Use of a nucleoside compound in preparing products related to anti-respiratory syncytial virus infection

Abstract

The present invention belongs to the field of medicine, and specifically provides use of nucleoside compounds in preparing drugs for resisting respiratory syncytial virus infection, a method for reducing the toxicity of nucleoside compounds in resisting respiratory syncytial virus infection, a method for improving the bioavailability of nucleoside compounds in resisting respiratory syncytial virus infection, a method for prolonging the action time of nucleoside compounds in resisting respiratory syncytial virus infection, and a method for accelerating the onset of action of nucleoside compounds in resisting respiratory syncytial virus infection.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to use of a nucleoside compound in preparing products related to resistance to respiratory syncytial virus infection. Background Art

[0002] Respiratory syncytial virus (RSV) can cause lower respiratory tract infections in infants and young children. 60% to 70% of infants under 1 year old will suffer from RSV infection, and almost all infants and young children will be infected with RSV at least once within 2 years old. RSV infection is global, and local outbreaks or even epidemics may occur. In 2019, there were approximately 33 million cases of acute lower respiratory tract diseases in children caused by RSV worldwide, and approximately 3.57 million children required hospitalization, of which approximately 1.4 million occurred in infants aged 0 to 6 months, and more than 100,000 children died as a result. More than 95% of cases occurred in low-income and middle-income countries. RSV infection has now become a global public health problem. To date, the FDA has approved only two drugs for the prevention and treatment of RSV infection: inhaled ribavirin and palivizumab, a humanized mouse monoclonal antibody to the human RSV F protein. However, the safety and scope of application of both drugs are poor, and they are not recommended for widespread clinical use. There is currently no vaccine or specific drug for the prevention and treatment of RSV infection. Its treatment mainly focuses on symptomatic supportive treatment such as maintaining airway patency, relieving cough and asthma, and oxygen inhalation. In view of the high morbidity rate in infants and young children and the high mortality rate in high-risk children, domestic and foreign countries are constantly developing preventive and therapeutic drugs for RSV infection. For the prevention and treatment of RSV infection, the development of antiviral drugs has attracted much attention. At present, the development of a variety of drugs targeting RSV F, L and N proteins for the prevention or treatment of RSV infection has entered the clinical research stage, mainly monoclonal antibodies, immunoglobulins, fusion inhibitors, nucleoprotein inhibitors, viral polymerase inhibitors, small molecule inhibitors, etc.

[0003] Viral polymerases remain the main therapeutic target for the treatment of viral infections. Various polymerase inhibitors have been approved for herpes, hepatitis B, hepatitis C, and HIV infections, which are essential for improving the quality of life of patients. L (Large) protein is a protein encoded by negative-strand RNA viruses with multiple biological activities. Its RNA polymerase activity is responsible for replicating the viral genome and transcribing mRNA. L protein is also a capping enzyme that guanylate and methylate the 5′ end of viral mRNA transcripts. In addition, it has polyadenylation activity that can protect the 3′ end of viral mRNA. Ribavirin is the only licensed drug for the treatment of RSV, which acts on the L protein. Due to its toxicity (including bone marrow suppression and potential carcinogenicity and teratogenicity) and marginal effects, it is not often used in infants hospitalized with RSV infection. It is also difficult to administer (it needs to be used with an aerosol) and has high drug costs. The antiviral mechanism of ribavirin is to inhibit the activity of the enzyme 5-phosphoinosine dehydrogenase (IMPDH), immunomodulate antiviral natural and cellular responses, terminate the synthesis of viral RNA, inhibit the capping reaction of viral mRNA, increase the accumulation of mutations in the viral genome, etc. In addition, RdRp inhibitors Monapinavir, Remdesivir and VV116 have entered clinical research as anti-RSV drugs. All three are oral prodrugs, and the prototype drugs of Remdesivir and VV116 are GS-441524 and its deuterated derivatives, respectively. Summary of the invention

[0004] Based on the shortcomings of GS-441524, the present invention has developed a series of 5'hydroxy ester-type prodrugs, including simple esters and amino acid esters, and the obtained nucleoside analogs were evaluated for anti-RSV activity at the cellular level. Since the recipients of the drug are mostly infants and young children, a variety of toxicity evaluations were also conducted in order to find highly effective and low-toxic nucleoside RSV therapeutic drugs.

[0005] In one aspect, the present invention provides the use of the compound of formula I or its salt in the preparation of a drug for resisting respiratory syncytial virus infection;

[0006]

[0007] R 1 is H, D or halogen;

[0008] R 2is C1-12 alkyl, 3-7 membered monocyclic heterocyclic group, 5-12 membered polycyclic heterocyclic group, C3-C7 monocyclic carbocyclic group, C5-12 polycyclic carbocyclic group, C6-10 aryl, C1-9 heteroaryl, 3-7 membered monocyclic heterocyclic group C1-6 alkyl, 5-12 membered polycyclic heterocyclic group C1-6 alkyl, C3-C7 monocyclic carbocyclic group C1-6 alkyl, C5-12 polycyclic carbocyclic group C1-6 alkyl, C6-10 aryl C1-6 alkyl, C1-9 heteroaryl C1-6 alkyl; wherein, optionally, R 2 The alkyl, monocyclic heterocyclic group, polycyclic heterocyclic group, monocyclic carbocyclic group, polycyclic carbocyclic group, aryl group and heteroaryl group in the above-mentioned group are each independently substituted by one or more substituents selected from deuterium, amino group, halogen group, methyl group and ethyl group.

[0009] In some embodiments, the compound described in Formula I has one of the following structures or a salt thereof:

[0010]

[0011]

[0012] On the other hand, the present invention provides a method for reducing the toxicity of nucleoside compound drugs in anti-respiratory syncytial virus infection, using a compound shown in formula I or a salt thereof as the nucleoside compound drug, wherein the compound shown in formula I has the structure described in the present invention.

