylamino)-2-hydroxycyclopentanecarboxylic acid) is a neuraminidase inhibitor, and its chemical structure is as follows:

[0006]

[0007] Peramivir has been marketed in several countries, including the United States, Japan, and China, for the prevention and treatment of influenza A or B virus infection. Due to its high polarity and low oral bioavailability, currently available peramivir formulations are only available for intravenous administration, resulting in a limited route of administration and failing to meet the increasingly urgent clinical needs.

[0008] Peramivir has low solubility, and its solubility exhibits a pH-dependent trend, increasing as the pH decreases. The solubility at pH 1.0 is approximately 59 mg / ml, while at pH ≥ 5, the solubility is ≤ 20 mg / ml. Currently, the highest concentration of peramivir injection used clinically is only 10 mg / ml. The US FDA-approved peramivir injection strength is 200 mg / 20 ml (10 mg / ml), Japan approves 300 mg / 60 ml (5 mg / ml) and 150 mg / 15 ml (10 mg / ml), and China approves 300 mg / 100 ml (3 mg / ml) and 150 mg / 100 ml (1.5 mg / ml). Based on a clinically used dose of 600 mg, at least 60 ml of solution volume is required. This high volume limits the development, administration methods, and clinical use of peramivir formulations. Furthermore, with the continuous development of drug resistance, the inhibitory effect of influenza virus will weaken at lower drug loads. Summary of the Invention

[0009] On the one hand, this application provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0010]

[0011] in

[0012] R in equation (I) 1 It is a substituted or unsubstituted C2-C6 alkyl group;

[0013] R in equation (II) 2 It is a substituted or unsubstituted C2-C6 alkyl group;

[0014] Optionally, the pharmaceutical composition may further comprise one or more pharmaceutically acceptable excipients.

[0015] In some embodiments of this application, the drug loading concentration of compound (I) or its pharmaceutically acceptable salt or compound (II) or its pharmaceutically acceptable salt is 2.5%-20% by weight / volume, which translates to a specific concentration of 25-200 mg / ml, wherein the weight is based on the weight of compound (I) or compound (II) and the volume is based on the volume of the administration liquid prepared from the pharmaceutical composition.

[0016] In some embodiments of this application, the drug loading concentration of compound (I) or its pharmaceutically acceptable salt or compound (II) or its pharmaceutically acceptable salt is 3%-15% by weight / volume, which translates to a specific concentration of 30-150 mg / ml, wherein the weight is based on the weight of compound (I) or compound (II) and the volume is based on the volume of the administration liquid prepared from the pharmaceutical composition.

[0017] In some embodiments of this application, the drug loading concentration of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the compound of formula (II) or a pharmaceutically acceptable salt thereof, is greater than or equal to 2.5% by weight / volume, for example, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20% by weight / volume.

[0018] In some embodiments of this application, the drug loading concentration of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the compound of formula (II) or a pharmaceutically acceptable salt thereof, is greater than or equal to 25 mg / ml, for example, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 mg / ml.

[0019] In some embodiments of this application, the substituted C2-C6 alkyl group is a C2-C6 alkyl group substituted with one or more substituents selected from hydroxyl, methoxy, ethoxy, amino, acetamido, chlorine, bromine, cyano, and succinimide.

[0020] In some embodiments of this application, R 1 and R 2 Independently ethyl, propyl, butyl, or isopropyl; or, R 1 and R 2 Independently ethyl, isopropyl, or butyl; or, R 1 and R 2 It is independently an ethyl group.

[0021] In some embodiments of this application, the pharmaceutically acceptable salts of the compound of formula (I) and the pharmaceutically acceptable salts of the compound of formula (II) are independently selected from hydrochloride, sulfate, hydrobromide, acetate, methanesulfonate, nitrate, phosphate, maleate, fumarate, tartrate, citrate, succinate, hydroxyethanesulfonate, trifluoroacetate, benzenesulfonate, toluenesulfonate, borate, lactate, benzoate, ascorbate, and salicylate; or, the pharmaceutically acceptable salts of the compound of formula (I) and the pharmaceutically acceptable salts of the compound of formula (II) are independently hydrochloride or sulfate; or, the pharmaceutically acceptable salts of the compound of formula (I) and the pharmaceutically acceptable salts of the compound of formula (II) are hydrochloride, such as monohydrochloride or dihydrochloride; or, the pharmaceutically acceptable salt of the compound of formula (I) is monohydrochloride and the pharmaceutically acceptable salt of the compound of formula (II) is dihydrochloride.

[0022] In some embodiments of this application, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds:

[0023]

[0024]

[0025] In some embodiments of this application, the compound of formula (II) or a pharmaceutically acceptable salt thereof is:

[0026]

[0027] In some embodiments of this application, the pharmaceutical composition is present in the form of an atomized inhalation solution, a sterile dispensed powder, a sterile lyophilized formulation, a sterile aqueous solution, a dry powder inhaler, a spray, and a nasal spray; or, the pharmaceutical composition is present in the form of a sterile dispensed powder or a sterile lyophilized formulation; or, the pharmaceutical composition is present in the form of a sterile lyophilized formulation.

[0028] In some embodiments of this application, the nebulized inhalation solution, sterile aqueous solution, spray, and nasal spray are aqueous formulations; and / or the sterile dispensed powder, sterile lyophilized formulation, and dry powder inhaler are recombined to form an aqueous solution, wherein the pH value of the aqueous solution is 4-8, or the pH value is 6.0-7.5.

[0029] In some embodiments of this application, when the pharmaceutical composition comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof, the solvent in the aqueous formulation and the aqueous solution formed after recombination is selected from water for injection, physiological saline, glucose solution or physiologically acceptable buffer solution; when the pharmaceutical composition comprises a compound of formula (II) or a pharmaceutically acceptable salt thereof, the solvent in the aqueous formulation and the aqueous solution formed after recombination is selected from physiologically acceptable aqueous solutions of inorganic or organic bases.

[0030] In some embodiments of this application, the physiologically acceptable buffer solution is selected from citrate buffer, phosphate buffer, and tromethorphan-hydrochloric acid buffer; and / or the physiologically acceptable inorganic or organic base aqueous solution is selected from one or more of tromethorphan aqueous solution, meglumine aqueous solution, ethylenediamine aqueous solution, lysine aqueous solution, arginine aqueous solution, sodium citrate aqueous solution, citric acid aqueous solution, sodium dihydrogen phosphate aqueous solution, disodium hydrogen phosphate aqueous solution, sodium carbonate aqueous solution, sodium bicarbonate aqueous solution, and sodium hydroxide aqueous solution.

[0031] In some embodiments of this application, the pharmaceutically acceptable excipients are pH adjusters and / or lyophilized excipients.

[0032] In some embodiments of this application, the pH adjuster includes, but is not limited to, one or more of sodium hydroxide, tromethorphan, hydrochloric acid, sodium citrate, citric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, and phosphoric acid.

[0033] In some embodiments of this application, the lyophilization excipients include, but are not limited to, one or more of sodium chloride, glucose, glycine, cysteine, and lysine.

[0034] In some embodiments of this application, the pH adjuster is selected from one or more of sodium hydroxide, hydrochloric acid, sodium citrate, and citric acid.

[0035] In some embodiments of this application, the lyophilization excipient is selected from one or both of sodium chloride and cysteine.

[0036] In some embodiments of this application, the drug composition is administered via intravenous, oral nebulization, nasal, subcutaneous, intradermal, or intramuscular routes.

[0037] In some embodiments of this application, the route of administration of the pharmaceutical composition is oral nebulization.

