WO2023030347A1 - Use of purine nucleoside drugs for preventing or treating coronavirus infectious diseases - Google Patents

Abstract

Use of a compound as shown in formula (I) or a pharmaceutically acceptable salt thereof in the preparation of drugs for preventing or treating coronavirus infectious diseases. The compound of formula (I) shows significant activity in inhibiting the replication of novel coronavirus in vitro tests, and has no significant cytotoxicity in the dose range of the test.

Description

Use of purine nucleoside drugs to prevent or treat coronavirus infectious diseases technical field

The invention relates to a synthesis process of purine nucleoside drugs and its application in the preparation of drugs for preventing or treating coronaviruses, including novel coronavirus (Covid-19) infectious diseases.

Background technique

Coronavirus (CoV) is a class of enveloped positive-sense single-stranded RNA viruses that spread widely among humans, other mammals, and birds, and can cause respiratory, intestinal, liver, and nervous system diseases. Seven CoVs are currently known to cause disease in humans, four of which, HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1, circulate in humans and commonly cause common cold symptoms. The other three SARS-CoV, MERS-CoV and new coronavirus (COVID-19) all have serious hazards of fast onset, strong infectivity and high lethality. From the outbreak of COVID-19 around the world at the end of 2019 to March 17, 2021, the number of global infections has reached 117 million, and the death toll has exceeded 2.6 million, becoming a huge challenge to global public health. Although several vaccines have been authorized for emergency use, there is currently no effective clinical treatment drug, and the clinical efficacy of remdesivir approved by the US FDA for emergency use is limited. Therefore, it is very urgent to develop drugs that directly resist the new coronavirus COVID-19.

Contents of the invention

The present invention provides the purposes of the compound represented by formula (I) or pharmaceutically acceptable salt thereof in the preparation of the medicament for preventing or treating coronavirus infectious disease;

in,

X and Y are independently selected from: alkyl or substituted alkyl, halogen, -CN, -N ₃ , -OH, -NH ₂ , -NHR ³ , -NR ³ (R ³ ), -OR ³ or -SR ³ ;

R' and R are each independently selected from: -H, -COR ³ , -COOR ³ , -CONHR ³ or

Ar is selected from: aryl or substituted aryl, heteroaryl or substituted heteroaryl;

R ^and R are ^{independently} selected from: -H, alkyl or substituted alkyl, alkynyl or substituted alkynyl, alkenyl or substituted alkenyl, or 1-3 haloalkyl;

Or R ¹ and R ² form a 3-6 membered ring saturated aliphatic ring;

^Each R is independently selected from -H, alkyl or substituted alkyl, alkynyl or substituted alkynyl, alkenyl or substituted alkenyl, 1-3 haloalkyl or substituted alkyl, aryl or substituted aryl, or Heteroaryl or substituted heteroaryl;

The substituents in the "substituted alkyl", "substituted aryl", "substituted heteroaryl", "substituted alkynyl" and "substituted alkenyl" are each independently selected from: alkyl, halogen, -CN, -N ₃ , -OH, -NH ₂ , -NHR ³ , -NR ³ (R ³ ), -OR ³ , -SR ³ ;

The halogen atoms in the "halogen" and "haloalkyl" are selected from F, Cl, Br or I;

The alkyl in the "alkyl" and "haloalkyl" is a C _1-20 straight chain or branched chain alkyl, optionally a C _1-10 straight chain or branched chain alkyl, optionally a C _1-6 Straight chain or branched chain alkyl; alternatives are: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, or isopentyl ; Optionally, the haloalkyl group is 2-chloroethyl, 2-fluoroethyl or trifluoroethyl;

The "alkynyl" is C ₂ -C ₂₀ alkynyl; optional C ₂ -C ₁₀ alkynyl; optional acetylene;

The "alkenyl" is C ₂ -C ₂₀ alkenyl; optionally C ₂ -C ₁₀ alkenyl; optionally vinyl;

The "aryl" is a 6-10 membered aryl group; it can be phenyl or naphthyl;

The "heteroaryl" is a 5-10 membered heteroaryl group containing at least one heteroatom selected from N, O or S in the ring.

Optionally, in formula (I),

X is selected from -H, -NH ₂ , -NHR ³ , -NR ³ (R ³ ), -OR ³ or -SR ³ ;

Y is -NH ₂ ;

R' is -H;

R is -H or

in,

R ¹ and R ² are independently selected from: -H or C _1-6 straight or branched chain alkyl;

Each R ³ is independently C _1-6 straight chain or branched chain alkyl;

Ar is selected from phenyl or substituted phenyl, naphthyl or substituted naphthyl;

Optionally: the C _1-6 straight chain or branched chain alkyl is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-butyl Pentyl, or isopentyl.

Optionally, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is a compound of formula (II) or a pharmaceutically acceptable salt thereof,

Wherein, X, Y, R' and R are as defined above.

Optionally, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof,

In formula (Ia) and (Ib), X is -H, -NH ₂ , -NHMe, -N(Me) ₂ , -N(Me)(Et), -N(Et) ₂ , -OMe, -OEt , -O(iPr), -SMe, -SEt, or -S(iPr);

In formula (I-b), Ar is phenyl or naphthyl.

Another aspect of the present invention provides the compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof, wherein the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof is used for the preparation of Drugs for the prevention or treatment of coronavirus infectious diseases.

Another aspect of the present invention provides a method for preventing or treating coronavirus infectious diseases, which includes administering a therapeutically or preventively effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof to a patient in need.

Optionally, the pharmaceutically acceptable salts include salts formed by compounds of formula (I) and inorganic acids or organic acids;

Optionally, the inorganic acid includes hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, hemisulfuric acid, nitric acid, phosphoric acid, or carbonic acid;

Optionally, the organic acids include formic acid, ascorbic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, citric acid, citric acid, tartaric acid, glucose acid, tartaric acid, glucuronic acid, carbonic acid, picric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzoic acid, benzenesulfonic acid, p-bromobenzenesulfonic acid, glutamic acid, salicylic acid, or pamoic acid;

Optionally, the pharmaceutically acceptable salt is the hydrochloride salt of the compound of formula (I);

Alternatively, the pharmaceutically acceptable salt is the hemisulfate salt of the compound of formula (I).

