CN118304315A

CN118304315A - Application of nucleoside compound in preparation of respiratory syncytial virus infection resistant related products

Info

Publication number: CN118304315A
Application number: CN202410451148.0A
Authority: CN
Inventors: 周启璠; 李官官; 杨斯迪; 陈远广; 文原梅; 方彩凤; 李硕
Original assignee: Shenzhen Antai Weishengwu Pharmaceutical Co ltd
Current assignee: Shenzhen Antai Weishengwu Pharmaceutical Co ltd
Filing date: 2024-04-12
Publication date: 2024-07-09

Abstract

The invention belongs to the field of medicines, and particularly provides an application of a nucleoside compound in preparing a medicine for resisting respiratory syncytial virus infection, a method for reducing toxicity of the nucleoside compound in resisting respiratory syncytial virus infection, a method for improving bioavailability of the nucleoside compound in resisting respiratory syncytial virus infection, a method for prolonging acting time of the nucleoside compound in resisting respiratory syncytial virus infection and a method for accelerating acting of the nucleoside compound in resisting respiratory syncytial virus infection.

Description

Application of nucleoside compound in preparation of respiratory syncytial virus infection resistant related products

Technical Field

The invention relates to the technical field of medicines, in particular to application of a nucleoside compound in preparation of a product related to respiratory syncytial virus infection resistance.

Background

Respiratory syncytial virus (respiratory syncytial virus, RSV) can cause lower respiratory tract infections in infants and young children, 60% to 70% of which are affected by RSV infection within 1 year of age, and almost all infants are infected at least 1 time with RSV within 2 years of age. RSV infection is global and localized and may occur in outbreaks or even epidemic. In 2019, about 3300 tens of thousands of children's acute lower respiratory diseases caused by global RSV, about 357 tens of thousands of infants need hospitalization, of which about 140 tens of thousands occur among infants of 0 to 6 months, more than 10 tens of thousands of infants die, and more than 95% of cases occur in low-and medium-income countries, and RSV infection has become a worldwide public health problem. To date, the FDA approves only 2 drugs for the prophylactic treatment of RSV infection: inhalation ribavirin and human RSVF protein humanized murine monoclonal antibody Palivizumab (Palivizumab). But the safety and the application range of the two are not good, and the two are not recommended to be widely used in clinic. At present, no vaccine and specific medicine for preventing and treating RSV infection exist, and the treatment mainly comprises symptomatic support treatment such as keeping the respiratory tract smooth, relieving cough and asthma, absorbing oxygen and the like. Aiming at the high prevalence rate of infants and the high mortality rate of high-risk children, preventive and therapeutic drugs for RSV infection are continuously developed at home and abroad. Antiviral drug development is of great concern for the prevention and treatment of RSV infection. At present, the F, L and N proteins of RSV are taken as targets to develop various medicaments for preventing or treating RSV infection, and the medicaments mainly comprise monoclonal antibodies, immunoglobulins, fusion inhibitors, nucleoprotein inhibitors, virus polymerase inhibitors, small molecule inhibitors and the like.

Viral polymerase remains the primary therapeutic target for the treatment of viral infections. Various polymerase inhibitors have been approved for herpes, hepatitis b, hepatitis c virus and aids infections, which are critical to improving the quality of life of the patient. The L (Large) protein is a protein encoded by negative strand RNA viruses that has a variety of biological activities, the RNA polymerase activity of which is responsible for replication of the viral genome and transcription of mRNA. The L protein is also a capping enzyme that guanylates and methylates the 5' end of the viral mRNA transcript. In addition, it has polyadenylation activity and can protect the 3' end of viral mRNA. Ribavirin is the only approved drug for the treatment of RSV and acts on the L protein, and is not commonly used in infants hospitalized with RSV infection due to its toxicity (including myelosuppression and potential carcinogenicity and teratogenicity) and marginal effects, and is difficult to administer (requiring aerosol administration), and is costly to administer. The antiviral mechanism of action of ribavirin is to inhibit the enzyme inosine 5 phosphate dehydrogenase (IMPDH) activity, immunomodulate antiviral natural and cellular responses, terminate viral RNA synthesis, inhibit viral mRNA capping responses, increase mutation accumulation in viral genomes, and the like. In addition, rdRp inhibitors Monapinavir, remdesivir and VV116 are currently being investigated clinically as anti-RSV drugs, all of which are oral prodrugs, wherein the proto-drugs of REMDESIVIR and VV116 are GS-441524 and deuterated thereof, respectively.

Disclosure of Invention

Based on the shortcomings of GS-441524, a series of 5' hydroxy ester prodrugs including simple esters, amino acid esters and the like are developed, and the obtained nucleoside analogues are subjected to cell level anti-RSV activity evaluation. Since most of the drug administration audiences are infants, various toxicity evaluations are also performed so as to find out the nucleoside RSV therapeutic drugs with high efficiency and low toxicity.

In one aspect, the invention provides the use of a compound of formula I or a salt thereof for the manufacture of a medicament for the treatment of respiratory syncytial virus infection;

R ¹ is H, D or halogen;

R ² is C1-12 alkyl, 3-7 membered monocyclic heterocyclyl, 5-12 membered polycyclic heterocyclyl, C3-C7 monocyclic carbocyclyl, C5-12 polycyclic carbocyclyl, C6-10 aryl, C1-9 heteroaryl, 3-7 membered monocyclic heterocyclyl C1-6 alkyl, 5-12 membered polycyclic heterocyclyl C1-6 alkyl, C3-C7 monocyclic carbocyclyl C1-6 alkyl, C5-12 polycyclic carbocyclyl C1-6 alkyl, C6-10 arylC 1-6 alkyl, C1-9 heteroaryl C1-6 alkyl; wherein, optionally, alkyl, monocyclic heterocyclyl, polycyclic heterocyclyl, monocyclic carbocyclyl, polycyclic carbocyclyl, aryl, heteroaryl in R ² are each independently substituted with 1 or more substituents selected from deuterium, amino, halogen, methyl, ethyl.

In some embodiments, the compound of formula I has one of the following structures or a salt thereof:

On the other hand, the invention provides a method for reducing the toxicity of nucleoside compounds in resisting respiratory syncytial virus infection, which adopts a compound shown in a formula I or salt thereof as the nucleoside compounds, wherein the compound shown in the formula I has the structure disclosed by the invention.

In some embodiments, a compound of formula I or a salt thereof reduces toxicity by reducing cytotoxicity and/or reducing mutagenicity and/or reducing chromosome or mitotic damage.

On the other hand, the invention provides a method for improving the bioavailability of nucleoside compounds in resisting respiratory syncytial virus infection, which adopts a compound shown in a formula I or salt thereof as the nucleoside compounds, wherein the compound shown in the formula I has the structure disclosed by the invention.

On the other hand, the invention provides a method for prolonging the acting time of nucleoside compound medicines in resisting respiratory syncytial virus infection, which adopts a compound shown in a formula I or salt thereof as the nucleoside compound medicines, wherein the compound shown in the formula I has the structure disclosed by the invention.

On the other hand, the invention provides a method for accelerating the effect of nucleoside compound medicines in resisting respiratory syncytial virus infection, which adopts a compound shown in a formula I or salt thereof as the nucleoside compound medicines, wherein the compound shown in the formula I has the structure disclosed by the invention.

In some embodiments, the medicament is an oral medicament.

In some embodiments, the oral drug is a tablet, dispersible tablet, fast-dissolving tablet, fast-melting tablet, orodispersible tablet, lyophilized unit, porous tablet, conventional tablet, coated tablet, uncoated tablet, enteric-coated tablet, effervescent tablet, dissolvable tablet, chewable tablet, oral lyophilisate, powder, oral powder, pill, capsule, and/or granule.

In some embodiments, the respiratory syncytial virus is RSVA type and/or rsvp type.

Description of the terms

Reference will now be made in detail to certain embodiments of the application, examples of which are illustrated in the accompanying structural and chemical formulas. The application is intended to cover all alternatives, modifications and equivalents, which may be included within the scope of the application as defined by the appended claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in the practice of the present application. The present application is in no way limited to the methods and materials described herein. In the event of one or more of the incorporated references, patents and similar materials differing from or contradictory to the present application (including but not limited to defined terms, term application, described techniques, etc.), the present application controls.

It should further be appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

Unless defined otherwise, 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. All patents and publications referred to herein are incorporated by reference in their entirety.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

In the following, all numbers disclosed herein are approximate, whether or not the word "about" or "about" is used. The numerical value of each number may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15% or 20%. Whenever a number is disclosed having a value of N, any number having a value of N+/-1%, N+/-2%, N+/-3%, N+/-5%, N+/-7%, N+/-8%, N+/-10%, N+/-15% or N+/-20% is explicitly disclosed, where "+/-" means plus or minus.

"Oral formulation" refers to a formulation form that is orally administered and the drug is absorbed into the blood in the gastrointestinal tract, including tablets, granules, capsules, oral solutions, and the like.

