CN112771048A - Inhibitors of influenza virus replication and intermediates and uses thereof - Google Patents

Inhibitors of influenza virus replication and intermediates and uses thereof Download PDF

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CN112771048A
CN112771048A CN201980062669.4A CN201980062669A CN112771048A CN 112771048 A CN112771048 A CN 112771048A CN 201980062669 A CN201980062669 A CN 201980062669A CN 112771048 A CN112771048 A CN 112771048A
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compound
pyrrolo
influenza
amino
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CN112771048B (en
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李进
王利莎
王琳
杨民民
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Nanjing Maisheng Technology Co.,Ltd.
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Pharmablock Sciences (nanjing) Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/06—Peri-condensed systems

Abstract

The application discloses a compound capable of being used as an influenza virus replication inhibitor, an intermediate for preparing the compound and application of the compound in preparing a medicament for preventing or treating virus infectious diseases, in particular to a medicament for preventing influenza A virus infection.

Description

Inhibitors of influenza virus replication and intermediates and uses thereof Technical Field
The application belongs to the field of chemical medicine, and particularly relates to an influenza virus replication inhibitor, an intermediate and application thereof.
Background
Influenza (Flu for short) is a respiratory disease caused by Influenza virus (Influenza virus). According to the difference of antigenic determinants of virus nucleoprotein and matrix protein, influenza viruses can be divided into three types of A (A), B (B) and C (C), wherein the A type influenza is the most common, has strong pathogenicity, is easy to cause large-scale epidemics and seriously threatens human life and health. For preventing and treating influenza, two methods, namely vaccination and anti-influenza drug treatment, are generally adopted. Vaccination is currently an effective measure to prevent influenza. The inoculation can achieve a better prevention effect for adults, but the effect is not ideal for infants, old people and other people with lower immunity. Furthermore, influenza viruses are constantly mutated and old vaccines are difficult to fight against new viruses. Chemicals are another important means for treating influenza, but to date, the number of anti-influenza chemicals on the market is small, and M2 ion channel inhibitors, Neuraminidase (NA) inhibitors and nucleoside antiviral drugs are more widely used.
In recent years, influenza RNA polymerase (RdRp) has received much attention. RdRp is a heterotrimer composed of three subunits, PA, PB1 and PB2, and plays an important role in the transcription and replication of influenza virus genomes. Transcription of influenza virus RNA has a special "cap-robbing" mechanism in which the PB2 subunit is responsible for recognizing and binding the "cap structure" of the host precursor mRNA, and then the PA subunit cleaves the "cap" as a primer, initiating the transcription process. Inhibition of cap-robbing can block the transcription process, achieving the effect of inhibiting the proliferation of influenza virus. Thus, PB2 is considered a promising anti-influenza drug target, and has attracted high attention from pharmaceutical companies and academic research institutions.
Currently, the globally pioneering influenza virus polymerase complex PB2 subunit inhibitor drug candidate is picotivir, which was obtained global development interest from Vertex pharmaceuticals by qiangsheng in 2014 and is currently in phase III clinical use. Experiments have shown that picosivir can significantly reduce the viral load in patients compared to placebo. The structure is as follows:
Figure PCTCN2019126277-APPB-000001
international patent publication WO2017133664a1, published as 2.3.2017, also discloses a class of compounds useful for the preparation of anti-influenza drugs, some of which exhibit superior antiviral activity, such as:
Figure PCTCN2019126277-APPB-000002
at present, the clinically applicable anti-influenza drugs are few in types and quantity, and have the problems of drug resistance, poor clinical effect and the like, so that clinical candidate compounds based on the new action mechanism still need to be developed urgently.
Disclosure of Invention
The application discloses compounds serving as influenza virus replication inhibitors and application thereof in preparing medicaments for preventing or treating virus infectious diseases.
In one aspect, the present application provides a compound of formula (I), formula (II), or formula (III):
Figure PCTCN2019126277-APPB-000003
wherein R is1、R 2Each independently selected from hydrogen, C1-C6 alkyl, cyano, halogen, nitro or amino;
x is selected from C or N.
In some embodiments, R1、R 2Each independently selected from hydrogen, cyano or halogen.
In other embodiments, R1Selected from hydrogen, cyano, fluorine or chlorine.
In other embodiments, R2Selected from hydrogen, fluorine or chlorine.
In other embodiments, the present application relates to the following compounds and pharmaceutically acceptable salts, esters, solvates, polymorphs, prodrugs, stereoisomers, or tautomers thereof, but is not limited to these compounds:
Figure PCTCN2019126277-APPB-000004
Figure PCTCN2019126277-APPB-000005
in another aspect, the application provides the use of the compound in preparing a medicament for preventing or treating viral infectious diseases.
In some embodiments, the viral infection is an influenza viral infection.
In other embodiments, the viral infection is an influenza a virus infection.
In another aspect, the present application provides compounds of formula (IV), formula (V) or formula (VI) useful as intermediates in the preparation of the aforementioned compounds having antiviral activity:
Figure PCTCN2019126277-APPB-000006
wherein R is1Selected from hydrogen, C1-C6 alkyl, cyano, halogen, nitro or amino;
R 3selected from chlorine, bromine or iodine.
In some embodiments, R1Selected from hydrogen, cyano, fluorine, chlorine or bromine; r3Is chlorine.
In other embodiments, the compound of formula (IV), formula (V), or formula (VI) has the structure:
Figure PCTCN2019126277-APPB-000007
Drawings
FIG. 1 is a graph of survival of mice dosed 2 hours prior to infection with H1N1 Puerto Rico/8/34 virus;
FIG. 2 survival plots of mice dosed 24 hours after infection with H1N1 Puerto Rico/8/34 virus.
The following acronyms are used throughout this application:
THF: tetrahydrofuran;
DIPEA: n, N-diisopropylethylamine;
Pd(dppf)Cl 2: [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride;
MgSO 4: magnesium sulfate;
Zn(CN) 2: zinc cyanide;
DPPF: 1,1' -bis (diphenylphosphino) ferrocene;
Pd(PPh 3) 4: tetrakis (triphenylphosphine) palladium;
DCM: dichloromethane;
MeCN: acetonitrile;
K 3PO 4: potassium phosphate;
DMF: n, N-dimethylformamide;
DMSO, DMSO: dimethyl sulfoxide;
DMAC: n, N-dimethylacetamide.
