RSV virus inhibitor

Abstract

The invention relates to a compound of an RSV (Respiratory Syndrome Virus) inhibitor and application of the compound in preventing and treating diseases caused by RSV infection.

Description

Technical Field

[0001] The present invention belongs to the field of chemical drugs, and particularly relates to a compound having inhibitory activity against RSV virus, and the application of the compound in RSV virus infection. Background Art

[0002] Respiratory syncytial virus (RSV) is an RNA virus with a genome length of about 15.2 kb, containing 10 genes and encoding 11 proteins. Among them, the adhesion protein G and fusion protein F embedded in the virus envelope play important roles in the virus entry process. When RSV infects host cells, it first adheres to the surface of host cells under the adsorption of G protein, and then, under the fusion mediated by F protein, fuses with host cells, enabling the RSV genome to enter the cells and cause infection. Viral membrane fusion inhibitors play a role in the early stage of virus replication by blocking virus entry into target cells, and have obvious advantages in treatment and prevention.

[0003] RSV shows seasonal epidemics, can be transmitted through the respiratory tract and close contact, mainly infects infants and young children, and causes acute respiratory infections, pneumonia, and even death.

[0004] RSV also infects adults. In this population, RSV mainly causes upper respiratory tract diseases, but also occurs in elderly patients, as well as immunosuppressed adults, especially bone marrow transplant patients. Other at-risk patients include patients with congestive heart failure and patients with chronic obstructive pulmonary disease (i.e., COPD).

[0005] Therefore, RSV-related respiratory infections have become one of the major public health problems worldwide, bringing a great burden to the healthcare systems of countries around the world. Effective prevention and treatment are the keys to controlling RSV infection. However, although people have started developing RSV vaccines and therapeutic drugs since the 1960s, unfortunately, so far, only one RSV vaccine was approved by the FDA for marketing on May 3, 2023, and antiviral therapeutic drugs for RSV infection are very limited. Among them, the two drugs ribavirin and palivizumab are greatly restricted in their clinical applications due to inherent defects such as large side effects and high prices. Clinically, there is an urgent need for a safe and effective treatment plan for RSV infection. Therefore, it is particularly important to develop effective drugs against RSV infection. Summary of the Invention

[0006] The object of the present invention is to provide a novel compound having inhibitory activity against RSV virus, or its stereoisomer or pharmaceutically acceptable salt, and the compound has the following structure:

[0007]

[0008] X is selected from hydrogen or halogen;

[0009] R1 is selected from hydroxyl, halogen, carbonyl, carbonyloxy, alkylcarbonyl, alkoxy, formyl, carboxyl, amino or substituted amino, azide, optionally substituted 4- to 7-membered cycloalkyl, optionally substituted 4- to 7-membered heterocycloalkyl, optionally substituted 4- to 7-membered aromatic ring group, optionally substituted 4- to 7-membered heteroaromatic ring group;

[0010] n is 0, 1, 2 or 3;

[0011] R2 is selected from halogen, hydroxyl, carboxyl, amino, azide or cyano.

[0012] In a specific embodiment, the present invention has a compound represented by the following structural formula

[0013]

[0014] X is selected from hydrogen or halogen;

[0015] R3 is selected from optionally substituted 4- to 7-membered cycloalkyl, optionally substituted 4- to 7-membered heterocycloalkyl, optionally substituted 4- to 7-membered aromatic ring group, optionally substituted 4- to 7-membered heteroaromatic ring group; preferably pyridyl or phenyl;

[0016] n is 0, 1 or 2;

[0017] R2 is selected from azide or cyano.

[0018] In a specific embodiment, the present invention is a compound represented by the following structural formula

[0019]

[0020] X is selected from hydrogen or halogen;

[0021] R3 is pyridyl or phenyl;

[0022] R2 is selected from azide or cyano.

[0023] Specifically, the present invention relates to a compound having the following structure, or a stereoisomer or pharmaceutically acceptable salt thereof:

[0024]

[0025]

[0026]

[0027] The present invention also relates to a pharmaceutical composition comprising the compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0028] The pharmaceutical compositions of the disclosure of the present invention can be administered orally, parenterally or by implantation of a depot. As used again, parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal and injection or infusion techniques in the area of injury.