[0013] In some embodiments, the compound of Formula I or a salt thereof reduces toxicity by reducing cytotoxicity and / or reducing mutagenicity and / or reducing chromosome or mitotic apparatus damage.

[0014] On the other hand, the present invention provides a method for improving the bioavailability of nucleoside compound drugs in anti-respiratory syncytial virus infection, using a compound shown in formula I or a salt thereof as the nucleoside compound drug, wherein the compound shown in formula I has the structure described in the present invention.

[0015] On the other hand, the present invention provides a method for prolonging the action time of nucleoside compound drugs in resisting respiratory syncytial virus infection, using a compound shown in formula I or a salt thereof as the nucleoside compound drug, wherein the compound shown in formula I has the structure described in the present invention.

[0016] On the other hand, the present invention provides a method for accelerating the effectiveness of nucleoside compound drugs in resisting respiratory syncytial virus infection, using a compound shown in formula I or a salt thereof as the nucleoside compound drug, wherein the compound shown in formula I has the structure described in the present invention.

[0017] In some embodiments, the drug is an oral drug.

[0018] In some embodiments, the oral medication is a tablet, a dispersible tablet, a fast-dissolving tablet, a quick-dissolving tablet, a fast-melting tablet, an orally dissolving tablet, an orally melting tablet, an orally dispersible tablet, a lyophilized unit, a porous tablet, a conventional tablet, a coated tablet, an uncoated tablet, an enteric-coated tablet, an effervescent tablet, a soluble tablet, a chewable tablet, an oral lyophilisate, a powder, an oral powder, a pill, a capsule and / or a granule.

[0019] In some embodiments, the respiratory syncytial virus is RSVA type and / or RSVB type.

[0020] Terminology

[0021] Certain embodiments of the present invention are now described in detail, examples of which are illustrated by the accompanying structural formulas and chemical formulae. The present invention is intended to encompass all substitutions, modifications, and equivalent technical solutions, which are all included within the scope of the present invention as defined in the claims. It should be appreciated by those skilled in the art that many methods and materials similar or equivalent to those described herein can be used to practice the present invention. The present invention is in no way limited to the methods and materials described herein. In the event that one or more of the combined documents, patents, and similar materials differ from or contradict the present application (including but not limited to defined terms, term applications, described technologies, etc.), the present application shall prevail.

[0022] It should be further appreciated that certain features of the invention, which for clarity are described in multiple separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which for brevity are described in a single embodiment, may also be provided separately or in any suitable sub-combination.

[0023] Unless otherwise specified, all technical terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. All patents and publications related to the present invention are incorporated herein by reference in their entirety.

[0024] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0025] In the following, all numbers disclosed herein are approximate, regardless of whether the words "about" or "approximately" are used. The value of each number may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15% or 20%. Whenever a number having a value of N is disclosed, 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.

[0026] "Oral preparations" refer to preparations that are administered orally and are absorbed into the blood in the gastrointestinal tract, including tablets, granules, capsules, oral solutions, etc.

[0027] In some embodiments, "oral preparation" is a "solid oral preparation", which refers to tablets, dispersible tablets, fast-dissolving tablets, fast-dissolving tablets, fast-melting tablets, oral dissolving tablets, oral melting tablets, orodispersible tablets, lyophilized units, porous tablets, conventional tablets, coated tablets, uncoated tablets, enteric-coated tablets (gastro-resistant tablets), effervescent tablets, soluble tablets, chewable tablets, oral lyophilisates, powders, oral powders, pills, capsules and / or granules. In some embodiments, the solid oral preparation is a capsule. In some embodiments, the solid oral preparation is a tablet.

[0028] “(C 1-n )alkyl”, where n is an integer, alone or in combination with another group, is intended to mean a non-cyclic, straight-chain or branched alkyl group containing 1 to n carbon atoms. 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), 1,1-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 a 1,1-dimethylethyl group.

[0029] "Aryl" includes aromatic hydrocarbon monocyclic or bicyclic rings having 6 to 10 carbon atoms, including benzene ring and naphthalene ring.

[0030] "Arylalkyl" refers to an alkyl group as defined herein in which one of the hydrogen atoms bonded to a carbon atom is replaced with an aryl group (ie, an aryl-alkyl-moiety) as described herein.

[0031] "Aryl-(C 1-n)alkyl-", wherein n is an integer, alone or in combination with another group, is intended to mean an alkyl group having 1 to n carbon atoms as defined herein, which itself is substituted by an aryl group as defined above.

[0032] "Heterocycle" is synonymous and, unless otherwise stated, refers to monocyclic and fused bicyclic, saturated or partially unsaturated rings having 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms, of which 1, 2, 3 or 4 ring atoms are heteroatoms independently selected from N, O and S and all remaining ring atoms are C.

[0033] "Heteroaryl" refers to a single aromatic ring or multiple fused 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, as long as the ring is aromatic. The ring includes, but is not limited to, pyridyl, pyrimidyl, oxazolyl or furanyl.

[0034] "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 "heteroarylalkyl" is typically 1-6 carbon atoms (i.e., heteroaryl (C1-C6) alkyl). Heteroarylalkyl groups include, but are not limited to, heteroaryl-CH2-, heteroaryl-CH(CH3)-, heteroaryl-CH2CH2-, 2-(heteroaryl)eth-1-yl, and the like, wherein the "heteroaryl" moiety includes any heteroaryl group as described herein.

[0035] Halogen and halogen refer to a halogen atom selected from F, Cl, Br and I.

[0036] One or more means 1 or 2, 3, 4, 5 or 6.