[0038] In some embodiments of this application, the pharmaceutical composition is administered via oral nebulization, and the drug loading concentration of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the compound of formula (II) or a pharmaceutically acceptable salt thereof, is 27–176 mg / ml, with an administration volume not exceeding 4 ml. In some embodiments of this application, the drug loading concentration of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the compound of formula (II) or a pharmaceutically acceptable salt thereof, is 27–150 mg / ml, with an administration volume not exceeding 2 ml.

[0039] In some embodiments of this application, the pharmaceutical composition comprises one of the following compounds:

[0040]

[0041] And one of citric acid, sodium dihydrogen phosphate, sodium citrate, tromethamine, and sodium hydroxide or hydrochloric acid;

[0042] Optionally, the pharmaceutical composition is present in the form of a sterile aqueous solution or a sterile lyophilized preparation, and / or the concentration of the compound is 100 mg / ml-200 mg / ml, or 160 mg / ml-180 mg / ml, based on the free base of the compound.

[0043] In some embodiments of this application, the pharmaceutical composition comprises one of the following compounds:

[0044]

[0045] The pharmaceutical composition is present in the form of a sterile powder, and contains citric acid and sodium hydroxide or hydrochloric acid;

[0046] Optionally, the resolvating solvent of the pharmaceutical composition comprises sodium citrate and water, as well as sodium hydroxide or hydrochloric acid; and / or the concentration of the compound in the resulting resolvating solution is 40 mg / ml-80 mg / ml, or 40 mg / ml-70 mg / ml, based on the free base of the compound.

[0047] In some embodiments of this application, the pharmaceutical composition comprises

[0048] Optionally, the pharmaceutical composition is present in the form of a sterile aqueous solution or a sterile lyophilized formulation, and / or

[0049] The pharmaceutical composition further comprises one of sodium chloride, cysteine, sodium citrate, or citrate, and sodium hydride or hydrochloric acid; and / or

[0050] The concentration of the compound is 25 mg / ml-180 mg / ml, or 50 mg / ml-150 mg / ml, based on the free base of the compound.

[0051] On the other hand, this application provides the use of the pharmaceutical composition described above in the preparation of a medicament for the prevention or treatment of influenza.

[0052] On the other hand, this application provides the pharmaceutical composition described above for the prevention or treatment of influenza.

[0053] On the other hand, this application provides a method for preventing or treating influenza, including administering an effective amount of the pharmaceutical composition described above to an individual in need.

[0054] On the other hand, this application provides the use of the pharmaceutical composition described above in the prevention or treatment of influenza.

[0055] On the other hand, this application provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a compound of formula (II) or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the prevention or treatment of influenza.

[0056]

[0057] in

[0058] R in equation (I) 1 It is a substituted or unsubstituted C2-C6 alkyl group;

[0059] R in equation (II) 2 It is a substituted or unsubstituted C2-C6 alkyl group.

[0060] On the other hand, this application provides compounds of formula (I) or pharmaceutically acceptable salts thereof for the prevention or treatment of influenza and / or compounds of formula (II) or pharmaceutically acceptable salts thereof.

[0061] On the other hand, this application provides a method for preventing or treating influenza, comprising administering to an individual in need an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and / or a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0062] On the other hand, this application provides the use of compound (I) or a pharmaceutically acceptable salt thereof and / or compound (II) or a pharmaceutically acceptable salt thereof in the prevention or treatment of influenza.

[0063] In some embodiments of this application, the substituted C2-C6 alkyl group is a C2-C6 alkyl group substituted with one or more substituents selected from hydroxyl, methoxy, ethoxy, amino, acetamido, chlorine, bromine, cyano, and succinimide.

[0064] In some embodiments of this application, R 1 and R 2 Independently ethyl, propyl, butyl, or isopropyl; or, R 1 and R 2 Independently ethyl, isopropyl, or butyl; or, R 1 and R 2 It is independently an ethyl group.

[0065] In some embodiments of this application, the pharmaceutically acceptable salts of the compound of formula (I) and the pharmaceutically acceptable salts of the compound of formula (II) are independently selected from hydrochloride, sulfate, hydrobromide, acetate, methanesulfonate, nitrate, phosphate, maleate, fumarate, tartrate, citrate, succinate, hydroxyethanesulfonate, trifluoroacetate, benzenesulfonate, toluenesulfonate, borate, lactate, benzoate, ascorbate, and salicylate; or, the pharmaceutically acceptable salts of the compound of formula (I) and the pharmaceutically acceptable salts of the compound of formula (II) are independently hydrochloride or sulfate; or, the pharmaceutically acceptable salts of the compound of formula (I) and the pharmaceutically acceptable salts of the compound of formula (II) are hydrochloride; or, the pharmaceutically acceptable salt of the compound of formula (I) is a monohydrochloride and the pharmaceutically acceptable salt of the compound of formula (II) is a dihydrochloride.

[0066] In some embodiments of this application, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds:

[0067] In some embodiments of this application, the compound of formula (II) or a pharmaceutically acceptable salt thereof is:

[0068]

[0069] In some embodiments of this application, the influenza (hereinafter referred to as flu) is an acute respiratory infectious disease caused by influenza A or B viruses. Influenza viruses include the H1N1 and H3N2 subtypes of influenza A viruses and the Victoria and Yamagata lineages of influenza B viruses.

[0070] Unless otherwise stated, the following terms as used in this application shall have the following meanings. A particular term should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with its ordinary meaning in the art. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0071] The term "substituted" refers to the substitution of one or more hydrogen atoms on a particular group by a substituent, provided that the valence state of the particular group is normal and the substituted compound is stable.

[0072] The term “optional” or “optionally” means that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0073] The term "alkyl" refers to a compound with the general formula C1. n H 2n+1 The alkyl group. The alkyl group can be straight-chain or branched. For example, the term "C1-C6 alkyl" refers to an alkyl group containing 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, etc.).

[0074] The term "treatment" means administering the compound or preparation described in this application to improve or eliminate a disease or one or more symptoms related to said disease, and includes:

[0075] (i) Suppress the disease or disease state, that is, curb its development;

[0076] (ii) Relieve the disease or disease state, even if the disease or disease state subsides.

[0077] The term “prevention” means administering the compound or formulation described in this application to prevent a disease or one or more symptoms associated with the disease, and includes: preventing the occurrence of a disease or disease state in mammals, particularly when such mammals are susceptible to the disease state but have not yet been diagnosed with the disease state.

[0078] The term "effective amount" means (i) the amount of the compound of this application used to treat or prevent a particular disease, condition, or disorder; (ii) to reduce, improve, or eliminate one or more symptoms of a particular disease, condition, or disorder; or (iii) to prevent or delay the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of the compound of this application constituting a "therapeutic effective amount" varies depending on the compound, the disease state and its severity, the route of administration, and the age of the mammal to be treated, but may routinely be determined by a person skilled in the art based on their own knowledge and the present disclosure.

[0079] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0080] The term "pharmaceutical composition" refers to a mixture of one or more compounds of this application or their salts with pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the compounds of this application to an organism.

[0081] The term "pharmaceuticalally acceptable excipient" refers to those excipients that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound.

[0082] The term "drug loading" refers to the weight of the drug component contained in a pharmaceutical composition. "Drug loading concentration" refers to the ratio of the weight of the drug component in a liquid formulation of a solid pharmaceutical composition to the volume of the liquid, or the ratio of the weight of the drug component in a liquid formulation to the volume of the liquid, wherein the weight is based on the weight of the free base drug and the volume is based on the volume of the liquid formulation. In this application, the weight of the drug component is calculated as the free base of compound (I) or compound (II).

[0083] The word “comprise” or “include” and its English variants such as comprises or comprising should be understood in an open, non-exclusive sense, meaning “including but not limited to”.

[0084] In some embodiments of this application, the pharmaceutical composition may use a compound of formula (II) as an active pharmaceutical ingredient, in addition to using a compound of formula (I) directly as an active pharmaceutical ingredient.