In some embodiments, the "oral formulation" is a "solid oral formulation" that refers to a tablet, dispersible tablet, fast-dissolving tablet, orodispersible tablet, lyophilized unit, porous tablet, conventional tablet, coated tablet, uncoated tablet, enteric-coated tablet (gastro-resistanttablet), effervescent tablet, dissolvable tablet, chewable tablet, oral lyophilisate, powder, oral powder, pill, capsule, and/or granule. In some embodiments, the solid oral formulation is a capsule. In some embodiments, the solid oral formulation is a tablet.

"(C _1-n) alkyl", wherein n is an integer, alone or in combination with another group, is intended to mean an acyclic, straight-chain or branched alkyl group containing from 1 to n carbon atoms. "(C _1-12) alkyl" includes, but is not limited to, methyl, ethyl, propyl (n-propyl), butyl (n-butyl), 1-methylethyl (isopropyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (isobutyl), 1-dimethylethyl (tert-butyl), pentyl, hexyl, heptyl, and octyl. The abbreviation Me represents a methyl group; et represents an ethyl group, pr represents a propyl group, iPr represents a 1-methylethyl group, bu represents a butyl group and tBu represents a1, 1-dimethylethyl group.

"Aryl" includes aromatic monocyclic or bicyclic rings having 6 to 10 carbon atoms, including benzene and naphthalene rings.

"Arylalkyl" refers to an alkyl group, as defined herein, wherein one of the hydrogen atoms bonded to a carbon atom is replaced with an aryl group (i.e., aryl-alkyl-moiety) as described herein.

"Aryl- (C _1-n) alkyl-", where n is an integer, alone or in combination with another group, is intended to mean an alkyl group having 1 to n carbon atoms as defined herein, itself substituted with an aryl group as defined above.

"Heterocycle" is synonymous and unless otherwise mentioned, refers to monocyclic and fused bicyclic, saturated or partially unsaturated rings having 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms (wherein 1,2,3 or 4 ring atoms are heteroatoms independently selected from N, O and S and all remaining ring atoms are C).

"Heteroaryl" refers to a single aromatic ring or multiple condensed rings. The term includes a single aromatic ring having about 1-6 carbon atoms and about 1-4 heteroatoms selected from oxygen, nitrogen and sulfur in the ring. The sulfur and nitrogen atoms may also be present in oxidized form, provided that the ring is aromatic. The ring includes, but is not limited to, pyridyl, pyrimidinyl, oxazolyl, or furanyl.

"Heteroarylalkyl" refers to an alkyl group, as defined herein, wherein one of the hydrogen atoms bonded to a carbon atom is replaced by a heteroaryl group as described herein (i.e., a heteroaryl-alkyl-moiety). The alkyl group of a "heteroarylalkyl" is typically 1 to 6 carbon atoms (i.e., heteroaryl (C1-C6) alkyl). Heteroarylalkyl groups include, but are not limited to, heteroaryl-CH ₂ -, heteroaryl-CH (CH ₃) -, heteroaryl-CH ₂CH₂ -, 2- (heteroaryl) ethan-1-yl, and the like, wherein the "heteroaryl" moiety includes any heteroaryl described herein.

Halogen and halogen refer to halogen atoms selected from F, cl, br and I.

1 Or more means 1 or 2, 3, 4, 5 or 6.

The salt of the present invention is a pharmaceutically acceptable salt, which refers to a salt of the compound of the present invention that is pharmaceutically acceptable and has pharmacological activity of the parent compound. Such salts include: salts added with inorganic acids such as nitric acid, phosphoric acid, carbonic acid, etc., or with organic acids; such as propionic acid, caproic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, gluconic acid, stearic acid, muconic acid, and the like; or salts formed when acidic protons present on the parent compound are replaced with metal ions, such as alkali metal ions or alkaline earth metal ions; or with organic bases such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, and the like. Pharmaceutically acceptable salts of the invention can be synthesized from the parent compound containing an acid or base by conventional chemical methods. In general, the preparation of such salts is as follows: prepared via reaction of these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications.

The reagents used in the present invention are all commercially available or can be prepared by the methods described herein.

Example 1: preparation of((2R, 3S,4R, 5R) -5- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-yl) methyltetrahydrofuran-3-carboxylate (ATV 001)

5.62G of Compound 1-1 was dissolved in 30mL of acetone, 11.50mL of 2, 2-dimethoxypropane and 1.34mL of concentrated sulfuric acid were added, stirred at 45℃for 30min, cooled to room temperature, and the organic solvent was removed by rotary evaporation. Extraction with 100mL of ethyl acetate and 100mL of saturated sodium bicarbonate solution was repeated three times, the organic layers were combined, dried over anhydrous sodium sulfate, suction filtered and evaporated to dryness, and column chromatography was performed to give 6.20g of compound 1-2 (white solid, yield) 97％).¹H NMR(400MHz,Chloroform-d)δ7.95(s,1H),7.11(d,J＝4.7Hz,1H),6.69(dd,J＝4.8,2.4Hz,1H),5.77(s,2H),5.42(d,J＝6.6Hz,1H),5.24(dd,J＝6.6,2.4Hz,1H),4.67(q,J＝1.9Hz,1H),3.99(dd,J＝12.5,1.9Hz,1H),3.84(dd,J＝12.5,1.7Hz,1H),1.81(s,3H),1.40(s,3H).

1.50G of compound 1-2 is dissolved in 15ml of dichloromethane, 0.52g of tetrahydrofuran-3-formic acid and 55.40mg of 4-dimethylaminopyridine are added, after stirring for 10min, 1.02g of dicyclohexylcarbodiimide is added, stirring is carried out for 24h at room temperature, the reaction solution is diluted with dichloromethane, then is washed with saturated citric acid solution, saturated sodium carbonate solution and saturated saline solution respectively, the organic layer is dried by anhydrous sodium sulfate, and then is filtered by suction and evaporated to dryness to obtain crude product 1-3, which is directly used for the next reaction.

1.50G of Compound 1-3 was dissolved in 3mL of concentrated hydrochloric acid and 15mL of tetrahydrofuran, stirred under ice bath for 6 hours, then sodium carbonate solid was added to adjust pH to 8, the organic solvent was removed by rotary evaporation, and 0.86g of Compound ATV001 was obtained by column chromatography (white solid, yield) 49％).¹H NMR(600MHz,DMSO-d₆)δ7.94(s,1H),7.90(br,2H),6.93(d,J＝4.4Hz,1H),6.82(d,J＝4.4Hz,1H),6.33(t,J＝5.5Hz,1H),5.40(d,J＝5.8Hz,1H),4.71(t,J＝5.3Hz,1H),4.37-4.34(m,1H),4.25-4.20(m,2H),3.97-3.96(m,1H),3.81-3.78(m,1H),3.75-3.69(m,2H),3.66-3.62(m,1H),3.15-3.10(m,1H),2.08-1.95(m,2H).¹³C NMR(151MHz,DMSO-d₆)δ150.1,148.4,124.0,117.4,117.1,110.8,101.3,81.6,81.5,79.5,74.5,70.7,70.6,69.8,69.7,67.8,63.9,63.8,43.4,29.5,29.4.

Example 2: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-nicotinic acid methyl ester (ATV 002)

Synthesis of Compound ATV002 as a white solid in a total of 0.75g Using 1-2 as a starting material and replacing tetrahydrofuran-3-carboxylic acid with pyridine-3-carboxylic acid according to the procedure described in example 1, the overall yield in two steps was 42％.¹H NMR(600MHz,Methanol-d₄)δ9.05(d,J＝2.2Hz,1H),8.73(dd,J＝5.0,1.7Hz,1H),8.35-8.25(m,1H),7.76(s,1H),7.53(dd,J＝8.0,4.9Hz,1H),6.83(dd,J＝32.8,4.6Hz,2H),4.97(d,J＝5.3Hz,1H),4.82-4.74(m,1H),4.61-4.45(m,2H),4.39-4.30(m,1H).¹³C NMR(151MHz,Methanol-d₄)δ164.5,155.8,152.8,149.8,146.9,137.6,126.2,124.0,123.8,116.6,116.2,110.8,101.1,81.9,80.2,74.1,70.6,63.4.

Example 3: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-pentadecanoic acid methyl ester (ATV 003)

Synthesis of Compound ATV003 in total 0.35g of white solid starting from 1-2 and replacing tetrahydrofuran-3-carboxylic acid with pentadecanoic acid according to the procedure described in example 1, the overall yield in two steps was 15％.¹HNMR(600MHz,Methanol-d₄)δ7.86(s,1H),6.89(q,J＝4.6Hz,2H),4.42(dd,J＝12.0,3.1Hz,1H),4.37(m,1H),4.31(dd,J＝11.9,5.2Hz,1H),4.17-4.12(m,1H),2.29(m,2H),1.55(p,J＝7.2Hz,2H),1.39-1.19(m,24H),0.89(t,J＝7.0Hz,3H).¹³C NMR(151MHz,Methanol-d₄)δ175.1,157.3,148.3,125.7,118.0,117.6,112.1,102.6,83.4,81.5,75.7,72.1,64.2,35.0,33.1,30.8,30.8,30.8,30.7,30.6,30.5,30.4,30.2,26.0,23.8,14.5.