[0037] The salts described in the present invention are pharmaceutically acceptable salts, which refer to salts of the compounds of the present invention that are pharmaceutically acceptable and have the pharmacological activity of the parent compound. Such salts include: acid addition salts formed with inorganic acids or organic acids, such as nitric acid, phosphoric acid, carbonic acid, etc.; organic acids such as propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, gluconic acid, stearic acid, muconic acid, etc.; or salts formed when the acidic protons present on the parent compound are replaced by metal ions, such as alkali metal ions or alkaline earth metal ions; or coordination compounds formed with organic bases, such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, etc. The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. In general, the preparation method of such salts is: in water or an organic solvent or a mixture of the two, these compounds in free acid or base form are reacted with a stoichiometric amount of an appropriate base or acid to prepare. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0039] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0040] Example 1: Preparation of ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyltetrahydrofuran-3-carboxylate (ATV001)

[0041]

[0042] 5.62 g of compound 1-1 was dissolved in 30 mL of acetone, 11.50 mL of 2,2-dimethoxypropane and 1.34 mL of concentrated sulfuric acid were added, stirred at 45 ° C for 30 min, cooled to room temperature, and the organic solvent was removed by rotary evaporation. Extraction was performed with 100 mL of ethyl acetate and 100 mL of saturated sodium bicarbonate solution, and the extraction was repeated three times. The organic layers were combined, dried over anhydrous sodium sulfate, filtered and evaporated to dryness, and 6.20 g of compound 1-2 (white solid, yield 97%) was obtained by column chromatography. 1 H 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),5.2 4(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).

[0043] 1.50 g of compound 1-2 was dissolved in 15 ml of dichloromethane, 0.52 g of tetrahydrofuran-3-carboxylic acid and 55.40 mg of 4-dimethylaminopyridine were added, and after stirring for 10 min, 1.02 g of dicyclohexylcarbodiimide was added, and stirred at room temperature for 24 h. The reaction solution was diluted with dichloromethane, washed with saturated citric acid solution, saturated sodium carbonate solution and saturated brine respectively, and the organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to dryness to obtain the crude product 1-3, which was directly used in the next step reaction.

[0044] 1.50 g of compound 1-3 was dissolved in 3 mL of concentrated hydrochloric acid and 15 mL of tetrahydrofuran. After stirring for 6 hours in an ice bath, solid sodium carbonate was added to adjust the pH to 8. The organic solvent was removed by rotary evaporation and separated by column chromatography to obtain 0.86 g of compound ATV001 (white solid, yield 49%). 1 H 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,1 H),3.75-3.69(m,2H),3.66-3.62(m,1H),3.15-3.10(m,1H),2.08-1.95(m,2H). 13 C 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.

[0045] Example 2: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-nicotinic acid methyl ester (ATV002)

[0046]

[0047] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with pyridine-3-carboxylic acid to synthesize compound ATV002, a total of 0.75 g of white solid, with a two-step total yield of 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). 13 C 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.

[0048] Example 3: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-pentadecanoic acid methyl ester (ATV003)

[0049]

[0050] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with pentadecanoic acid to synthesize compound ATV003, a total of 0.35 g of white solid, with a total yield of 15% in two steps. 1 HNMR (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). 13 C 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.

[0051] Example 4: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-palmitic acid methyl ester (ATV004)

[0052]

[0053] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with hexadecanoic acid to synthesize compound ATV004, a total of 0.45 g of white solid, with a two-step total yield of 19%. 1 HNMR (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). 13 C NMR(151MHz,Methanol-d4)δ173.7,155.8,146.9,124.3,116.5,116.2,110.7,101.1,82.0,80.1,7 4.2,70.7,62.8,33.5,31.7,29.4,29.4,29.4,29.4,29.3,29.2,29.1,29.0,28.7,24.6,22.3,13.0.

[0054] Example 5: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-1-methylcyclohexane-1-carboxylic acid)methyl ester (ATV005)

[0055]

[0056] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with 1-methylcyclohexane-1-carboxylic acid to synthesize compound ATV005, a total of 1.0 g of white solid, with a total yield of 55% in two steps. 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,3 H), 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). 13 CNMR(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.

[0057] Example 6: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-isobutyric acid)methyl ester (ATV006)

[0058]

[0059] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with isobutyric acid to synthesize compound ATV006, a total of 0.96 g of white solid, with a two-step total yield of 59%. 1 HNMR (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). 13 C NMR(101MHz,Methanol-d4)δ176.96,155.82,146.92,124.25,116.54,116.29,1 10.75,101.20,82.04,80.00,74.27,70.68,62.93,33.58,25.00,17.95,17.87.

[0060] Example 7: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-acetic acid methyl ester (ATV007)

[0061]

[0062] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with acetic acid to synthesize compound ATV007, a total of 0.69 g of white solid, with a two-step total yield of 46%. 1 HNMR (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). 13 C 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.

[0063] Example 8: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-propionic acid)methyl ester (ATV008)

[0064]

[0065] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with propionic acid to synthesize compound ATV008, a total of 0.77 g of white solid, with a two-step total yield of 49%. 1 HNMR (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). 13 C 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.

[0066] Example 9: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-butyric acid methyl ester (ATV009)

[0067]

[0068] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with butyric acid to synthesize compound ATV009, a total of 0.83 g of white solid, with a total yield of 51% in two steps. 1 HNMR (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). 13 C 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.

[0069] Example 10: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-nonanoic acid methyl ester (ATV010)

[0070]

[0071] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with nonanoic acid to synthesize compound ATV010, a total of 0.56 g of white solid, with a two-step total yield of 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). 13 C 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.

[0072] Example 11: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-2-ethylbutanoate)methyl ester (ATV011)

[0073]

[0074] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with 2-ethylbutyric acid to synthesize compound ATV011, a total of 0.93 g of white solid, with a two-step total yield of 53%. 1 HNMR (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). 13 C 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.

[0075] Example 12: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-cyclopropanecarboxylic acid)methyl ester (ATV012)

[0076]

[0077] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with cyclopropanecarboxylic acid to synthesize compound ATV012, a total of 0.89 g of white solid, with a two-step total yield of 55%. 1 HNMR (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). 13 C 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.

[0078] Example 13: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-benzoic acid methyl ester (ATV013)

[0079]

[0080] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with benzoic acid to synthesize compound ATV013, a total of 0.93 g of white solid, with a total yield of 52% in two steps. 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). 13 C 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.