[0085] An aqueous solution of compound (II) or its pharmaceutically acceptable salt thereof will rapidly convert to compound (I) or its pharmaceutically acceptable salt under pH conditions of 4–8. Dissolving compound (II) or its pharmaceutically acceptable salt in water and adjusting the pH to 4–8 will convert it to compound (I) or its pharmaceutically acceptable salt. In fact, compound (II) can be considered an equivalent compound of compound (I). It can be inferred that if compound (II) or its pharmaceutically acceptable salt is administered directly in solid form (e.g., by dry powder inhalation), compound (II) will similarly and rapidly convert to compound (I) in the blood or tissue fluid, having the same effect as a pharmaceutical composition containing compound (I) directly.

[0086]

[0087] According to one aspect of this application, this application provides a method for preparing a pharmaceutical composition of a compound of formula (I) in the form of a sterile powder formulation, a lyophilized formulation, and an aqueous solution. The method includes: when producing a sterile powder formulation, directly dispensing the active pharmaceutical ingredient under sterile conditions; when producing a lyophilized formulation and a sterile aqueous solution, dissolving a compound of formula (I) or a compound of formula (II) or a pharmaceutically acceptable salt thereof in water for injection, adding a pH adjuster, optionally adding a lyophilization excipient, and after complete dissolution, sterile filtration, aseptic filling, and / or lyophilization.

[0088] The pharmaceutical compositions and their various dosage forms disclosed herein can be prepared using conventional methods in the art. For example, conventional mixing, dissolving, grinding, and freeze-drying methods.

[0089] According to one aspect of this application, this application provides a method for preparing the compound of formula (I) or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0090] Peramivir is prepared by adding substances that can create an acidic environment, such as thionyl chloride, hydrogen chloride, hydrogen bromide, and sulfuric acid, to a small molecule fatty alcohol solvent system (including but not limited to methanol, ethanol, propanol, isopropanol, etc.) to convert peramivir into the corresponding short-chain carboxylic acid ester. Then, after neutralization and separation purification, the salt formed by peramivir ester and pharmaceutically acceptable acid radical is obtained.

[0091]

[0092] During the conversion of peramivir to compound (I), partial formation of compound (II) can occur under acidic conditions. Compound (I) can also be converted to compound (II) under strongly acidic conditions. Separating and purifying these compounds yields the salts formed by compound (II) and acid, typically salts formed from diacid anions, as shown below:

[0093]

[0094]

[0095] Where X represents an acid radical, including but not limited to chloride ions, sulfate ions, etc.

[0096] Typical compounds of formula (I) or formula (II) include the following compounds:

[0097]

[0098]

[0099] According to one aspect of this application, the pharmaceutical composition provided by this application, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is converted into the active drug peramivir in vivo; or the compound of formula (II) or a pharmaceutically acceptable salt thereof is rapidly converted into the compound of formula (I) under pH 4 to 8 conditions, and the compound of formula (I) is slowly converted into the active drug peramivir in vivo, thereby exerting a long-term inhibitory effect on influenza virus neuraminidase.

[0100]

[0101] What is particularly encouraging is that, although compound (I) is an ester compound, it also has extremely low tissue permeability, resulting in very low systemic exposure levels after local administration. This characteristic also provides extremely high safety assurance for compound (I) as a drug.

[0102] This application also provides the use of the above-described pharmaceutical composition for the prevention or treatment of influenza.

[0103] This application provides pharmacokinetic characteristics of the compound of formula (I) or its pharmaceutically acceptable salt in rats to illustrate that the compound of formula (I) can be converted into the active drug peramivir in vivo and can be enriched in the lungs, thus having the use of preventing or treating diseases caused by influenza viruses, and that the enrichment of the active drug in the lungs can be achieved by nebulized inhalation.

[0104] In some embodiments of this application, the compound of formula (I) or a pharmaceutically acceptable salt thereof described in this application exhibits unexpected solubility (up to 200 mg / ml) within a suitable pH range for administration, approximately 10 times that of peramivir, and is metabolized in vivo into the active drug peramivir. Formulating the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the compound of formula (II) or a pharmaceutically acceptable salt thereof, into an aqueous formulation with a high drug loading capacity overcomes the disadvantages of volume-restricted administration methods inherent in peramivir formulations.

[0105] In some embodiments of this application, the volume of the injection prepared by this application can be reduced to 3 ml (200 mg / ml). Reducing the volume can improve the sterility assurance level in the production process, while reducing the scale and production costs; it also facilitates the production, transportation and storage process, and is more conducive to ensuring its quality; in clinical use, it can also reduce the infusion volume and time, and improve patient compliance.

[0106] In some embodiments of this application, the pharmaceutical compositions of this application improve antiviral efficacy. For example, in oral inhalation formulations, high-load aqueous formulations can be inhaled at a rate of 50–400 mg (based on a 2 ml inhalation volume), thereby effectively inhibiting the virus and avoiding drug inactivation due to viral resistance, while also improving patient compliance.

[0107] In some embodiments of this application, the pharmaceutical composition and its aqueous formulation also have a long-lasting effect, being absorbed and converted into the active drug peramivir in vivo for at least 8 hours. For example, via oral nebulization, the pharmaceutical composition and its aqueous formulation are absorbed and converted into peramivir in vivo for at least 72 hours. Influenza is a self-limiting disease with a generally short duration. Using the pharmaceutical composition provided in this application, even once a week, can achieve antiviral efficacy, reducing the frequency of administration and greatly improving patient compliance.

[0108] In some embodiments of this application, the pharmaceutical composition and its aqueous formulation also possess safety. In clinical use, the drug is completely dissolved by a water-for-injection system, avoiding the use of excipients such as surfactants, solubilizers, and cosolvents to improve solubility. This also avoids the hemolytic and irritant toxic reactions caused by surfactants and organic solvents.

[0109] In some embodiments of this application, the pharmaceutical composition contains a high concentration of drug and can be administered via multiple routes, such as by nebulized inhalation or by intravenous injection, for the prevention or treatment of diseases caused by influenza viruses. Attached Figure Description

[0110] Figure 1 Showing the X-ray diffraction pattern of compound 7 single crystal;

[0111] Figure 2 The pharmacokinetic curves of SD rats administered via nebulization and injection are shown.

[0112] Figure 3 The pharmacokinetic curves of plasma and lung tissue after nebulized inhalation in SD rats are shown. Detailed Implementation

[0113] The following examples are provided to help understand the content of this application, but do not limit the scope of this application. Experimental methods in the following examples that do not specify specific conditions are generally performed under conventional conditions or conditions recommended by the manufacturer. All reagents used in this application are commercially available and can be used without further purification.

[0114] The peramivir trihydrate (CAS#:1041434-82-5) used in the preparation and formulation examples was purchased from Jiangsu Zhengji Pharmaceutical Co., Ltd.; the D301 resin used refers to the styrene series macroporous weak base anion exchange resin D301 (Styrene Series Macroporous Weak Base Anion Exchange Resin, D301), purchased from Bengbu Liaoyuan New Materials Co., Ltd.

