A nucleoside analog and its deuterated product and preparation method and use thereof

Abstract

The present invention provides a nucleoside analog and a deuterated product thereof and a preparation method and use thereof, belonging to the field of pharmaceutical chemistry. The nucleoside analog and the deuterated product thereof are compounds shown in formula I, or salts thereof, or stereoisomers thereof, or prodrugs thereof, or solvates thereof; wherein R 1 ~R 34 Each independently selected from hydrogen or deuterium. The compounds of the present invention have good inhibitory effects on hepatitis C virus, MERS virus, SARS virus, and novel coronavirus (SARS‑CoV‑2); and the compounds of the present invention have a long elimination half-life, which can reduce the frequency of drug administration, increase safety of use, and have better pharmacokinetics and good drugability. Therefore, the compounds of the present invention can be used to prepare drugs for treating related viral infections, and have good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to a nucleoside analog and a deuterated product thereof, and a preparation method and use thereof. Background Art

[0002] Coronaviruses are a large class of viruses that are widely present in nature, and some of them can cause illness in humans. Human diseases caused by coronaviruses are mainly respiratory infections. Currently, a total of 7 types of coronaviruses that can infect humans have been discovered, of which MERS-CoV, SARS-CoV, and SARS-CoV-2 are the most well-known. They are contagious, spread quickly and widely, and pose a great threat to the health and even life of the infected. At present, there is no specific drug treatment for diseases caused by these coronaviruses, such as Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS), and Novel Coronavirus Pneumonia (COVID-19).

[0003] Nucleoside analogues (Nu) are an important class of chemical substances. Their structural characteristics are similar to those of nucleosides. Studies have found that some nucleoside analogues have pharmacological activities, such as antiviral effects, immunosuppressive effects, and anti-tumor effects. Sofosbuvir is a nucleoside analogue, which is a new drug developed by Gilead for the treatment of chronic hepatitis C. It was approved for marketing in the United States by the U.S. Food and Drug Administration (FDA) on December 6, 2013, and was approved for marketing in EU countries by the European Medicines Agency (EMEA) on January 16, 2014.

[0004]

[0005] Nucleoside analogs have not been used to treat coronavirus. Summary of the invention

[0006] The purpose of the present invention is to provide a nucleoside analog and a deuterated product thereof and a preparation method and use thereof.

[0007] The present invention provides a compound represented by formula I, or a salt thereof, or a stereoisomer thereof, or a prodrug thereof, or a solvate thereof:

[0008]

[0009] Among them, R 1 ~R 34 are each independently selected from hydrogen or deuterium.

[0010] Furthermore, the compound is as shown in Formula II:

[0011]

[0012] Among them, R 1 ~R 21 are each independently selected from hydrogen or deuterium.

[0013] Furthermore, the compound is as shown in Formula III:

[0014]

[0015] Among them, R 12 ~R 20 are each independently selected from hydrogen or deuterium.

[0016] Further,

[0017] R 12 ~R 20 All are selected from hydrogen;

[0018] Or, R 12 ~R 16 All deuterium, R 17 ~R 20 are each independently selected from hydrogen or deuterium;

[0019] Or, R 18 ~R 20 All deuterium, R 12 ~R 17 are each independently selected from hydrogen or deuterium;

[0020] Or, R 17 Selected from deuterium, R 12 ~R 16 and R 18 ~R 20 are each independently selected from hydrogen or deuterium.

[0021] Furthermore, the compound is selected from the following structures:

[0022]

[0023] The present invention also provides a method for preparing the aforementioned compound, which comprises the following steps:

[0024]

[0025] (1) Compound A and compound B undergo esterification reaction to obtain compound C;

[0026] (2) Compound C and compound D undergo N-phosphorylation reaction; then compound E is added to undergo nucleophilic substitution reaction to obtain compound F;

[0027] (3) Compound F reacts with compound G in the presence of a catalyst and an acid-binding agent to generate compound H;

[0028] (4) deprotecting compound H to obtain the target product, compound of formula III;

[0029] Among them, R 12 ~R 20 The definition of R is as above. 35 Selected from hydrogen or deuterium.