Optionally, the coronavirus infectious diseases include diseases caused by infecting humans or other animals;

Optionally, the coronavirus includes HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV or COVID-19.

Optionally, the dosage form of the drug is an immediate-release dosage form, a sustained-release dosage form, or a controlled-release dosage form;

Optionally, the dosage form of the drug is tablet, capsule, aqueous or oily suspension, granule, emulsion, syrup, elixir, injection, or powder injection.

The present invention also provides a preparation method for the compound of formula (I) or a pharmaceutically acceptable salt thereof, comprising subjecting compound 5 to the dihydroxylation reaction of step e, then to the cyclic sulfoesterification reaction of step f, and then to the step g adopting triethylamine trihydrofluoride to open the ring, and then undergoing the post-hydrolysis cyclization reaction and benzoylation of step h to obtain the step of intermediate compound 9;

Optionally, in step e, the following reagents and conditions are used: KMnO ₄ , NaHCO ₃ , acetone;

Optionally, in step f, the following reagents and conditions are used: SOCl ₂ , EtN ₃ , NaClO;

Optionally, in step g, the following reagents and conditions are used: Et ₃ N·3HF, Et ₃ N, HCl;

Optionally, in step h, the following reagents and conditions are used: BaCl, BzCl, DMAP, Et ₃ N, CH ₃ CN;

然后化合物11与

反应得到

嘌呤核苷，然后经过酯化的步骤获得

Optionally, the method further includes: using intermediate compound 9 to obtain compound 11 through the reduction reaction of step i and the bromination reaction of step j in sequence,

Then compound 11 with

response to get

Purine nucleosides, which are then obtained through an esterification step

Optionally, in step i, the following reagents and conditions are used: LiAlH[OC(CH ₃ ) ₃ ] ₃ , THF;

Optionally, in step j, the following reagents and conditions are used: PPh ₃ , CBr ₄ , DCM;

Optionally, the reagents and conditions for the reaction step of formula (IV) to obtain formula (VI): t-BuOK, t-BuOH/CH ₃ CN;

Optionally, the method also includes: reacting compound 1 and ethyl acrylate to obtain compound 2 through step a, then brominating step b, hydrolyzing step c, and fluorination reaction step d to obtain compound 5. :

Optionally, in step a, the following reagents and conditions are used: triethylenediamine (DABCO);

Optionally, in step b, the following reagents and conditions are used: NBS, DMS, DCM;

Optionally, in step c, the following reagents and conditions are used: sodium acetate, reflux conditions;

Optionally, in step d, the following reagents and conditions are used: perfluorobutylsulfonyl fluoride, Et ₃ N·3HF.

Detailed ways

Specific embodiments of the present invention will be described in detail below. It should be understood that the specific implementations described here are only used to illustrate the present invention, and are not intended to limit the present invention.

Embodiment 1. The synthesis of compound

1. Synthesis of Compound 2

Dissolve R-acetone glyceraldehyde (compound 1, 500g, 3.85mol) in 1500mL of ethyl acrylate, add DABCO (215g, 1.93mol) to the above solution, react at 0°C for 12h, then turn to room temperature Continue to react for 6 days. After the reaction was detected by TLC, concentrate under reduced pressure to no distillate (ethyl acrylate), dissolve the residue in 2L ethyl acetate, wash the organic phase with 0.5M dilute hydrochloric acid until the water phase is colorless, and then water, washed with saturated saline in sequence. The organic phase was dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain a pale yellow oil (Compound 2) (637 g, 2.77 mol), with a yield of 72%. ¹ H NMR (400MHz, CDCl ₃ )δ6.37-6.36(m,1H),5.97-5.96(m,1H),4.52-4.51(m,1H),4.36-4.29(m,1H),4.26-4.21 (m,2H),3.93-3.92(m,2H),1.44(s,3H),1.35(s,3H),1.31(t,J=7.2Hz,3H)ppm;

2. Synthesis of compound 3

To a suspension of NBS (124 g, 0.70 mol) in DCM (1200 mL), 200 mL of dimethyl sulfide (47 g, 0.76 mol) in DCM was added dropwise at 0°C. After the addition, continue to stir for 30 minutes, add 200 mL of the DCM solution of compound 2 (146 g, 0.64 mol) dropwise to the reaction solution obtained above, move to room temperature, and after TLC detects that the reaction of compound 2 is complete, evaporate the DCM under reduced pressure at 30 ° C. , the residue was extracted with 500mL ether and 500mL saturated brine, the organic phase was washed with 0.5mol NaOH aqueous solution until the organic phase was colorless, and then washed with water and saturated brine in sequence. The organic phase was thoroughly dried over anhydrous sodium sulfate. Concentrate under reduced pressure at 25-30°C to distill off ether to obtain a colorless to pale yellow oil (Compound 3) (126 g, 0.56 mol), with a yield of 80%. ¹ H NMR (400MHz, CDCl ₃ ) δ6.84(d, J=8.4Hz, 1H), 4.96-4.90(m, 1H), 4.32-4.19(m, 5H), 3.74-3.70(m, 1H), 1.46(s,3H),1.42(s,3H),1.32(t,J＝6.8Hz,3H)ppm.