Example 4: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-palmitic acid methyl ester (ATV 004)

The procedure as described in example 1 was followed starting from 1-2 and replacing tetrahydrofuran-3-carboxylic acid with hexadecanoic acid to give compound ATV004 as a total of 0.45g of white solid in two steps with overall yield 19％.¹HNMR(600MHz,Methanol-d₄)δ7.86(s,1H),6.89(q,J＝4.6Hz,2H),4.42(dd,J＝11.9,3.1Hz,1H),4.38-4.34(m,1H),4.32-4.27(m,1H),4.16-4.08(m,1H),2.36-2.24(m,2H),1.60-1.50(m,2H),1.36-1.24(m,24H),0.89(t,J＝7.0Hz,3H).¹³C NMR(151MHz,Methanol-d₄)δ173.7,155.8,146.9,124.3,116.5,116.2,110.7,101.1,82.0,80.1,74.2,70.7,62.8,33.5,31.7,29.4,29.4,29.4,29.4,29.3,29.2,29.1,29.0,28.7,24.6,22.3,13.0.

Example 5: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-1-methylcyclohexane-1-carboxylic acid) methyl ester (ATV 005)

According to the method described in example 1, starting from 1-2 and replacing tetrahydrofuran-3-carboxylic acid with 1-methylcyclohexane-1-carboxylic acid, the compound ATV005 was synthesized as a white solid in a total of 1.0g, with a total yield of two steps 55％.¹H NMR(400MHz,Methanol-d₄)δ7.86(s,1H),6.95-6.80(m,2H),4.90-4.86(m,1H),4.42-4.32(m,3H),4.17(t,J＝5.7Hz,1H),2.03-1.88(m,2H),1.55-1.42(m,3H),1.34-1.13(m,5H),1.09(s,3H).¹³C NMR(101MHz,Methanol-d₄)δ177.5,155.9,146.9,124.3,116.6,116.2,110.7,101.1,82.0,79.8,74.3,70.7,62.9,43.1,35.2,25.3,22.9.

Example 6: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-isobutyryl) methyl ester (ATV 006)

Synthesis of Compound ATV006 as a white solid in a total of 0.96g Using 1-2 as a starting material and replacing tetrahydrofuran-3-carboxylic acid with isobutyric acid according to the method described in example 1, the overall yield in two steps was 59％.¹HNMR(400MHz,Methanol-d₄)δ7.76(s,1H),6.78(s,2H),4.78(d,J＝5.3Hz,1H),4.40-4.24(m,2H),4.24-4.11(m,1H),4.10-4.01(m,1H),2.42(p,J＝7.0Hz,1H),0.99(dd,J＝7.0,4.1Hz,6H).¹³C NMR(101MHz,Methanol-d₄)δ176.96,155.82,146.92,124.25,116.54,116.29,110.75,101.20,82.04,80.00,74.27,70.68,62.93,33.58,25.00,17.95,17.87.

Example 7: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-acetic acid methyl ester (ATV 007)

Synthesis of Compound ATV007 as a white solid in total with 1-2 starting from 1-2 and replacing tetrahydrofuran-3-carboxylic acid with acetic acid according to the procedure described in example 1, the overall yield in two steps was 46％.¹HNMR(600MHz,CD₃OD)δ(ppm):7.86(s,1H),6.89(t,J＝5.0Hz,2H),4.87(s,1H),4.43-4.41(dd,J＝12Hz,2.8Hz,1H),4.37-4.34(m,1H),4.30-4.27(m,1H),4.13(t,J＝5.7Hz,1H),2.03(s,3H).¹³C NMR(150MHz,CD₃OD)δ(ppm):171.0,155.8,146.9,124.2,116.6,116.2,110.7,101.1,81.9,80.2,74.1,70.7,63.1,19.3.

Example 8: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-propanoic acid) methyl ester (ATV 008)

Synthesis of Compound ATV008 as a white solid according to the method of example 1 starting from 1-2 and replacing tetrahydrofuran-3-carboxylic acid with propionic acid, total yield in two steps was 0.77g 49％.¹HNMR(600MHz,CD₃OD)δ(ppm):7.86(s,1H),6.90-6.88(q,J＝4.5Hz,2H),4.87-4.86(m,1H),4.46-4.43(dd,J＝12Hz,2.8Hz,1H),4.37-4.36(m,1H),4.31-4.28(m,1H),4.15(t,J＝5.8Hz,1H),2.38-2.28(m,2H),1.08(t,J＝7.5Hz,3H).¹³C NMR(150MHz,CD₃OD)δ(ppm):174.3,155.8,146.9,124.2,116.5,116.2,110.7,101.1,82.0,80.1,74.2,70.7,62.9,26.7,7.9.

Example 9: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-butyric acid methyl ester (ATV 009)

Synthesis of Compound ATV009, 0.83g of white solid, starting from 1-2 and replacing tetrahydrofuran-3-carboxylic acid with butyric acid, according to the procedure described in example 1, the overall yield in two steps is 51％.¹HNMR(600MHz,CD₃OD)δ(ppm):7.86(s,1H),6.90-6.88(q,J＝4.5Hz,2H),4.87-4.86(m,1H),4.44-4.42(dd,J＝12Hz,2.8Hz,1H),4.37-4.35(m,1H),4.31-4.28(m,1H),4.14(t,J＝5.8Hz,1H),2.32-2.23(m,2H),1.62-1.56(m,2H),0.91(t,J＝7.4Hz,3H).¹³C NMR(150MHz,CD₃OD)δ(ppm):174.3,155.9,146.9,124.3,116.5,116.2,110.7,101.1,82.0,80.1,74.2,70.7,62.8,35.4,17.9,12.5.

Example 10: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-nonanoic acid methyl ester (ATV 010)

Synthesis of Compound ATV010 in total of 0.56g of white solid, starting from 1-2 and replacing tetrahydrofuran-3-carboxylic acid with nonanoic acid according to the procedure described in example 1, the overall yield in two steps was 29％.¹HNMR(600MHz,CD₃OD)δ(ppm):7.86(s,1H),6.90-6.88(q,J＝4.5Hz,2H),4.87-4.86(m,1H),4.43-4.41(dd,J＝12Hz,2.8Hz,1H),4.37-4.35(m,1H),4.32-4.29(m,1H),4.14(t,J＝5.8Hz,1H),2.38-2.23(m,2H),1.56-1.53(m,2H),1.29-1.27(m,10H),0.87(t,J＝7.0Hz,3H).¹³C NMR(150MHz,CD₃OD)δ(ppm):173.7,155.9,146.9,124.3,116.5,116.2,110.7,101.1,82.0,74.2,70.7,62.8,33.5,31.5,28.8,28.7,24.6,22.3.

Example 11: ((2R, 3S,4R, 5R) -5- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-2-ethylbutanoic acid) methyl ester (ATV 011)

According to the method described in example 1, starting from 1-2 and replacing tetrahydrofuran-3-carboxylic acid with 2-ethylbutyric acid, the synthetic compound ATV011 amounted to 0.93g of a white solid, overall yield in two steps 53％.¹HNMR(600MHz,CD₃OD)δ(ppm):7.86(s,1H),6.89(s,2H),4.87-4.86(m,1H),4.39-4.43(dd,J＝12Hz,2.8Hz,1H),4.37-4.35(m,1H),4.14(t,J＝5.8Hz,1H),2.38-2.22(m,1H),1.60-1.45(m,4H),0.86-0.82(m,6H).¹³C NMR(150MHz,CD₃OD)δ(ppm):176.1,155.9,146.9,124.3,116.6,116.2,110.7,101.1,81.9,79.9,74.2,70.7,62.8,48.9,24.7,24.6.10.7,10.6.

Example 12: ((2R, 3S,4R, 5R) -5- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-cyclopropanecarboxylic acid) methyl ester (ATV 012)

According to the method described in example 1, starting from 1-2 and replacing tetrahydrofuran-3-carboxylic acid with cyclopropanecarboxylic acid, the synthetic compound ATV012 was found to be a total of 0.89g of a white solid in two steps in total yield 55％.¹HNMR(600MHz,CD₃OD)δ(ppm):7.86(s,1H),6.89(t,J＝4.5Hz,2H),4.87-4.86(m,1H),4.46-4.44(dd,J＝12Hz,2.8Hz,1H),4.36-4.34(m,1H),4.29-4.26(m,1H),4.15(t,J＝5.8Hz,1H),1.64-1.60(m,1H),0.92-0.87(m,4H).¹³C NMR(150MHz,CD₃OD)δ(ppm):174.9,155.9,146.9,124.2,116.6,116.2,110.7,101.1,80.2,80.1,74.2,70.6,63.0,12.1,7.5,7.4.