[0030] Further,

[0031] In step (1), the esterification reaction is carried out by stirring compound A and compound B at 0-4° C. for 5-10 minutes and then adding a catalyst for reaction;

[0032] In step (2), the N-phosphorylation reaction is to dissolve compound C in an organic solvent, cool to -78°C, add compound D and triethylamine, and react;

[0033] In step (2), the nucleophilic substitution reaction is to cool the reaction solution after the N-phosphorylation reaction to 0-4°C, and then add compound E and triethylamine to react;

[0034] In step (3), the reaction is to dissolve compound F, compound G and a catalyst in an organic solvent at 50-80° C. and stir for 10-20 min, then add an acid binding agent for reaction;

[0035] In step (4), the deprotection is performed by dissolving compound H in an organic solvent and then adding an acid at 0-5°C for reaction;

[0036] Preferably,

[0037] In step (1), the mass volume ratio of compound A to compound B is (1-5) g:10 mL;

[0038] And / or, in step (1), the catalyst is thionyl chloride, and the molar ratio of thionyl chloride to compound A is 1.0 to 2.0;

[0039] And / or, in step (2), the molar ratio of compound C, compound D and compound E is (1-1.2):(1-1.2):1; in the N-phosphitylation reaction, the molar ratio of triethylamine to compound C is (1-1.2):1; in the nucleophilic substitution reaction, the molar ratio of triethylamine to compound C is (1-1.2):1;

[0040] And / or, in step (2), the organic solvent is dichloromethane;

[0041] And / or, in step (3), the molar ratio of compound F, compound G, catalyst and acid binding agent is 1:(1-2):(1-2):(3-5);

[0042] And / or, in step (3), the organic solvent is acetonitrile, tetrahydrofuran or toluene, preferably acetonitrile; the catalyst is magnesium chloride, zinc chloride, tin chloride or stannous chloride, preferably magnesium chloride; the acid binding agent is triethylamine, pyridine or N,N-diisopropylethylamine, preferably N,N-diisopropylethylamine;

[0043] And / or, in step (4), the molar ratio of the compound H to the acid is 1:(1-1.5);

[0044] And / or, in step (4), the organic solvent is acetonitrile, tetrahydrofuran or acetone, preferably tetrahydrofuran; the acid is hydrochloric acid;

[0045] More preferably,

[0046] In step (1), the mass volume ratio of compound A to compound B is 1 g:10 mL;

[0047] And / or, in step (2), the molar ratio of compound C, compound D and compound E is 1.2:1.2:1; in the N-phosphitylation reaction, the molar ratio of triethylamine to compound C is 1:1; in the nucleophilic substitution reaction, the molar ratio of triethylamine to compound C is 1:1;

[0048] And / or, in step (3), the molar ratio of compound F, compound G, catalyst and acid binding agent is 1:1:1:3;

[0049] And / or, in step (4), the molar ratio of compound H to acid is 1:1.

[0050] Further,

[0051] In step (1), the reaction temperature of the esterification reaction is 20-80°C, and the reaction time is 4-10h; preferably, the reaction temperature of the esterification reaction is 60°C, and the reaction time is 10h;

[0052] And / or, in step (2), the reaction temperature of the N-phosphitylation reaction is 0-25°C, and the reaction time is 3-6h; preferably, the reaction temperature of the N-phosphitylation reaction is 20°C, and the reaction time is 5h;

[0053] And / or, in step (2), the reaction temperature of the nucleophilic substitution reaction is 10-20°C, and the reaction time is 2-4h; preferably, the reaction temperature of the nucleophilic substitution reaction is 20°C, and the reaction time is 3h;

[0054] And / or, in step (3), the reaction temperature of the reaction is 50-80°C, and the reaction time is 20-120 min; preferably, the reaction temperature of the reaction is 50°C, and the reaction time is 20 min;