3. Synthesis of Compound 4

Dissolve compound 3 (118 g, 0.40 mol) obtained in the previous step in 1 L of ethanol, add 100 g of anhydrous sodium acetate, and heat to reflux until the reaction of the raw materials is complete. The insoluble matter was filtered off, the temperature of the reaction solution was lowered to 0° C., sodium ethoxide (8.3 g, 0.10 mol) was added, and the temperature was kept until the reaction of the raw materials was complete. Add 0.3eq acetic acid to neutralize, distill ethanol out under reduced pressure, add 200mL water to dissolve, extract with ethyl acetate until there is no product in the water phase, combine the organic phases, wash with water, saturated sodium bicarbonate solution, and brine in turn, and dry with anhydrous sodium sulfate . Concentration under reduced pressure, separation by silica gel column chromatography, petroleum ether: ethyl acetate = 10:1-3:1, a light yellow oil (compound 4) (55.7 g, 0.24 mol) was obtained, yield 60%. ¹ H NMR (400MHz, CDCl ₃ ) δ6.80 (d, J=7.6Hz, 1H), 4.96 (dd, J=14.0, 7.2Hz, 1H), 4.43-4.30 (m, 2H), 4.27-4.17( m,3H),3.70-3.66(m,1H),1.45(s,3H),1.40(s,3H),1.32(t,J=6.8Hz,3H)ppm;

4. Synthesis of Compound 5

Compound 4 (200g, 0.87mol), perfluorobutylsulfonyl fluoride (525g, 1.74mol) and Et ₃ N 3HF (210g, 130mol) were dissolved in 1700mL of anhydrous tetrahydrofuran, and triethylamine was added dropwise under ice cooling (440g, 4.35mol), the temperature of the reaction solution was controlled at 20°C during the dropwise addition, and kept at 20-25°C after addition, and monitored by TLC until the reaction of the raw materials was complete. Add 2L petroleum ether and stir for 5min, let stand to separate the layers, and collect the upper organic phase. The brown insoluble oil in the lower layer was extracted with a mixture of petroleum ether/ethyl acetate (10:1), the upper organic phases were combined, concentrated under reduced pressure, and separated by silica gel column chromatography, petroleum ether: ethyl acetate = 80:1 to obtain a light yellow liquid (Compound 5) (121 g, 0.52 mol), yield 60%. ¹ H NMR (400MHz, CDCl ₃ ) δ=7.02(ddd, J=8,3.6,0.8Hz,1H),5.28(dd,J=46.4,11.2Hz,1H),5.12(ddd,J=48.4,11.6 ,1.2Hz,1H),5.02-4.96(m,1H),4.28-4.18(m,3H),3.71(dd,J＝8.4,7.2Hz,1H),1.47(s,3H),1.42(s, 3H), 1.32(t, J=7.2Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ=165.19(J=3.8Hz), 145.70(J=4.6Hz), 129.60(J=15.4Hz), 110.42, 76.85 (J=163.5Hz), 72.36, 69.24 (J=3.4Hz), 61.38, 26.54, 25.68, 14.17. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₁ H ₁₇ FNaO ₄ 255.1003, found 255.1006.

5. Synthesis of Compound 6

Compound 5 (81 g, 0.35 mol), NaHCO ₃ (88 g, 1.05 mol) and ethylene glycol (87 g, 1.40 mol) were dissolved in 1500 mL of acetone. Add KMnO4 (66g, 0.42mol) in batches at 0°C, keep the temperature in the reaction solution not exceeding 5°C, and keep warm for 30min after adding. Slowly add 300 mL of saturated aqueous sodium bisulfite solution to quench the reaction, and stir at room temperature for 2 h until the brown color subsides. Suction filtration, washing the filter cake with acetone. The acetone in the filtrate was evaporated under reduced pressure, extracted 3-4 times with ethyl acetate, and the organic phases were combined. The organic phase was successively washed with water and brine, dried over anhydrous sodium sulfate, concentrated and evaporated to dryness. Add 150 mL of toluene and evaporate to dryness, heat to dissolve with 100 mL of toluene and 500 mL of n-heptane, cool to precipitate a white solid, and filter with suction to obtain a white solid (compound 6) (40 g, 0.45 mol) (purity 75%-80%), yield 43 %. ¹ H NMR (CDCl ₃ , 400MHz): δ4.87–4.56(m,2H), 4.41–4.33(m,1H), 4.21–4.08(m,4H), 4.06–4.00(m,1H), 3.82( t, J＝7.6Hz, 1H), 2.89(d, J＝9.2Hz, 1H), 1.36–1.31(m, 9H) ppm; ¹³ C NMR (CDCl ₃ , 100MHz): δ171.5(d, J _CF ＝3.8Hz), 109.7, 86.5(d, J _CF ＝172.8Hz), 78.5(d, J _CF ＝17.9Hz), 74.1, 73.9(d, J _CF ＝5.7Hz), 67.0, 62.8, 26.4, 25.1, 14.0ppm; HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₁ H ₁₉ FNaO ₆ 289.1058, found 289.1060.

6. Synthesis of Compound 7

Compound 6 (8.0g, 30mmol) was dissolved in a mixed solution of 15mL acetonitrile and 60mL isopropyl acetate, triethylamine (7.28g, 72mmol) was added, and SOCl ₂ (4.12g, 34.6mmol) was slowly added dropwise, and the addition process Keep the temperature of the reaction system at no more than 20°C, and keep it warm for 30 minutes after the addition is complete. After the reaction was completed, 30 mL of water was added and stirred, then left to stand, the aqueous phase was discarded, and the organic phase was washed with 30 mL of water and 30 mL of saturated NaHCO ₃ successively. Add 15mL of acetonitrile and _5.04g of NaHCO to the washed organic phase Solid, the resulting mixed solution is cooled to 10°C, slowly add 60mL of NaClO solution (about 2 times the amount) of 8-10% available chlorine, and transfer Reaction at room temperature for 2h. After the reaction, the aqueous phase was discarded, and the organic phase was washed twice with 30 mL saturated Na ₂ SO ₃ aqueous solution, and dried over anhydrous sodium sulfate. Added 0.5mL triethylamine as a stabilizer, evaporated the solvent under reduced pressure, and separated by silica gel column chromatography, petroleum ether: ethyl acetate = 6:1, to obtain a colorless oil (Compound 7) (8g, 24.3mmol), The yield was 81%. ¹ H NMR (CDCl ₃ , 400MHz): δ5.14–5.04(m, 1H), 5.03–4.92(m, 2H), 4.59–4.55(m, 1H), 4.36(d, J=7.2Hz, 2H) ¹³ C NMR ( CDCl ₃ , 100MHz): δ164.7(d, J _CF ＝7.4Hz), 111.6, 88.4(d, J _CF ＝17.8Hz), 82.5(d, J _CF ＝1.8Hz), 81.3(d, J _CF ＝ 179.1Hz), 71.7(d, J _CF =2.7Hz), 67.2, 64.1, 26.8, 24.9, 14.0ppm; HRMS (ESI) m/z: [M+H] ⁺ calcd for C ₁₁ H ₁₇ FNaO ₈ S 351.0520 , found 351.0519.