Example 13: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-benzoic acid methyl ester (ATV 013)

Synthesis of Compound ATV013 as a white solid in total 0.93g Using 1-2 as a starting material and replacing tetrahydrofuran-3-carboxylic acid with benzoic acid according to the procedure described in example 1, the overall yield in two steps was 52％.¹H NMR(600MHz,DMSO-d₆)δ(ppm):7.92(br,2H),7.90(d,J＝7.4Hz,2H),7.86(s,1H),7.68(t,J＝7.4Hz,1H),7.52(t,J＝7.7Hz,2H),6.87(d,J＝4.5Hz,1H),6.81(d,J＝4.5Hz,1H),6.36(d,J＝5.9Hz,1H),5.46(d,J＝5.9Hz,1H),4.79(t,J＝5.3Hz,1H),4.61-4.58(dd,J＝12.2Hz,2.6Hz,1H),4.45-4.42(dd,J＝12.3Hz,4.8Hz,1H),4.39-4.37(m,1H),4.14-4.10(m,1H).¹³C NMR(150MHz,DMSO-d₆)δ(ppm):166.0,156.1,148.4,134.0,129.8,129.7,129.2,123.9,117.4,117.1,110.8,101.3,81.7,79.7,74.5,70.6,63.9.

Example 14: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-cyclohexanecarboxylic acid) methyl ester (ATV 014)

Synthesis of Compound ATV014 as a white solid 1.14g, starting from 1-2 and replacing tetrahydrofuran-3-carboxylic acid with cyclohexane-carboxylic acid according to the procedure described in example 1, the overall yield in two steps was 63％.¹H NMR(600MHz,DMSO-d₆)δ(ppm):7.92(s,1H),7.86(br,1H),6.92(d,J＝4.5Hz,1H),6.81(d,J＝4.5Hz,1H),6.33(d,J＝5.9Hz,1H),5.38(d,J＝5.9Hz,1H),4.70(t,J＝5.3Hz,1H),4.32-4.29(dd,J＝12.2Hz,2.6Hz,1H),4.24-4.21(m,1H),4.16-4.13(dd,J＝12.3Hz,4.8Hz,1H),3.98-3.95(q,J＝5.9Hz,1H),2.26-2.22(m,1H),1.75-1.72(m,2H),1.64-1.56(m,3H),1.30-1.12(m,5H).¹³C NMR(150MHz,DMSO-d₆)δ(ppm):175.34,156.06,148.4,124.0,117.4,117.0,110.7,101.2,81.7,79.4,74.5,70.6,63.0,42.6,29.0,28.9,25.7,25.2,25.1.

Example 15: ((2R, 3S,4R, 5R) -5- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-cyclopentanecarboxylic acid) methyl ester (ATV 015)

Synthesis of Compound ATV015 as a white solid in total, starting from 1-2 and replacing tetrahydrofuran-3-carboxylic acid with cyclopentanecarboxylic acid according to the procedure described in example 1, with a total yield of two steps of 51％.¹HNMR(600MHz,CD₃OD)δ(ppm):7.86(s,1H),6.90-6.87(q,J＝4.6Hz,2H),4.85-4.83(m,1H),4.39-4.43(dd,J＝12.1Hz,3.1Hz,1H),4.37-4.35(m,1H),4.14(t,J＝5.7Hz,1H),2.75-2.70(m,1H),1.87-1.80(m,2H),1.75-1.53(m,6H).¹³C NMR(150MHz,CD₃OD)δ(ppm):176.5,155.9,146.9,124.3,116.5,116.2,110.7,101.1,82.0,80.0,74.3,70.7,62.8,43.5,29.5,29.4,25.3.

Example 16: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-3, 3-trifluoropropionate) methyl ester (ATV 016)

According to the procedure described in example 1, starting from 1-2 and replacing tetrahydrofuran-3-carboxylic acid by 3, 3-trifluoropropionic acid, the synthetic compound ATV016 amounted to 0.90g of a white solid, the overall yield in two steps being 50％.¹H NMR(600MHz,CD₃OD)δ(ppm):7.86(s,1H),6.90-6.88(q,J＝4.6Hz,2H),4.89(d,J＝5.3Hz,1H),4.54-4.50(m,1H),4.42-4.38(m,2H),4.15(t,J＝5.7Hz,1H),3.45-3.35(m,2H).¹³C NMR(150MHz,CD₃OD)δ(ppm):164.3(J＝4.0Hz),155.5,146.9,123.8(q,J＝273.6Hz),124.1,116.6,116.2,110.8,101.2,81.7,80.2,74.0,70.6,64.1.

Example 17: ((2R, 3S,4R, 5R) -5- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-3-methylbutanoate) methyl ester (ATV 017)

According to the method described in example 1, starting from 1-2 and replacing tetrahydrofuran-3-carboxylic acid with 3-methylbutanoic acid, the synthetic compound ATV017 amounted to 0.75g of a white solid, overall yield in two steps 44％.¹HNMR(600MHz,CD₃OD)δ(ppm):7.86(s,1H),6.90-6.88(q,J＝4.6Hz,2H),4.87(d,J＝5.3Hz,1H),4.43-4.40(m,1H),4.39-4.35(m,2H),4.31-4.29(m,1H),4.14(t,J＝5.7Hz,1H),2.18-2.16(m,2H),2.04-1.97(m,1H),0.91-0.90(q,J＝3.2Hz,6H).¹³C NMR(150MHz,CD₃OD)δ(ppm):155.9,146.9,124.3,116.5,116.2,110.7,101.1,82.0,80.0,74.2,70.7,70.6,62.8,62.7,42.6,25.4,21.3,21.2.

Example 18: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-pivalate) methyl ester (ATV 018)

Synthesis of Compound ATV018 as a white solid in a total of 0.36g, starting from 1-2 and replacing tetrahydrofuran-3-carboxylic acid with pivalic acid according to the method described in example 1, the overall yield in two steps is 21％.¹HNMR(600MHz,CD₃OD)δ(ppm):7.86(s,1H),6.89-6.87(q,J＝4.6Hz,2H),4.86(d,J＝5.3Hz,1H),4.39-4.36(m,2H),4.32-4.29(m,1H),4.16(t,J＝5.6Hz,1H),1.15(s,9H).¹³C NMR(150MHz,CD₃OD)δ(ppm):155.9,146.9,124.3,116.6,116.2,110.7,101.1,82.0,79.9,74.2,70.6,63.0,38.5,26.1.

Example 19: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-D-valine yl) methyl ester (ATV 019)

1.80G of 1-2 was dissolved in 15mL of methylene chloride, followed by addition of 1.18g of (D) -Boc-valine and then addition of 66.48mg of 4-dimethylaminopyridine, followed by stirring for 10min, 1.22g of dicyclohexylcarbodiimide and stirring at room temperature for 24h. Separation by column chromatography gave 2.81g of compound 19-1 (white solid, yield) 97％).¹HNMR(600MHz,Methanol-d₄)δ7.79(s,1H),6.79(s,2H),5.39(s,1H),4.90(dd,J＝6.5,3.4Hz,1H),4.51(q,J＝4.1Hz,1H),4.29(dd,J＝12.0,3.8Hz,1H),4.24(dd,J＝12.1,5.2Hz,1H),3.77(d,J＝6.0Hz,1H),3.27-3.11(m,1H),1.61(s,4H),1.32(d,J＝2.5Hz,9H),1.24(s,3H),0.73(dd,J＝19.0,6.8Hz,6H).

2.50G of Compound 19-1 was dissolved in 3mL of 37% by mass aqueous hydrochloric acid and 15mL of tetrahydrofuran, stirred for 6 hours, then sodium carbonate was added to adjust pH to 8, the organic solvent was removed by rotary evaporator, and the mixture was separated by column chromatography (eluent: methanol/ethyl acetate (V/V) =1:20) to give 0.99g of Compound ATV019 (white solid, yield) 54％).¹H NMR(400MHz,Methanol-d₄)δ7.76(s,1H),6.80(s,2H),4.79(s,1H),4.42-4.24(m,3H),4.08(d,J＝5.5Hz,1H),3.23(d,J＝11.1Hz,1H),1.90-1.76(m,1H),0.82(d,J＝6.9Hz,3H),0.74(d,J＝6.9Hz,3H).

Example 20: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-L-valine yl) methyl ester (ATV 020)

According to the method of example 19, N-Boc-D-valine was replaced with N-Boc-L-valine to synthesize Compound ATV020 as a white solid in an amount of 1.1g, with a total yield of two steps 21％.¹H NMR(600MHz,CD₃OD)δ7.76(s,1H),6.80(d,J＝1.6Hz,2H),4.81(d,J＝5.3Hz,1H),4.42-4.26(m,3H),4.04(t,J＝5.8Hz,1H),3.25(d,J＝4.9Hz,1H),1.97-1.84(m,1H),0.83(d,J＝6.9Hz,3H),0.79(d,J＝6.9Hz,3H).¹³C NMR(151MHz,CD₃OD)δ173.76,155.85,146.93,124.12,116.62,116.21,110.86,101.11,81.75,80.16,74.04,70.76,63.66,59.27,31.62,17.75,16.46.