[0055] And / or, in step (4), the reaction temperature of the deprotection is 0-15°C, and the reaction time is 1-3h; preferably, the reaction temperature of the deprotection is 5°C, and the reaction time is 2h;

[0056] Preferably,

[0057] In step (1), compound C is purified by: concentrating under reduced pressure, adding ethyl acetate at room temperature and stirring, and filtering;

[0058] In step (2), compound F is purified by: concentrating under reduced pressure to remove the organic solvent, separating the concentrate by silica gel column chromatography to obtain a colorless semi-solid, stirring the semi-solid with diisopropyl ether and then filtering;

[0059] In step (3), compound H is purified by: concentrating under reduced pressure to remove the organic solvent, adding ethyl acetate, and then washing with 5% citric acid aqueous solution, saturated ammonium chloride aqueous solution, 5% sodium bicarbonate aqueous solution and saturated brine in sequence, and the organic phase is concentrated under reduced pressure and then separated and purified by column chromatography;

[0060] In step (4), the compound of formula III is purified by adding water, adjusting the pH to 7-8 with a saturated sodium bicarbonate solution, extracting with ethyl acetate, washing with saturated brine, concentrating under reduced pressure, and separating by column chromatography.

[0061] The present invention also provides the use of the aforementioned compound, or a salt thereof, or a stereoisomer thereof, or a prodrug thereof, or a solvate thereof in the preparation of a drug for inhibiting hepatitis virus and / or inhibiting coronavirus;

[0062] Preferably, the drug is a drug for preventing and / or treating diseases caused by hepatitis virus infection and / or diseases caused by coronavirus infection;

[0063] More preferably, the hepatitis virus is hepatitis C virus; and / or, the coronavirus is MERS-CoV, SARS-CoV, SARS-CoV-2;

[0064] Further preferably, the disease caused by hepatitis virus infection is hepatitis C; and / or, the disease caused by coronavirus infection is MERS, SARS, or COVID-19.

[0065] The present invention also provides a drug, which is a preparation prepared by using the aforementioned compound, or its salt, or its stereoisomer, or its prodrug, or its solvate as an active ingredient and adding pharmaceutically acceptable excipients.

[0066] The "deuterium" mentioned in the present invention is an isotope of hydrogen (H), also known as heavy hydrogen, and its element symbol is generally D or 2 H, deuterium has one proton and one neutron in its nucleus, and its relative atomic weight is 2.

[0067] The "deuterated product" mentioned in the present invention refers to a product in which at least one hydrogen in a molecule is replaced by deuterium.

[0068] The "salt of the compound" described in the present invention refers to a "pharmaceutically acceptable salt", which is a product obtained by direct salt-forming reaction of the free base of the compound with an inorganic or organic acid. The inorganic or organic acid can be selected from hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, picric acid, citric acid, maleic acid, methanesulfonic acid, trifluoromethanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, etc.

[0069] The compounds of the present invention have good inhibitory effects on hepatitis C virus, MERS virus, SARS virus, and novel coronavirus (SARS-CoV-2); and the compounds of the present invention have a long elimination half-life, which can reduce the frequency of drug administration and increase the safety of use. The pharmacokinetics are better and have good drugability, among which compound 8 has the best effect. The compounds of the present invention can be used to prepare drugs for treating related viral infections and have good application prospects.

[0070] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and conventional means in the art, other various forms of modification, replacement or change may be made without departing from the above basic technical concept of the present invention.

