CN113461760A

CN113461760A - 4-thiodeoxythymidine derivative and anti-hepatitis B virus pharmaceutical application thereof

Info

Publication number: CN113461760A
Application number: CN202111036007.5A
Authority: CN
Inventors: 冯力; 杨子峰; 陆冬晓; 马丁; 陈锋; 杨丽; 彭盛; 周明; 唐秀春; 叶小新; 刘红宁
Original assignee: Guoqing Biomedical Shanghai Co ltd; Nanjing Yiyuan Biomedical Research Institute Co ltd
Current assignee: Guoqing Biomedical Shanghai Co ltd; Nanjing Yiyuan Biomedical Research Institute Co ltd
Priority date: 2021-09-06
Filing date: 2021-09-06
Publication date: 2021-10-01
Anticipated expiration: 2041-09-06
Also published as: CN113461760B

Abstract

The invention discloses a 4-thiodeoxythymidine derivative and anti-hepatitis B virus pharmaceutical application thereof. The invention provides a 4-thiodeoxythymidine derivative or a pharmaceutically acceptable salt thereof, wherein the structure of the derivative is shown as a structural formula I or a structural formula II. The invention proves that the 4-thiodeoxythymidine derivative or the derivative thereof is proved by in vitro cytotoxicity test and in vitro anti-HBV virus drug effect testThe pharmaceutically acceptable salt has anti-HBV activity and anti-hepatitis B drug development prospect, and provides a potential choice for treating viral hepatitis.

Description

4-thiodeoxythymidine derivative and anti-hepatitis B virus pharmaceutical application thereof

Technical Field

The invention belongs to the field of medicinal chemistry, and particularly relates to a 4-thiodeoxythymidine derivative and anti-hepatitis B virus pharmaceutical application thereof.

Background

Persistent infection with HBV is a major cause of hepatitis b chronicity and can lead to the development of disease conditions, exacerbation, and HBV-associated hepatocellular carcinoma. Thus, antiviral therapy is critical for the treatment of chronic hepatitis b. Currently, commonly used anti-HBV drugs are classified into immunomodulatory drugs and nucleoside analogs. In addition, the Chinese herbal medicine and the effective components thereof also play an important role in anti-HBV treatment. The interferon is a common immunomodulator for HBV treatment, has broad antiviral spectrum, has a lasting treatment effect and a high virus surface antigen clearance rate, but has a low response rate and a large side effect. Nucleoside analogs include primarily lamivudine, telbivudine, entecavir, adefovir, tenofovir, and the like, which competitively bind to viral DNA during viral replication and reduce viral DNA replication by inhibiting reverse transcriptase activity. The medicine can reduce incidence of hepatic failure and hepatocellular carcinoma, and improve survival rate. However, these drugs have a common disadvantage in that they are resistant to drugs after a certain period of time and cannot completely eliminate hepatitis B virus. In view of the drug resistance and the drug effect of the existing anti-hepatitis B virus drugs which need to be improved, the research and development of novel anti-hepatitis B virus drugs are still important issues for treating viral hepatitis at present.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the prior art, the invention provides 4-thiodeoxythymidine derivatives, wherein the 4-thiodeoxythymidine derivatives comprise D-configuration 4-thiodeoxythymidine derivatives and L-configuration 4-thiodeoxythymidine derivatives, have good inhibitory rate and treatment rate on HBV, and can be used for preparing medicaments for treating hepatitis B or liver cancer.

The invention also provides application of the 4-thiodeoxythymidine derivative.

The technical scheme is as follows: the structure of the 4-thiodeoxythymidine derivative or the pharmaceutically acceptable salt thereof is shown as a formula I or a formula II:

；

wherein R is₁Is selected from H, -CH₃，-CH₂CH₃-C3-C10 hydrocarbyl, methoxy, methylthio, -C2-C10 hydrocarbyloxy, C2-C10 hydrocarbylthio, aminomethyl, hydroxymethyl, -CH₂O(CH₂)nO(CH₂)nCH₃(n=1-3)，

-CH₂NH(CH₂)nNH(CH₂)nCH₃(n =1-3), an aromatic ring of C5-C10, a heteroaromatic ring of C5-C10, an alicyclic ring of C3-C10, an alicyclic ring of C3-C10, -F, -Cl, -Br, -I, or cyanogenA group, etc.;

wherein R is₂Is selected from H, -CH₃，-CH₂CH₃-C3-C10 hydrocarbyl, methoxy, methylthio, -C2-C10 hydrocarbyloxy, C2-C10 hydrocarbylthio, aminomethyl, hydroxymethyl, -CH₂O(CH₂)nO(CH₂)nCH₃(n=1-3)，-CH₂NH(CH₂)nNH (CH₂)nCH₃(n =1-3), an aromatic ring of C5-C10, a heteroaromatic ring of C5-C10, an alicyclic ring of C3-C10, an alicyclic ring of C3-C10, -F, -Cl, -Br, -I, or a cyano group, etc.;