7. Synthesis of Compound 9

A mixture of compound 7 (8g, 24.4mmol), Et ₃ N·3HF (1.96g, 12.2mmol) and triethylamine (2.96g, 29.3mmol) was heated to 90°C for 3-4h, cooled to room temperature and concentrated After adding 4 mL of hydrochloric acid, react at 90°C for 30 min to obtain compound 8. Compound 8 was directly added to 30 mL of saturated BaCl ₂ solution without treatment, and the reaction was continued at 90°C for 4 h, 200 mL of n-propanol was added to azeotropically evaporate the solvent, and after evaporation to dryness, 150 mL of toluene was added and evaporated to dryness with water twice. The resulting mixture was dissolved in 60 mL of acetonitrile, phenyl acid chloride (10.29 g, 73.2 mmol), DMAP (298 mg, 2.44 mmol) and triethylamine (7.41 g, 73.2 mmol) were added sequentially, and the temperature was controlled not to exceed 45°C. After the reaction was completed, 30 mL of water was added to quench the reaction, the aqueous phase was discarded after standing, and the organic phase was washed successively with saturated NaHCO ₃ aqueous solution and saturated brine, and dried over anhydrous sodium sulfate. Concentrate under reduced pressure to distill off the solvent, separate by silica gel column chromatography, petroleum ether/ethyl acetate = 5:1, and obtain compound 9 (3.8 g, 9.76 mmol) as a white solid with a yield of 40%. ¹ H NMR (CDCl ₃ , 400MHz): δ8.08–8.99 (m, 4H), 7.66–7.55 (m, 2H), 7.50–7.40 (m, 4H), 5.88 (ddd, J=13.2, 7.2, 2.0 ¹³ C NMR (CDCl ₃ ,100MHz):δ167.1(d,J _CF ＝17.9Hz),165.8,165.2,134.3,133.6,130.2,129.8,128.9,128.8,128.6,127.8,89.1(dd,J _CF ＝194.1,24.2 Hz), 79.0(dd, J _CF ＝174.4, 35, 2Hz), 78.2, 68.0(dd, J _CF ＝14.3, 2.1Hz), 62.2ppm; HRMS(ESI) m/z: [M+H] ⁺ calcd for C ₂₀ H ₁₇ F ₂ O ₆ 391.0988,found 391.0987.

8. Synthesis of Compound 10

Weigh compound 9 (10g, 25.6mmol) and dissolve it in 100mL of anhydrous THF, add 31mL of 1M (t-BuO) ₃ AlH THF solution dropwise at -20°C, maintain the temperature for 4h, TLC detects that there are remaining raw materials, add Add 7.4mL of 1M (t-BuO) ₃ AlH THF solution, continue the reaction for 2h, add 100mL of water to quench the reaction, let it stand and discard the water phase, steam off most of the tetrahydrofuran under pressure, add 150mL of ethyl acetate to dissolve, and use Wash with saturated brine, dry the organic phase over anhydrous sodium sulfate, and concentrate to dryness under pressure to obtain a colorless oil, which was placed at 45°C for 8 h to obtain compound 10 as a white solid, which was directly used in the following step.

9. Synthesis of Compound 11

The crude compound 10 obtained in the previous step was dissolved in dichloromethane, and PPh ₃ (8.0g, 30.7mmol) was added at -20°C. After stirring for 20 minutes, CBr ₄ (11.0g, 33.18mmol) was added, and the temperature was raised to 0°C to continue the reaction After 2 hours, after monitoring the completion of the reaction, 30 mL of water was added to quench the reaction, extracted with dichloromethane, and dried over anhydrous sodium sulfate. The solvent was evaporated by concentration under reduced pressure, separated by silica gel column chromatography, petroleum ether/ethyl acetate = 5:1, and compound 11 (6.16 g, 13.57 mmol) was obtained as a white solid, and the total yield of the two steps was 53%. ¹ H NMR (CDCl ₃ , 400MHz): δ8.12(d, J=7.6Hz, 2H), 8.00(d, J=8.0Hz, 2H), 7.64–7.57(m, 2H), 7.50–7.43(m ,4H),6.64(s,1H),5.32–5.29(m,1H),4.91(dt,J＝6.0,3.2Hz,1H),4.87–4.71(m,2H),4.69–4.51(m,2H ) ppm; ¹³ C NMR (CDCl ₃ , 100MHz): δ166.0, 165.7, 134.1, 133.7, 130.2, 129.7, 129.2, 128.8, 128.5, 95.0 (dd, J _CF = 214.5, 17.8 Hz), 87.1 (dd, J _CF ＝23.6,6.3Hz), 81.5(d, J _CF ＝1.3Hz), 81.3(dd, J _CF ＝181.9,27.5Hz), 68.5(dd, J _CF ＝14.9,5.0Hz), 62.1ppm; HRMS(ESI )m/z:[M+H] ⁺ calcd for C ₂₀ H ₁₈ BrF ₂ O ₅ 455.0300,found 455.0302.