[0071] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. DETAILED DESCRIPTION

[0072] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0073] Example 1. Preparation of Compound 1 of the Invention

[0074] The synthetic route is:

[0075]

[0076] The specific preparation method includes:

[0077] L-alanine (10.00 g, 0.11 mol) and 2-ethylbutanol (100 mL) were added to the reaction flask, the reaction system was stirred at 0 ° C for 5 min, dichlorothionyl (14.4 g, 0.12 mol) was slowly added dropwise, heated to 60 ° C and stirred for 10 h, and most of the solvent was removed by concentration under reduced pressure. After the concentrate was cooled to room temperature, 200 mL of ethyl acetate was added and stirred for 60 min. The off-white solid, compound C-1 (18.4 g, yield 80.0%, LCMS m / z 174.1 [M+H]) was obtained by filtration.

[0078] Take compound C-1 (10.0g, 0.048mol) and 120mL of dichloromethane, stir and cool the system to -78℃, add benzyloxy dichlorophosphate (10.1g, 0.048mol), then slowly drop triethylamine (4.9g, 0.048mol), heat the system to 20℃ and stir for 5h. Cool the system to 0℃ and add 4-nitrophenol (5.6g, 0.040mol), then slowly drop triethylamine (4.9g, 0.048mol), heat the reaction to 20℃ and stir for 3h. After the reaction is completed, concentrate under reduced pressure to remove the solvent, and separate the concentrate by silica gel column chromatography (petroleum ether: ethyl acetate = 1:2, v / v) to obtain a colorless semisolid (15.7g, yield 73.0%). The obtained semi-solid was stirred with 100 mL of diisopropyl ether for 24 h and then filtered. The filter cake was compound F-1, which was dried under vacuum at 40 °C for 4 h to obtain 5.0 g, with a yield of 32.0%. 1H-NMR (400 MHz, DMSO-d6): δ8.37-8.29 (m, 2H), 7.55-7.49 (m, 2H), 7.48-7.40 (m, 2H), 7.29-7.21 (m, 3H), 4.12-4.07 (m, 1H), 4.01 (d, J = 5.7 Hz, 2H), 3.90-3.78 (m, 1H), 1.50-1.43 (m, 1H), 1.39-1.05 (m, 7H), 0.80 (t, J = 7.4 Hz, 6H); LCMS m / z 451.2 [M+H].

[0079] Take compound F-1 (0.9 g, 0.002 mol) and compound G (0.7 g, 0.002 mol), magnesium chloride (0.20 g, 0.002 mol) and 15 mL of acetonitrile, stir at 50 ° C for 10 min, then add N, N-diisopropylethylamine (0.8 g, 0.006 mol), stir for 20 min, cool to 20 ° C, and concentrate under reduced pressure at 30-40 ° C to remove acetonitrile, then add 150 mL of ethyl acetate, the reaction solution is washed with 5% citric acid aqueous solution (20 ml), saturated ammonium chloride aqueous solution (20 mL), 5% sodium bicarbonate aqueous solution (20 mL), and saturated brine (20 mL), the organic phase is concentrated under reduced pressure and purified by column chromatography (petroleum ether: ethyl acetate = 1: 2, v / v) to obtain an off-white solid, namely compound H-1 (1.0 g, yield 78.0%). 1H-NMR (400MHz, DMSO-d6): δ8.35(s,1H),8.30(d,1H),7.84(s,1H),7.46-7.40(m,2H), 7.29-7.20(m,3H),7.01(s,2H),6.89(m,2H),5.34(m,1H),4.99(m,1H),4. 60-4.53(m,1H),4.36-4.24(m,2H),3.78(m,1H),4.02(m,1H),3.92(m,1H), 3.84(m,1H),1.70(s,3H),1.50–1.42(m,1H),1.40(s,3H),1.35–1.26(m,4H), 0.90(t,J=7.2Hz,6H); LCMS m / z 642.1[M+H].