[0081] Example 14: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-cyclohexanecarboxylic acid)methyl ester (ATV014)

[0082]

[0083] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with cyclohexanecarboxylic acid to synthesize compound ATV014, a total of 1.14 g of white solid, with a total yield of 63% in two steps. 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). 13 CNMR(150MHz,DMSO-d6)δ(ppm):175.34,156.06,148.4,124.0,117.4,117.0,1 10.7,101.2,81.7,79.4,74.5,70.6,63.0,42.6,29.0,28.9,25.7,25.2,25.1.

[0084] Example 15: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-cyclopentanecarboxylic acid)methyl ester (ATV015)

[0085]

[0086] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with cyclopentanecarboxylic acid to synthesize compound ATV015, a total of 0.89 white solid, with a total yield of 51% in two steps. 1 HNMR (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 = 12.1 Hz, 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.

[0087] Example 16: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-3,3,3-trifluoropropanoyl)methyl ester (ATV016)

[0088]

[0089] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with 3,3,3-trifluoropropionic acid to synthesize compound ATV016, a total of 0.90 g of white solid, with a two-step total yield of 50%. 1 H NMR (600MHz, CD3OD) δ (ppm): 7.86 (s, 1H), 6.90-6.88 (q, J = 4.6Hz, 2H), 4.89 (d, J = 5.3H) z,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). 13 C 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.

[0090] Example 17: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-3-methylbutanoyl)methyl ester (ATV017)

[0091]

[0092] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with 3-methylbutyric acid to synthesize compound ATV017, a total of 0.75 g of white solid, with a two-step total yield of 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). 13 C 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.

[0093] Example 18: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-pivaloyl)methyl ester (ATV018)

[0094]

[0095] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with pivalic acid to synthesize compound ATV018, a total of 0.36 g of white solid, with a total yield of 21%. 1 HNMR (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). 13 C 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.

[0096] Example 19: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-D-valine)methyl ester (ATV019)

[0097]

[0098] 1.80 g of 1-2 was dissolved in 15 mL of dichloromethane, followed by the addition of 1.18 g of (D)-Boc-valine and 66.48 mg of 4-dimethylaminopyridine, and stirred for 10 min, followed by the addition of 1.22 g of dicyclohexylcarbodiimide, and stirred at room temperature for 24 h. After column chromatography, 2.81 g of compound 19-1 (white solid, yield 97%) was obtained. 1 HNMR(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),1.6 1(s,4H),1.32(d,J=2.5Hz,9H),1.24(s,3H),0.73(dd,J=19.0,6.8Hz,6H).

[0099] 2.50 g of compound 19-1 was dissolved in 3 mL of 37% hydrochloric acid aqueous solution and 15 mL of tetrahydrofuran. After stirring for 6 hours, sodium carbonate was added to adjust the pH to 8. The organic solvent was removed by rotary evaporator and separated by column chromatography (eluent: methanol / ethyl acetate (V / V) = 1:20) to obtain 0.99 g of compound ATV019 (white solid, yield 54%). 1 HNMR(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).

[0100] Example 20: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-L-valine)methyl ester (ATV020)

[0101]

[0102] According to the method described in Example 19, N-Boc-D-valine was replaced with N-Boc-L-valine to synthesize compound ATV020 as a white solid (1.1 g) with a total yield of 21% for two steps. 1 H 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 = 5.8 Hz, 1H), 3.25 (d, J = 4.9 Hz, 1H), 1.97-1.84 (m, 1H), 0.83 (d, J = 6.9 Hz, 3H), 0.79 (d, J = 6.9 Hz, 3H). 13 C 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.

[0103] Example 21: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-L-phenylalanine methyl ester (ATV021)

[0104]

[0105] According to the method described in Example 19, N-Boc-D-valine was replaced with N-Boc-L-phenylalanine to synthesize compound ATV021, a total of 0.1 g of white solid, with a two-step total yield of 17%. 1 H 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,1 H),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.

[0106] Example 22: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-D-phenylalanine methyl ester (ATV022)

[0107]

[0108] According to the method described in Example 19, N-Boc-D-valine was replaced with N-Boc-D-phenylalanine to synthesize compound ATV022, a total of 0.1 g of white solid, with a two-step yield of 15%. 1 H 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, 1 H), 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). 13 C NMR(150MHz,DMSO-d6)δ(ppm):175.2,156.1,148.4,138.2,129.7,128.6,12 6.8,124.0,117.4,117.1,110.8,101.3,81.7,79.5,74.5,70.7,63.9,56.1.

[0109] Example 23: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-(2S)-2-amino-3-methylpentanoyl)methyl ester (ATV023)

[0110]

[0111] According to the method described in Example 19, N-Boc-D-valine was replaced with N-Boc-L-isoleucine to synthesize compound ATV023 as a white solid (0.06 g) with a two-step yield of 10%. 1 H 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). 13 C 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.

[0112] Example 24: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-(2R)-2-amino-3-methylpentanoyl)methyl ester (ATV024)

[0113]

[0114] According to the method described in Example 19, N-Boc-D-valine was replaced with N-Boc-D-isoleucine to synthesize compound ATV024 as a white solid (0.06 g), with a two-step yield of 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 (b r,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). 13 C 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.

[0115] Example 25: ((2R,3S,4R,5R)-5-(4-amino-5-fluoropyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl isobutyrate (ATV025)

[0116]

[0117] Take ATV006 (1 g, 2.77 mmol), Selectfluor (1-chloromethyl-4-fluoro-1,4-diazobicyclo 2.2.2 octane bis(tetrafluoroborate) salt, 1.4 g, 5.5 mmol) and DMAP (0.34 g, 2.77 mmol), add 20 ml of a mixed solvent of acetonitrile-water (v / v=9:1), stir at room temperature for 24 h, monitor by TLC (mobile phase: DCM:MeOH=10:1), until ATV006 is basically reacted, remove acetonitrile by distillation under reduced pressure, add water and ethyl acetate, stir to separate the organic layer, extract the aqueous layer twice with ethyl acetate, combine the organic layers, wash the combined organic phases with saturated sodium carbonate solution and saturated sodium chloride solution in turn, dry over anhydrous sodium sulfate, filter and evaporate to dryness to obtain a dark red oil, which is separated and purified by column chromatography to obtain 100 mg of nearly white solid ATV025 with a yield of 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). 13 C 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. 19 F NMR(600MHz,CD3OD)δ(ppm):-160.8.