Detailed Description
Example 1 (2S, 3S) -3- ((2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrrolo [2,1-f ] [1,2,4] triazin-4-yl) amino) bicyclo [2.2.2] octane-2-carboxylic acid (Compound 1)
Figure PCTCN2019126277-APPB-000008
Step 1 preparation of intermediate 1.1
Adding 2, 4-dichloropyrrolo [2,1-f ] into a reaction bottle][1,2,4]Triazine (10.00g,53.18mmol,1.0eq.) and THF (100mL), cooling in an ice-water bath, adding DIPEA (13.75g,106.4mmol,2.0eq.) dropwise with stirring, adding (2S, 3S) -3-aminobicyclo [2.2.2] to the reaction system in portions after adding]Octane-2-carboxylic acid ethyl ester (12.59g,63.82mmol,1.2eq.) was reacted with stirring at room temperature for 2 hours. The reaction mixture was concentrated, ethyl acetate and water were added to the concentrate, and the mixture was separated into layers, the organic phase was dried over anhydrous magnesium sulfate, and concentrated to give sand, and column chromatography (petroleum ether: ethyl acetate: 5: 1) was performed to obtain intermediate 1.1 as a yellow oily substance 18.47g, yield: 100 percent.1HNMR(400MHz,CDCl 3)δ(ppm)7.51(s,1H),6.60(m,2H),5.58(brs,1H),4.68(m,1H),4.22(q,J＝7.2Hz,2H),2.45(d,J＝4.8Hz,1H),1.45-2.05(m,10H),1.26(t,J＝7.2Hz,3H)。
Step 2 preparation of intermediate 1.2
To a reaction flask was added intermediate 1.1(1.15g,3.31mmol,1.2eq.), 5-fluoro-3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-pyrrolo [2,3-b ]]Pyridine-1-carboxylic acid tert-butyl ester (1.00g,2.76mmol,1.0eq.) and 1, 4-dioxane (30mL), to which was added a solution of potassium carbonate (1.14g,8.28mmol,3.0eq.) in water (10mL), and to which was added a catalytic amount of Pd (dppf) Cl2Heating to reflux reaction for 8 hours under the protection of nitrogen,water was added to the reaction mixture, followed by extraction with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate, concentrated to give sand, and subjected to column chromatography (petroleum ether: ethyl acetate: 3: 1) to give intermediate 1.2 as an off-white solid (0.43 g), yield: 35 percent.1HNMR(400MHz,CDCl 3)δ(ppm)9.52(s,1H),8.62(dd,J＝2.8Hz,9.3Hz,1H),8.31(d,J＝2.6Hz,1H),8.26(t,J＝1.8Hz,1H),7.62(dd,J＝1.6Hz,2.5Hz,1H),6.63(dd,J＝2.6Hz,4.4Hz,1H),6.56(t,J＝1.2Hz,1H),5.41(d,J＝6.7Hz,1H),4.93(m,1H),4.04-4.24(m,2H),2.49-2.51(m,1H),2.07(m,2H),1.62-1.93(m,8H),1.15(t,J＝7.2Hz,3H)。
Step 3 preparation of Compound 1
Dissolving the intermediate 1.2(430mg,0.96mmol,1.0eq.) in tetrahydrofuran (30mL), placing in a reaction bottle, adding a water (10mL) solution of lithium hydroxide monohydrate (80mg,1.92mmol,2.0eq.), heating the reaction system to reflux reaction for 16 hours, concentrating the reaction solution, adding water, adjusting the pH to 5-6 with 6N hydrochloric acid aqueous solution, extracting with ethyl acetate, drying the organic phase with anhydrous magnesium sulfate, concentrating to prepare sand, and performing column chromatography (dichloromethane: methanol: 30: 1) to obtain the compound 1 as an off-white solid 200mg, wherein the yield is as follows: 50 percent.1HNMR(400MHz,DMSO-d 6)δ(ppm)12.31(brs,1H),12.18(brs,1H),8.52(d,J＝8.6Hz,1H),8.28(s,1H),8.18(s,1H),7.88(s,1H),7.68(s,1H),6.97(s,1H),6.60(s,1H),4.81(s,1H),2.78(s,1H),1.42-2.03(m,10H)；LC-MS(m/z):421[M+H] +。
Example 2 (2S, 3S) -3- ((5-fluoro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrrolo [2,1-f ] [1,2,4] triazin-4-yl) amino) bicyclo [2.2.2] octane-2-carboxylic acid (Compound 2)
Figure PCTCN2019126277-APPB-000009
Step 1 preparation of intermediate 2.1
Dissolving 3-fluoro-1H-pyrrole-2-carboxylic acid ethyl ester (7.20g,45.8mmol,1.0eq.) in DMF, placing in a reaction flask, adding sodium hydride (60%, 2.40g,59.6mmol,1.3eq.) in portions under the condition of ice-water bath, and stirring after addingStirring for 1 hour. O- (2, 4-dinitrophenyl) hydroxylamine (13.67g,68.7mmol,1.5eq.) was added in portions, and after the addition, the mixture was stirred at room temperature for 17 hours. The reaction mixture was poured into cold water and extracted with ethyl acetate. The organic phase was washed with brine, MgSO4Dried and concentrated to dryness. Column chromatography (petroleum ether: ethyl acetate: 30: 1) gave 2.1, 7.00g of intermediate in 89% yield as a yellow oil.
Step 2 preparation of intermediate 2.2
A solution of intermediate 2.1(7.00g,40.7mmol,1.0eq.) in saturated methanolic ammonia (200mL) was heated to 150 ℃ in a sealed tube for 48 hours. The reaction mixture was concentrated to dryness and column chromatographed (petroleum ether: ethyl acetate: 30: 1) to give intermediate 2.2, 2.30g, 40% yield as an off-white solid.
Step 3 preparation of intermediate 2.3
Compound 2.2(2.30g,16.0mmol,1.0eq.) was suspended in toluene (20mL), oxalyl chloride (3.4mL,40.2mmol,2.5eq.) was added, and the reaction mixture was heated under reflux for 17 h. The reaction was concentrated to dryness, washed with methanol, filtered and the filter cake collected to give intermediate 2.3, 1.80g, 67% yield as an off-white solid.
Step 4 preparation of intermediate 2.4
Intermediate 2.3(1.00g,6.0mmol,1.0eq.), POCl was added to a closed tube3(4.60g,30.0mmol,5.0eq.), DIPEA (2.33g,18.0mmol,3.0eq.), and the reaction mixture was heated to 120 ℃ for 12 hours. The reaction mixture was poured into cold water and the pH was adjusted to 7-8 with saturated aqueous sodium carbonate solution. The mixture was extracted with ethyl acetate. The organic phase was washed with brine, MgSO4Dried and concentrated to dryness. Column chromatography (petroleum ether: ethyl acetate 100: 1) afforded intermediate 2.4, 0.97g, 79% yield as a yellow oil.
Step 5 preparation of Compound 2
Compound 2 was obtained according to the synthesis method in example 1, using compound 2.4 as a starting material.1HNMR(400MHz,DMSO-d 6)δ(ppm)12.24-12.27(m,2H),8.49(dd,J＝2.8Hz,9.6Hz,1H),8.30(s,1H),8.18(d,J＝2.8Hz,1H),7.61-7.63(m,1H),7.14(d,J＝6.4Hz,1H),6.50(d,J＝3.1Hz,1H),4.82(t,J＝6.5Hz,1H),2.96(d,J＝6.7Hz,1H),1.41-2.00(m,10H)；LC-MS(m/z):439[M+H] +。
Example 3 (2S, 3S) -3- ((7-cyano-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrrolo [2,1-f ] [1,2,4] triazin-4-yl) amino) bicyclo [2.2.2] octane-2-carboxylic acid (Compound 3)
Figure PCTCN2019126277-APPB-000010
Step 1 preparation of intermediate 3.1
Intermediate 3.1 was synthesized using a method analogous to compound 1.1 in example 1, starting with 7-bromo-2, 4-dichloropyrrolo [2,1-f ] [1,2,4] triazine.