[0115] The chemical purity of the preparation examples was determined by high performance liquid chromatography (HPLC). Chromatographic conditions:

[0116] HPLC Method A:

[0117] Chromatographic column: octadecylsilane-bonded silica gel 250×4.6mm 5μm;

[0118] Column temperature: 35℃;

[0119] Flow rate: 1.0 ml / min;

[0120] Detection wavelength: 200nm

[0121] Mobile phase:

[0122] A: 5 mmol / L potassium dihydrogen phosphate + 10 mmol / L sodium perchlorate + 1% triethylamine (adjust pH to 3.0 with phosphoric acid) / acetonitrile (85 / 15)

[0123] B: Acetonitrile

[0124] Gradient procedure table:

[0125] Time (min) A(％) B(％) 0.01 100 0 5 100 0 20 35 65 30 35 65 31 100 0 40 100 0

[0126] Preparation Example 1: Preparation of (1S,2S,3R,4R)-3-[(1S)-1-(acetamido)-2-ethylbutyl]-4-guanidino-2-hydroxycyclopentanecarboxylic acid methyl ester hydrochloride (Compound 1, peramivir methyl ester hydrochloride)

[0127] Peramivir trihydrate (4.1 g, 10.7 mmol) and anhydrous methanol (40 ml) were added to a reaction flask. SOCl2 (2.6 g, 21.4 mmol) was added dropwise while stirring and cooling in an ice bath. After the addition was complete, the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated to dryness under vacuum, then reconstituted with methanol (40 ml). The pH was adjusted to 7.0–7.5 using D301 resin. The resin was removed by filtration, and the solution was washed with methanol. The filtrate was concentrated to dryness under vacuum at 45 °C. The resulting solid residue was dissolved in methanol (10 ml), and methyl tert-butyl ether (90 ml) was slowly added dropwise while stirring. The solid precipitated, and the mixture was stirred to crystallize overnight. The solution was filtered, and the filter cake was washed with methyl tert-butyl ether. The filter cake was dried under vacuum at 40 °C to obtain 1.1 g of white solid, which was the title compound, with a yield of 27.1%.

[0128] 1H NMR (400MHz, D2O): δ4.40-4.30(m,2H),3.84(m,1H),3.66(s,3H,-OCH3),2.90(m, 1H),2.57(m,1H),2.15(m,1H),1.91(s,3H,-CH3CO),1.77(m,1H),1.50–1.29(m,3H),1.06-0.90(m,2H),0.86(t,3H,-CH3),0.81(t,3H,-CH3).

[0129] ESI(+) m / z: 343.31 [M+H] +

[0130] Preparation Example 2: Preparation of (1S,2S,3R,4R)-3-[(1S)-1-(acetamido)-2-ethylbutyl]-4-guanidino-2-hydroxycyclopentanecarboxylic acid ethyl ester hydrochloride (Compound 2, peramivir ethyl ester hydrochloride)

[0131] Peramivir trihydrate (17.9 g, 46.8 mmol) and anhydrous ethanol (180 ml) were added to a reaction flask. SOCl2 (11.2 g, 94.1 mmol) was added dropwise under stirring and ice bath cooling. After the addition was complete, the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated under vacuum to an oily substance, then dissolved in anhydrous ethanol (500 ml) to obtain an ethanol solution. The pH was adjusted to 7.0–7.5 using D301 type resin (180 ml) that had been washed with ethanol. The resin was filtered off, and the solution was washed with anhydrous ethanol. The filtrate was concentrated to dryness under vacuum at 45 °C to obtain 22.5 g of crude oily product.

[0132] The crude product (22.5 g) was dissolved in anhydrous ethanol (45 ml), and methyl tert-butyl ether (200 ml) was added with stirring. The system began to become turbid, and crystals were stirred overnight. After filtration, the filter cake was washed with methyl tert-butyl ether (15 ml * 2), and the filter cake was dried under vacuum at 40 °C to obtain 14.6 g of white solid, which was the title compound, with a yield of 79.4% and a chemical purity of 98.86% (HPLC-Method A).

[0133] 1 H NMR(400MHz,D2O): δ4.40-4.30(m,2H),4.13(m,2H),3.85(m,1H),2.88(m,1H),2.58(m ,1H),2.14(m,1H),1.90(s,3H,-CH3CO),1.78(m,1H),1.46–1.30(m,3H),1.19(t,3H), 1.06-0.90(m,2H), 0.87(t,3H,-CH3), 0.81(t,3H,-CH3).

[0134] ESI(+) m / z: 357.35 [M+H] +

[0135] Preparation Example 3: Preparation of (1S,2S,3R,4R)-3-[(1S)-1-(acetamido)-2-ethylbutyl]-4-guanidino-2-hydroxycyclopentanecarboxylic acid ethyl ester sulfate (Compound 3, peramivir ethyl ester sulfate)

[0136] Peramivir trihydrate (1.4 g, 3.66 mmol) and anhydrous ethanol (40 ml) were added to a reaction flask, and concentrated sulfuric acid (0.6 g, 6 mmol) was added dropwise with stirring at room temperature. After the system clarified, it was heated to 50 °C and stirred overnight. After cooling to room temperature, the pH was adjusted to 7.0–7.5 using D301 resin washed with ethanol. The resin was filtered off, and the mixture was washed with anhydrous ethanol. The filtrate was concentrated to dryness under vacuum at 45 °C to obtain 1.5 g of crude oil. The crude product was dissolved in anhydrous ethanol (5 ml), and methyl tert-butyl ether (25 ml) was added with stirring. Crystallization was carried out overnight. After filtration, the filter cake was washed with methyl tert-butyl ether (5 ml * 2), and the filter cake was dried under vacuum at 40 °C to obtain 1.0 g of white solid, which was the title compound, with a yield of 67.5% and a chemical purity of 98.25% (HPLC-Method A).

[0137] ESI(+) m / z: 357.35 [M+H] +

[0138] Preparation Example 4: Preparation of (1S,2S,3R,4R)-3-[(1S)-1-(acetamido)-2-ethylbutyl]-4-guanidino-2-hydroxycyclopentanecarboxylic acid isopropyl hydrochloride (Compound 4, peramivir isopropyl hydrochloride)

[0139] Peramivir trihydrate (2.0 g, 5.2 mmol) and isopropanol (20 ml) were added to a reaction flask. SOCl2 (1.3 g, 10.5 mmol) was added dropwise while stirring and cooling in an ice bath. After the addition was complete, the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated under vacuum to dryness to obtain an oily substance, which was then redissolved in isopropanol (20 ml). The pH was adjusted to 7.0–7.5 using D301 resin. The resin was removed by filtration, and the mixture was washed with isopropanol. The filtrate was concentrated under vacuum to dryness at 45 °C to obtain 2.5 g of crude solid. The crude product was dissolved in isopropanol (4 ml), and methyl tert-butyl ether (20 ml) was slowly added dropwise while stirring. The solid precipitated, and the mixture was stirred to crystallize overnight. The mixture was filtered, and the filter cake was washed with methyl tert-butyl ether. The filter cake was dried under vacuum at 40 °C to obtain 0.44 g of white solid, which was the title compound, with a yield of 22.7% and a chemical purity of 98.37% (HPLC-Method A).

[0140] 1 H NMR(400MHz,D2O):δ5.02(m,1H,-C H Me2),4.48-4.37(m,2H),3.93(m,1H),2.93(m, 1H),2.66(m,1H),2.20(m,1H),1.99(s,3H,-CH3CO),1.86(m,1H),1.55–1.38(m,3H),1.27(d, 6H,-CH(C H 3)2),1.14-1.00(m,2H),0.96(t,3H,-CH3),0.89(t,3H,-CH3).