[0029] The pharmaceutical composition can be in the form of a sterile injectable preparation, for example, in the form of a sterile injectable aqueous or oily suspension. Such suspensions can be formulated using suitable dispersing or wetting agents and suspending agents according to techniques known in the art. Details of the preparation of these compounds are known to those skilled in the art.

[0030] When administered orally, the pharmaceutical compositions of the disclosure of the present invention can be administered in any orally acceptable dosage form, including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of oral use of tablets, carriers commonly used include lactose and corn starch. Lubricants such as magnesium stearate can also be added. For oral administration in the form of capsules, useful carriers / diluents include lactose, high and low molecular weight polyethylene glycols, and dry corn starch. When an aqueous suspension is administered orally, the active ingredient is mixed with emulsifying and suspending agents. If desired, certain sweetening and / or flavoring and / or coloring agents can be added.

[0031] Other suitable carriers for the above compositions can be found in standard pharmaceutical textbooks, such as in "Remington’s Pharmaceutical Sciences", 19th ed., Mack Publishing Company, Easton, Penn., 1995. Those skilled in the art know more details about the design and preparation of suitable delivery forms of the pharmaceutical compositions of the disclosure.

[0032] In the present invention, in addition to comprising the compound of the present invention or a pharmaceutically acceptable salt thereof, other anti-RSV virus compounds can also be comprised.

[0033] When used for preventing and / or treating RSV virus, the dosage levels of the compounds in the disclosure of the present invention are typically from about 1 to about 500 milligrams per kilogram (mg / kg) of body weight per day, and more specifically, from about 1 to about 50 mg / kg of body weight per day. Typically, the pharmaceutical compositions in the disclosure of the present invention can be administered about 1 to about 3 times a day, preferably once before or after the occurrence of RSV. Or administered in the form of continuous infusion, and such administration can be used as a chronic or acute therapy. The amount of active ingredient that can be mixed with the carrier material to prepare a single dosage form will vary depending on the host being treated and the specific mode of administration.

[0034] On the other hand, the present invention relates to an article or kit, comprising a container and a package insert, wherein the container contains a compound having the structure described in the present invention, its isomers or pharmaceutically acceptable salts, or a composition containing the compound having the structure described in the present invention, and the package insert carries instructions for use of the drug. In a preferred embodiment, the article or kit further comprises one or more containers, which contain one or more other antiviral drugs for preventing or treating RSV virus infection, such as ribavirin, interferon, etc.

[0035] Term definitions:

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0037] The term "unsubstituted", when used to define a certain group, means that the defined group is not substituted by any other group except a hydrogen atom, and at this time the certain group has the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For example, an unsubstituted heterocyclic group means that the hydrogen atoms on the heterocycle are not substituted by any other group, such as furan, pyridine, dihydropyridine, etc.

[0038] The term "substituted", when used to define a certain group, means that one, two, three or more hydrogen atoms on the defined group are substituted by substituents. One, two, three or more hydrogen atoms can be hydrogen atoms on the same carbon (or nitrogen) atom, or hydrogen atoms on different carbons (or nitrogens). At this time, the meaning of the certain group should be understood in combination with the substituents. In the present invention, unless otherwise specified, when referring to "substituted", it means that the hydrogen atoms in the group defined by it are substituted by one, two, three or more substituents selected from the following:

[0039] Cyano, halogen, hydroxyl, carboxyl, ester group, amide group, sulfonamide group, amino group, formyl, lower alkyl, lower hydrocarbon group, lower alkynyl, halogenated lower alkyl, hydroxyl-substituted lower alkyl, cycloalkyl, aryl, heterocyclic group, aryl lower alkyl, heterocyclic group lower alkyl, lower alkyloxy, halogenated lower alkyloxy, lower alkyloxy lower alkyl, lower alkyloxy lower alkyloxy, lower alkylcarbonyl, lower alkyloxycarbonyl, lower alkylamino, lower alkylcarbonylamino, lower alkylaminocarbonyl, lower alkylsulfonyl, lower alkylsulfonylamino, carboxy lower alkyloxy, aryl lower alkyloxy.