10. Synthesis of Compound 12

Potassium tert-butoxide (3.0g, 26.43mmol) was added in batches to a solution of 2-chloro-6-aminopurine (4.5g, 26.43mmol) in 30mL of tert-butanol at room temperature, and after stirring for 1 hour at 30°C, the mixture The solution was poured into the acetonitrile solution of compound 11 (4.0 g, 8.81 mmol) obtained in the previous step, and the temperature was gradually raised to 50 ° C. After 16 hours, TLC monitored that the reaction was complete. After cooling down to room temperature, water was added to quench the reaction, and extracted with ethyl acetate ( 50mL x 3), combined organic phases, dried over anhydrous sodium sulfate, column chromatography, petroleum ether/ethyl acetate=5:1, isolated to obtain white solid β-configuration compound 12 (2.2g, 4.14mmol), yield 47%. ¹ H NMR (CDCl ₃ , 400MHz): δ7.96–7.93(m,2H),7.89–7.87(m,2H),7.84(s,1H),7.58–7.54(m,1H),7.47–7.43( m,1H),7.40–7.36(m,2H),7.28–7.24(m,2H,overlapped with the peak of chloroform),6.67(dd,J＝22.0,8.8Hz,1H),6.26(d,J＝ 18.0Hz, 1H), 5.60(s, 2H), 5.21–5.16(m, 1H), 4.86(dt, J=10.8, 5.6Hz, 1H), 4.78–4.44(m, 3H) ppm; ¹³ C NMR ( CDCl ₃ , 100MHz): δ166.4, 165.7, 159.2, 152.5, 152.4, 141.8, 134.0, 133.4, 130.0, 129.7, 129.1, 128.6, 128.4, 128.0, 126.0, 100.7 (dd, _J5Hz ) ＝190.4, 189. (dd, J _CF ＝39.6, 1.8Hz), 81.9 (dd, J _CF ＝174.6, 24.8Hz), 78.2, 71.4 (dd, J _CF ＝15.6, 5.2Hz), 62.9ppm; HRMS(ESI) m/z :[M+H] ⁺ calcd for C ₂₅ H ₂₁ ClF ₂ N ₅ O ₅ 544.1194,found 544.1197.

11. Synthesis of Compound 13

Weigh compound 12 (1.5g, 2.76mmol) and dissolve it in methanol, add 3mL of 33% methylamino alcohol solution, react at 85°C for 16 hours, after the reaction, directly evaporate to dryness, separate by silica gel column chromatography, dichloromethane: methanol =15:1, white solid compound 13 (740mg, 2.23mmol) was obtained with a yield of 81%. ¹ H NMR (CD ₃ OD, 400MHz): δ7.92(s, 1H), 6.23(d, J=18.4Hz, 1H), 4.75(dd, J=23.6, 9.2Hz, 2H), 4.60(dt, J＝47.6, 11.2Hz, 1H), 4.44–4.23(m, 1H), 4.09–4.00(m, 2H), 3.86(dd, J＝12.4, 2.8Hz, 1H), 2.99(s, 3H)ppm; ¹³ C NMR (CD ₃ OD, 100MHz): δ161.9, 157.2, 151.0, 137.8, 114.9, 101.6 (dd, J _CF = 184.9, 17.0 Hz), 89.3 (d, J _CF = 39.5 Hz), 83.6, 82.0 (dd , J _CF ＝173.5, 27.6Hz), 69.2 (dd, J _CF ＝17.1, 4.7Hz), 60.9, 27.5ppm; HRMS (ESI) m/z: [M+H] ⁺ calcd for C ₁₂ H ₁₇ F ₂ N ₆ O ₃ 331.1325,found 331.1328.

12. Synthesis of Compound III

Under nitrogen protection, weigh compound 13 (50mg, 0.15mmol) and dissolve it in THF at 0°C, slowly add 270μL of 1M tert-butylmagnesium chloride solution dropwise, keep warm for 15 minutes, then move the reaction system to room temperature for 15 minutes, and then transfer the system to After reaching 0°C, add N-[(S)-(2,3,4,5,6-pentafluorophenoxy)phenoxyphosphoryl]-L-alanine isopropyl ester (100mg, 0.22mmol ), moved to room temperature and continued to react for 18 hours after being incubated for 30 minutes, the basic reaction of monitoring reaction was complete, adding saturated ammonium chloride to quench the reaction, extraction with ethyl acetate (50mL x 3), merging the organic phases, drying over anhydrous sodium sulfate, column Chromatography separation, dichloromethane:methanol=15:1-10:1, gave white solid compound III (50mg, 0.087mmol), the yield was 58%. ¹ H NMR (CD ₃ OD, 400MHz): δ7.77, 7.76(ss, 1H), 7.28(t, J=7.6Hz, 2H), 7.23–7.17(m, 2H), 7.12(t, J=7.2 Hz,1H),6.23(d,J＝18.8Hz,1H),4.97–4.89(m,1H),4.84–4.80(m,1H),4.72–4.40(m,4H),4.23–4.21(m, 1H),3.90–3.80(m,1H),2.99(s,3H),1.25–1.22(m,3H),1.13–1.10(m,6H)ppm; ¹³ C NMR(CD ₃ OD,100MHz):δ174 .4(d, J _CP ＝5.4Hz), 162.1, 157.2, 152.1(d, J _CP ＝6.8Hz), 138.0, 130.8, 130.7, 126.1, 121.4(d, J _CP ＝4.8Hz), 121.3(d, J _CP ＝4.8Hz), 115.1, 101.5(dd, J _CF ＝184.8, 16.7Hz), 89.3(d, J _CF ＝40.3Hz), 82.3(dd, J _CF ＝173.5, 26.4Hz), 81.9(d, J _CF ＝8.1Hz),70.8(dd,J _CF ＝17.0,4.7Hz),70.12,70.08,67.3(d,J _CP ＝4.4Hz),51.6(d,J _CP ＝1.5Hz),21.9,21.8, 20.5(d,J _CP ＝6.4Hz)ppm; HRMS(ESI)m/z:[M+H] ⁺ calcd for C ₂₄ H ₃₃ F ₂ N ₇ O ₇ P 600.2142,found 600.2141.