[0118] Example 26: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-(1R,4R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate)methyl ester (ATV026)

[0119]

[0120] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with (1R,4R)-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid to synthesize compound ATV026, a total of 1.0 g of white solid, with a total yield of 61% for two steps. 1 H 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.

[0121] Example 27: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-(3R,5R,7R)-adamantane-1-carboxylate)methyl ester (ATV027)

[0122]

[0123] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with (3R,5R,7R)-adamantane-1-carboxylic acid to synthesize compound ATV027, a total of 1.23 g of white solid, with a two-step total yield of 67%. 1 H 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). 13 C 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.

[0124] Example 28: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl-5-d)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methylcyclohexanecarboxylate (ATV028)

[0125]

[0126] 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 2 h, and then the solvent was removed under reduced pressure. The residue was purified by column chromatography (ethyl acetate / petroleum ether = 1 / 2 (V / V)) to obtain compound 28-2 (red solid, 0.4 g, yield 64%).

[0127] PdCl2(dppf)2 (32 mg, 0.04 mmol) was added to a solution of D2O-DMSO-d6 (10 mL, D2O:DMSO = 1:9 (V / V)) containing compound 28-2 (200 mg, 0.35 mmol) and cesium carbonate (247 mg, 0.76 mmol) under argon conditions, and the reaction was stirred at 80°C for 10 hours. The mixture was cooled to room temperature and slowly poured into water (10 mL), and then extracted with ethyl acetate (30 mL×2). The organic layers were combined, washed with water and concentrated in vacuo to give a red oil. The red oil was purified by column chromatography (eluent: ethyl acetate / petroleum ether = 1 / 2 (V / V)) to give compound 28-3 (light red oil, 80 mg, yield 51.3%).

[0128] Compound 28-3 (80 mg, 0.18 mmol) was dissolved in a mixed solution of 6N aqueous hydrochloric acid solution (1 mL) and tetrahydrofuran (1.5 mL), stirred at 0-5°C for 7 hours, the pH was adjusted to 8 with Na2CO3, the solvent was removed in vacuo, and the residue was purified by column chromatography to obtain compound ATV028 (off-white solid, 45 mg, yield 62.1%). 1 H 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).

[0129] Example 29: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-1-methylpiperidine-2-carboxylate)methyl ester (ATV029)

[0130]

[0131] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with 1-methylpiperidine-2-carboxylic acid to synthesize compound ATV029, a total of 0.40 g of white solid, with a two-step total yield of 37%. 1 HNMR (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). 13 C NMR(151MHz,Methanol-d4)δ174.1,174.0,157.3,148.4,125.7,118.0,117.6,112.2,102.6,1 02.5,83.3,81.5,81.5,75.6,72.3,72.2,68.6,64.7,64.6,55.9,44.4,30.7,30.6,26.0,23.8.

[0132] Example 30: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyltetrahydro-2H-pyran-4-carboxylate (ATV030)

[0133]

[0134] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with 2H-pyran-4-carboxylic acid to synthesize compound ATV030, a total of 0.96 g of white solid, with a two-step total yield of 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),4.3 4-4.32(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). 13 C 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.

[0135] Example 31: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-cyclobutanecarboxylic acid)methyl ester (ATV031)

[0136]

[0137] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with cyclobutylcarboxylic acid to synthesize compound ATV031, a total of 0.7 g of white solid, with a two-step total yield of 42%. 1 H 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.

[0138] Example 32: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-cycloheptanecarboxylic acid)methyl ester (ATV032)

[0139]

[0140] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with cycloheptanecarboxylic acid to synthesize compound ATV032, a total of 0.67 g of white solid, with a two-step total yield of 36%. 1 H 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). 13 C NMR(151MHz,DMSO-d6)δ176.1,156.1,148.4,124.1,117.4,117.0,110.6,10 1.2,81.7,79.4,74.6,70.6,63.1,60.2,44.4,30.7,30.6,28.2,28.1,26.2.

[0141] Example 33: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-1-methylpiperidine-4-carboxylate)methyl ester (ATV033)

[0142]

[0143] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with 1-methylpiperidine-4-carboxylic acid to synthesize compound ATV033, a total of 0.38 g of white solid, with a total yield of 30% in two steps.1 HNMR (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). 13 C 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.

[0144] Example 34: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-4-fluorocyclohexane-1-carboxylic acid)methyl ester (ATV034)

[0145]

[0146] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with 4-fluorocyclohexylcarboxylic acid to synthesize compound ATV034, a total of 0.91 g of white solid, with a two-step total yield of 48%. 1 H NMR(600MHz,DMSO-d6)δ7.98(br,2H),7.87(s,1H),6.93(d,J=4.5Hz,1H),6.8 2(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).

[0147] Example 35: ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-4,4-difluorocyclohexane-1-carboxylic acid)methyl ester (ATV035)

[0148]

[0149] According to the method described in Example 1, 1-2 was used as the raw material, and tetrahydrofuran-3-carboxylic acid was replaced with 4,4-difluorocyclohexylcarboxylic acid to synthesize compound ATV035, a total of 0.87 g of white solid, with a two-step total yield of 44%. 1 H 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).