[0040] When it comes to specific naming, the substituent is usually placed before the group to be substituted. For example, "carboxymethyloxy" means that the oxy group is substituted by a methylene group, and the methylene group is further substituted by a carboxyl group. Its structure can be represented as:

[0041]

[0042] The term "stereoisomer" refers to isomers generated by different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers, and conformational isomers. All stereoisomers fall within the scope of the present invention. The compounds of the present invention can be individual stereoisomers or mixtures of other isomers, such as racemates, or mixtures of all other stereoisomers.

[0043] The term "salt" refers to a pharmaceutically acceptable salt formed by the compounds of the present invention and an acid, which can be an organic or inorganic salt, such as selected from: hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, fumaric acid, citric acid, maleic acid, benzenesulfonic acid, sulfonic acid, malic acid, succinic acid, lactic acid, acetic acid, malonic acid, etc.

[0044] The term "hydrocarbon group" includes an alkyl group or a hydrocarbon group (such as an alkenyl group or an alkynyl group).

[0045] Alkyl refers to a saturated substituent composed of carbon and hydrogen that is straight-chain, branched-chain, or cyclic. Lower alkyl refers to an alkyl group composed of 1-6 carbon atoms. Alkenyl refers to an unsaturated substituent composed of carbon and hydrogen that is straight-chain, branched-chain, or cyclic. Lower alkenyl refers to an alkenyl group composed of 1-6 carbon atoms; alkynyl refers to an unsaturated substituent composed of carbon and hydrogen that is straight-chain or branched-chain. Lower alkynyl refers to an alkynyl group composed of 1-6 carbon atoms. Substituted alkyl (or substituted hydrocarbon group) means that one or more hydrogen atoms on the alkyl group are substituted by other groups such as halogen, hydroxyl, carboxyl, cyano, cycloalkyl, aryl, heteroaryl, oxo, heterocyclic alkoxy, etc.

[0046] The term "cycloalkyl" refers to a saturated or unsaturated monocyclic hydrocarbon group, generally containing 3-20 carbon atoms. The cycloalkyl group can be monocyclic, or can be spirocyclic, bridged cyclic, fused cyclic, or annulated cyclic.

[0047] The term "aryl" includes not only carbocyclic aryl but also heterocyclic aryl. Carbocyclic aryl refers to a 6- to 10-membered all-carbon ring or polycyclic aromatic group, including phenyl, naphthyl, biphenyl, etc., and the carbocyclic aryl can also be substituted or unsubstituted. Heterocyclic aryl refers to a heteroaromatic system group containing at least one heteroatom, including monocyclic heterocyclic aryl or fused heterocyclic aryl, and the heteroatom is selected from oxygen, sulfur or nitrogen, including but not limited to furan, thiophene, pyrrole, thiazole, etc., and the heteroaryl can be substituted or unsubstituted.

[0048] The term "heterocyclic group" refers to a cycloalkyl saturated or unsaturated monocyclic hydrocarbon group containing at least one heteroatom Detailed implementation mode

[0049] The following lists the general manufacturing methods of the compounds of the present invention. In addition, extraction, purification, etc. only need to be processed in the usual organic chemistry experiments.

[0050] The synthesis of the compounds of the present invention can be carried out with reference to the steps known in the art.

[0051] The starting compounds can be commercially available compounds, the compounds described in this specification, the compounds described in the documents cited in this specification, and other known compounds.

[0052] In the compounds of the present invention, tautomers may exist, and the present invention includes these compounds, including all possible isomers and their mixtures.

[0053] When the salts of the compounds of the present invention are to be obtained, the compounds of the present invention can be prepared in a suitable salt form.