Step 2 preparation of intermediate 3.2
Intermediate 3.1(800mg,1.87mmol,1.0eq.) was dissolved in DMAC (15mL) and Zn (CN) was added2(164mg,1.40mmol,0.75eq.), and a catalytic amount of DPPF and Pd (PPh) added thereto3) 4The resulting mixture was heated to reflux under nitrogen for 12 hours. The reaction mixture was poured into cold water, and the mixture was extracted with ethyl acetate. The organic phase was washed with brine, MgSO4Dried and concentrated to dryness. Column chromatography (petroleum ether: ethyl acetate 2: 1) gave intermediate 3.2, 300mg, 43% yield as a pale yellow solid.
Step 3 preparation of Compound 3
Compound 3 was prepared according to a similar procedure to that used for the synthesis of compound 1 in example 1, starting from intermediate 3.2.1HNMR (400MHz,DMSO-d 6)δ(ppm)12.39(br,2H),8.46(dd,J＝2.6Hz,9.6Hz,1H),8.40(d,J＝6.4Hz,1H),8.33(s,1H),8.28(s,1H),7.35(d,J＝4.7Hz,1H),7.11(d,J＝4.7Hz,1H),4.80(t,J＝6.4Hz,1H),2.75(d,J＝6.6Hz,1H),1.43-2.02(m,10H)；LC-MS(m/z):446[M+H] +。
Example 4 (2S, 3S) -3- ((6-cyano-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrrolo [2,1-f ] [1,2,4] triazin-4-yl) amino) bicyclo [2.2.2] octane-2-carboxylic acid (Compound 4)
Figure PCTCN2019126277-APPB-000011
Compound 4 according to a similar procedure to that used for the synthesis of Compound 3 in example 3, 6-bromo-2, 4-dichloropyrrolo [2,1-f ]][1,2,4]Triazine is used as the starting material.1HNMR(400MHz,DMSO-d 6)δ(ppm)12.34(br,2H),8.49(d,J＝8.2Hz,1H),8.44(s,1H),8.25-8.31(m,3H),7.43(s,1H),4.80(m,1H),2.73(d,J＝5.9Hz,1H),1.44-2.03(m,10H)；LC-MS(m/z):446[M+H] +。
Example 5 (1R, 2S, 4R, 5S, 6S, 7S) -7- ((2- (5-fluoro-1H-pyrrolo [2, 3-b)]Pyridin-3-yl) pyrrolo [2,1-f][1,2,4]Triazin-4-yl) amino) tricyclo [3.2.2.02,4]Nonane-6-carboxylic acid (Compound 5)
Figure PCTCN2019126277-APPB-000012
Step 1 preparation of intermediate 5.1
Reacting (1S, 2S, 3S, 4R) -3 (((benzyloxy) carbonyl) amino) bicyclo [2.2.2]Ethyl oct-5-ene-2-carboxylate (40.00g,121.4mmol,1.0eq.) was dissolved in DCM and a 2M solution of diethyl zinc in n-hexane (121.4mL,242.8mmol,2.0eq.) was added dropwise at ice water bath temperature under nitrogen protection. CH is added dropwise to the system2I 2(130.1g,485.7mmol,4.0eq.) a further 2M solution of diethylzinc in n-hexane (121.4mL,242.8mmol,2.0eq.) was added dropwise at the temperature of the ice-water bath. The reaction mixture was allowed to warm to room temperature for overnight, quenched with saturated aqueous ammonium chloride and extracted with DCM. The organic phase was washed with brine, over anhydrous MgSO4Dried and concentrated to dryness. Column chromatography (petroleum ether: ethyl acetate: 10: 1) gave 5.1, 21.00g of intermediate in 50% yield as a colorless oil.
Step 2 preparation of intermediate 5.2
Intermediate 5.1(21.00g,61.1mmol,1.0eq.) was dissolvedTrimethyliodosilane (26.91g,134.5mmol,2.2eq.) was added dropwise to MeCN at the temperature of the ice-water bath, and after 2 hours of reaction triethylamine (15.46g,152.8mmol,2.5eq.) was added and stirred for 15 minutes. The reaction mixture was concentrated to dryness, and extracted with ethyl acetate. The organic phase was washed with water and anhydrous MgSO4Drying and concentrating to dryness to obtain intermediate 5.2, 9.50g, yield 75%.
Step 3 preparation of intermediate 5.3
2, 4-dichloro pyrrolo [2, 1-f)][1,2,4]Triazine (8.48g,45.4mmol,1.0eq.) was dissolved in tetrahydrofuran (95mL), DIPEA (17.59g,136.17mmol,3.0eq.) was added dropwise at the temperature of the ice-water bath, and then intermediate 5.2(9.50g,45.4mmol,1.0eq.) was added and stirred at room temperature for 2 hours. The reaction mixture was concentrated to dryness, and extracted with ethyl acetate. The organic phase was washed with water and anhydrous MgSO4Dried and concentrated to dryness. Column chromatography (petroleum ether: ethyl acetate: 8: 1) afforded intermediate 5.3, 12.00g, 73% yield as a yellow solid.
Step 4 preparation of intermediate 5.4
Reacting 5-fluoro-3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1-p-toluenesulfonyl-1H-pyrrolo [2,3-b ]]Pyridine (28.04g,67.4mmol,2.2eq.) and intermediate 5.3(11.10g,30.6mmol,1.0eq.) were dissolved in 1, 4-dioxane (300mL), and K was added to the system3PO 4(21.62g,101.8mmol,3.0eq.) in water (60mL) and a catalytic amount of Pd (dppf) Cl2The mixture was heated to reflux under nitrogen for 8 hours. The reaction was diluted with water and extracted with ethyl acetate. The organic phase was washed with brine, over anhydrous MgSO4Dried and concentrated to dryness. Column chromatography (petroleum ether: ethyl acetate: 10: 1) afforded intermediate 5.4, 15.00g, 71% yield as a white solid.