[0150] Example 36: Antiviral activity and cytotoxicity test of compounds on respiratory syncytial virus

[0151] HEp-2 cells were seeded into 48-well plates and cultured overnight. When the cell confluence reached 80%, the cells were treated with different drug concentrations (MOI of 0.5) and then infected with RSV A2 virus for 1 hour. Subsequently, the virus-drug mixture was removed and the cells continued to be cultured in medium containing drugs. 48 hours after seeding, total RNA was extracted from the cells and then reverse transcribed using the PrimeScript RT Kit with gDNAEraser (from TaKaRa). To determine the viral copies, TB Absolute quantitative RT-PCR was performed using Premix Ex TaqTMII (from TaKaRa). The RSVA2 F fragment was quantified using primers 5'-CGAGCCAGAAGAGAACTACCA-3' and 5'-CCTTCTAGGTGCAGGAACCTTA-3'.

[0152] Each concentration was tested for cell viability in triplicate in a 96-well plate. All drugs were diluted 2-fold in 9 serial steps starting from 500 μM in maintenance medium (DMEM containing 2% FBS). After 48 hours of culture, the supernatant was removed, and 10 μL of maintenance medium containing WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazolyl monosodium salt) was added to the culture medium. After 2 hours of culture, the plate was measured at a wavelength of 450 nm using a spectrophotometer (from BioTek), and cell viability was calculated.

[0153] Table 1. Anti-RSV activity and cytotoxicity of some compounds in HEp-2 cells

[0154]

[0155]

[0156] As shown in Table 1, although Remdesivir has the best in vitro activity in inhibiting RSV, EC 50 It reaches 0.04μM, but it also has great cytotoxicity. Compared with the anti-RSV drug Monapinavir that has entered clinical research, all compounds show more than 10 times the inhibitory activity. It is 50 times more active than the clinical drug Ribavirin in anti-RSV activity. VV116 is a tri-esterified form of deuterated GS441524. Most compounds are slightly better than VV116, and some show the characteristics of high efficiency and low toxicity, such as ATV017.

[0157] Example 37: Pharmacokinetic properties of compounds ATV014 and GS-441524 in rats

[0158] 16 SD rats (male) weighing 220-250g were divided into 4 groups, namely ATV014 intravenous injection group, ATV014 oral group, GS441524 intravenous injection group, and GS441524 oral group, with 4 rats in each group, and were administered and blood was collected from the jugular vein. About 0.3mL of blood was collected at 0.083h (no blood was collected in the oral group), 0.16h (no blood was collected in the oral group), 0.25h, 0.5h, 1h (no blood was collected in the intravenous injection group), 2h, 4h, 8h, 24h, and 48h after administration into a heparin tube, centrifuged at 4000r / min for 10min at 4℃, and the upper plasma was transferred to a refrigerator (about -20℃) for temporary storage until the measurement. Take 50μL plasma sample, add 100L 90% methanol aqueous solution, vortex mix; then add 350μL methanol acetonitrile mixed solution (1:1, V / V), vortex mix; centrifuge at 10000rpm for 10min, take the supernatant and filter it through 0.22μm filter membrane before sampling and detection; blood samples taken 0.5 hours after intravenous administration and 4 hours after oral administration were diluted 10 times and then sampled for detection. High performance liquid chromatography (HPLC) / mass spectrometry (MS) was used to determine the drug concentration in each sample. Waters UPLC / XEVO TQ-S column and InertSustainAQ-C18HP column (3.0mm×50mm,3.0μm,GL) were used to separate the analytes. DAS (Drug and Statistics) 3.0 software was used to calculate the pharmacokinetic parameters.

[0159] The results are shown in Tables 2 and 3. The oral bioavailability of ATV014 was 49.08%, and the oral bioavailability of GS-441524 was 22.63%, indicating that compared with GS-441524, the oral bioavailability of ATV014 was significantly improved, the drug plasma exposure AUC was doubled, the half-life of the oral group was prolonged, and the 5' ester prodrug had better oral drugability than the prototype drug.

[0160] Table 2. Pharmacokinetic parameters of SD rats after administration of ATV014 (detection of GS-441524, mean ± SD, n = 3)

[0161]

[0162] Table 3. Pharmacokinetic parameters of GS-441524 administered to SD rats (mean ± standard deviation, n = 4)

[0163]

[0164] The compound of the present invention has better bioavailability and can achieve a larger Cmax, and the drug absorption speed is faster.

[0165] Example 38: Study on the single maximum tolerated dose of ATV014 administered orally to SD rats

[0166] ATV014 was administered orally to 3 rats of each sex in each group at increasing doses of 1000 and 2000 mg / kg using a 20% SBE-β-CD + 5% TPGS deionized water solution as the solvent. After a single dose, the rats were observed for 4 consecutive days. Evaluation indicators included cage-side observation, detailed clinical observation and clinical observation after administration, body weight and body weight changes.

[0167] The experimental results showed that SD rats were well tolerated by a single oral gavage of 1000mg / kg / day and 2000mg / kg / day of ATV014, and all animals survived until the planned end date. No clinical observations or abnormal body weight changes related to ATV014 were observed in the experimental animals. Therefore, the MTD of ATV014 for SD rats administered by oral gavage is 2000mg / kg.

[0168] Example 39: Dose range exploration of ATV014 administered orally to SD rats for 14 days

[0169] The experimental animals were orally gavaged with different doses (200, 400 or 800 mg / kg / day) of ATV014 or vehicle (20% SBE-β-CD + 5% TPGS in deionized water) at a dosing volume of 10.0 mL / kg once a day for 14 consecutive days. The experimental evaluation indicators included cage-side observation, detailed clinical observation, detailed clinical observation after administration, body weight, food intake and clinical examination (blood biochemistry, hematology, coagulation function). On the 15th day, clinical pathology samples were collected from all surviving experimental animals before dissection. A gross autopsy was performed on all animals with planned and unplanned deaths, including gross examination of the body surface, orifices, cranial cavity, brain surface, chest cavity, abdominal cavity, pelvic cavity, internal organs, trunk and reproductive organs, and organ weighing was performed.