[0054] The meanings of each abbreviation

[0055] MsCl: Methanesulfonyl chloride

[0056] Et3N: Triethylamine

[0057] DCM: Dichloromethane

[0058] EtOAc: Ethyl acetate

[0059] NaCN: Sodium cyanide

[0060] NaN3: Sodium azide

[0061] 18-crown-6: 18-Crown-6

[0062] DMSO: Dimethyl sulfoxide

[0063] Na2CO3: Sodium carbonate

[0064] 1,4-dioxane: 1,4-Dioxane

[0065] PdCl2(PPh3)2: Bis(triphenylphosphine)palladium(II) chloride

[0066] MeOH: Methanol

[0067] DMF: N,N - Dimethylformamide

[0068] HATU: 2-(7 - Azabenzotriazol - 1 - yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0069] DIPEA: N,N - Diisopropylethylamine

[0070] Example 1: Synthesis of the following compound

[0071]

[0072]

[0073] Synthesis of Compound 1

[0074]

[0075] In a 500 mL eggplant - shaped flask, 3-(4 - bromophenoxy)propan - 1 - ol (15.0 g, 64.9 mmol) was dissolved in 120 mL of dichloromethane. Then, triethylamine (7.2 g, 71.4 mmol) was added. The temperature was cooled to 0 °C in an ice bath. While maintaining 0 °C, a dichloromethane solution of MsCl (14.1 g, 68.1 mmol in 30 mL DCM) was added dropwise. The addition was completed in 30 min. Then, the temperature was allowed to rise to room temperature naturally, and the reaction was carried out overnight. 150 mL of water was added to quench the reaction. After liquid - liquid separation, the organic phase was washed with water (100 mL × 1) and saturated brine (50 mL × 1), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was separated by normal - phase column chromatography and eluted with a DCM / EtOAc system. The eluate was concentrated under reduced pressure to obtain the target compound (off - white solid, 19.1 g, 95.1%).

[0076] ESI(310.0, 312.0, [M + H] + ), 1 1H - NMR(400 MHz, CDCl3): δ ppm 7.40(d, 2H), 6.82(d, 2H), 4.42(t, 2H), 4.13(t, 2H), 3.22(s, 3H), 2.02(m, 2H).

[0077]

[0078] A similar operation process was applied to the synthesis of 3-(4 - bromo - 2 - fluorophenoxy)propyl methanesulfonate, and an off - white solid product of 8.5 g (96.3%) was obtained.

[0079] ESI(327.0, 329.0, [M+H] + ), 1 H-NMR(400 MHz, CDCl3): δ ppm 7.40 (m, 1H), 7.36 (m, 1H), 6.78 (m, 1H), 4.39 (t, 2H), 4.12 (t, 2H), 3.22 (s, 3H), 2.11 (m, 2H).

[0080]

[0081] 3-(4-Bromophenoxy)propyl methanesulfonate (3.4 g, 10.9 mmol), DMSO (20 mL), 18-crown-6 (0.3 g) and NaCN (1.60 g, 32.6 mmol) were added to a 100 mL eggplant-shaped flask. The reaction was carried out overnight at 75 °C. After cooling, it was poured into 200 mL of deionized water. The insoluble matter was collected by suction filtration and then purified by normal-phase column chromatography, eluted with DCM / EtOAc to obtain a off-white solid (1.68 g, 64.3%).

[0082] ESI(240.0, 242.0, [M+H] + ), 1 H-NMR(400 MHz, CDCl3): δ ppm 7.38 (d, 2H), 6.90 (d, 2H), 4.11 (t, 2H), 2.21 (m, 2H), 1.98 (m, 2H).

[0083] Similar operations were applied to

[0084]

[0085] an off-white solid, 1.72 g, 61.5%.

[0086] ESI(258.0, 260.0, [M+H] + ), 1 H-NMR(400 MHz, CDCl3): δ ppm 7.36 (m, 2H), 6.78 (m, 1H), 4.00 (t, 2H), 1.96 - 2.10 (m, 4H).

[0087]

[0088] Add 3-(4-bromophenoxy)propyl methanesulfonate (4.1 g, 13.3 mmol), DMSO (30 mL), 18-crown-6 (0.4 g) and NaN3 (1.04 g, 16.0 mmol) into a 100 mL eggplant-shaped flask. React overnight at 50 °C. After cooling, pour it into 300 mL of deionized water. Filter to collect the insoluble matter, wash the filter cake with 100 mL of purified water, and then purify by normal-phase column chromatography, eluting with DCM / EtOAc to obtain an off-white solid (2.67 g, 78.6%).

[0089] ESI(256.0,258.0,[M+H] + ), 1 H-NMR(400MHz,CDCl3):δppm 7.38(d,2H),6.90(d,2H),4.11(t,2H),2.21(m,2H),1.98(m,2H).