Step 5 preparation of Compound 5
Intermediate 5.4(15.00g,21.3mmol,1.0eq.) was dissolved in DCM (150mL), trifluoroacetic acid (24.30g,213.1mmol,10.0eq.) and triethylsilane (12.38g,106.6mmol,5.0eq.) were added and stirred at rt overnight. The reaction mixture was concentrated to dryness, dissolved in methanol (80mL), water (15mL) and tetrahydrofuran (20mL), and then added lithium hydroxide monohydrate (2.18g,52.0mmol,3.0eq.) the reaction mixture was stirred at 50 ℃ for 5 hours. The reaction mixture was concentrated, tetrahydrofuran and methanol were removed, and the residue was diluted with water. The resulting mixture was adjusted to pH 5-6 with 6N HCl, and the solid was collected by filtration and washed with ethyl acetate (10mL) to give compound 5, 3.85g, yield 51% as a white solid.1HNMR(400MHz,DMSO-d 6)δ(ppm)14.15(brs,1H),12.56(brs,1H),8.65(s,1H),8.55(d,J＝8.6Hz,1H),7.79(s,1H),7.73(d,J＝4.1Hz,1H),7.06(d,J＝3.2Hz,1H),6.70(dd,J＝2.6Hz,4.2Hz,1H),4.62(brs,1H),2.85(d,J＝6.4Hz,1H),2.76(s,1H),1.51-1.69(m,4H),1.10-1.12(m,2H),0.75-0.85(m,1H),0.30-0.39(m,1H)；LC-MS(m/z):434[M+H] +；[α]＝-26.6°(c＝1.08g/100mL,MeOH)。
Example 6 (1R, 2S, 4R, 5S, 6S, 7S) -7- ((5-fluoro-2- (5-fluoro-1H-pyrrolo [2, 3-b)]Pyridin-3-yl) pyrrolo [2,1-f][1,2,4]Triazin-4-yl) amino) tricyclo [3.2.2.02,4]Nonane-6-carboxylic acid (Compound 6)
Figure PCTCN2019126277-APPB-000013
Compound 6 was prepared according to a similar preparation to that for compound 5 in example 5.1HNMR(400MHz,DMSO-d 6)δ(ppm)12.28(s,1H),12.23(d,1H),8.50-8.47(dd,1H),8.29(d,1H),8.23(d,1H),7.64(t,1H),6.63(d,1H),6.51(d,1H),4.53(brs,1H),2.72(d,1H),2.51(m,1H),2.35(d,1H),1.69-1.67(m,2H),1.59-1.54(m,2H),1.10(m,1H),0.91-0.82(m,2H),0.49-0.37(m,1H)；LC-MS(m/z):451[M+H] +。
Example 7 (2S, 3S) -3- ((2- (1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrrolo [2,1-f ] [1,2,4] triazin-4-yl) amino) bicyclo [2.2.2] octane-2-carboxylic acid (Compound 7)
Figure PCTCN2019126277-APPB-000014
Compound (I)7 according to a similar preparation as that for the synthesis of Compound 1 in example 1, 3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-pyrrolo [2,3-b]Pyridine-1-carboxylic acid tert-butyl ester is used as a starting material.1HNMR(400MHz,DMSO-d 6)δ(ppm)12.30(s,1H),11.98(s,1H),8.75(d,J＝1.36Hz,1H),8.27(d,J＝1.6Hz,1H),8.09(d,J＝2.7Hz,1H),7.84(d,J＝6.6Hz,1H),7.65(t,J＝2.3Hz,1H),7.18(d,J＝4.7Hz,1H),6.95(d,J＝1.4Hz,1H),6.58(d,J＝2.6Hz,1H),4.78(t,J＝6.7Hz,1H),2.76(d,J＝7.0Hz,1H),2.03(s,2H),1.39-1.80(m,7H)；LC-MS(m/z):404[M+H] +。
Example 8 (2S, 3S) -3- ((5-fluoro-2- (1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrrolo [2,1-f ] [1,2,4] triazin-4-yl) amino) bicyclo [2.2.2] octane-2-carboxylic acid (Compound 8)
Figure PCTCN2019126277-APPB-000015
Compound 8 was prepared according to a similar procedure to that used to synthesize compound 7 in example 7, starting from intermediate 2.4 in example 2.1HNMR(400MHz,DMSO-d 6)δ(ppm)12.25(brs,1H),12.07(brs,1H),8.73(d,1H),8.29(d,1H),8.10(s,1H),7.58(t,1H),7.20(q,1H),7.08(d,1H),6.49(d,1H),4.82(m,1H),2.96(d,1H),2.03-1.99(m,2H),1.78-1.60(m,3H),1.58-1.40(m,5H)；LC-MS(m/z):422[M+H] +。
Example 9 (1R, 2S, 4R, 5S, 6S, 7S) -7- ((2- (1H-pyrrolo [2, 3-b))]Pyridin-3-yl) pyrrolo [2,1-f][1,2,4]Triazin-4-yl) amino) tricyclo [3.2.2.02,4]Nonane-6-carboxylic acid (Compound 9)
Figure PCTCN2019126277-APPB-000016
Compound 9 was prepared according to a similar procedure to that used to synthesize compound 7 in example 7, starting from intermediate 5.2 in example 5.1HNMR(400MHz,DMSO-d 6)δ(ppm)12.37(s,1H),11.98(s,1H),8.74-8.76(d,J＝7.9Hz,2H),8.29(s,1H),8.12(s,1H),7.64-7.66(d,J＝8.4Hz,1H),7.20(s,1H),7.00(s,1H),6.58(s,1H),4.46(s,1H),2.68(s,2H),2.35(s,1H),1.71(s,1H),1.55(s,1H),1.01-1.06(d,J＝18.1,2H),0.84(s,1H),0.36(s,1H)；LC-MS(m/z):416[M+H] +。
Example 10 (2S, 3S) -3- ((2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrrolo [2,1-f ] [1,2,4] triazin-4-yl) amino) bicyclo [2.2.2] octane-2-carboxylic acid (Compound 10)
Figure PCTCN2019126277-APPB-000017
Compound 10 according to a similar preparation as that used for the synthesis of Compound 7 in example 7, 5-chloro-3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1-p-toluenesulfonyl-1H-pyrrolo [2,3-b ] is used]Pyridine is used as a starting material.1HNMR(400MHz,DMSO-d 6)δ(ppm)12.32(s,1H),12.28(s,1H),8.78(d,J＝2.4Hz,1H),8.29(d,J＝2.4Hz,1H),8.17(d,J＝2.7Hz,1H),7.90(d,J＝6.7Hz,1H),7.69(t,J＝2.2Hz,1H),6.97(q,1H),6.59(q,1H),4.80(t,1H),2.77(d,1H),2.03(s,2H),1.59-1.87(m,8H)；LC-MS(m/z):438[M+H] +。
Example 11 (2S, 3S) -3- ((2- (5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) -5-fluoro-pyrrolo [2,1-f ] [1,2,4] triazin-4-yl) amino) bicyclo [2.2.2] octane-2-carboxylic acid (Compound 11)
Figure PCTCN2019126277-APPB-000018