Example 21: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-L-phenylalanine methyl ester (ATV 021)

According to the method of example 19, and substituting N-Boc-D-valine for N-Boc-L-phenylalanine, the synthetic compound ATV021 was 0.1g of a white solid in total, the overall yield in two steps was 17％.¹H NMR(600MHz,DMSO-d₆)δ(ppm):7.96(br,1H),7.95(s,1H),7.87(br,1H),7.21-7.13(m,5H),6.93(d,J＝4.5Hz,1H),6.81(d,J＝4.5Hz,1H),6.33(d,J＝6.2Hz,1H),5.36(br,1H),4.70(t,J＝5.0Hz,1H),4.28-4.24(m,2H),4.19-4.16(m,1H),3.88(t,J＝5.5Hz,1H),3.57(t,J＝6.7Hz,1H),2.84-2.73(m,2H),1.85(br,2H).¹³C NMR(150MHz,DMSO-d₆)δ(ppm):174.5,155.4,147.8,137.5,129.0,127.9,126.1,123.4,116.8,116.4,110.1,100.7,81.1,78.9,73.8,70.0,63.1,55.6,40.4.

Example 22: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-D-phenylalanine methyl ester (ATV 022)

The procedure as in example 19 was followed, substituting N-Boc-D-valine for N-Boc-D-phenylalanine, to synthesize compound ATV022 in a total of 0.1g of white solid in two-step yield 15％.¹H NMR(600MHz,DMSO-d₆)δ(ppm):7.92(s,1H),7.85(br,1H),7.25-7.14(m,5H),6.90(d,J＝4.5Hz,1H),6.80(d,J＝4.5Hz,1H),6.33(d,J＝5.9Hz,1H),5.39(d,J＝5.6Hz,1H),4.71(t,J＝5.3Hz,1H),4.25-4.17(m,3H),3.95-3.94(m,1H),3.56(t,J＝6.7Hz,1H),2.86-2.71(m,2H),1.75(br,2H).¹³C NMR(150MHz,DMSO-d₆)δ(ppm):175.2,156.1,148.4,138.2,129.7,128.6,126.8,124.0,117.4,117.1,110.8,101.3,81.7,79.5,74.5,70.7,63.9,56.1.

Example 23: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2- (2S) -2-amino-3-methylpentanoic acid) methyl ester (ATV 023)

According to the method of example 19, N-Boc-D-valine was replaced with N-Boc-L-isoleucine to synthesize compound ATV023 in a total of 0.06g of a white solid in two steps of yield 10％.¹H NMR(600MHz,DMSO-d₆)δ(ppm):7.95(br,1H),7.92(s,1H),7.87(br,1H),6.92(d,J＝5.8Hz,1H),6.83(d,J＝5.8Hz,1H),6.35(br,1H),5.40(br,1H),4.73(d,J＝4.6Hz,1H),4.29-4.24(m,3H),3.96(t,J＝5.0Hz,1H),3.18(d,J＝4.2Hz,1H),1.53-1.51(m,1H),1.39-1.32(m,1H),1.11-1.04(m,1H),0.80-0.74(m,6H).¹³C NMR(150MHz,DMSO-d₆)δ(ppm):175.6,156.1,148.4,124.0,117.4,117.0,110.8,101.3,81.6,79.5,74.5,70.7,63.5,59.1,39.1,24.6,16.0,11.8.

Example 24: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2- (2R) -2-amino-3-methylpentanoic acid) methyl ester (ATV 024)

The procedure as in example 19 was followed, substituting N-Boc-D-valine for N-Boc-D-isoleucine, to give the compound ATV024 as a white solid in a total of 0.06g in two-step yield 9％.¹H NMR(600MHz,DMSO-d₆)δ(ppm):7.92(s,1H),7.86(br,2H),6.92(d,J＝5.8Hz,1H),6.83(d,J＝5.8Hz,1H),6.33(d,J＝4.7Hz,1H),5.39(br,1H),4.71(br,1H),4.30-4.19(m,3H),3.97(t,J＝5.1Hz,1H),3.15(d,J＝5.3Hz,1H),1.53-1.50(m,1H),1.39-1.34(m,1H),1.11-1.04(m,1H),0.80-0.75(m,6H).¹³C NMR(150MHz,DMSO-d₆)δ(ppm):175.6,156.1,148.4,124.0,117.4,117.1,110.8,101.3,81.7,79.5,74.5,70.8,63.8,59.1,39.0,24.6,16.1,11.8.

Example 25: ((2R, 3S,4R, 5R) -5- (4-amino-5-fluoropyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-yl) methyl isobutyrate (ATV 025)

ATV006 (1 g,2.77 mmol), selectFluor (1-chloromethyl-4-fluoro-1, 4-diazotized bicyclo 2.2.2 octane bis (tetrafluoroboric acid) salt, 1.4g,5.5 mmol) and DMAP (0.34 g,2.77 mmol) were taken, a mixed solvent of 20ml acetonitrile-water (v/v=9:1) was added, stirring was carried out at room temperature for 24h, TLC monitoring (mobile phase: DCM: meOH=10:1) was carried out until the ATV006 was substantially reacted completely, acetonitrile was distilled off under reduced pressure, water and ethyl acetate were further added, the organic layer was separated by stirring, the aqueous layer was extracted twice with ethyl acetate, the combined organic layers were successively washed with saturated sodium carbonate solution, saturated sodium chloride solution and dried with anhydrous sodium sulfate, suction filtration was evaporated to dryness, 100mg of off-white solid ATV025 was obtained by column chromatography, yield was 100mg of off-white solid ATV025 9.5％.¹H NMR(600MHz,CD₃OD)δ(ppm):7.79(s,1H),6.65(s,1H),4.79(d,J＝5.0Hz,1H),4.40-4.30(m,3H),4.09(t,J＝5.6Hz,1H),2.59-2.54(m,1H),1.14-1.13(m,6H).¹³C NMR(150MHz,CD₃OD)δ(ppm):176.9,154.5,147.6,144.0,142.3,121.0,115.7,102.7,102.5,97.0,96.9,81.9,79.6,74.5,70.5,62.7,33.7,17.9,17.8.¹⁹F NMR(600MHz,CD₃OD)δ(ppm):-160.8.

Example 26: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2- (1R, 4R) -bicyclo [2.2.1] hept-5-ene-2-carboxylic acid) methyl ester (ATV 026)

According to the method described in example 1, starting from 1-2 and replacing tetrahydrofuran-3-carboxylic acid with (1R, 4R) -bicyclo [2.2.1] hept-5-ene-2-carboxylic acid, the compound ATV026 was synthesized in a total of 1.0g of a white solid in a two-stage total yield 61％.¹H NMR(400MHz,DMSO-d₆)δ7.93(s,1H),8.0(br,2H),6.93-6.91(m,1H),6.85-6.82(m,1H),6.35-6.32(m,1H),6.17-6.11(m,1H),5.85-5.78(m,1H),5.40-5.37(m,1H),4.73-4.69(m,1H),4.25-4.09(m,3H),3.99-3.93(m,1H),3.07-2.84(m,3H),1.87-1.75(m,1H),1.35-1.16(m,3H).¹³C NMR(101MHz,DMSO-d₆)δ174.0,156.1,148.4,138.0,136.1,132.8,124.0,117.4,117.0,110.7,101.3,81.7,79.3,74.5,70.6,63.5,49.5,45.5,43.0,42.4,29.2.

Example 27: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2- (3R, 5R, 7R) -adamantane-1-carboxylic acid) methyl ester (ATV 027)

Synthesis of Compound ATV027 as a white solid in total 1.23g starting from 1-2 and replacing tetrahydrofuran-3-carboxylic acid with (3R, 5R, 7R) -adamantane-1-carboxylic acid according to the procedure described in example 1, the overall yield in two steps was 67％.¹H NMR(600MHz,DMSO-d₆)δ7.93(s,1H),7.90(br,2H),6.93(d,J＝4.5Hz,1H),6.83(d,J＝4.5Hz,1H),6.37(d,J＝5.9Hz,1H),5.37(d,J＝5.9Hz,1H),4.71(t,J＝5.3Hz,1H),4.29-4.23(m,2H),4.16-4.13(m,1H),4.01-3.98(m,1H),1.94-1.91(m,3H),1.71-1.60(m,12H).¹³C NMR(151MHz,DMSO-d₆)δ176.7,156.1,148.4,124.1,117.4,117.0,110.5,101.2,81.6,79.2,74.7,70.4,62.7,38.7,36.3,27.7.

Example 28: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl-5-d) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-yl) methylcyclohexane carboxylic acid ester (ATV 028)

Compound 28-1 (0.5 g,1.1 mmol) and N-iodosuccinimide (0.28 g,1.2 mmol) were mixed with dichloromethane (10 mL), stirred at room temperature for 2h and then the solvent was removed under reduced pressure, and the residue was purified by column chromatography (ethyl acetate/petroleum ether=1/2 (V/V)) to give compound 28-2 (red solid, 0.4g, yield 64%).