Example 2. In vitro anti-coronavirus activity and cytotoxicity test of compound III

(1) Purpose of the experiment

In order to study the anti-coronavirus efficacy of the compound in vitro, the cytopathic effect (CPE) assay was used to determine its cytotoxicity (CC ₅₀ ) to H460 cells and the half maximal inhibitory concentration (IC ₅₀ ) to the coronavirus HCoV-OC43. Ribavirin (RBV) was used as a positive control drug.

(2) Experimental materials and reagents

1. The name of the test product: compound Ⅲ (synthesized in this laboratory), this product is white to light yellow solid. Seal and store in a cool place.

2. Positive control drug

Positive control drug: Ribavirin injection was purchased from Tianjin Jinyao Group Hubei Tianyao Pharmaceutical Co., Ltd., the batch number is 31712252, the specification is 100mg/ml, diluted to the required concentration when used, and stored in a refrigerator at 4°C.

3. Cells: Passage human lung cancer cells H460 cells are all preserved in this laboratory, in DMEM medium containing 10% fetal bovine serum (inactivated fetal bovine serum) and 1% double antibodies (penicillin and streptomycin), at 37°C , cultured in a 5% CO ₂ incubator. Passage once every 2-3 days.

4. Strains: HCoV-OC43 was passaged in HCT-8 cells and stored in a -80°C refrigerator.

5. Reagents: DMEM liquid medium, fetal bovine serum (fetal bovine serum), penicillin and streptomycin solution (penicillin-streptomycin), PBS (pH=7.4) and 0.25% Trypsin-EDTA were purchased from Invitrogen.

6. Experimental supplies and instruments: cell culture flasks, 96-well culture plates and pipettes are products of Corning Company in the United States; carbon dioxide incubators (Model 3111) are products of Thermo Company in the United States; biological safety cabinets are products of NUAIRE Company in the United States; inverted microscopes, The product of Olympus; the vacuum pump is the product of INTEGRA Biosciences; the 12-channel pipette and single-channel pipette are the products of Eppendorf.

(3) Experimental steps

1. Cell culture

Add 3ml of 0.25% Trypsin-EDTA to a culture flask full of H460 cells, digest at 37°C for 1-2 minutes, discard the digestion solution, add culture solution and pipette, 1:3 passaging, passaging once every 2-3 days, prepare when seeding the plate 150,000 cells per milliliter were inoculated into a 96-well cell culture plate, 0.1 ml per well, cultured overnight at 37°C, 5% CO ₂ , and the experiment was performed after the cells grew into a monolayer.

2. Determination of drug toxicity to cells by CPE method

H460 cells were inoculated in a 96-well plate at 1.5× ¹⁰⁴ cells/well, and after overnight culture, a maintenance solution containing the drug to be tested was added, and the drug to be tested was tested with 8 doses of three-fold dilution samples, and the culture was continued. After 3 days of administration, the toxicity of the drug to the cells under an inverted microscope (CPE method), and the half toxic concentration TC ₅₀ was calculated by the Reed-Muench method, and the calculation formula was as follows:

Where: A = drug concentration with cumulative inhibitory rate < 50%, B = cumulative inhibitory rate > 50% inhibitory rate, C = cumulative inhibitory rate < 50% inhibitory rate, D = log dilution factor

3. Anti-HCoV-OC43 activity assay (CPE method)

The experiment was carried out in the passaged H460 cells. The cells were seeded in 96-well plates at 1.5× ¹⁰⁴ /well. After overnight culture, 100TCID ₅₀ HCoV-OC43 virus liquid was used to infect the cells in the 96-well plates. The drug to be tested was diluted with culture medium. Measuring at the same time as the infection, the drug to be tested was tested with three times diluted samples of 8 doses, each dose was set with 2 parallel wells, and the results were observed when the virus control group had lesions of 4+, recorded and analyzed by Reed-Muench The half-inhibitory concentration (the formula is as follows) and the selection index (SI=CC ₅₀ /IC ₅₀ ) of the drug on the virus were calculated by the method.

Wherein: A=drug concentration with cumulative inhibition rate<50%, B=inhibition rate with cumulative inhibition rate>50%, C=inhibition rate with cumulative inhibition rate<50%, D=log dilution factor

(4) Calculation of experimental results

1. Toxicity of compound III in H460 cells and its inhibitory effect on HCoV-OC43 strain

In H460 cells, the CC ₅₀ of compound III determined by CPE method was >100 μM, and its IC ₅₀ against HCoV-OC43 was 0.09 μM; the selection index SI was >1111; the positive control drug RBV half toxic concentration CC ₅₀ was >0.36±0.06 μM, the _IC50 against HCoV-OC43 is 0.015±0.01μM, and the selection index SI is >24. The anti-HCoV-OC43 activity of the positive drug RBV was comparable to the results in the literature, indicating that the experimental system was established.

The results showed that compound III had obvious inhibitory activity against HCoV-OC43.

Example 3. Test of compound III anti-new coronavirus 2019-nCoV (COVID-19) activity and cytotoxicity in vitro

(1) Purpose of the experiment

To evaluate the inhibitory effect of compound III on 2019-nCoV (COVID-19) at the cellular level.

(2) Experimental materials and reagents

1. Cell line: Huh7 cells, preserved in our laboratory;

2. Virus strain: 2019-nCoV (COVID-19);

3. Test drug: compound Ⅲ

4. Positive control drug: remdesivir;

5. Reagents: DMEM medium (Gibco), fetal bovine serum (Gibco), double antibody, trypsin, MTT (Amresco), etc.;

6. Kit: QIAamp viral RNA mini kit (52906, Qiagen), One Step TB Green PrimeScript PLUS RT-PCR Kit (Perfect Real Time) (RR096A TaKaRa)

7. Consumables: cell culture plates, 96-well microtiter plates, etc.;

8. Instruments: Multifunctional microplate reader, StepOnePlus fluorescence quantitative PCR instrument, carbon dioxide incubator, etc.