Compound 11 was prepared according to a similar procedure to that used to synthesize compound 10 in example 10, starting from intermediate 2.4 in example 2.1HNMR(400MHz,DMSO-d 6)δ(ppm)12.34(brs,1H),12.26(brs,1H),8.74(s,1H),8.30(s,1H),8.17(s,1H),7.63(m,1H),7.16(m,1H),6.50(s,1H),4.84(m,1H),2.98(d,1H),2.01(m,2H),1.79-1.91(m,3H),1.40-1.57(m,5H)；LC-MS(m/z):456[M+H] +。
Example 12 (1R, 2S, 4R, 5S, 6S, 7S) -7- ((2- (5-chloro-1H-pyrrolo [2, 3-b))]Pyridin-3-yl) pyrrolo [2,1-f][1,2,4]Triazin-4-yl) amino) tricyclo [3.2.2.02,4]Nonane-6-carboxylic acid (Compound 12)
Figure PCTCN2019126277-APPB-000019
Compound 12 was prepared according to a similar procedure to that used to synthesize compound 10 in example 10, starting from intermediate 5.2 in example 5.1HNMR(400MHz,DMSO-d 6)δ(ppm)12.38(s,1H),12.27(s,1H),8.76(s,1H),8.29(s,1H),8.20(s,1H),7.68-7.71(t,2H,J＝14.0Hz),7.01-7.02(d,1H,J＝2.6Hz),6.60(s,1H),4.47(s,1H),2.66-2.70(t,2H,J＝16.0),2.40(s,1H),1.71-1.75(t,2H,J＝15.8),1.55(s,2H),1.18-1.19(d,2H,J＝6.8),0.82-0.96(d,2H),0.36(s,1H)；LC-MS(m/z):450[M+H] +。
Example 13 (1R, 2S, 3S, 4R) -3- ((6-chloro-2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrrolo [2,1-f ] [1,2,4] triazin-4-yl) amino) bicyclo [2.2.2] octane-2-carboxylic acid (Compound 13)
Figure PCTCN2019126277-APPB-000020
Compound 13 was prepared according to a similar preparation to that used to synthesize compound 2 in example 2, starting from ethyl 4-chloro-1H-pyrrole-2-carboxylate.1HNMR(400MHz,DMSO-d 6)δ(ppm)12.34(s,1H),12.24(s,1H),8.48(d,J＝2.8Hz,1H),8.29(d,J＝1.4Hz,1H),8.19(d,J＝2.8Hz,1H),7.93(d,J＝6.7Hz,1H),7.88(d,J＝1.8Hz,1H),7.04(d,J＝1.8Hz,1H),4.76(t,J＝6.1Hz,1H),2.67(m,1H),2.03(s,2H),1.62-1.81(m,7H)；LC-MS(m/z):456[M+H] +。
Example 14 (1S, 2S, 3S, 4R) -3- ((2- (5-fluoro-1H-pyrrolo [2,3-b ] pyridin-3-yl) pyrrolo [2,1-f ] [1,2,4] triazin-4-yl) amino) bicyclo [2.2.2] oct-5-ene-2-carboxylic acid (Compound 14)
Figure PCTCN2019126277-APPB-000021
Compound 14 according to a similar preparation as that for the synthesis of compound 1 in example 1, was prepared from (1S, 2S, 3S, 4R) -3-aminobicyclo [2.2.2]The octyl-5-alkene-2-carboxylic acid ethyl ester is prepared by taking the starting material.1HNMR(400MHz,DMSO-d 6)δ(ppm)12.45(s,1H),12.19(s,1H),8.52(d,J＝2.8Hz,1H),8.29(d,J＝1.5Hz,1H),8.28(d,J＝1.4Hz,1H),8.23(d,J＝2.7Hz,1H),7.68(t,J＝1.7Hz,1H),7.58(d,J＝6.3Hz,1H),6.94(d,J＝1.5Hz,1H),6.52-6.57(m,2H),6.27(t,J＝7.0Hz,1H),4.76(s,1H),3.08(d,J＝3.0Hz,1H),2.91(d,J＝3.52Hz,1H),1.61-1.78(m,2H),1.13-1.34(m,3H)；LC-MS(m/z):420[M+H] +。
Example 15 (2S, 3S) -3- ((2- (5-fluoro-1H-pyrazolo [3,4-b ] pyridin-3-yl) pyrrolo [2,1-f ] [1,2,4] triazin-4-yl) amino) bicyclo [2.2.2] octane-2-carboxylic acid (compound 15)
Figure PCTCN2019126277-APPB-000022
Compound 15 according to a similar preparation as that for the synthesis of Compound 1 in example 1, 5-fluoro-3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1-trityl-1H-pyrazolo [3,4-b]Pyridine is used as a starting material.1HNMR(400MHz,DMSO-d 6)δ(ppm)14.13(brs,1H),12.41(brs,1H),8.64(s,1H),8.58(d,J＝8.6Hz,1H),8.11(d,J＝7.7Hz,1H),7.77(s,1H),7.04(d,J＝3.8Hz,1H),6.69(t,J＝2.4Hz,1H),4.92(m,1H),2.85(t,J＝7.0Hz,1H),1.57-2.07(m,10H)；LC-MS(m/z):422[M+H] +；[α]＝-22.6°(c＝0.87g/100mL,DMSO)。
Example 16 (2S, 3S) -3- ((5-fluoro-2- (5-fluoro-1H-pyrazolo [3,4-b ] pyridin-3-yl) pyrrolo [2,1-f ] [1,2,4] triazin-4-yl) amino) bicyclo [2.2.2] octane-2-carboxylic acid (compound 16)
Figure PCTCN2019126277-APPB-000023
Compound 16 analogously to compound 15 synthesized in example 15, the procedure was followed to obtain (2S, 3S) -3- ((2-chloro-5-fluoropyrrolo [2, 1-f)][1,2,4]Triazin-4-yl) amino) bicyclo [2.2.2]Octane-2-carboxylic acid ethyl ester is used as a starting material.1HNMR(400MHz,DMSO-d 6)δ(ppm)14.16(brs,1H),12.31(brs,1H),8.64(s,1H),8.57(d,J＝8.7Hz,1H),7.70(s,1H),7.43(d,J＝6.9Hz,1H),6.60(s,1H),4.98(m,1H),3.11(d,J＝6.3Hz,1H),1.46-2.04(m,10H)；LC-MS(m/z):440[M+H] +；[α]＝-19.0°(c＝0.89g/100mL,DMSO)。
Example 17 (1R, 2S, 4R, 5S, 6S, 7S) -7- ((2- (5-fluoro-1H-pyrazolo [3, 4-b)]Pyridin-3-yl) pyrrolo [2,1-f][1,2,4]Triazin-4-yl) amino) tricyclo [3.2.2.02,4]Nonane-6-carboxylic acid (Compound 17)
Figure PCTCN2019126277-APPB-000024
Mixing 5-fluoro-3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1-trityl-1H-pyrazolo [3, 4-b)]Pyridine (28.04g,67.4mmol,2.2eq.) and intermediate 5.3(11.10g,30.6mmol,1.0eq.) were dissolved in 1, 4-dioxane (300mL) and K was added3PO 4(21.62g,101.8mmol,3.0eq.) in water (60mL) followed by a catalytic amount of Pd (dppf) Cl2The resulting mixture was heated to reflux under nitrogen for 8 hours. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic phase was washed with brine, over anhydrous MgSO4Dried and concentrated to dryness. Column chromatography (petroleum ether: ethyl acetate: 10: 1) afforded intermediate 17.1, 15.00g, 71% yield as a white solid.