[0141] ESI(+) m / z: 371.36 [M+H] +

[0142] Preparation Example 5: Preparation of (1S,2S,3R,4R)-3-[(1S)-1-(acetamido)-2-ethylbutyl]-4-guanidino-2-hydroxycyclopentanecarboxylic acid propyl ester hydrochloride (Compound 5, peramivir propyl ester hydrochloride)

[0143] Peramivir trihydrate (4.0 g, 10.5 mmol) and n-propanol (40 ml) were added to a reaction flask. SOCl2 (2.5 g, 21.0 mmol) was added dropwise while stirring and cooling in an ice bath. After the addition was complete, the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated to dryness under vacuum to obtain an oily substance. Then, n-propanol (30 ml) was added to redissolve the oil. The pH was adjusted to 7.0–7.5 using D301 resin. The resin was filtered off, and the mixture was washed with n-propanol. The filtrate was concentrated to dryness under vacuum at 45°C. The resulting oily substance was dissolved in n-propanol (10 ml), and methyl tert-butyl ether (90 ml) was slowly added dropwise while stirring. A solid precipitated, and the mixture was stirred to allow crystals to crystallize overnight. The mixture was filtered, and the filter cake was washed with methyl tert-butyl ether. The filter cake was dried under vacuum at 40°C to obtain 0.76 g of white solid, which was the title compound, with a yield of 18.1%.

[0144] 1 H NMR (400MHz, D2O): δ4.41-4.30(m,2H),4.05(m,2H,-OC H 2Et),3.85(m,1H),2.90(m, 1H),2.58(m,1H),2.13(m,1H),1.90(s,3H,-CH3CO),1.79(m,1H),1.60(m,2H,-OCH2C H 2CH3), 1.46-1.30(m,3H),1.05-0.91(m,2H),0.91-0.75(m,9H,3×-CH3).

[0145] ESI(+) m / z: 371.36 [M+H] +

[0146] Preparation Example 6: Preparation of (1S,2S,3R,4R)-3-[(1S)-1-(acetamido)-2-ethylbutyl]-4-guanidino-2-hydroxycyclopentanecarboxylate butyl hydrochloride (Compound 6, peramivir butyl hydrochloride)

[0147] Peramivir trihydrate (2.0 g, 5.2 mmol) and n-butanol (20 ml) were added to a reaction flask. SOCl2 (1.3 g, 10.5 mmol) was added dropwise while stirring and cooling in an ice bath. After the addition was complete, the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated to dryness under vacuum to obtain an oily substance. Then, n-butanol (20 ml) was added to redissolve the oil. The pH was adjusted to 7.0–7.5 using D301 resin. The resin was filtered off, and the mixture was washed with n-butanol. The filtrate was concentrated to dryness under vacuum at 60 °C. The resulting oily substance was dissolved in n-butanol (4 ml), and methyl tert-butyl ether (25 ml) was slowly added dropwise while stirring. A solid precipitated, and the mixture was stirred to allow crystallization to occur overnight. The mixture was filtered, and the filter cake was washed with methyl tert-butyl ether. The filter cake was dried under vacuum at 40 °C to obtain 0.90 g of white solid, which was the title compound, with a yield of 41.1%.

[0148] 1 H NMR (400MHz, D2O): δ4.40-4.30(m,2H),4.10(m,2H,-OC H 2 n Pr),3.85(m,1H),2.90(m, 1H),2.58(m,1H),2.12(m,1H),1.90(s,3H,-CH3CO),1.79(m,1H),1.57(m,2H,-OCH2C H 2Et), 1.47-1.24(m,5H),1.06-0.91(m,2H),0.91-0.75(m,9H,3×-CH3).

[0149] ESI(+) m / z: 385.37 [M+H] +

[0150] Preparation Example 7: Preparation of (1S,2S,3S,4R)-3-[(1S)-1-amino-2-ethylbutyl]-4-guanidino-2-acetoxycyclopentane carboxylic acid ethyl ester dihydrochloride (Compound 7)

[0151] Peramivir trihydrate (2.0 g, 5.2 mmol) was added to a reaction flask, followed by a 2M ethanol solution of hydrogen chloride (2 M, 20 ml). The mixture was stirred at room temperature for 6 hours. The reaction solution was concentrated under vacuum to obtain an oily substance, which was then dissolved by adding isopropanol (20 ml) and heating to reflux. After natural cooling and crystallization for 72 hours, the solution was filtered to obtain needle-like crystals. These crystals were dried under vacuum at 40 °C to obtain 0.45 g of solid, which was the title compound, with a yield of 20.5%. X-ray single-crystal diffraction was performed on suitable crystals using a Rigaku synergy-SX diffractometer.

[0152] 1 H NMR(400MHz,D2O): δ5.36(d,1H),4.23-4.10(m,3H),3.65(m,1H),3.02(m,1H),2.78-2 .64(m,2H),2.08(s,3H,-CH3COO),1.86(m,1H),1.64-1.52(m,1H),1.52-1.39(m,1H), 1.38-1.28(m,1H),1.28-1.16(m,1H),1.20(t,3H),1.15-1.04(m,1H),0.91(t,3H,-CH3),0.83(t,3H,-CH3).

[0153] ESI(+) m / z: 357.35 [M+H] +

[0154] The X-ray single-crystal diffraction data are as follows:

[0155]

[0156]

[0157] The X-ray single-crystal diffraction pattern is attached. Figure 1 .

[0158] Preparation Example 8: Preparation of (1S,2S,3S,4R)-3-[(1S)-1-amino-2-ethylbutyl]-4-guanidino-2-acetoxycyclopentane carboxylic acid ethyl ester dihydrochloride (Compound 7)

[0159] Compound 2 (Preparation Example 2) (10.0 g) and hydrogen chloride ethanol solution (2.6 M, 100 ml) were added to the reaction flask and refluxed for 5 h. After cooling, the reaction solution was concentrated under vacuum to dryness to obtain a bubbly solid. The obtained solid was then dissolved in anhydrous ethanol (10 ml) at 60 °C. 150 ml of isopropanol was added, and the solvent was evaporated under reduced pressure until 30 ml remained. The mixture was stirred overnight at room temperature, filtered, and the filter cake was washed with isopropanol. The solid was dried under vacuum at 40 °C to obtain 4.89 g of solid, which is the title compound, with a yield of 44.7% and a chemical purity of 97.90% (HPLC-Method A). 1 The H NMR and ESI data are the same as those in Preparation Example 7.

[0160] Formulation Example 1: High-drug-loading sterile concentrated solution or lyophilized formulation

[0161] Prescription 1 is peramivir injection (300mg:30ml peramivir), which is a comparative prescription for peramivir with a lower drug loading.

[0162] Prescriptions 2, 4 and 6 are high-drug-loading aqueous solutions or lyophilized preparations (calculated as peramivir, strength 300mg: 2ml); Prescriptions 3, 5 and 7 are high-drug-loading aqueous solutions or lyophilized preparations (calculated as peramivir, strength 600mg: 4ml).

[0163]

[0164]

[0165] Note: "Specifications (in terms of peramivir)" refers to the amount of peramivir added, converted to the weight of anhydrous peramivir.

[0166] "Drug loading concentration (calculated as free alkali)" is the ratio of the weight of the feed amount converted into the weight of free alkali without acid radicals to the volume of the solution after reconstitution.

[0167] The above description also applies to subsequent formulation examples.

[0168] Preparation method: According to the above prescription, pour water for injection into the solution preparation vessel, add the title compound and excipients of the preparation example while stirring, stir until completely dissolved, then adjust the pH of the solution to 7.2 with sodium hydroxide or hydrochloric acid, filter through two stages of 0.22μm sterile filter, and aseptically fill to obtain a sterile aqueous solution; the obtained sterile aqueous solution can be used directly in clinical applications, or it can be further partially stoppered and lyophilized to obtain a lyophilized preparation, which should be reconstituted with water for injection to the concentration before lyophilization before use.

[0169] Formulation Example 2: Dispensing of medium-load sterile powder

[0170]

[0171] Preparation method: According to the above prescription, the sterile powder of the title compound of the preparation example is dispensed into 2ml borosilicate glass vials for injection, and capped to obtain sterile powder; water for injection is poured into the solution preparation bottle, and excipients are added while stirring. The solution is stirred until completely dissolved, and then the pH of the solution is adjusted to 8.5-9.5 with sodium hydroxide or hydrochloric acid. The solution is sterilely filtered, filled with sterile BFS, and sterilized by moist heat at 121℃ for 15min to obtain the reconstituted solvent; before clinical use, the pH value after reconstitution is in the range of 4-8.