[0170] The test results are shown in Table 4. The results show that rats were given ATV014 by oral gavage once a day for 14 consecutive days, with doses of 200, 400 and 800 mg / kg / day. All animals tolerated it well and all animals survived until the planned end date. After oral gavage of ATV014, the plasma was mainly in the form of GS441524 at each time point in each dose group, so only the toxicity parameters of GS441524 were calculated. On the first day of the test, as the ATV014 dose increased from 200 mg / kg / day to 800 mg / kg / day, the plasma C max Slight decrease in AUC last Slightly elevated, C max and AUC lastThe increase in the values ​​was lower than the dose increase ratio. Except for the 200 mg / kg / day group on day 14, the exposure of male animals was 0.499 times that of female animals. max and AUC last The gender difference in the value was less than 2 times. After multiple administration, no accumulation of GS441524 was observed in SD rats (C max , AUC 0-t value <2 times).

[0171] Therefore, the MTD of ATV014 administered to rats by repeated oral gavage for 14 days is 800 mg / kg / day. ATV014 is rapidly metabolized to GS441524 in rats after oral administration. At this dose, the C max and AUC last The values ​​were 8977 ng / mL and 72861 hr*ng / mL (male), and 11457 ng / mL and 77670 hr*ng / mL (female).

[0172] Table 4. Toxicokinetic parameter test of ATV014 (calculated based on metabolite GS441524)

[0173]

[0174] Example 40: In vitro non-mammalian cell system - Salmonella typhimurium and Escherichia coli reverse mutation assay

[0175] According to the results of the dose-finding experiment, the ability of the test substance ATV014 to induce the production of reverse mutations in the test strains TA98, TA100, TA1535, TA1537 and WP2 uvrA was evaluated under two conditions with or without the addition of an exogenous metabolic activation system (β-naphthoflavone and phenobarbital-induced rat liver S9). The test doses were 5000, 2000, 1000, 500 and 200 μg / dish (corresponding to the test substance concentration of 50, 20, 10, 5, 2 mg / mL). The corresponding negative (solvent) control and positive control were also tested.

[0176] In the main test of bacterial reverse mutation, no obvious bacterial toxicity was observed at all doses of ATV014 in the test strains TA98, TA1535, TA1537 and WP2 uvrA, in the presence or absence of S9; in the test strain TA100, in the absence of S9, the number of bacterial reverse mutation colonies was significantly reduced by 50%, accompanied by a certain dose-effect relationship, so the strain TA100 was determined to be toxic at a dose of 5000μg / dish without the addition of S9 mixture; precipitation was observed at a dose of 5000μg / dish.

[0177] With or without the addition of S9 mixture, the average number of reverse mutant colonies induced by ATV014 at the tested doses was not greater than 2 times (for TA98, TA100 and WP2 uvrA) or 3 times (for TA1535 and TA1537) the mean of the corresponding solvent control group, and there was no concentration-dependent increase in the number of reverse mutant colonies.

[0178] The average number of reverse mutation colonies in the positive control group was more than 3 times that of the corresponding solvent control group under the conditions of adding and not adding S9 mixture. The average number of reverse mutation colonies in the solvent control group and the positive control group of all tested strains was comparable with the laboratory historical data. This bacterial reverse mutation test was effective, and ATV014 was inferred to be negative under this test condition. The results are shown in Table 5.

[0179] Table 5. ATV014 Salmonella typhimurium and Escherichia coli reverse mutation test

[0180]

[0181]

[0182] Example 41: In vivo mammalian cell system - rat micronucleus test

[0183] Female and male SD rats were given the test substance ATV014 twice by oral gavage at a dose of 200, 800 and 2000 mg / kg / day, respectively, with an interval of 24 hours. The vehicle control was administered in the same manner as ATV014. The positive control (cyclophosphamide monohydrate, CP) was administered once by intraperitoneal injection at a dose of 20 mg / kg. The main experimental design of micronucleus in SD rats is as shown in Table 6 below:

[0184] Table 6. Experimental design of the main experiment of SD rat micronucleus test

[0185]

[0186] *7 animals per sex were included to allow for drug-induced mortality. The number of animals used for analysis was 5 (first 5 surviving animals in the high dose group).

[0187] Compared with the parallel vehicle control, a statistically significant decrease in the percentage of PCE (polychromatophilic erythrocytes) in the total number of red blood cells was observed in male rats at a dose of 2000 mg / kg / day, but the percentage of PCE in the total number of red blood cells was within the historical data range of the laboratory negative control, and no obvious dose-effect relationship was observed, suggesting that ATV014 had no cytotoxicity to rat bone marrow at the measured doses. Compared with the parallel vehicle control, the test substance ATV014 did not induce a statistically significant increase in micronuclei at all dosage levels. The results of Cochran-Armitage analysis showed that the micronucleus formation rate did not show a dose-effect relationship (p>0.05). In short, this micronucleus test is valid and the test results meet the negative judgment criteria. Therefore, the test substance ATV014 was judged to be negative in this rat bone marrow cell micronucleus test. The results are shown in Table 7.

[0188] Table 7. Rat in vivo micronucleus test

[0189]

[0190]

[0191] *.Significantly different from the parallel solvent control, analysis of variance (DUNNETT), p≤0.05;

[0192] # .Significantly different from the parallel solvent control, analysis of variance (ANOVA), p ≤ 0.05;

[0193] ## .Significantly different from the parallel vehicle control, analysis of variance (ANOVA), p≤0.01.

[0194] Example 42: Effects of oral administration of ATV014 on the respiratory system of SD rats

[0195] 64 SD rats (32 / sex) were randomly divided into 4 groups (8 / sex / group) according to body weight, and were orally administered with a single dose of vehicle control (20% SBE-β-CD + 5% TPGS in deionized water) and 100, 200, 400 mg / kg of ATV014 formulation (prepared with vehicle). During the in vivo experiment, cage-side observation, detailed clinical observation, body weight and respiratory function indicators (tidal volume, minute tidal volume and respiratory rate) were evaluated for all rats.