Intermediate 17.1(15.00g,21.3mmol,1.0eq.) was dissolved in DCM (150mL), trifluoroacetic acid (24.30g,213.1mmol,10.0eq.) and triethylsilane (12.38g,106.6mmol,5.0eq.) were added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated to dryness, and the obtained residue was dissolved with methanol (80mL), water (15mL) and tetrahydrofuran (20mL), and lithium hydroxide monohydrate (2.18g,52.0mmol,3.0eq.) was added. The reaction mixture was stirred at 50 ℃ for 5 hours. The reaction mixture was concentrated to remove tetrahydrofuran and methanol, and the residue was diluted with water. The resulting mixture was adjusted to pH 5-6 with 6N HCl, and the solid was collected by filtration and washed with ethyl acetate (10ml) to give compound 17, 3.85g, yield 51% as a white solid.1HNMR(400MHz,DMSO-d 6)δ(ppm)14.15(brs,1H),12.56(brs,1H),8.65(s,1H),8.55(d,J＝8.6Hz,1H),7.79(s,1H),7.73(d,J＝4.1Hz,1H),7.06(d,J＝3.2Hz,1H),6.70(dd,J＝2.6Hz,4.2Hz,1H),4.62(brs,1H),2.85(d,J＝6.4Hz,1H),2.76(s,1H),1.51-1.69(m,4H),1.10-1.12(m,2H),0.75-0.85(m,1H),0.30-0.39(m,1H)；LC-MS(m/z):434[M+H] +；[α]＝-26.6°(c＝1.08g/100mL,MeOH)。
Example 18 (1R, 2S, 4R, 5S, 6S, 7S) -7- ((5-fluoro-2- (5-fluoro-1H-pyrazolo [3, 4-b)]Pyridin-3-yl) pyrrolo [2,1-f][1,2,4]Triazin-4-yl) amino) tricyclo [3.2.2.02,4]Nonane-6-carboxylic acid (Compound 18)
Figure PCTCN2019126277-APPB-000025
Compound 18 analogously to compound 15 synthesized in example 15, 2, 4-dichloro-5-fluoro- [2,1-f][1,2,4]Triazine and intermediate 5.2 in example 5 were prepared as starting materials.1HNMR(400MHz,DMSO-d 6)δ(ppm)8.57(s,1H),8.49-8.47(d,1H),7.66(s,1H),4.77(s,1H),2.43(s,1H),2.35(s,1H),1.68-1.63(m,3H),1.41-1.39(m,1H),0.98(s,1H),0.79(s,2H),0.33-0.32(m,1H)；LC-MS(m/z):452[M+H] +。
Example 19 (1S, 2S, 3S, 4R) -3- ((2- (5-fluoro-1H-pyrazolo [3,4-b ] pyridin-3-yl) pyrrolo [2,1-f ] [1,2,4] triazin-4-yl) amino) bicyclo [2.2.2] oct-5-ene-2-carboxylic acid (Compound 19)
Figure PCTCN2019126277-APPB-000026
Compound 19 was prepared according to a similar procedure to that used to synthesize compound 15 in example 15.1HNMR(400MHz,DMSO-d 6)δ(ppm)8.70-8.60(m,2H),7.80-7.75(m,2H),7.03-7.02(d,1H),6.66-6.64(dd,1H),6.59-6.55(t,1H),6.24-6.21(t,1H),4.92(s,1H),3.14(s,1H),2.98(m,1H),2.63(s,1H),1.78-1.75(m,2H),1.62(m,1H),1.36-1.29(m,1H),1.16-1.14(m,1H)；LC-MS(m/z):421[M+H] +。
Example 20 (1R, 2S, 3S, 4R) -3- ((2- (1H-pyrazolo [3,4-b ] pyridin-3-yl) pyrrolo [2,1-f ] [1,2,4] triazin-4-yl) amino) bicyclo [2.2.2] octane-2-carboxylic acid (Compound 20)
Figure PCTCN2019126277-APPB-000027
Compound 20 was prepared according to a similar procedure to that used to synthesize compound 15 in example 15.1HNMR(400MHz,DMSO-d 6)δ(ppm)13.91(s,1H),12.40(s,1H),8.84(d,J＝1.5Hz,1H),8.57(d,J＝1.5Hz,1H),8.06(d,J＝7.0Hz,1H),7.74(d,J＝1.8Hz,1H),7.30(d,J＝4.4Hz,1H),7.03(d,J＝1.4Hz,1H),6.68(d,J＝2.6Hz,1H),4.89(t,J＝6.8Hz,1H),2.84(d,J＝7.0Hz,1H),2.06(s,1H),1.63-1.79(m,7H)；LC-MS(m/z):405[M+H] +。
Example 21 (1R, 2S, 3S, 4R) -3- ((5-fluoro-2- (1H-pyrazolo [3,4-b ] pyridin-3-yl) pyrrolo [2,1-f ] [1,2,4] triazin-4-yl) amino) bicyclo [2.2.2] octane-2-carboxylic acid (Compound 21)
Figure PCTCN2019126277-APPB-000028
Compound 21 was prepared according to a similar procedure to that used to synthesize compound 15 in example 15.1HNMR(400MHz,DMSO-d 6) δ(ppm)13.98(brs,1H),12.31(brs,1H),8.84-8.82(d,1H),8.58(d,1H),7.68(t,1H),7.38(d,1H),7.32(q,1H),6.60(d,1H),4.96(m,1H),3.10(d,1H),2.04-1.99(m,2H),1.82-1.72(m,3H),1.61-1.38(m,5H)；LC-MS(m/z):423[M+H] +。
Example 22 (1R, 2S, 4R, 5S, 6S, 7S) -7- ((2- (1H-pyrazolo [3,4-b ] pyridin-3-yl) pyrrolo [2,1-f ] [1,2,4] triazin-4-yl) amino) tricyclo [3.2.2.02,4] nonane-6-carboxylic acid (Compound 22)
Figure PCTCN2019126277-APPB-000029
Compound 22 was prepared according to a similar procedure to that used to synthesize compound 15 in example 15.1HNMR(400MHz,DMSO-d 6)δ(ppm)8.82-8.85(m,1H),8.57-8.89(m,1H),7.74-7.76(d,2H),7.30-7.34(m,1H),7.07-7.08(m,1H),6.68-6.70(m,1H),4.61(s,1H),2.86-2.87(t,1H),2.77(s,1H),1.68(s,2H),1.50-1.52(d,2H),1.11-1.13(m,1H),1.08(m,1H),0.82(s,1H),0.33(d,1H)；LC-MS(m/z):417[M+H] +。
Example 23 (1S, 2S, 3S, 4R) -3- ((2- (1H-pyrazolo [3,4-b ] pyridin-3-yl) pyrrolo [2,1-f ] [1,2,4] triazin-4- (yl) amino) bicyclo [2.2.2] oct-5-ene-2-carboxylic acid (Compound 23)
Figure PCTCN2019126277-APPB-000030
Compound 23 was prepared according to a similar procedure to that used to synthesize compound 15 in example 15.1HNMR(400MHz,DMSO-d 6)δ(ppm)13.94(s,1H),12.60(s,1H),8.87-8.90(m,1H),8.58-8.59(m,1H),7.74-7.77(m,2H),7.31-7.34(m,1H),7.00-7.01(m,1H),6.65-6.67(m,1H),6.55-6.59(m,1H),6.22-6.25(m,1H),4.91(s,1H),2.97-3.17(m,2H),2.67(s,1H),1.59-1.63(m,2H)；LC-MS(m/z):403[M+H] +。
Example 24 (1R, 2S, 3S, 4R) -3- ((2- (5-chloro-1H-pyrazolo [3,4-b ] pyridin-3-yl) pyrrolo [2,1-f ] [1,2,4] triazin-4-yl) amino) bicyclo [2.2.2] octane-2-carboxylic acid (compound 24)
Figure PCTCN2019126277-APPB-000031
Compound 24 was prepared according to a similar procedure to that used to synthesize compound 15 in example 15.1HNMR(400MHz,DMSO-d 6)δ(ppm)14.20(s,1H),12.40(s,1H),8.90(d,J＝2.1Hz,1H),8.61(d,J＝2.2Hz,1H),8.13(d,J＝6.9Hz,1H),7.78(s,1H),7.05(d,J＝3.2Hz,1H),6.69(q,J＝2.7Hz,1H),4.91(t,J＝6.4Hz,1H),2.85(d,J＝6.8Hz,1H),2.04(d,J＝13.9Hz,2H),1.78-1.87(m,3H),1.57-1.66(m,5H)；LC-MS(m/z):439[M+H] +。
Example 25 (1R, 2S, 3S, 4R) -3- ((2- (5-chloro-1H-pyrazolo [3,4-b ] pyridin-3-yl) -5-fluoropyrrole [2,1-f ] [1,2,4] triazin-4-yl) amino) bicyclo [2.2.2] octane-2-carboxylic acid (compound 25)
Figure PCTCN2019126277-APPB-000032
Compound 25 was prepared according to a similar procedure to that used to synthesize compound 15 in example 15.1HNMR(400MHz,DMSO-d 6)δ(ppm)14.25(brs,1H),12.32(brs,1H),8.88(d,1H),8.62(d,1H),7.71(t,1H),7.47(d,1H),6.61(d,1H),4.99(t,1H),3.11(d,1H),2.04-2.02(m,2H),1.83-1.62(m,3H),1.60-1.31(m,5H)；LC-MS(m/z):457[M+H] +。