[0172] Formulation Example 3: Sterile concentrated solution or lyophilized formulation with medium drug loading

[0173]

[0174]

[0175] Preparation method: According to the above prescription, pour water for injection into the solution preparation vessel, add compound 2 of Preparation Example 2 and the excipients while stirring, stir until completely dissolved, then adjust the pH of the solution to 7.4 with sodium hydroxide or hydrochloric acid, perform two-stage 0.22μm sterile filtration, and aseptically fill to obtain a sterile aqueous solution; the obtained sterile aqueous solution can be used directly in clinical practice, or it can be further partially stoppered and lyophilized to obtain a lyophilized preparation. Before use, reconstitute with water for injection to the concentration before lyophilization.

[0176] Formulation Example 4: Dry Powder Inhaler

[0177]

[0178] Preparation method: Prepare 20,000 capsules according to the above-mentioned dosage. The sterile powder of the title compound from the preparation examples is pulverized using an air jet mill to obtain a dry powder. The dry powder is then filled into hydroxypropyl methylcellulose capsules, each capsule containing 30 mg (calculated as peramivir). The particle size D50 of the dry powder is 1–3 μm, and the D90 is 3–5 μm. For clinical use, the capsules are directly inserted into a dry powder inhaler.

[0179] Compound performance testing

[0180] Test Example 1

[0181] Solubility test

[0182] Prepared sodium chloride hydrochloride solution (pH 1.0), acetate solution (pH 4.5), phosphate solution (pH 6.8), and phosphate solution (pH 7.4) according to the United States Pharmacopeia (USP) 40-NF35. At 37±1℃, excess of the representative compound (I) was added to each of the above pH 1.0, pH 4.5, pH 6.8, pH 7.4, and aqueous media, and the mixture was dispersed by shaking. The shake-flask method was used, and three samples were measured in parallel for each medium. After shaking in a water bath for 24 hours, samples were taken to determine the solubility.

[0183]

[0184]

[0185] As can be seen from the results in the table above, compared with the low solubility of peramivir (solubility of about 59 mg / ml at pH 1.0 and ≤20 mg / ml at pH ≥ 5), the compound of formula (I) and its pharmaceutically acceptable salt provided in this application have good solubility (up to 200 mg / ml) and are suitable for making aqueous formulations with high drug loading of more than 30 mg / ml, with a drug loading of up to 20% by weight / volume.

[0186] Test Example 2

[0187] Compound transformation stability test

[0188] 1. Preparation of buffer solutions with different pH values

[0189] (1) Acetate buffer

[0190] Preparation of 2 mol / L acetic acid solution: Take 120.0 g of glacial acetic acid and dilute it with purified water to 1000 ml.

[0191] Preparation of pH 4.5 acetate buffer: Dissolve 2.99g sodium acetate in purified water, add 14.0ml of 2mol / L acetic acid solution, and dilute with purified water to 1000ml.

[0192] Preparation of pH 5.5 acetate buffer: Dissolve 5.98g sodium acetate in purified water, add 3.0ml of 2mol / L acetic acid solution, and dilute with purified water to 1000ml.

[0193] (2) Phosphate buffer

[0194] Preparation of 0.2 mol / L potassium dihydrogen phosphate solution: Take 27.22 g of potassium dihydrogen phosphate and dilute it with purified water to 1000 ml.

[0195] Preparation of 0.2 mol / L sodium hydroxide solution: Take 8.0 g of sodium hydroxide and dilute it with purified water to 1000 ml.

[0196] Preparation of pH 6.8 acetate buffer: Take 250 ml of 0.2 mol / L potassium dihydrogen phosphate solution, add 112.0 ml of 0.2 mol / L sodium hydroxide solution, and dilute with purified water to 1000 ml.

[0197] Preparation of pH 7.4 acetate buffer: Take 250 ml of 0.2 mol / L potassium dihydrogen phosphate solution, add 195.5 ml of 0.2 mol / L sodium hydroxide solution, and dilute with purified water to 1000 ml.

[0198] Preparation of pH 8.0 acetate buffer: Take 250 ml of 0.2 mol / L potassium dihydrogen phosphate solution, add 230.5 ml of 0.2 mol / L sodium hydroxide solution, and dilute with purified water to 1000 ml.

[0199] 2. Stability Testing Methods

[0200] Accurately weigh 20.0 mg of compound 7 (Preparation Example 8) sample and place it in a 20.0 ml volumetric flask. Dissolve the sample in the above-mentioned pH buffer solutions (pH 4.5, pH 5.5, pH 6.8, pH 7.4, and pH 8.0), and dilute to the mark with the same buffer solution to obtain a 1.0 mg / ml sample solution. Filter the solution using a 0.45 μm nylon filter membrane and collect the filtrate. Inject 15 μl of the sample into the liquid chromatograph at room temperature at corresponding injection times of 5 min, 1 h, 1 h, 2 h, etc., and record the liquid chromatogram (HPLC-Method A). The retention time of compound 7 was 13.2 min, and the retention time of compound 2 was 14.6 min. The results are shown in the table below:

[0201]

[0202]

[0203] The above experimental results indicate that even at room temperature, compound 7 rapidly transforms into compound 2 under physiological pH conditions (6.8–8.0), and it can be predicted that the transformation rate of compound 7 into compound 2 will be even faster at physiological ambient temperature (37°C). Under physiological conditions, compound 7 exhibits characteristics equivalent to or nearly equivalent to compound 2.

[0204] Test Example 3

[0205] Accelerated stability test of the formulation (40℃ / 75%RH)

[0206] Lyophilized formulations (in controlled injection vials) of representative prescriptions, including prescriptions 1, 2, 4, 6, and 18, were placed under accelerated conditions (40°C / 75% RH) and samples were taken for testing at 1 month, 3 months, and 6 months, respectively.

[0207]

[0208] The above test results show that the pH value of the formulations prepared from various peramivir derivatives did not change significantly within 6 months under accelerated conditions (40℃ / 75%RH); the known impurities, other single impurities, and total impurities did not show a significant trend of change compared to 0 months, indicating that the formulations of this application can meet the requirements for long-term storage stability.

[0209] Test Example 4: Pharmacokinetic Study of Plasma and Lung Tissue in SD Rats After Nebulized Inhalation of Different Doses

[0210] Experimental Methods: Fifty-four SD rats (average weight 250g, purchased from Beijing Vital River Laboratory Technology Co., Ltd.) were randomly divided into three groups. Two groups were administered Formulation 17 of Example 3 (dose 12.76mg / kg, peramivir, Group 1, 0.638mg / kg / min inhalation for 20 minutes) and Formulation 19 of Example 3 (dose 57.89mg / kg, peramivir, Group 2, 2.895mg / kg / min inhalation for 20 minutes) via nebulization using a single-concentration small animal oral-nasal exposure inhalation system (Beijing Huironghe Technology Co., Ltd.), respectively. The third group was administered peramivir injection of Example 1 (dose 50mg / kg, concentration 10mg / ml, peramivir, Group 3) intravenously. Blood and lung tissue samples were collected at 0.5h, 1h, 2h, 4h, 6h, and 8h after administration (3 rats per time point). The concentrations of peramivir and compound 2 in plasma and tissues were analyzed by HPLC-MS / MS.

[0211] Experimental results: see the table below.

[0212] Mean concentration data of plasma and lung tissue at each time point (n=3)

[0213]

[0214] Pharmacokinetic characteristic curves are attached. Figure 2 .