[0090] Similar operations were applied to

[0091]

[0092] an off-white solid, 2.47 g, 80.2%.

[0093] ESI(274.0,276.0,[M+H] + ), 1 H-NMR(400MHz,CDCl3):δppm 7.35(m,2H),6.84(m,1H),4.08(t,2H),1.75(m,2H),1.53(m,2H).

[0094]

[0095] Add 4-(4-bromophenoxy)butyronitrile (1.0 g, 4.2 mmol), (4-aminothiophen-2-yl)boronic acid (655 mg, 4.6 mmol), sodium carbonate (801 mg, 7.6 mmol), dioxane / water (20 mL / 1 mL) and PdCl2(PPh3)2 (295 mg, 0.42 mmol) into a 50 mL eggplant-shaped flask. React under reflux for 6 h. Then concentrate under reduced pressure. Purify the residue by column chromatography, eluting with a DCM / MeOH system to obtain a pale yellow solid (470 mg, 43.4%).

[0096] ESI(369.3,[M+H] + ), 1H-NMR (400 MHz, CDCl3): δ ppm 8.42 (s, 1H), 7.55 (d, 2H), 6.94 (d, 2H), 6.27 (s, 1H), 4.28 (brs, 2H), 4.12 (t, 2H), 2.12 (m, 2H), 1.85 (t, 2H).

[0097] Similar operations are applied to

[0098]

[0099] Pale yellow solid (342 mg, 38.6%)

[0100] ESI (277.1, [M+H] + ), 1 H-NMR (400 MHz, CDCl3): δ ppm 8.40 (s, 1H), 7.50 (m, 2H), 7.24 (m, 1H), 6.30 (s, 1H), 4.57 (brs, 2H), 4.03 (t, 2H), 2.10 (m, 2H), 1.88 (t, 2H).

[0101]

[0102] Pale yellow solid (408 mg, 41.5%)

[0103] ESI (275.2, [M+H] + ), 1 H-NMR (400 MHz, CDCl3): δ ppm 8.40 (s, 1H), 7.51 (m, 2H), 7.24 (m, 1H), 6.30 (s, 1H), 4.57 (brs, 2H), 4.03 (t, 2H), 1.72 (m, 2H), 1.47 (t, 2H).

[0104]

[0105] Pale yellow solid (277 mg, 29.8%)

[0106] ESI (293.2, [M+H] + ), 1 H-NMR (400 MHz, CDCl3): δ ppm 8.25 (s, 1H), 7.50 (m, 2H), 7.28 (m, 1H), 6.41 (s, 1H), 4.77 (brs, 2H), 4.14 (t, 2H), 1.72 (m, 2H), 1.48 (t, 2H).

[0107]

[0108] Add 4-(4-(4-aminothiophen-2-yl)phenoxy)butyronitrile (70 mg, 0.27 mmol), picolinic acid (37 mg, 0.30 mmol), HATU (114 mg, 0.30 mmol), DIPEA (42 mg, 0.32 mmol) and anhydrous DMF (5 mL) into a 25 mL eggplant-shaped flask. React at room temperature for 6 h under N2 protection, then concentrate under reduced pressure. Purify the residue by Pre-TLC, using DCM / MeOH as the developing solvent, to obtain the target compound (light brown solid, 71 mg, 72.5%).

[0109] ESI(363.1,[M+H] + ), 1 1H-NMR(400 MHz, CDCl3): δ ppm 11.2 (s, 1H), 8.72 (d, 1H), 8.33 (s, 1H), 8.37 (s, 1H), 7.95 (m, 2H), 7.76 (d, 2H), 6.99 (d, 2H), 6.33 (s, 1H), 3.98 (t, 2H), 2.12 (m, 2H), 1.85 (t, 2H).

[0110]

[0111] Add 4-(4-(4-aminothiophen-2-yl)phenoxy)butyronitrile (70 mg, 0.27 mmol), benzoic acid (36.6 mg, 0.30 mmol), HATU (114 mg, 0.30 mmol), DIPEA (42 mg, 0.32 mmol) and anhydrous DMF (5 mL) into a 25 mL eggplant-shaped flask. React at room temperature for 6 h under N2 protection, then concentrate under reduced pressure. Purify the residue by Pre-TLC, using DCM / MeOH as the developing solvent, to obtain the target compound.