[0080] Take compound H-1 (0.8 g, 0.001 mol) and 10 mL of THF, stir the system and cool it to 5°C, then add concentrated hydrochloric acid (0.13 g, 0.001 mol) dropwise, keep it at 5°C and stir for 2 h, add 5 mL of water, adjust the pH to 7-8 with saturated sodium bicarbonate solution, extract the system with ethyl acetate (10 mL*2), wash with saturated brine (5 mL), and concentrate under reduced pressure to obtain an off-white solid, namely, a crude compound 1, which was purified by column chromatography (petroleum ether: ethyl acetate = 1:2, v / v) to obtain 0.47 g of compound 1 with a yield of 78.0%. 1HNMR (400MHz, DMSO-d6):8.38(brs,1H),8.28(d,1H),7.78(brs,1H),7.35-7.37(m, 2H),7.24-7.18(m,3H),6.04(m,1H),5.96(d,1H),5.65(brs,1H),5.29(t,1H), 5.11(brs,1H),4.23(m,1H),3.85-4.13(m,3H),3.80-3.82(m,2H),3.55-3.58(m,2H),1.34-1.26(m,7H),0.91(t,J=7.2Hz,6H).LCMS m / z 601.1[M+H].

[0081] Example 2-8, Preparation of Compound 2-8 of the Present Invention

[0082] Preparation of compound 2-8: Compound 2-8 was prepared by the synthetic route and preparation method of compound 1, and only the structures of substrates A-1 and D-1 were changed during the preparation process. The specific substrate selection and the structural formula of compound 2-8 are shown in Table 1.

[0083] Table 1. Preparation of compounds 2-8 of the present invention

[0084]

[0085]

[0086]

[0087]

[0088]

[0089] Example 9: Results of the compounds of the present invention inhibiting hepatitis C virus in vitro

[0090] The Huh7-Luc cell line containing HCV genotype 1b replicon was selected. The solubility of the compound in DMSO is greater than 18.8 mg / mL. If a solution with a higher concentration is prepared, the general steps are as follows: heat the test tube at 37°C for 10 minutes and / or place it in an ultrasonic bath for shaking.

[0091] Take the Huh7-Luc cell line containing HCV genotype 1b replicon in the logarithmic phase and prepare 1 × 10 4 The cell suspension was prepared by incubating for 24 hours until the cells adhered to the wall, and then adding compounds 1-8 at a concentration of 0.1 to 1000 nM, respectively. After adding, the cells were incubated for 72 hours, and then the activity of the cells after 72 hours was detected by the MTT method, and the EC50 value was calculated. The EC50 values ​​of the compounds 1-8 of the present invention are shown in Table 2. The compound sofosbuvir was used as the control group, and its structural formula is as follows:

[0092]

[0093] Table 2. EC50 values ​​of compounds 1-8 of the present invention against HCV

[0094]

[0095]

[0096] The test results show that the compounds of the present invention have a good inhibitory effect on hepatitis C virus; the effect is similar to that of sofosbuvir and favipiravir in the control group, and some compounds (compounds 1, 2, 5, 7 and 8) are even better than sofosbuvir and favipiravir in the control group.

[0097] Example 10: Inhibitory Effects of the Compounds of the Invention on Coronavirus

[0098] The virus strains were derived from the MERS-CoV, SARS-CoV and SARS-CoV-2 virus strains isolated and purified from clinical practice in a certain research institute, and the cells used were the HEK293 cell line.

[0099] The solubility of this compound in DMSO is greater than 18.8 mg / mL. To prepare a solution with a higher concentration, the general steps are as follows: Heat the test tube at 37°C for 10 minutes and / or place it in an ultrasonic bath for a period of time.

[0100] HEK293 cell lines infected with MERS-CoV, SARS-CoV and SARS-CoV-2 in the logarithmic phase were prepared into 1×10 4The cells were incubated for 24 hours until the cells attached to the wall, and then 0.1 to 1000 nM of compounds 1-8 were added. The cells were incubated for 72 hours, and then the MTT method was used to detect the activity of the cells after 72 hours, and the EC50 values ​​were calculated. The EC50 values ​​of compounds 1-8 of the present invention are shown in Table 3.