[0215] Experimental Conclusions: The above results indicate that different doses of compound 2 nebulized inhalation solution exhibited favorable linear pharmacokinetic characteristics in rats; systemic circulatory exposure and lung tissue exposure were positively correlated with the inhaled dose. Furthermore, it was observed that both compound 2 and peramivir maintained high concentrations in lung tissue for at least 8 hours, but the blood concentrations of both components were relatively low, suggesting that nebulized inhalation of this product provides high local exposure and low systemic exposure, which can be expected to increase antiviral efficacy and reduce systemic adverse reactions.

[0216] Test Example 5: Pharmacokinetic Study of Plasma and Lung Tissue in SD Rats After Nebulized Inhalation

[0217] Experimental Methods: Thirty-six SD rats (approximately 250g each, purchased from Beijing Vital River Laboratory Technology Co., Ltd.) were administered formulation 18 (11.02mg / kg, calculated as peramivir, 0.55mg / kg / min, inhaled for 20 minutes) via nebulization using a single-concentration small animal oral-nasal exposure inhalation system (Beijing Huironghe Technology Co., Ltd.). Blood, alveolar perfusion fluid, and lung tissue samples were collected at 0.5h, 1h, 4h, 8h, 10h, 12h, 14h, 16h, 24h, 36h, 48h, and 72h after administration (3 rats per time point). The concentrations of peramivir and compound 2 in the samples were analyzed using HPLC-MS / MS.

[0218] Experimental results: see the table below.

[0219] Mean plasma and lung tissue concentrations after inhalation administration of prescription 18 (n=3)

[0220]

[0221]

[0222] Comparison of pharmacokinetic parameters in rats administered with nebulized inhalation formulation 18 and intravenous injection formulation 1 (control group)

[0223]

[0224] Note: AUC is ng / mL*h or ng / g*h, Cmax is ng / mL or ng / g.

[0225] Pharmacokinetic characteristic curves are attached. Figure 3 .

[0226] Experimental Conclusion: The above data show that after administration of the nebulized inhalation solution of compound 2 (Formula 18) to rats, the plasma concentration of compound 2 was low, while the alveolar concentration remained high (>1000 ng / g) for more than 36 hours. More surprisingly, the active drug peramivir maintained a concentration above 1000 ng / g for up to 72 hours. In contrast, the peramivir injection group (50 mg / kg) in Test Example 5 could only maintain a lung concentration of 1000 ng / g for no more than 2 hours. Therefore, the composition provided in this application has the advantage of a long in vivo residence time and the ability to maintain an effective active drug concentration for a prolonged period, suggesting a more prolonged antiviral effect in humans.

[0227] Inhaled (I) formulations of compounds exhibit superior pharmacokinetic advantages, with significantly higher local lung concentrations than those administered via injection, and a much lower risk of systemic circulatory exposure compared to direct injection of peramivir.

Claims

1. A pharmaceutical composition comprising a compound of formula (II) or a pharmaceutically acceptable salt thereof, Equation (II) in R in equation (II) 2 It is an unsubstituted C2-C6 alkyl group; and, The pharmaceutical composition also includes one or more pharmaceutically acceptable excipients.

2. The pharmaceutical composition of claim 1, wherein the drug loading concentration of the compound of formula (II) or a pharmaceutically acceptable salt thereof is 2.5%-20% by weight / volume.

3. The pharmaceutical composition of claim 2, wherein the drug loading concentration of the compound of formula (II) or a pharmaceutically acceptable salt thereof is 3%-15% by weight / volume.

4. The pharmaceutical composition according to any one of claims 1-3, wherein R 2 It is independently ethyl, propyl, butyl or isopropyl.

5. The pharmaceutical composition of claim 4, wherein R 2 It can be ethyl, isopropyl, or butyl independently.

6. The pharmaceutical composition of claim 5, wherein R 2 It is independently an ethyl group.

7. The pharmaceutical composition according to any one of claims 1-3 and 5-6, wherein the pharmaceutically acceptable salt of the compound of formula (II) is independently selected from hydrochloride, sulfate, hydrobromide, acetate, methanesulfonate, nitrate, phosphate, maleate, fumarate, tartrate, citrate, succinate, hydroxyethanesulfonate, trifluoroacetate, benzenesulfonate, toluenesulfonate, borate, lactate, benzoate, ascorbate, and salicylate.

8. The pharmaceutical composition of claim 4, wherein the pharmaceutically acceptable salt of the compound of formula (II) is independently selected from hydrochloride, sulfate, hydrobromide, acetate, methanesulfonate, nitrate, phosphate, maleate, fumarate, tartrate, citrate, succinate, hydroxyethanesulfonate, trifluoroacetate, benzenesulfonate, toluenesulfonate, borate, lactate, benzoate, ascorbate, and salicylate.

9. The pharmaceutical composition of claim 7, wherein the pharmaceutically acceptable salt of the compound of formula (II) is independently a hydrochloride or a sulfate.

10. The pharmaceutical composition of claim 9, wherein the pharmaceutically acceptable salt of the compound of formula (II) is a hydrochloride salt.

11. The pharmaceutical composition of claim 10, wherein the pharmaceutically acceptable salt of the compound of formula (II) is a dihydrochloride.

12. The pharmaceutical composition according to any one of claims 1-3, wherein a pharmaceutically acceptable salt of the compound of formula (II) is: 。 13. The pharmaceutical composition according to any one of claims 1-3, 5-6 and 8-11, wherein the pharmaceutical composition is in the form of an atomized inhalation solution, a sterile dispensed powder, a sterile lyophilized formulation, a sterile aqueous solution, a dry powder inhaler, and a spray.

14. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition is in the form of an atomized inhalation solution, a sterile dispensed powder, a sterile lyophilized formulation, a sterile aqueous solution, a dry powder inhaler, and a spray.

15. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is in the form of an atomized inhalation solution, a sterile dispensed powder, a sterile lyophilized formulation, a sterile aqueous solution, a dry powder inhaler, and a spray.

16. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is in the form of an atomized inhalation solution, a sterile dispensed powder, a sterile lyophilized formulation, a sterile aqueous solution, a dry powder inhaler, and a spray.

17. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition is in the form of a sterile dispensed powder or a sterile lyophilized formulation.

18. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition is in the form of a sterile lyophilized formulation.

19. The pharmaceutical composition of claim 13, wherein the spray is a nasal spray.

20. The pharmaceutical composition of claim 13, wherein the nebulized inhalation solution, sterile aqueous solution, and spray are aqueous formulations; and / or the sterile dispensed powder, sterile lyophilized formulation, and dry powder inhaler are recombined to form an aqueous solution, wherein the pH of the aqueous solution is 4-8.

21. The pharmaceutical composition of any one of claims 14-16, wherein the nebulized inhalation solution, sterile aqueous solution, and spray are aqueous formulations; and / or the sterile dispensed powder, sterile lyophilized formulation, and dry powder inhaler are recombined to form an aqueous solution, wherein the pH of the aqueous solution is 4-8.

22. The pharmaceutical composition of claim 20, wherein the pH of the aqueous solution is 6.0-7.

5.

23. The pharmaceutical composition of claim 21, wherein the pH of the aqueous solution is 6.0-7.

5.

24. The pharmaceutical composition of claim 20, wherein the spray is a nasal spray.

25. The pharmaceutical composition of claim 21, wherein the spray is a nasal spray.

26. The pharmaceutical composition of claim 20, wherein the solvent in the aqueous formulation and the aqueous solution formed after recombination is selected from physiologically acceptable aqueous solutions of inorganic or organic bases.

27. The pharmaceutical composition of claim 21, wherein the solvent in the aqueous formulation and the aqueous solution formed after recombination is selected from physiologically acceptable aqueous solutions of inorganic or organic bases.