To a solution of compound 28-2 (200 mg,0.35 mmol) and cesium carbonate (247 mg,0.76 mmol) in D ₂O-DMSO-d6(10mL,D₂ O: dmso=1:9 (V/V)) under argon was added PdCl ₂(dppf)₂ (32 mg,0.04 mmol), the reaction was stirred at 80 ℃ for 10 hours, cooled to room temperature and slowly poured into water (10 mL), extracted with ethyl acetate (30 ml×2), the organic phase layers were combined, the combined organic phase layers were washed with water and concentrated in vacuo to give a red oil which was purified by column chromatography (eluent ethyl acetate/petroleum ether=1/2 (V/V)) to give compound 28-3 (pale red oil, 80mg, yield 51.3%).

Compound 28-3 (80 mg,0.18 mmol) was dissolved in a mixed solution of 6N aqueous hydrochloric acid (1 mL) and tetrahydrofuran (1.5 mL), stirred at 0-5℃for 7 hours, pH was adjusted to 8 with Na ₂CO₃, the solvent was removed in vacuo, and the residue was purified by column chromatography to give compound ATV028 (off-white solid, 45mg, yield) 62.1％).¹H NMR(600MHz,DMSO-d₆)δ7.92(s,1H),7.86(br,1H),6.80(s,1H),4.69(d,J＝4.8Hz,1H),4.30(dd,J＝12.2,2.9Hz,1H),4.25-4.18(m,1H),4.14(dd,J＝12.2,4.9Hz,1H),3.99-3.90(m,1H),2.28-2.17(m,1H),1.79-1.68(m,2H),1.68-1.52(m,3H),1.34-1.07(m,5H).

Example 29: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-1-methylpiperidine-2-carboxylic acid) methyl ester (ATV 029)

Synthesis of Compound ATV029 as a white solid in a total of 0.40g Using 1-2 as a starting material and replacing tetrahydrofuran-3-carboxylic acid with 1-methylpiperidine-2-carboxylic acid according to the method described in example 1, the overall yield in two steps was 37％.¹HNMR(600MHz,Methanol-d₄)δ7.86(s,1H),6.90(d,J＝2.5Hz,2H),4.91-4.87(m,1H),4.49-4.34(m,3H),4.22-4.12(m,1H),2.89(dd,J＝11.9,3.3Hz,1H),2.79-2.68(m,1H),2.19(s,1H),2.17(s,2H),2.13-2.06(m,1H),1.82-1.49(m,5H),1.35-1.24(m,1H).¹³C NMR(151MHz,Methanol-d₄)δ174.1,174.0,157.3,148.4,125.7,118.0,117.6,112.2,102.6,102.5,83.3,81.5,81.5,75.6,72.3,72.2,68.6,64.7,64.6,55.9,44.4,30.7,30.6,26.0,23.8.

Example 30: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-yl) methyltetrahydro-2H-pyran-4-carboxylate (ATV 030)

Synthesis of Compound ATV030 as a white solid in a total of 0.96g Using 1-2 as a starting material and replacing tetrahydrofuran-3-carboxylic acid with 2H-pyran-4-carboxylic acid according to the procedure described in example 1, the overall yield in two steps was 67％.¹H NMR(600MHz,DMSO-d₆)δ7.93(s,1H),7.90(br,1H),6.92(d,J＝4.6Hz,1H),6.82(d,J＝4.6Hz,1H),6.35(d,J＝5.7Hz,1H),5.41(d,J＝5.6Hz,1H),4.71(t,J＝5.3Hz,1H),4.34-4.32(m,1H),4.25-4.22(m,1H),4.19-4.16(m,1H),3.99-3.96(m,1H),3.79-3.77(m,2H),3.33-3.29(m,2H),2.55-2.50(m,1H),1.68-1.63(m,2H),1.53-1.45(m,2H).¹³C NMR(151MHz,DMSO-d₆)δ156.2,148.5,124.0,117.4,117.1,110.7,101.2,81.6,79.4,74.5,70.6,66.5,63.3,28.8,28.7.

Example 31: ((2R, 3S,4R, 5R) -5- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-cyclobutanecarboxylic acid) methyl ester (ATV 031)

Synthesis of Compound ATV031 as a white solid in a total of 0.7g Using 1-2 as a starting material and replacing tetrahydrofuran-3-carboxylic acid with cyclobutylformic acid according to the method described in example 1, the overall yield in two steps was 42％.¹H NMR(400MHz,DMSO-d₆)δ7.93(s,1H),7.90(br,2H),6.93(d,J＝4.5Hz,1H),6.80(d,J＝4.5Hz,1H),6.34(d,J＝5.9Hz,1H),5.38(d,J＝5.8Hz,1H),4.68(t,J＝5.2Hz,1H),4.35-4.31(m,1H),4.25-4.15(m,2H),3.96-3.92(m,1H),3.19-3.11(m,1H),2.15-2.09(m,4H),1.97-1.73(m,2H).¹³C NMR(101MHz,DMSO-d₆)δ174.7,156.1,148.4,124.0,117.4,117.0,110.7,101.3,81.6,79.5,74.5,70.6,63.3,37.6,25.2,25.1,18.2.

Example 32: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-cycloheptanecarboxylic acid) methyl ester (ATV 032)

Synthesis of Compound ATV032 as a white solid in a total of 0.67g Using 1-2 as a starting material and replacing tetrahydrofuran-3-carboxylic acid with cycloheptanecarboxylic acid according to the procedure described in example 1, the overall yield in two steps was 36％.¹H NMR(600MHz,DMSO-d₆)δ7.93(s,1H),7.90(br,1H),6.92(d,J＝4.5Hz,1H),6.81(d,J＝4.5Hz,1H),4.70(d,J＝4.8Hz,1H),4.31-4.29(m,1H),4.24-4.22(m,1H),4.17-4.14(m,1H),3.97-3.95(m,1H),2.46-2.41(m,1H),1.80-1.77(m,2H),1.63-1.37(m,10H).¹³C NMR(151MHz,DMSO-d₆)δ176.1,156.1,148.4,124.1,117.4,117.0,110.6,101.2,81.7,79.4,74.6,70.6,63.1,60.2,44.4,30.7,30.6,28.2,28.1,26.2.

Example 33: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-1-methylpiperidine-4-carboxylic acid) methyl ester (ATV 033)

Synthesis of Compound ATV033 as a white solid in a total of 0.38g Using 1-2 as a starting material and replacing tetrahydrofuran-3-carboxylic acid with 1-methylpiperidine-4-carboxylic acid according to the method described in example 1, the overall yield in two steps was 30％.¹HNMR(600MHz,Methanol-d₄)δ7.86(s,1H),6.89(q,J＝4.6Hz,2H),4.90-4.88(m,1H),4.46-4.39(m,1H),4.39-4.30(m,2H),4.16(t,J＝5.7Hz,1H),3.02-2.86(m,2H),2.45-2.33(m,6H),1.97-1.86(m,2H),1.82-1.65(m,2H).¹³C NMR(151MHz,Methanol-d₄)δ175.3,157.3,148.4,125.6,118.0,117.7,112.2,102.6,83.4,81.5,75.6,72.2,64.7,55.1,49.5,49.3,49.2,49.0,48.9,48.8,48.6,45.5,40.3,28.1.

Example 34: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-4-fluorocyclohexane-1-carboxylic acid) methyl ester (ATV 034)

Synthesis of Compound ATV034 as a white solid in total 0.91g starting from 1-2 and replacing tetrahydrofuran-3-carboxylic acid with 4-fluorocyclohexylcarboxylic acid according to the procedure described in example 1, the overall yield in two steps is 48％.¹H NMR(600MHz,DMSO-d₆)δ7.98(br,2H),7.87(s,1H),6.93(d,J＝4.5Hz,1H),6.82(d,J＝4.5Hz,1H),6.74-6.49(m,1H),5.88-5.49(m,1H),4.71(d,J＝4.8Hz,1H),4.34-4.27(m,1H),4.26-4.20(m,1H),4.19-4.11(m,1H),3.97(t,J＝5.8Hz,1H),2.37-2.20(m,1H),2.01-1.90(m,2H),1.88-1.76(m,2H),1.50-1.31(m,4H).

Example 35: ((2R, 3S,4R, 5R) -5- (4-Aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-4, 4-difluorocyclohexane-1-carboxylic acid) methyl ester (ATV 035)

According to the method described in example 1, starting from 1-2 and replacing tetrahydrofuran-3-carboxylic acid with 4, 4-difluorocyclohexylcarboxylic acid, the synthesis of compound ATV035 amounted to 0.87g of a white solid, the overall yield in the two steps being 44％.¹H NMR(600MHz,DMSO-d₆)δ8.47(s,1H),7.96(d,J＝44.6Hz,4H),6.87(dd,J＝63.7,4.5Hz,4H),4.71(d,J＝4.9Hz,1H),4.41-4.11(m,5H),3.97(t,J＝5.7Hz,2H),2.06-1.69(m,9H),1.55(t,J＝11.8Hz,3H).