Example 26 (1R, 2S, 4R, 5S, 6S, 7S) -7- ((2- (5-chloro-1H-pyrazolo [3, 4-b)]Pyridin-3-yl) pyrrolo [2,1-f][1,2,4]Triazin-4-yl) amino) tricyclo [3.2.2.02,4]Nonane-6-carboxylic acid (Compound 26)
Figure PCTCN2019126277-APPB-000033
Compound 26 was prepared according to a similar procedure to that used to synthesize compound 15 in example 15.1HNMR(400MHz,DMSO-d 6)δ(ppm)14.21(s,1H),12.53(s,1H),8.85(s,1H),8.61(s,1H),7.79(s,1H),7.74(s,1H),7.07(s,1H),6.70(s,1H),4.63(s,1H),2.85-2.86(d,J＝5.20Hz,2H),2.75(s,1H),1.70-1.80(d,J＝38.6Hz,2H),1.55-1.57(d,J＝9.9Hz,2H),1.10-1.15(d,2H),0.85-0.89(m,2H),0.36(d,2H)；LC-MS(m/z):451[M+H] +。
Experimental example 1 inhibition of influenza Virus (influenza A/CA/07/2009) Activity
Test article: the structural formulas and preparation methods of the compounds 1,2 and 3 in the part of the application are shown in preparation examples of the compounds.
Control substance: pimodivir, the structural formula of which is described in the background section.
The experimental method is briefly described as follows:
MDCK cells were cultured in MEM medium containing 10% fetal bovine serum. Virus-infected cells were cultured in MEM medium containing 0.42% bovine serum albumin and 5. mu.g/mL of trypsin. On the day before virus inoculation, MDCK cells were arranged at 3X 104Density per well was seeded onto 96-well cell culture plates. The next day, MDCK cells were infected with 50PFU of influenza virus (influenza A/CA/07/2009) added to 100. mu.L of MEM medium containing bovine serum albumin, and incubated at 37 ℃ for 1 hour. After virus removal, virus-infected cells were diluted in 100 μ L MEM medium in two-fold gradients and incubated in media containing different concentrations of test compound (0-10 μ M) in each case, three replicate wells per concentration setting. Three MDCK cell wells not infected with virus served as control groups. 20 μ L of resazurin solution at 0.15mg/mL was added to each well of cells infected with the virus for 72 hours, and incubated for 4 hours. Fluorescence quantification experiments were performed with fluorescent agents under 560nm excitation/590 nm emission conditions. The MDCK cell protection rate was calculated according to the following equation. Test Compounds inhibit influenza Virus EC50Obtained by cell protection rate transformation.
Figure PCTCN2019126277-APPB-000034
Preparation of resazurin solution:
1. dissolving resazurin with DPBS solution with pH of 7.4 to a concentration of 0.15 mg/mL;
2. filtering the resazurin solution by using a 0.2 mu m filter into a sterile light-proof test tube;
3. the resazurin solution is stored in a refrigerator at-20 ℃ in the dark.
The experimental results are as follows:
TABLE 1 influenza Virus inhibiting Activity of some of the Compounds of the present application
Figure PCTCN2019126277-APPB-000035
Figure PCTCN2019126277-APPB-000036
As can be seen from table 1, compounds 1,2 and 3 exhibited sufficiently good inhibitory activity against influenza virus in cells, and were superior to the control.
EXPERIMENTAL EXAMPLE 2 inhibition of influenza Virus (A/PR/8/34(H1N1)) Activity
Test article: the structural formula and the preparation method of the compound are shown in the preparation examples of each compound.
Control substance: pimodivir, and the compounds disclosed in WO2017133664a 1:
Figure PCTCN2019126277-APPB-000037
cell: canine kidney cells MDCK were purchased from ATCC, cat # CCL-34. Cells were cultured in EMEM (Sigma) medium supplemented with 10% fetal bovine serum (Hyclone), 1% double antibody (Hyclone), 1% L-glutamine (Gibco) and 1% non-essential amino acids (Gibco). The culture solution of OptiPRO SFM (Gibco) supplemented with 1% of double antibody, 1% of L-glutamine and 1% of non-essential amino acid was used as the experimental culture solution. The experimental culture medium to which pancreatin (Invitrogen) was added was a virus infection culture medium.
Virus: influenza A/PR/8/34(H1N1) strain was purchased from ATCC under accession number VR-1469.
The experimental steps are as follows:
MDCK cells were seeded at a density of 2,000 cells per well in 384-well test plates and incubated in 5% CO2And cultured overnight in an incubator at 37 ℃. The next day, compounds (8 concentration points, duplicate wells) and virus were added to 384-well cell culture plates. The final concentrations of DMSO and pancreatin in the culture were 0.5% and 2.5. mu.g/mL, respectively. Cells in 5% CO2And culturing in an incubator at 37 ℃ for 5 days until the cytopathy in the virus control hole without the compound reaches 80-95 percent. Cell viability was measured using the Cell counting kit 8 kit (Shanghai Liji). The antiviral activity of the compound is represented by the inhibition ratio (%) of the compound at various concentrations against the cytopathic effect caused by the virus. The calculation formula is as follows:
inhibition (%) (test well read-virus control average)/(cell control average-virus control average) × 100 inhibition and cell viability of compounds were analyzed by nonlinear fitting using GraphPad Prism software, and half Effective Concentration (EC) of compounds was calculated50) The value is obtained.
The experimental results are as follows:
TABLE 2 influenza Virus inhibiting Activity of some of the Compounds of the present application
Figure PCTCN2019126277-APPB-000038
Figure PCTCN2019126277-APPB-000039
Figure PCTCN2019126277-APPB-000040
Figure PCTCN2019126277-APPB-000041
Figure PCTCN2019126277-APPB-000042
It can be seen that the compounds of the present application all have good antiviral activity, wherein the antiviral activity of compounds 15,16,17, 19, 22, 24, 25, 26 is significantly better than that of the control Pimodivir.