28. The pharmaceutical composition of claim 26 or 27, wherein the physiologically acceptable aqueous inorganic or organic base is selected from one or more of the following: aqueous tromethamine, aqueous meglumine, aqueous ethylenediamine, aqueous lysine, aqueous arginine, aqueous sodium citrate, aqueous citric acid, aqueous sodium dihydrogen phosphate, aqueous disodium hydrogen phosphate, aqueous sodium carbonate, aqueous sodium bicarbonate, and aqueous sodium hydroxide.

29. The pharmaceutical composition of any one of claims 1-3, 5-6, 8-11, 14-20 and 22-27, wherein the pharmaceutically acceptable excipient is a pH adjuster and / or a lyophilized excipient.

30. The pharmaceutical composition of claim 4, wherein the pharmaceutically acceptable excipient is a pH adjuster and / or a lyophilized excipient.

31. The pharmaceutical composition of claim 7, wherein the pharmaceutically acceptable excipient is a pH adjuster and / or a lyophilized excipient.

32. The pharmaceutical composition of claim 12, wherein the pharmaceutically acceptable excipient is a pH adjuster and / or a lyophilized excipient.

33. The pharmaceutical composition of claim 13, wherein the pharmaceutically acceptable excipient is a pH adjuster and / or a lyophilized excipient.

34. The pharmaceutical composition of claim 21, wherein the pharmaceutically acceptable excipient is a pH adjuster and / or a lyophilized excipient.

35. The pharmaceutical composition of claim 28, wherein the pharmaceutically acceptable excipient is a pH adjuster and / or a lyophilized excipient.

36. The pharmaceutical composition of claim 29, wherein the pH adjuster is selected from one or more of sodium hydroxide, tromethorphan, hydrochloric acid, sodium citrate, citric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, and phosphoric acid; and / or the lyophilization excipient is selected from one or more of sodium chloride, glucose, glycine, cysteine, and lysine.

37. The pharmaceutical composition according to any one of claims 30-35, wherein the pH adjuster is selected from one or more of sodium hydroxide, tromethorphan, hydrochloric acid, sodium citrate, citric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, and phosphoric acid; and / or the lyophilization excipient is selected from one or more of sodium chloride, glucose, glycine, cysteine, and lysine.

38. The pharmaceutical composition of claim 36, wherein the pH adjuster is selected from one or more of sodium hydroxide, hydrochloric acid, sodium citrate, and citric acid; and / or the lyophilization excipient is selected from one or two of sodium chloride and cysteine.

39. The pharmaceutical composition according to any one of claims 1-3, 5-6, 8-11, 14-20, 22-27, 30-36 and 38, wherein the pharmaceutical composition is administered via intravenous, oral nebulization, nasal, subcutaneous, intradermal or intramuscular route.

40. The pharmaceutical composition of claim 4, wherein the route of administration of the pharmaceutical composition is intravenous, oral nebulization, nasal administration, subcutaneous administration, intradermal administration, or intramuscular administration.

41. The pharmaceutical composition of claim 7, wherein the route of administration of the pharmaceutical composition is intravenous, oral nebulization, nasal administration, subcutaneous administration, intradermal administration, or intramuscular administration.

42. The pharmaceutical composition of claim 12, wherein the route of administration of the pharmaceutical composition is intravenous, oral nebulization, nasal administration, subcutaneous administration, intradermal administration, or intramuscular administration.

43. The pharmaceutical composition of claim 13, wherein the route of administration of the pharmaceutical composition is intravenous, oral nebulization, nasal administration, subcutaneous administration, intradermal administration, or intramuscular administration.

44. The pharmaceutical composition of claim 21, wherein the route of administration of the pharmaceutical composition is intravenous, oral nebulization, nasal administration, subcutaneous administration, intradermal administration, or intramuscular administration.

45. The pharmaceutical composition of claim 28, wherein the route of administration of the pharmaceutical composition is intravenous, oral nebulization, nasal administration, subcutaneous administration, intradermal administration, or intramuscular administration.

46. ​​The pharmaceutical composition of claim 29, wherein the route of administration of the pharmaceutical composition is intravenous, oral nebulization, nasal administration, subcutaneous administration, intradermal administration, or intramuscular administration.

47. The pharmaceutical composition of claim 37, wherein the route of administration of the pharmaceutical composition is intravenous, oral nebulization, nasal administration, subcutaneous administration, intradermal administration, or intramuscular administration.

48. The pharmaceutical composition of claim 39, wherein the route of administration of the pharmaceutical composition is oral nebulization.

49. The pharmaceutical composition of claim 39, wherein the route of administration is oral nebulization, and the drug loading concentration of the compound of formula (II) or a pharmaceutically acceptable salt thereof is 27-176 mg / ml, and the administration volume is not greater than 4 ml.

50. The pharmaceutical composition according to any one of claims 40-48, wherein the route of administration of the pharmaceutical composition is oral nebulization, and the drug loading concentration of the compound of formula (II) or a pharmaceutically acceptable salt thereof is 27-176 mg / ml, and the administration volume is not greater than 4 ml.

51. The pharmaceutical composition of claim 49, wherein the route of administration of the pharmaceutical composition is oral nebulization, and the drug loading concentration of the compound of formula (II) or a pharmaceutically acceptable salt thereof is 27-150 mg / ml, and the administration volume is not greater than 2 ml.

52. The pharmaceutical composition of claim 50, wherein the route of administration of the pharmaceutical composition is oral nebulization, and the drug loading concentration of the compound of formula (II) or a pharmaceutically acceptable salt thereof is 27-150 mg / ml, and the administration volume is not greater than 2 ml.

53. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition comprises the following compounds: ， The pharmaceutical composition is present in sterile powder form, and The resolvation solvent of the pharmaceutical composition comprises sodium citrate and water, as well as sodium hydroxide or hydrochloric acid; and / or the concentration of the compound in the resulting resolvation solution is 40 mg / ml to 80 mg / ml, based on the free base of the compound.

54. The pharmaceutical composition of claim 53, wherein the concentration of the compound in the resulting reconstituted solution is 40 mg / ml to 70 mg / ml, based on the free base of the compound.

55. Use of the pharmaceutical composition of any one of claims 1-54 in the preparation of a medicament for the prevention or treatment of influenza.

56. Use of a compound of formula (II) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of influenza. Equation (II) in R in equation (II) 2 It is an unsubstituted C2-C6 alkyl group.

57. The use as described in claim 56, wherein R 2 It is independently ethyl, propyl, butyl or isopropyl.

58. The use as described in claim 57, wherein R 2 It can be ethyl, isopropyl, or butyl independently.

59. The use as described in claim 58, wherein R 2 It is independently an ethyl group.

60. The use according to any one of claims 56-59, wherein the pharmaceutically acceptable salt of the compound of formula (II) is independently selected from hydrochloride, sulfate, hydrobromide, acetate, methanesulfonate, nitrate, phosphate, maleate, fumarate, tartrate, citrate, succinate, hydroxyethanesulfonate, trifluoroacetate, benzenesulfonate, toluenesulfonate, borate, lactate, benzoate, ascorbate, and salicylate.

61. The use as described in claim 60, wherein the pharmaceutically acceptable salt of the compound of formula (II) is independently a hydrochloride or a sulfate.

62. The use as described in claim 61, wherein the pharmaceutically acceptable salt of the compound of formula (II) is a hydrochloride salt.

63. The use as described in claim 62, wherein the pharmaceutically acceptable salt of the compound of formula (II) is a dihydrochloride.

64. The use as described in claim 56, wherein the pharmaceutically acceptable salt of the compound of formula (II) is: 。 65. The use as described in any one of claims 55-56, wherein the influenza is common influenza, influenza A, or influenza B caused by an influenza virus.

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