Example 36: antiviral Activity and cytotoxicity test of Compounds on respiratory syncytial Virus

HEp-2 cells were seeded into 48-well plates and cultured overnight. When the cell confluency reached 80%, the cells were treated at different drug concentrations (MOI 0.5) and then infected with RSVA2 virus for 1 hour. Subsequently, the virus-drug mixture is removed and the cells continue to be cultured in the drug-containing medium. 48 hours after inoculation, total RNA was extracted from the cells and then reverse transcribed using PRIMESCRIPT RT kit with GDNAERASER (from TaKaRa). To determine viral copies, TB is usedPremix Ex TaqTMII (from TaKaRa) were subjected to absolute quantitative RT-PCR. The RSV A2F fragment was quantified using primers 5'-CGAGCCAGAAGAGAACTACCA-3' and 5'-CCTTCTAGGTGCAGGAACCTTA-3'.

Three cell viability assays were performed in 96-well plates at each concentration. All drugs were diluted 2-fold in maintenance medium (DMEM with 2% fbs) starting at 500 μm in 9 gradients. After 48 hours of incubation, the supernatant was removed, 10. Mu.L of maintenance medium containing WST-8 (2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazolium monosodium salt) was added to the medium, after 2 hours of incubation, the plates were measured using a spectrophotometer (from BioTek) at a wavelength of 450nm, and cell viability was calculated.

TABLE 1 anti-RSV Activity and cytotoxicity of partial Compounds in HEp-2 cells

As is clear from Table 1, REMDESIVIR has the optimal in vitro RSV inhibitory activity, and EC ₅₀ reached 0.04. Mu.M, but also had a large cytotoxicity. All compounds showed more than 10-fold inhibition compared to the anti-RSV drug Monapinavir entered into the clinical study. The activity of the Ribavirin is 50 times higher than that of the clinical administration of the Ribavirin. VV116 is a tri-esterified form of deuterated GS441524, most of the compounds are slightly better than VV116, and some exhibit highly efficient and low-toxic properties, such as ATV017.

Example 37: pharmacokinetic properties of Compounds ATV014 and GS-441524 in rats

16 SD rats (males) weighing 220-250g were divided into 4 groups, namely ATV014 intravenous injection group, ATV014 oral administration group, GS441524 intravenous injection group, GS441524 oral administration group, and 4 animals each, each group was administered and jugular vein was collected. After administration, about 0.083 mL of collected blood (not collected in oral group), 0.16h (not collected in oral group), 0.25h, 0.5h, 1h (not collected in intravenous group), 2h, 4h, 8h, 24h, 48h was introduced into heparin tube, centrifuged at 4000r/min for 10min at 4℃and the upper plasma was transferred to refrigerator for freezing (about-20 ℃) and temporarily stored for measurement. Taking 50 mu L of plasma sample, adding 100L of 90% methanol aqueous solution, and mixing uniformly by vortex; then adding 350 mu L of methanol acetonitrile mixed solution (1:1, V/V) and mixing uniformly by vortex; centrifuging at 10000rpm for 10min, collecting supernatant, filtering with 0.22 μm filter membrane, and detecting by sample injection; blood samples within 0.5 hours after intravenous administration and 4 hours after oral administration were subjected to 10-fold dilution and then subjected to sample injection detection. The drug concentration in each sample was determined by High Performance Liquid Chromatography (HPLC)/Mass Spectrometry (MS). Analytes were separated using a Waters UPLC/XEVO TQ-S chromatography column, inertSustainAQ-C18HP column (3.0 mm. Times.50 mm,3.0 μm, GL). Pharmacokinetic parameters were calculated using DAS (Drug AND STATISTICS) 3.0 software.

The results are shown in tables 2 and 3, the oral bioavailability of ATV014 is 49.08%, the oral bioavailability of GS-441524 is 22.63%, and the results show that compared with GS-441524, the oral bioavailability of ATV014 is obviously improved, the plasma exposure dose AUC of the drug is doubled, the half-life period of an oral group is prolonged, and the 5' -ester prodrug has better oral drug property than the original drug.

Table 2. Drug generation parameters after administration of ATV014 to sd rats (detection GS-441524, mean ± standard deviation, n=3)

TABLE 3 drug generation parameters (mean ± standard deviation, n=4) after administration of GS-441524 to SD rats

The compound has better bioavailability, can reach larger Cmax, and has faster absorption speed.

Example 38: single maximum tolerated dose study of SD rats given ATV014 by oral gavage

ATV014 was given orally and gastrically to 3 rats per sex in each group at ascending dose levels of 1000 and 2000mg/kg with deionized water solution of 20% SBE-. Beta. -CD+5% TPGS as vehicle, and was observed continuously for 4 days after a single administration. Evaluation indexes include caged observations, detailed clinical observations, and post-administration clinical observations, body weight, and body weight changes.

Experimental results show that SD rats were given 1000mg/kg/day and 2000mg/kg/day of ATV014 by single oral gavage, all animals were well tolerated, all animals survived to the planned end day, and the experimental animals did not see changes in clinical observations, weight abnormalities, etc. associated with ATV 014. Thus SD rats were given a single oral gavage of ATV014 with an MTD of 2000mg/kg.

Example 39: dose range exploration of ATV014 given to SD rats by oral gavage for 14 days

The experimental animals were dosed orally with different doses (200, 400 or 800 mg/kg/day) of ATV014 or vehicle (20% SBE-beta-CD+5% TPGS in deionized water) at a dosing volume of 10.0mL/kg once daily for 14 consecutive days. The experimental evaluation indexes comprise cage side observation, detailed clinical observation after administration, weight, food intake and clinical examination (blood biochemistry, hematology and blood coagulation function). On day 15, all surviving test animals were subjected to clinical pathology sample collection prior to dissection. All planned and unplanned dead animals were subjected to gross section examinations including gross examination of body surfaces, orifices, cranial cavities, brain surfaces, thoracic cavities, abdominal cavities, pelvic cavities, viscera, bodies, and reproductive organs, etc., and viscera were weighed.

The test results are shown in Table 4, and the results show that ATV014 was given orally by gavage 1 time per day for 14 consecutive days to all animals well tolerated at doses of 200, 400 and 800mg/kg/day, with all animals surviving to the planned end day. After oral gavage of ATV014, plasma was mainly present as GS441524 at each time point in each dose group, so only the toxic parameters of GS441524 were calculated. On trial 1, plasma C _max of GS441524 was slightly decreased with increasing ATV014 dose from 200mg/kg/day to 800mg/kg/day, AUC _last was slightly increased, the extent of increase in C _max and AUC _last values was lower than the dose increase ratio, and the sex differences between the other doses C _max and AUC _last values were less than 2-fold except that on day 14 the 200mg/kg/day dose group had a 0.499-fold exposure to males. After multiple dosing, no accumulation of GS441524 was seen in SD rats (C _max、AUC_0-t values < 2-fold).

Thus, the MTD of ATV014 was 800mg/kg/day by repeated oral gavage for 14 days in the rat. ATV014 was rapidly metabolized to GS441524 in rats following oral administration, at which dose the C _max and AUC _last values for GS441524 on day 14 were 8977ng/mL and 72861hr ng/mL (male), 11457ng/mL and 77670hr ng/mL (female), respectively.

Table 4.Atv014 toxicological parameters test (calculated as metabolite GS 441524)

Example 40: in vitro non-mammalian cell system- -reverse mutation test of Salmonella typhimurium and Escherichia coli

Based on the results of the dose-discovery test, test subjects ATV014 were evaluated for their ability to induce reverse mutation in test subjects TA98, TA100, TA1535, TA1537 and WP2 uvrA at doses of 5000, 2000, 1000, 500 and 200 μg/dish (corresponding to subject concentrations of 50, 20, 10, 5, 2 mg/mL) with or without exogenous metabolic activation systems (β -naphthoflavone and phenobarbital-induced rat liver S9). The corresponding negative (vehicle) control and positive control were simultaneously tested.

In the bacterial back mutation main trial, ATV014 observed no significant bacterial toxicity at all doses administered in the presence or absence of S9 in the test strains TA98, TA1535, TA1537 and WP2 uvrA; under the condition that the test strain TA100 does not have S9, the colony number of the bacterial back mutation is obviously reduced by 50 percent and is accompanied by a certain dose response relation, so that the strain TA100 is toxic when the administration dose is 5000 mug/dish under the condition that the S9 mixture is not added; precipitation was observed at a dose of 5000 μg/dish.

ATV014 neither showed a 2-fold (for TA98, TA100 and WP2 uvrA) nor a 3-fold (for TA1535 and TA 1537) increase in the average number of the induced back mutant colonies in the doses tested, with or without the S9 mixture, relative to the average of the vehicle control group.

The average number of the reverse mutation colonies of the positive control group exceeds the corresponding vehicle control group by more than 3 times under the condition of adding and not adding the S9 mixture. The average number of revertant colonies from all test strain vehicle control and positive control groups were comparable to laboratory historical data. The bacterial back mutation test was effective, and ATV014 was inferred to be negative under the test conditions. The results are shown in Table 5.