[0196] The test results showed that the ATV014 dose was as high as 400 mg / kg, and no abnormal clinical observations, body weight, and tidal volume (RR) related to the test substance were observed. Three hours after administration, the minute tidal volume (MV) of male animals in the 200 and 400 mg / kg dose groups was lower than that of the control group, with statistically significant differences. This was mainly due to the fact that the TV and RR values ​​of the two groups at this time point were lower than those of the corresponding control group, resulting in a lower product MV, but the change trend and amplitude of the MV in the two groups were not significantly different from those in the control group. 24 hours after administration, the minute tidal volume of male animals in each dose group was lower than that of the control group during the same period. Although there was a statistically significant difference, it was equivalent to the level before administration, so it was considered to be unrelated to the test substance. 24 hours after administration, the respiratory rate of male animals in the 100 and 400 mg / kg groups was lower than that of the control group, with statistically significant differences, but the values ​​of the high-dose group were equivalent to their own pre-dose levels, and the values ​​of the respiratory rate in the low-dose group were also equivalent to their pre-dose levels except for the individual difference of one animal. The respiratory rate (RR) of female animals in the 100 and 200 mg / kg dose groups was significantly lower than that of the control group during the same period, but there was no significant difference compared with the pre-drug administration. In addition, 6 / 8 animals in each of these two groups showed a decrease in RR, which was similar to the frequency of 5 / 8 animals in the control group. Therefore, it is believed that the change in RR is mainly affected by the fluctuation of individual data.

[0197] In summary, under the conditions of this study, SD rats were given 100, 200 and 400 mg / kg ATV014 by oral gavage, and no significant changes in respiratory parameters related to the test substance were observed. The NOAEL is 400 mg / kg / day.

[0198] Example 43: Effects of oral administration of ATV014 on the central nervous system of SD rats

[0199] A total of 64 SD rats (32 / sex) were used in this study and randomly divided into 4 groups (8 / sex / group) according to body weight. The rats were given a single oral administration of vehicle control (20% SBE-β-CD + 5% TPGS in deionized water) and 100, 200, 400 mg / kg of ATV014 preparation (prepared with vehicle). During the in vivo experiment, all rats were evaluated by cage observation, detailed clinical observation, body weight and neurological functional behavioral observation and testing (cage observation, restraint observation, mine field observation, stimulation reflex and body temperature).

[0200] ATV014 was administered to rats by single oral gavage at a dose of 400 mg / kg, and no clinical observations or abnormal body weight related to the test substance were observed. The retention time of male animals in the 200 mg / kg dose group was shorter than that of the control group on the second day after administration, which was statistically different, but given that the trend and amplitude of changes in other dose groups were comparable to those in the control group, it was considered that the change was not related to the test substance. The number of standing times of female animals in the 100 mg / kg dose group on the day after administration was greater than that of the control group. Although there was a statistical difference, the trend and amplitude of changes were comparable to those in the control group, so it was considered not to be related to the test substance. A single oral gavage of ATV014 to 400 mg / kg to SD rats had no effect on other open field observation indicators, and had no effect on cage observations, restraint observations, stimulus reflexes, and body temperature.

[0201] In summary, under the conditions of this experiment, a single oral gavage of 100, 200 and 400 mg / kg ATV014 to SD rats had no significant effect on the rat central nervous system, and the NOAEL was 400 mg / kg / day.

[0202] Example 44: Effect of ATV014 on hERG potassium channels in human embryonic kidney cells

[0203] This experiment evaluated the effect of ATV014 on hERG current in human embryonic kidney cells (HEK293) expressing hERG potassium channels. The experiment used a manual patch clamp method to detect the effects of different concentrations of ATV014 (1, 3, 10, 30 μM), negative control (extracellular fluid containing 0.1% dimethyl sulfoxide) and positive control (0.1 μm cisapride) on the hERG potassium channel current expressed in HEK293 cells. The results showed that the average corrected inhibition rate of the positive control on the hERG current was 84.73%, indicating that this experimental system is established. Under the experimental conditions, when the concentration reached 30 μM, the corrected inhibition rate of the hERG potassium current was less than 50%, so the IC was not calculated. 50 Based on this, it is speculated that the IC 50 The value was greater than 30 μM.

[0204] The present invention develops an oral nucleoside drug, which shows a highly effective and low-toxic antiviral effect in an in vitro anti-RSV activity test experiment, and is superior to the anti-RSV drugs currently in clinical use and in clinical research. Pharmacokinetic studies have shown that ATV014 has good oral bioavailability. ATV014 showed good safety in single and repeated dosing toxicity experiments, Salmonella typhimurium and Escherichia coli reverse mutation tests, and in vivo micronucleus tests in rats. ATV014 toxicity was not found in the respiratory system, central nervous system, and cardiac pharmacology safety assessments, and is expected to be used to treat RSV infection in infants and young children.

[0205] The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A compound or a salt thereof, wherein the compound is selected from ATV034 or compound ATV035: 。 2. Use of a compound or a salt thereof in the preparation of a drug for resisting respiratory syncytial virus infection; characterized in that: The compound is ATV013, ATV034 or ATV035: 。 3. The use according to claim 2, characterized in that: The medicine is an oral medicine.

4. The use according to claim 3, characterized in that The oral medicine is a tablet, an oral lyophilisate, a powder, a pill, a capsule and / or a granule.

5. The use according to claim 4, wherein the tablet is selected from dispersible tablets, porous tablets, enteric-coated tablets, effervescent tablets, and chewable tablets.

6. The use according to claim 4, wherein the tablet is selected from the group consisting of a quick-dissolving tablet and an orodispersible tablet.

7. The use according to claim 4, wherein the tablet is selected from coated tablets and uncoated tablets.

8. The use according to claim 4, wherein the tablet is selected from orally disintegrating tablets.

9. The use according to claim 4, wherein the tablet is selected from soluble tablets.

10. The use according to claim 2, characterized in that: The respiratory syncytial virus is RSV A type.

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