[0101] Table 3. Inhibitory effects of the compounds of the present invention on coronavirus

[0102]

[0103] The test results show that the compounds of the present invention have good inhibitory effects on coronaviruses: MERS virus, SARS-CoV virus and SARS-CoV-2 virus; the effects are similar to those of the control group sofosbuvir and favipiravir, and some compounds (such as compounds 2 and 8) are even better than the control group sofosbuvir and favipiravir, especially compound 8 has the best inhibitory effect.

[0104] Example 11: Pharmacokinetic study of the compounds of the present invention

[0105] Favipiravir (lead substance) was orally administered to 10 SD rats at a dose of 12 mg / kg, and blood was collected at 3h, 6h, 9h, 12h, 18h, 24h, and 48h to measure the changes in blood drug concentration.

[0106] Compounds 1-8 of the present invention were orally administered to 10 SD rats at a dose of 12 mg / kg, and blood was collected at 3h, 6h, 9h, 12h, 18h, 24h and 48h to measure changes in blood drug concentrations.

[0107] The elimination half-life of the compounds of the present invention was calculated, and the results are shown in Table 4:

[0108] Table 4. Elimination half-life results of the compounds of the present invention

[0109] Compound <![CDATA[Elimination half-life T 1 / 2 > Compound 1 15h±3.2h Compound 2 12h±4.2h Compound 3 16h±5.5h Compound 4 13h±6.2h Compound 5 10h±2.2h Compound 6 11h±3.3h Compound 7 14h±6.1h Compound 8 17h±2h Favipiravir 9h±2.1h Sofobuwe 11h±5.3h

[0110] The test results show that compared with favipiravir, the elimination half-life of some compounds of the present invention is significantly increased compared with sofosbuvir, which can reduce the frequency of drug administration and increase the safety of use. The pharmacokinetics are better and the drugability is good.

[0111] In summary, the compounds of the present invention have good inhibitory effects on hepatitis C virus, MERS virus, SARS virus, and new coronavirus (SARS-CoV-2); and the compounds of the present invention have a long elimination half-life, which can reduce the frequency of drug administration and increase the safety of use. Its pharmacokinetics is better and has good drugability. Among them, compound 8 has the best effect. The compounds of the present invention can be used to prepare drugs for treating related viral infections and have good application prospects.

Claims

1. Use of a compound, a salt thereof, or a stereoisomer thereof in the preparation of a drug for inhibiting coronavirus; the coronavirus is MERS-CoV, SARS-CoV, or SARS-CoV-2; The compound is selected from the following structures:

2. The use according to claim 1, characterized in that: The medicine is a medicine for preventing and / or treating diseases infected by coronavirus; The diseases infected by the coronavirus are MERS, SARS, and COVID-19.

3. Use of the compound of formula I, or its salt, or its stereoisomer in the preparation of a drug for inhibiting coronavirus: in, R1~R 34 are each independently selected from hydrogen or deuterium; The coronaviruses are MERS-CoV, SARS-CoV, and SARS-CoV-2.

4. The use according to claim 3, characterized in that: The compound is shown in Formula II: Among them, R1~R 21 are each independently selected from hydrogen or deuterium.

5. The use according to claim 4, characterized in that: The compound is shown in formula III: Among them, R 12 ~R 20 are each independently selected from hydrogen or deuterium.

6. The use according to claim 5, characterized in that: R 12 ~R 20 All are selected from hydrogen; Or, R 12 ~R 16 All deuterium, R 17 ~R 20 are each independently selected from hydrogen or deuterium; Or, R 18 ~R 20 All deuterium, R 12 ~R 17 are each independently selected from hydrogen or deuterium; Or, R 17 Selected from deuterium, R 12 ~R 16 and R 18 ~R 20 are each independently selected from hydrogen or deuterium.

7. The use according to claim 3, characterized in that: The compound is selected from the following structures:

8. The use according to claim 3, characterized in that: The medicine is a medicine for preventing and / or treating diseases infected by coronavirus; The diseases infected by the coronavirus are MERS, SARS, and COVID-19.

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