TABLE 5 reverse mutation assay of ATV014 Salmonella typhimurium and Escherichia coli

Example 41: in vivo mammalian cell system- -rat micronucleus test in vivo

Female and male SD rats were given test ATV014 twice at doses of 200, 800 and 2000 mg/kg/day, respectively, orally gavaged for 24 hours. Vehicle control was administered in the same manner as ATV 014. The positive control (cyclophosphamide monohydrate, CP) was administered in a single dose of 20mg/kg by intraperitoneal injection. SD rat in vivo micronucleus primary assay design, table 6 below:

TABLE 6 SD rat micronucleus test Main test design

* 7 Animals per sex contained the potential to allow for drug-induced mortality. The number of animals used for analysis was 5 (5 surviving animals in the top of the high dose group).

Compared with the parallel vehicle control, the male mice observe a statistically significant reduction in PCE (multi-stained erythrocytes) percentage of total erythrocytes at 2000mg/kg/day dose, but PCE percentage of total erythrocytes is within the historical data range of the laboratory negative control, and no obvious dose-response relationship is seen, which suggests that ATV014 is not cytotoxic to rat bone marrow at the dose tested. The test ATV014 did not induce a statistically significant increase in micronuclei at all dosing levels compared to the parallel vehicle control. The results of the Cochran-Armitage analysis showed that the micronuclei formation rate also did not exhibit a dose-response relationship (p > 0.05). In conclusion, the micronucleus test is effective, and the test result accords with the negative judgment standard. Therefore, test substance ATV014 was judged negative in this rat bone marrow cell micronucleus test. The results are shown in Table 7.

TABLE 7 micronucleus assay in rats

* The comparison ratio of the solution to the parallel solvent is obviously different, the analysis of variance (DUNNETT) is carried out, and p is less than or equal to 0.05;

^# Significant differences compared to the parallel vehicle control, analysis of variance (ANOVA), p < 0.05;

^## Significant differences compared to the parallel vehicle control, analysis of variance (ANOVA), p.ltoreq.0.01.

Example 42: influence of ATV014 on respiratory system of SD rat by oral gavage

64 SD rats (32/sex) were used and randomly divided into 4 groups (8/sex/group) by body weight, each single oral administration of vehicle control (20% SBE-. Beta. -CD+5% TPGS in deionized water) and 100, 200, 400mg/kg dose of ATV014 formulation (formulated in vehicle). All rats were evaluated for caged observations, detailed clinical observations, body weight and respiratory function indicators (tidal volume, minute tidal volume and respiratory rate) during the body test.

The test results showed that ATV014 doses as high as 400mg/kg were found to be unoccupied by clinical observations, body weight, tidal volume (RR) abnormalities associated with the test subjects. The male animals in the 200 and 400mg/kg dose groups had a statistically significant difference in minute tidal volume (MV) lower than that in the control group at 3 hours after administration, mainly because the product MV was lower in both groups because the TV value and RR value at this time point were smaller than those of the corresponding control group, but the change trend and change amplitude of the MVs were not significantly different from those of the control group. The minute tidal volume of the males in each dose group was lower than that in the contemporaneous control group 24 hours after dosing, and was considered independent of the test substance, although there was a statistically significant difference, but at comparable levels as compared to the pre-dosing. The respiratory rate of the male animals in the 100 and 400mg/kg groups was lower than that of the control group at 24 hours after the administration, and the respiratory rate was statistically significantly different, but the respiratory rate of the high-dose group was comparable to the pre-administration level of the high-dose group, and the respiratory rate of the low-dose group was comparable to the pre-administration level of the low-dose group except for the individual difference of 1 animal. Female animals 100 and 200mg/kg dose group had a significantly different frequency of inhalation (RR) than the contemporaneous control group, but no significantly different from that before their own dosing, and 6/8 of each of these groups showed a reduction in RR, similar to the frequency of 5/8 of the RR reduction in the control group. The change in RR is therefore considered to be primarily an effect of individual data fluctuations.

In summary, under the present test conditions, 100, 200 and 400mg/kg ATV014 were administered to SD rats by single oral gavage, and no significant changes in respiratory parameters associated with the subjects were seen. NOAEL was 400mg/kg/day.

Example 43: effects of ATV014 on the Central nervous System of SD rats by oral gavage

The experiment used a total of 64 SD rats (32/sex), randomly divided into 4 groups by body weight (8/sex/group), and each single oral administration of vehicle control (20% SBE-. Beta. -CD+5% TPGS in deionized water) was performed with 100, 200, 400mg/kg doses of ATV014 formulation (formulated in vehicle). All rats were evaluated during the body test for paracage observations, detailed clinical observations, body weight and neuro-functional behavioral observations and tests (intra-cage observations, warranty observations, mine observations, stimulated reflex and body temperature).

A single oral gavage administration of ATV014 to 400mg/kg to rats did not reveal any clinical observations and weight abnormalities associated with the test subjects. The 200mg/kg male animals had a smaller residence time than the control group on day 2 post-dose, but the changes were not considered to be related to the subjects, since the trend and magnitude of the changes in the other animals were comparable to the control group level. The female animals 100mg/kg dose group had a larger number of stands on the day after administration than the control group, and the trend and magnitude of the change were comparable to those of the control group, though statistically different, and therefore were not considered to be related to the test substance. The single oral gavage administration of SD rat ATV014 to 400mg/kg has no effect on other open field observation indexes, and has no effect on in-cage observation, warranty observation, stimulated reflex and body temperature.

In summary, under the present test conditions, single oral gavage administration of 100, 200 and 400mg/kg ATV014 to SD rats had no significant effect on the central nervous system of the rats, with NOAEL of 400mg/kg/day.

Example 44: effects of ATV014 on hERG potassium ion channel on human embryonic kidney cells

The effect of ATV014 on hERG current was evaluated in human embryonic kidney cells (HEK 293) expressing hERG potassium ion channel. The effect of different concentrations of ATV014 (1, 3, 10, 30. Mu.M), negative control (extracellular fluid containing 0.1% dimethyl sulfoxide), positive control (0.1 μm cisapride) on hERG potassium channel current expressed in HEK293 cells was examined using manual patch clamp. The results showed that the positive control had an average corrected inhibition of 84.73% on hERG current, suggesting that the test system was valid. Under the experimental conditions, at concentrations up to 30 μm, the corrected inhibition rate for hERG potassium current was less than 50% and thus IC ₅₀ values were not calculated. It is speculated that the IC ₅₀ value for suppressing hERG potassium current is greater than 30 μm.

The invention develops an oral nucleoside drug, which shows high-efficiency low-toxicity antiviral effect in an in-vitro anti-RSV activity test experiment and is superior to the current clinical and clinical anti-RSV drugs. Pharmacokinetic studies showed that ATV014 has good oral bioavailability. ATV014 showed good safety in single and multiple repeat dosing toxicity experiments, salmonella typhimurium and Escherichia coli back mutation experiments, and micronucleus experiments in rats. ATV014 toxicity was also not found in respiratory, central and cardiac pharmacological profiling, and is expected to be useful in the treatment of infant RSV infection.

While the methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and combinations of the methods and applications described herein can be made and applied within the spirit and scope of the invention. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included within the present invention.

Claims

1. The use of a compound of formula I or a salt thereof for the manufacture of a medicament for the treatment of respiratory syncytial virus infection;

R ¹ is H, D or halogen;

2. Use according to claim 1, wherein the compound of formula I has one of the following structures or a salt thereof:

3. A method for reducing toxicity of nucleoside compounds in resisting respiratory syncytial virus infection, which is characterized in that a compound shown in a formula I or salt thereof is adopted as the nucleoside compounds, wherein the compound shown in the formula I has the structure as set forth in claim 1 or 2.

4. A method according to claim 3, wherein the compound of formula I or a salt thereof reduces toxicity by reducing cytotoxicity and/or reducing mutagenicity and/or reducing chromosome or mitotic damage.

5. A method for improving the bioavailability of a nucleoside compound drug against respiratory syncytial virus infection, which is characterized in that a compound shown in a formula I or a salt thereof is used as the nucleoside compound drug, wherein the compound shown in the formula I has the structure as set forth in claim 1 or 2.

6. A method for prolonging the acting time of nucleoside compound medicine in resisting respiratory syncytial virus infection, which is characterized in that a compound shown in a formula I or salt thereof is adopted as the nucleoside compound medicine, wherein the compound shown in the formula I has the structure as set forth in claim 1 or 2.

7. A method for accelerating the onset of action of nucleoside compounds in the treatment of respiratory syncytial virus infection, which is characterized in that a compound shown in formula I or a salt thereof is used as the nucleoside compound, wherein the compound shown in formula I has the structure as defined in claim 1 or 2.

8. The use according to claim 1 or 2, the method according to any one of claims 3-7, wherein the medicament is an oral medicament.

9. The use according to claim 1 or 2, the method according to any one of claims 3 to 7, wherein the oral medicament is a tablet, dispersible tablet, fast dissolving tablet, fast melting tablet, orodispersible tablet, lyophilized unit, porous tablet, conventional tablet, coated tablet, uncoated tablet, enteric tablet, effervescent tablet, dissolvable tablet, chewable tablet, oral lyophilisate, powder, oral powder, pill, capsule and/or granule.

10. The use according to claim 1 or 2, the method according to any one of claims 3-7, characterized in that the respiratory syncytial virus is of the RSVA type and/or the RSV type B.