[0112] Similar operations can be used to synthesize compounds with the following structures (compounds 2 to 12):

[0113]

[0114] Synthesis of Compound 13

[0115]

[0116] Add 4-(4-(4-aminothiophen-2-yl)phenoxy)butyronitrile (70 mg, 0.27 mmol), 2-(bromomethyl)pyridine hydrobromide (137 mg, 0.54 mmol), cesium carbonate (264 mg, 0.81 mmol) and anhydrous DMF (5 mL) into a 25 mL eggplant-shaped flask. React at 80 °C for 8 h under N2 protection, then separate the layers in DCM / water. Back-extract the aqueous phase with DCM (15 mL × 2). Combine the organic phases, wash with water (20 mL × 1), wash with saturated brine (20 mL × 1), dry over anhydrous sodium sulfate, and concentrate under reduced pressure. Purify the residue by Pre-TLC, using a DCM / MeOH developing plate to obtain the target compound (light brown solid, 78 mg, 42.3%).

[0117] ESI(350.1,[M+H] + ), 1 1H-NMR(400 MHz, CDCl3): δ ppm 8.62 (d, 1H), 8.24 (s, 1H), 7.84 (m, 1H), 7.78 (br s 1H), 7.70 (m, 2H), 7.23 (m, 2H), 7.01 (d, 2H), 6.37 (m, 1H), 4.83 (s, 2H), 4.24 (t, 2H), 1.85 - 2.10 (m, 4H).

[0118] Compounds 14 to 24 can be synthesized by a similar method.

[0119] Synthesis of Compound 33 in Example 4

[0120]

[0121] Add 4-(4-bromophenoxy)butyronitrile (1.0 g, 4.2 mmol), (4-aminoimidazol-2-yl)boronic acid (579 mg, 4.6 mmol), sodium carbonate (801 mg, 7.6 mmol), dioxane / water (20 mL / 1 mL) and PdCl2(PPh3)2 (295 mg, 0.42 mmol) into a 50 mL eggplant-shaped flask. React under reflux for 6 h, then concentrate under reduced pressure. Purify the residue by column chromatography, eluting with a DCM / MeOH system to obtain a pale yellow solid (420 mg, 41.4%).

[0122] ESI(242.2,[M+H] + ), 11H-NMR (400 MHz, CDCl3): δ ppm 12.52 (br s, 1H), 7.87 (d, 2H), 7.10 (d, 2H), 7.03 (s, 1H), 6.58 (br s, 2H), 6.52 (d, 1H), 4.11 (t, 2H), 2.21 (m, 2H), 1.87 (m, 2H).

[0123]

[0124] 4-(4-(4-Aminoimidazol-2-yl)phenoxy)butyronitrile (0.27 mmol), pyridinecarboxylic acid (37 mg, 0.30 mmol), HATU (114 mg, 0.30 mmol), DIPEA (42 mg, 0.32 mmol) and anhydrous DMF (5 mL) were added to a 25 mL eggplant-shaped flask. The reaction was carried out at room temperature for 6 h under N2 protection, and then concentrated under reduced pressure. The residue was purified by Pre-TLC, developed with DCM / MeOH, to obtain the target compound (light brown solid, 9.8 mg, 10.5%).

[0125] ESI (346.2, [M+H] + ), 1 1H-NMR (400 MHz, CDCl3): δ ppm 12.32 (br s, 1H), 10.52 (brs, 1H), 8.82 (d, 1H), 8.35 (d, 1H), 8.10 (m, 1H), 7.97 (m, 1H), 7.88 (d, 2H), 7.03 (d, 2H), 7.04 (d, 1H), 6.48 (d, 1H), 4.13 (t, 2H), 2.06 (m, 2H), 1.87 (t, 2H).

[0126]

[0127]

[0128]

[0129]

[0130] Example 5: Inhibitory Activity of the Compound against RSV Virus

[0131] Reagents and materials used: DMEM medium, FBS, PBS, 0.25% trypsin, all products of Thermo; DMSO and absolute ethanol are products of Sigma.