(3) Experimental steps

1. Determination of the cytotoxicity of compound Ⅲ

The toxicity of compound Ⅲ to Huh7 cells was detected by MTT assay. The full name of MTT is 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenyltetrazoliumromide, which is a yellow dye. MTT colorimetry is a method to detect cell survival and growth. The detection principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan (Formazan) and deposit in the cells, while dead cells do not have this function. 10% SDS (dissolved in 0.01mol/L HCl solution) can dissolve formazan in cells, and its light absorption value is measured at a wavelength of 570nm with a multifunctional microplate reader, which can indirectly reflect the number of living cells. Within a certain cell number range, the amount of MTT crystal formation is proportional to the cell number. By detecting the light absorption value at 570nm wavelength under different interferon concentrations, the survival rate of cells under the concentration of compound III can be calculated, thereby calculating the half-toxic concentration (CC ₅₀ ) of compound III.

Inoculate Huh7 cells in a 96-well plate one day in advance, 1× ¹⁰⁴ cells per well (note that the edge wells of the 96-well plate should not be used as experimental wells, add PBS to prevent the medium in other wells from volatilizing); observe the state of the cells, and reach about 50 %, compound III was diluted 2-fold with DMEM medium containing 2% FBS, and then added to the cell plate at 100 μL/well, with 6 replicates for each concentration; at the same time, a control group (without drug group) was set and the blank group (without cell group), placed in an incubator at 37°C with 5% CO ₂ for cultivation; 48 hours after adding the drug, 25 μL of MTT solution (5 mg/mL) was added to each well, and after continuing to cultivate for 4 hours, each well was Add 125 μL of 10% SDS (dissolved in 0.01mol/L HCl solution), pipette gently, and place it for 2 hours to fully dissolve the crystals. Set the blank group to zero, measure OD570, and calculate according to the following formula: Survival rate (%)= OD570 of drug-dosed group/OD570 of control group×100%. At the same time, the median toxic concentration (CC ₅₀ ) of the drug was calculated.

2. Evaluation of the inhibitory effect of compound Ⅲ on 2019-nCoV (COVID-19) virus

The antiviral activity was measured on the Huh7 cell model, and each experiment was set up with 3 replicate wells, which were repeated 3 times.

1) Inoculate 5×10 ⁴ Huh7 cells in each well of a 24-well cell culture plate. Under the culture conditions of 37°C and 5% CO ₂ , when the confluence reaches 60%, the MOI of infection is 0.005. Add 200 μL of 2019-nCoV (COVID-19) virus solution diluted with DMEM medium containing 2% FBS to the well, absorb in a 37°C, 5% _CO2 incubator for 1 hour, discard the virus solution, and mix the positive control drug and Compounds were diluted 2-fold from the maximum non-toxic concentration in DMEM medium containing 2% FBS, added to the cell plate at 500 μL/well, and a control group (without drug group) was set at the same time, and the supernatant was collected 48 hours after infection virus fluid.

2) Use real-time RT-PCR (qRT-PCR) to quantify the RNA of the collected virus:

Take 140 μL of the collected supernatant virus liquid, and perform RNA extraction according to the instructions of the QIAamp viral RNA mini kit kit. The One Step TB Green PrimeScript PLUS RT-PCR Kit (Perfect Real Time) kit was used for qRT-PCR detection, and the primers were RBD-qF1:5’-CAATGGTTTAACAGGCACAGG-3’; RBD-qR1:5’-CTCAAGTGTCTGTGGATCACG-3’. The total volume of the reaction system is 20 μL: 10 μL 2X One Step TB Green RT-PCR Buffer 4, 1.2 μL TaKaRa Ex Taq HS Mix, 0.4 μL PrimeScript PLUS RTase Mix, 0.8 μL each of RBD-qF1 and RBD-qR1, 0.4 μL ROX Reference Dye (50X), 2 μL viral RNA, 4.4 μL RNase Free dH2O. The reaction parameters were: reverse transcription at 42°C for 5min, pre-denaturation at 95°C for 10s, 40 cycles of PCR including denaturation at 95°C for 10s, annealing and extension at 60°C for 30s.

3) Calculate the drug inhibition rate at each concentration. Inhibition rate (%)=1-viral RNA copy number of experimental group/viral RNA copy number of drug-free group×100%. Simultaneously calculate half effective concentration (EC ₅₀ ) and therapeutic index (TI) = half toxic concentration (CC ₅₀ )/ half effective concentration (EC ₅₀ ).

(4) Calculation of experimental results

Table 1. Compound III inhibits the activity of novel coronavirus (COVID-19)

Test drug _EC50 CC ₅₀ Ti Compound III 0.08μM >100μM >1250 remdesivir 1.50μM >45.0μM >30.0

According to the results in Table 1, the concentration required for Compound III to inhibit 50% of the new coronavirus is 0.08 μM, while Redcivir requires 1.5 μM. The activity of Compound III in inhibiting the new coronavirus is 18 times that of Redcivir, and the cytotoxicity is less .

Therefore, compound III and its analogues have the effect of preventing or treating coronavirus infectious diseases.

Claims (8)

Compounds represented by formula (I) or pharmaceutically acceptable salts thereof are used in the preparation of medicines for preventing or treating coronavirus infectious diseases;

in, X and Y are independently selected from: -H, alkyl or substituted alkyl, halogen, -CN, -N ₃ , -OH, -NH ₂ , -NHR ³ , -NR ³ (R ³ ), -OR ³ or SR ³ ; R' and R are each independently selected from: -H, -COR ³ , -COOR ³ , -CONHR ³ or