R₃is selected from

；

R₅Is selected from-CH₃，-CH₂CH₃-C3-C10 hydrocarbyl, methoxy, methylthio, -C2-C10 hydrocarbyloxy, C2-C10 hydrocarbylthio, aminomethyl, hydroxymethyl, -CH₂O(CH₂)nO(CH₂)nCH₃(n=1-3)，-CH₂NH(CH₂)nNH (CH₂)nCH₃(n =1-3), an aromatic ring of C5-C10, an aromatic heterocycle of C5-C10, an alicyclic ring of C3-C10, or an alicyclic heterocycle of C3-C10, etc.;

R₆is composed of

Wherein R is₈，R₉Independently or simultaneously selected from H, -CH₃，-CH₂CH₃-C3-C10 hydrocarbyl, -CH₂CH₂O(CH₂)nO(CH₂)nCH₃(n=1-3)，-CH₂CH₂NH(CH₂)nNH (CH₂)nCH₃(n =1-3), an aromatic ring of C5-C10, an aromatic heterocycle of C5-C10, an alicyclic ring of C3-C10, or an alicyclic heterocycle of C3-C10, and the like, R₁₀Is selected from-CH₃，

-CH₂CH₃-a linear hydrocarbon group from C3 to C10, -a branched hydrocarbon group from C3 to C10, methoxy, methylthio, -a hydrocarbonoxy group from C2 to C10, a hydrocarbylthio group from C2 to C10, -CH₂O(CH₂)nO(CH₂)nCH₃(n=1-3)，-CH₂CH₂NH(CH₂)nNH (CH₂)nCH₃(n =1-3), an aromatic ring of C5-C10, an aromatic heterocycle of C5-C10, an alicyclic ring of C3-C10, or an alicyclic heterocycle of C3-C10, etc.;

ar is selected from phenyl, substituted phenyl, C5-C10 aromatic ring, or C5-C10 aromatic heterocycle, etc., R7 has the following structure

Wherein R is₁₁，R₁₂Independently or simultaneously selected from H, -CH₃，-CH₂CH₃-C3-C10 hydrocarbyl, -CH₂CH₂O(CH₂)nO(CH₂)nCH₃(n=1-3)，-CH₂CH₂NH(CH₂)nNH (CH₂)nCH₃(n =1-3), an aromatic ring of C5-C10, an aromatic heterocycle of C5-C10, an alicyclic ring of C3-C10, or an alicyclic heterocycle of C3-C10, and the like, R₁₃Is selected from-CH₃，-CH₂CH₃-a linear hydrocarbon group of C3-C10, -a branched hydrocarbon group of C3-C10, -CH₂O(CH₂)nO(CH₂)nCH₃(n=1-3)，-CH₂CH₂NH(CH₂)nNH (CH₂)nCH₃(n =1-3), an aromatic ring of C5-C10, an aromatic heterocycle of C5-C10, an alicyclic ring of C3-C10, or an alicyclic heterocycle of C3-C10, etc.;

R₄is selected from H, -CH₃，-CH₂CH₃-C3-C10 hydrocarbyl, methoxy, methylthio, -C2-C10 hydrocarbyloxy, C2-C10 hydrocarbylthio, aminomethyl, hydroxymethyl, -CH₂O(CH₂)nO(CH₂)nCH₃(n=1-3)，-CH₂NH(CH₂)nNH (CH₂)nCH₃(n =1-3), an aromatic ring of C5-C10, a heteroaromatic ring of C5-C10, an alicyclic ring of C3-C10, an alicyclic heterocyclic ring of C3-C10, -Cl, -Br, -I, or a cyano group, and the like.

Preferably, R₁Is selected from-CH₃，R₂Selected from H, R₄Is selected from H.

Preferably, R₃Selected from:

。

the derivative or the pharmaceutically acceptable salt thereof according to the present invention is preferably any one of the following:

preferably, the 4-thiodeoxythymidine derivative may be used as a pharmaceutically acceptable salt, including salts of the compound with metal ions or pharmaceutically acceptable amines or ammonium ions.

The invention relates to an application of 4-thiodeoxythymidine derivative or its pharmaceutically acceptable salt in preparing medicine for treating hepatitis or liver cancer.

Wherein the hepatitis is hepatitis B caused by HBV infection.

The pharmaceutical composition comprises an effective amount of the 4-thiodeoxythymidine derivative or its pharmaceutically acceptable salt, stereoisomer, active metabolite, prodrug, solvate or crystal form, and pharmaceutically acceptable excipient.