[0132] Cell preparation: Before use in antiviral experiments, HEp2 cells (human epithelial cells, obtained from ATCC) were passaged in T57 culture flasks with DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 1 mM sodium pyruvate, 0.1 mM NEAA. One day before the experiment, the cells were split 1:2 to ensure they were in the exponential growth phase at the time of infection. Cell counting and viability quantification were performed using a hemocytometer and trypan blue dye exclusion. For cells used in the experiment, the cell viability was greater than 95%. The cells were resuspended at 1x104 cells per well in tissue culture medium and added to flat-bottom microtiter plates in a volume of 100 μL. The plates were incubated overnight at 37°C / 5% CO2 to allow cell attachment.

[0133] The RSV strain 9302 was obtained from ATCC and grown in HEp2 cells to prepare a stock virus bank. A pre-titrated virus aliquot was removed from the freezer (-80°C) and allowed to thaw slowly to room temperature in a biosafety cabinet. The virus was resuspended and diluted in experimental medium (DMEM supplemented with 2% heat-inactivated FBS, 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 1 mM sodium pyruvate, 0.1 mM NEAA) such that the amount of virus added to each well in a volume of 100 μL was the amount that killed 85% to 95% of the cells on day 6 post-infection. The efficacy and toxicity XTT plate staining and analysis were performed.

[0134] Each plate contained cell control wells (cells only), virus control wells (cells plus virus), triplicate drug toxicity wells for each compound (cells plus drug only), and triplicate experimental wells (drug plus cells plus virus).

[0135] XTT Staining: After incubation at 37°C in a 5% CO2 incubator, the test plates were stained with the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide). XTT-tetrazolium is metabolized by mitochondrial enzymes of metabolically active cells to soluble formazan products, allowing rapid quantitative analysis of the inhibitory effect of antiviral test substances on virus-induced cell killing. An XTT solution was made as a 1 mg / mL stock solution in RPMI 1640 daily. A phenazine methosulfate (PMS) solution was prepared at 0.15 mg / mL in PBS and stored in the dark at -20°C. An XTT / PMS stock solution was prepared by adding 40 μL of PMS per mL of XTT solution immediately prior to use. 50 μL of XTT / PMS was added to each well of the plate, and the plate was incubated at 37°C for an additional 4 hours. The plate was sealed with a sticky plate sealer and gently shaken or inverted several times to mix the soluble formazan products, and the plate was read spectrophotometrically at 450 / 650 nm using a Molecular Devices Vmax plate reader.

[0136] Data Analysis - Raw data were collected from Softmax Pro 4.6 software and imported into a Microsoft Excel spreadsheet for analysis. For each compound, the percentage reduction in virus cytopathic effect compared to the untreated virus control was calculated. The cell control values percentage of different compounds was calculated by comparing the drug-treated uninfected cells with the uninfected cells in medium alone.

[0137] When the EC90 is between 10 μM and below, the activity is counted as A; when the EC90 is between 10 - 50 μM, the activity is counted as B; when the EC90 is in the range of 50 μM - 100 μM, the activity is counted as C. The results are shown in the following table.

[0138]

[0139]

[0140] "—" indicates that the activity data of EC90 was not detected.

Claims

1. Use of a compound of formula (I) below, its stereoisomers or pharmaceutically acceptable salts in the preparation of a medicament for preventing or treating RSV virus infection diseases, X is selected from hydrogen or halogen; R3 is selected from optionally substituted 4-7 membered cycloalkyl, optionally substituted 4-7 membered heterocycloalkyl, optionally substituted 4-7 membered aromatic ring group, optionally substituted 4-7 membered heteroaromatic ring group; preferably pyridyl or phenyl; n is 0, 1 or 2; R2 is selected from azido or cyano.

2. The use according to claim 1, wherein the compound has the following structure: X is selected from F or Cl; R3 is selected from pyridyl or phenyl; n is 0, 1 or 2; R2 is selected from azido or cyano. The use according to claim 2, wherein the compound structure is: X is selected from hydrogen or halogen; R3 is pyridyl or phenyl; R2 is selected from azido or cyano.

3. The use according to any one of claims 1-2, wherein the compound structure is:

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