Ar is selected from: aryl or substituted aryl, heteroaryl or substituted heteroaryl; R ^and R are ^{independently} selected from: -H, alkyl or substituted alkyl, alkynyl or substituted alkynyl, alkenyl or substituted alkenyl, or 1-3 haloalkyl; Or R ¹ and R ² form a 3-6 membered ring saturated aliphatic ring; ^Each R is independently selected from -H, alkyl or substituted alkyl, alkynyl or substituted alkynyl, alkenyl or substituted alkenyl, 1-3 haloalkyl or substituted alkyl, aryl or substituted aryl, or Heteroaryl or substituted heteroaryl; The substituents in the "substituted alkyl", "substituted aryl", "substituted heteroaryl", "substituted alkynyl" and "substituted alkenyl" are each independently selected from: alkyl, halogen, -CN, -N ₃ , -OH, -NH ₂ , -NHR ³ , -NR ³ (R ³ ), -OR ³ , -SR ³ ; The halogen atoms in the "halogen" and "haloalkyl" are selected from F, Cl, Br or I; The alkyl in the "alkyl" and "haloalkyl" is a C _1-20 straight chain or branched chain alkyl, optionally a C _1-10 straight chain or branched chain alkyl, optionally a C _1-6 Straight chain or branched chain alkyl; alternatives are: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, or isopentyl ; Optionally, the haloalkyl group is 2-chloroethyl, 2-fluoroethyl or trifluoroethyl; The "alkynyl" is C ₂ -C ₂₀ alkynyl; optional C ₂ -C ₁₀ alkynyl; optional acetylene; The "alkenyl" is C ₂ -C ₂₀ alkenyl; optionally C ₂ -C ₁₀ alkenyl; optionally vinyl; The "aryl" is a 6-10 membered aryl group; it can be phenyl or naphthyl; The "heteroaryl" is a 5-10 membered heteroaryl group containing at least one heteroatom selected from N, O or S in the ring. purposes according to claim 1, is characterized in that, in formula (I), X is selected from -H, -NH ₂ , -NHR ³ , -NR ³ (R ³ ), -OR ³ or -SR ³ ; Y is -NH ₂ ; R' is -H; R is -H or

in, R ¹ and R ² are independently selected from: -H or C _1-6 straight or branched chain alkyl; Each R ³ is independently C _1-6 straight chain or branched chain alkyl; Ar is selected from phenyl or substituted phenyl, naphthyl or substituted naphthyl; Optionally: the C _1-6 straight chain or branched chain alkyl is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-butyl Pentyl, or isopentyl. The use according to claim 1 or 2, characterized in that the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof is a compound of the formula (II) or a pharmaceutically acceptable salt thereof,

Wherein, X, Y, R' and R are as defined in claim 1 or 2. The use according to any one of claims 1-3, characterized in that the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof is selected from compounds (I-a), (I-b), and pharmaceutical acceptable salt,

In formula (Ia) and (Ib), X is -H, -NH ₂ , -NHMe, -N(Me) ₂ , -N(Me)(Et), -N(Et) ₂ , -OMe, -OEt , -O(iPr), -SMe, -SEt, or -S(iPr); In formula (I-b), Ar is phenyl or naphthyl. The use according to any one of claims 1-4, wherein the pharmaceutically acceptable salt comprises a salt formed of a compound of formula (I) and an inorganic acid or an organic acid; Optionally, the inorganic acid includes hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, hemisulfuric acid, nitric acid, phosphoric acid, or carbonic acid; Optionally, the organic acids include formic acid, ascorbic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, citric acid, citric acid, tartaric acid, glucose acid, tartaric acid, glucuronic acid, carbonic acid, picric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzoic acid, benzenesulfonic acid, p-bromobenzenesulfonic acid, glutamic acid, salicylic acid, or pamoic acid; Optionally, the pharmaceutically acceptable salt is the hydrochloride salt of the compound of formula (I); Alternatively, the pharmaceutically acceptable salt is the hemisulfate salt of the compound of formula (I). The use according to any one of claims 1-5, wherein the coronavirus infectious diseases include diseases caused by infecting humans or other animals; Optionally, the coronavirus includes HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV or COVID-19. The use according to any one of claims 1-6, wherein the dosage form of the drug is an immediate-release dosage form, a sustained-release dosage form, or a controlled-release dosage form; Optionally, the dosage form of the drug is tablet, capsule, aqueous or oily suspension, granule, emulsion, syrup, elixir, injection, or powder injection. The preparation method of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises the following steps: comprising subjecting compound 5 to the dihydroxylation reaction of step e, then the cyclic sulfoesterification reaction of step f, and then adopting step g Triethylamine trihydrofluoride ring-opening, and then undergoing hydrolysis post-cyclization reaction and benzoylation in step h to obtain the step of intermediate compound 9;

Optionally, in step e, the following reagents and conditions are used: KMnO ₄ , NaHCO ₃ , acetone; Optionally, in step f, the following reagents and conditions are used: SOCl ₂ , EtN ₃ , NaClO; Optionally, in step g, the following reagents and conditions are used: Et ₃ N·3HF, Et ₃ N, HCl; Optionally, in step h, the following reagents and conditions are used: BaCl, BzCl, DMAP, Et ₃ N, CH ₃ CN; Optionally, the method further includes: using intermediate compound 9 to obtain compound 11 through the reduction reaction of step i and the bromination reaction of step j in sequence,

Then compound 11 with

response to get

Purine nucleoside, and then through the step of esterification to get

Optionally, in step i, the following reagents and conditions are used: LiAlH[OC(CH ₃ ) ₃ ] ₃ , THF; Optionally, in step j, the following reagents and conditions are used: PPh ₃ , CBr ₄ , DCM; Optionally, the reagents and conditions for the reaction step of formula (IV) to obtain formula (VI): t-BuOK, t-BuOH/CH ₃ CN; Optionally, the method also includes: reacting compound 1 and ethyl acrylate to obtain compound 2 through step a, then brominating step b, hydrolyzing step c, and fluorination reaction step d to obtain compound 5. :

Optionally, in step a, the following reagents and conditions are used: triethylenediamine (DABCO); Optionally, in step b, the following reagents and conditions are used: NBS, DMS, DCM; Optionally, in step c, the following reagents and conditions are used: sodium acetate, reflux conditions; Optionally, in step d, the following reagents and conditions are used: perfluorobutylsulfonyl fluoride, Et ₃ N·3HF.