The medicine composition is prepared into any one of dosage forms in pharmacy, including capsules, powder, tablets, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories or patches.

The application of the 4-thiodeoxythymidine derivative and the pharmaceutical composition in preparing the drugs for treating hepatitis B or liver cancer is within the protection scope of the invention. The hepatitis B is caused by HBV infection.

Has the advantages that: the invention provides a group of 4-thiodeoxythymidine derivatives or pharmaceutically acceptable salts thereof, and in vitro cytotoxicity tests and in vitro anti-HBV virus pharmacodynamic tests prove that the 4-thiodeoxythymidine derivatives or the pharmaceutically acceptable salts thereof have anti-HBV activity, the effect is better than that of positive control, and the anti-hepatitis B drug development prospect is provided.

The 4-thiodeoxythymidine derivative or the pharmaceutically acceptable salt thereof has the advantages of ingenious design, simple structure, cheap and easily-obtained raw materials, safe and environment-friendly synthesis process and easy large-scale production.

Drawings

FIG. 1 is a drawing of compound Q15¹HNMR spectrogram;

FIG. 2 is a drawing of compound Q22¹HNMR spectrogram;

FIG. 3 is a drawing of compound Q23¹HNMR spectrogram;

FIG. 4 is of compound Q24¹HNMR spectrogram;

FIG. 5 is a drawing of compound Q21¹HNMR spectrogram;

FIG. 6 is of compound Q12¹HNMR spectrogram;

FIG. 7 is a drawing of compound Q11¹HMNR profile.

Detailed Description

The present invention will be described in detail with reference to specific examples. The compounds prepared in the examples are represented by the code Q and are within the scope of formula I or formula II.

Example 1

The synthetic route is as follows:

；

thymine (26 g, 205.7 mmol) was mixed in hexamethyldisilazane (320 mL, 1.48 mol) and trimethylchlorosilane (42 mL, 321 mmol), N₂Stirring for 3h at 130 ℃ under protection. After cooling to room temperature, the reaction was concentrated, pumped off by an oil pump, and dichloromethane (500 mL, anhydrous) was added to the crude product, followed by the addition of Compound A (CAS: 141846-57-3) (80 g, 205.7 mmol) and trimethylsilyl trifluoromethanesulfonate (2.25 mL, 12.3 mmol) in that order, and the mixture was stirred at room temperature for 1 h. After completion of the reaction, a saturated sodium bicarbonate solution was slowly added to the reaction solution with stirring, dichloromethane extraction was performed, the organic phase was washed with saturated brine, the organic phase was separated, dried over anhydrous sodium sulfate, filtered to remove anhydrous sodium sulfate, the filtrate was concentrated to obtain a crude product, and the crude product was separated by column chromatography (petroleum ether: ethyl acetate = 10/1-1/1) to obtain compound B (90.3 g, yellow solid).

Compound B (10.0 g, 20.9 mmol) was mixedIn toluene (100 mL), N₂Stirring at 120 ℃ under protection until the solution is clear, adding Lawson's reagent (9.3 g, 23.0 mmol), and stirring at 80 ℃ for 2 h. After completion of the reaction, it was cooled to room temperature, insoluble matter was removed by filtration, the filtrate was concentrated, and the crude product was isolated by column chromatography (petroleum ether: ethyl acetate = 10/1-1/1) to obtain compound C (9 g, yellow solid, impure).

Compound C (20 g, 41.6 mmol) was dissolved in MeOH (200 mL), and sodium methoxide in methanol (15 g, W/W = 30%) was added at room temperature, and stirred at room temperature overnight. After completion of the reaction, the reaction mixture was cooled in an ice-water bath, hydrochloric acid (1N) was added dropwise with stirring to adjust pH =7, and the reaction mixture was concentrated and separated by column chromatography (dichloromethane: methanol = 50/1-10/1) to obtain product D (9.66 g, yellow solid).

¹H NMR（400MHz, DMSO-d₆）δ12.71（s，1H），7.90（s，1H），6.11（t，1H），5.28-5.27（m，1H），5.10（t，1H），4.27-4.22（m，1H），3.81-3.79（q，1H），3.66-3.54（m，2H），3.40（t，1H），3.17（d，1H），1.95（s，3H）。

Compound D (9.66 g, 40.0 mmol) was dissolved in pyridine (150 mL), 0 deg.C, N₂Pivaloyl chloride (5.25 g, 43.5 mmol) was added under protection, stirred at room temperature overnight, the reaction was concentrated after completion of the reaction, and the crude product was isolated by column chromatography (dichloromethane: methanol = 50/1-20/1) to give compound E (12.3 g, yellow solid).