Experimental example 3 in vivo pharmacokinetic study
Male CD-1 mice (purchased from LC Laboratory Animal Co. LTD; 22-23 g; 6-8 weeks; N-18, 9 per route of administration, 3 animals per time point) were dosed by tail vein injection (5mg/kg) and oral feeding (10mg/kg) under single dose conditions of test compound, each in a physiological saline solution containing 5% N, N-dimethyl sulfoxide (DMSO) and 10% polyethylene glycol 15 hydroxystearate (Solutol HS 15). The experimental animals were fasted overnight the day before dosing and were fed 4 hours after dosing with free access to water. The study met the guidelines and standards of the institute for laboratory animal management evaluation and recognition (international AAALAC) and the american national institute of health. After anesthetizing the animals with isoflurane at the indicated sampling time points, approximately 110 μ L of blood samples were collected via facial vein or cardiac puncture under isoflurane inhalation anesthesia by staggered bleeding into EDTA-2K tubes for storage. The collected blood samples were kept in wet ice and centrifuged within 15 minutes after sampling to obtain plasma (2000g, 4 ℃,5 min). Plasma samples were stored at approximately-70 ℃ under refrigerated conditions until analysis. Prior to analysis of collected plasma samples, 20 μ L aliquots of undiluted plasma samples were added to 200 μ L IS (20ng/mL glipizide in acetonitrile). The mixture was vortexed at 750rpm for 10min and centrifuged at 5800rpm for 10 min. Aliquots of 2. mu.L of the supernatant were taken and the compound concentration was quantified using UPLC-MS/MS-22(Triple quad 6500). A standard calibration curve was constructed by analyzing a series of control plasma aliquots of 3.0-3,000ng/mL test compound containing glipizide (20ng/mL) as an internal reference. For a 10 fold dilution of the plasma sample, an aliquot of 2 μ L of the blood sample was added to 18 μ L of blank diluted plasma with a dilution factor of 10. The subsequent procedure was the same as for the undiluted plasma sample described above.
The results of the experiment are shown in table 3.
TABLE 3
Figure PCTCN2019126277-APPB-000043
Compared with a control compound Pimodivir, the compounds 15,16 and 17 have lower in vivo clearance rate, higher peak concentration and total exposure, and show obviously better druggability.
Experimental example 4 in vivo efficacy study of influenza A H1N1 Virus in mouse infection model
Female BALB/c mice were selected for the experiment at 6-8 weeks and infected with influenza A H1N1A/puerto Rico/8/34 virus via nasal drops. The day of infection was set to day 0 of the experimental cycle. Oral treatment with test compounds was started 2 hours before infection (PI-2) and 24 hours after infection (PI24), respectively, and the in vivo efficacy of the compounds against influenza a H1N1 was evaluated by observing survival and weight change in mice. The administration was continued for 10 days with an observation period of 20 days. All surviving mice were euthanized after 20 days.
Solutions of the compounds to be tested were prepared in physiological saline containing 5% N, N-dimethyl sulfoxide (DMSO) and 10% polyethylene glycol 15 hydroxystearate (Solutol HS 15). After the experimental mice were placed in an anesthesia chamber, they were anesthetized with 5% v/v isoflurane in oxygen (2.5L/min) and infected by slow nasal instillation of 50 microliters LD90 dose of H1N1 Puerto Rico/8/34 virus under oxygen maintenance anesthesia with 2-2.5% isoflurane. Oral administration was started two hours before infection and 24 hours after infection, respectively, according to experimental requirements. The positive control, Pimodivir, was dosed twice daily in two different infection time experiments. Whereas test compound 17 was administered twice daily in the PI-2 experiment, the PI24 experiment was administered once daily. The dose administered is shown in the results of fig. 1 and 2. Mice status was observed daily and body weight and survival were recorded. When the weight of the tested mouse is reduced to exceed the standard regulated by animal welfare organization, the health index reaches 6, and serious pathological states such as serious lethargy, paralysis and the like occur, the euthanasia is carried out. At the end of the experiment, all surviving mice were euthanized.
The experimental results are shown in FIGS. 1 and 2.
When the drug administration was started two hours before infection, the survival rate of mice with compound 17 in all three doses (1,3,10mg/kg, BID) reached 100%. Whereas the control compound Pimodivir survived 0% at low doses (1mg/kg, BID) and 80% at medium doses (3mg/kg, BID), with complete survival only being achieved at the highest dose (10mg/kg, BID). In response to the survival results, no significant weight loss occurred in animals treated with all three doses of compound 17. Whereas the control compound pimoivir, the low dose group showed weight loss starting on day 4 and more than 20% of mice starting on day 8, euthanization was performed according to animal welfare specifications until day 10, where all animals had weight loss of more than 20%. The medium dose group showed significant weight loss from day 6. As dosing continued, body weight gradually recovered from day 9. Wherein on day 10, 1 case of weight loss of more than 20% occurred.
Compound 17, when dosed once daily (1mg/kg, QD) at low doses and at medium and high doses (3,10mg/kg, QD), gave a 60% survival rate when dosed once daily, starting 24 hours post infection, and a 100% survival rate was achieved in mice. Whereas the control compound Pimodivir shows a survival rate of 40% at low doses given twice daily (1mg/kg, BID) and 80% at medium doses (3mg/kg, BID), complete survival is achieved only at the highest dose (10mg/kg, BID). The survival rate of the oseltamivir which is approved to be marketed at present is 80 percent under the condition of 10mg/kg and BID dose.
Compound 17 showed significantly superior efficacy against influenza a virus at different dosing times compared to the control compound Pimodivir. Compound 17 showed similar efficacy at a total daily dose (3mg/kg/Day) much lower than Pimodivir (20mg/kg/Day) when administered 24 hours after infection.

Claims (13)

  1. A compound of formula (I), formula (II) or formula (III):
    Figure PCTCN2019126277-APPB-100001
    wherein R is1、R 2Each independently selected from hydrogen, C1-C6 alkyl, cyano, halogen, nitro or amino;
    x is selected from C or N.
  2. The compound of claim 1, wherein R1、R 2Each independently selected from hydrogen, cyano or halogen.
  3. A compound according to claim 1 or 2, wherein R1Selected from hydrogen, cyano, fluorine or chlorine.
  4. A compound according to claim 1 or 2, wherein R2Selected from hydrogen, fluorine or chlorine.
  5. The compound of claim 1, having the formula any one of:
    Figure PCTCN2019126277-APPB-100002
    Figure PCTCN2019126277-APPB-100003
  6. the compound of any one of claims 1-2 and 5, wherein the compound is present in the form of a pharmaceutically acceptable salt, ester, solvate, polymorph, prodrug, stereoisomer, or tautomer.
  7. Use of a compound according to any one of claims 1-2 and 5 for the manufacture of a medicament for the prophylaxis or treatment of a viral infectious disease.
  8. The use of claim 7, wherein the viral infection is an influenza infection.
  9. The use of claim 8, wherein the viral infection is an influenza a virus infection.
  10. A compound of formula (IV), formula (V) or formula (VI):
    Figure PCTCN2019126277-APPB-100004
    wherein R is1Selected from hydrogen, C1-C6 alkyl, cyano, halogen, nitro or amino;
    R 3selected from chlorine, bromine or iodine.
  11. The compound of claim 10, wherein R1Selected from hydrogen, cyano, fluorine, chlorine or bromine; r3Is chlorine.
  12. The compound of claim 10 or 11, having the formula any one of:
    Figure PCTCN2019126277-APPB-100005
  13. use of a compound according to claim 10 for the preparation of a compound according to any one of claims 1-2 and 5.
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