¹H NMR (400 MHz, DMSO-d ₆) δ 12.77 (s, 1H), 7.54 (d, 1H), 7.54-7.37(m，1H), 6.12 (t, 1H), 5.45 (d, 1H), 4.22 (d, 2H), 4.13 – 3.94 (m, 1H), 2.21 (dd, 2H), 1.99 (d, 3H), 1.15 (s, 9H)。

Compound E (12.3 g, 37.6 mmol) was dissolved in dichloromethane (200 mL, anhydrous), silver nitrate (12.1 g, 71.4 mmol), 2,4, 6-trimethylpyridine (38.7 g, 319.4 mmol) and 4-methoxytriphenylchloromethane (34.8 g, 112.7 mmol) were added successively at room temperature, and stirred at room temperature overnight. After completion of the reaction, insoluble matter was removed by filtration, the filtrate was concentrated, and the crude product was isolated by column chromatography (petroleum ether: ethyl acetate = 10/1-1/1) to give compound F (16.8 g, yellow solid, impure).

Compound F (16.8 g, 28.0 mmol) was dissolved in MeOH (200 mL), and sodium methoxide in methanol (10.2 g, W/W = 30%) was added at room temperature, and stirred at room temperature overnight. After completion of the reaction, a small amount of water was added to quench, the reaction solution was concentrated, and the crude product was isolated by column chromatography (petroleum ether: ethyl acetate = 10/1-1/1) to give intermediate compound 1 (6.4 g, yellow solid).

¹H NMR (400 MHz, DMSO-d ₆) δ 12.71 (s, 1H), 7.77 (s, 1H), 7.43-7.28 (m, 12H), 6.94 (d, 2H), 5.76 (s, 1H), 5.00 (s, 1H), 4.26 (s, 1H), 4.06-4.01 (m, 4H), 3.18-3.16 (m, 1H), 1.91 (s, 3H), 1.74-1.68 (m, 2H)。

Intermediate compound 1 (2.4G, 4.5 mmol) and 4-dimethylaminopyridine (111 mg, 0.9 mmol) were dissolved in acetonitrile (50 mL, anhydrous), triethylamine (687 mg, 6.8 mmol) and isobutyric anhydride (787 mg, 5.0 mmol) were added at room temperature, stirring was carried out overnight at room temperature, after completion of the reaction, methanol (10 mL) was added, stirring was carried out for 30min, the reaction solution was concentrated, and the crude product was isolated by column chromatography (petroleum ether: ethyl acetate = 10/1-1/1) to give product G (2.3G, white solid).

¹H NMR (400 MHz, DMSO-d ₆) δ 12.75 (s, 1H), 7.47 – 7.24 (m, 13H), 6.97 – 6.90 (m, 2H), 6.11 (dd, 1H), 4.19 (dt, 1H), 4.08 – 3.94 (m, 1H), 3.98 – 3.80 (m, 2H), 3.75 (s, 3H), 2.50 – 2.37 (m, 1H), 1.92 (d, 3H), 1.88 – 1.73 (m, 2H), 0.99 (dd, 6H)。

Compound G (2.3G, 3.8 mmol) was dissolved in acetic acid solution (50 mL, V/V = 80%), stirred at room temperature overnight, after completion of the reaction, the reaction was concentrated, and the crude product was isolated by column chromatography (dichloromethane: methanol = 50/1-20/1) to give compound Q15 (1.09G, yellow solid).

¹The HNMR spectrum is shown in figure 1,¹H NMR (400 MHz, DMSO-d ₆) δ 12.76 (s, 1H), 7.56 (d, 1H), 6.12 (t, 1H), 5.45 (d, 1H), 4.24 (td, 3H), 3.96 (q, 1H), 2.56 (p, 1H), 2.30 – 2.14 (m, 2H), 2.01 – 1.96 (m, 3H), 1.09 (dd, 6H)。

example 2

The synthetic route is as follows:

；

intermediate compound 1 (2 g, 3.8 mmol) and compound I (3.4 g, 7.5 mmol, CAS: 1354823-36-1) were dissolved in MeCN (100 mL), magnesium chloride (359 mg, 3.8 mmol) was added, stirring was carried out at 50 ℃ for 10 min, DIEA (1.22 g, 9.4 mmol) was added, and stirring was carried out at 50 ℃ overnight. After completion of the reaction, the reaction mixture was cooled to room temperature, diluted with dichloromethane (200 mL), washed with citric acid (1N), the organic phase was washed successively with saturated ammonium chloride, then with saturated sodium bicarbonate, with saturated brine, the organic phase was dried over anhydrous sodium sulfate, anhydrous sodium sulfate was removed by filtration, the filtrate was concentrated, and the crude product was isolated by column chromatography (petroleum ether: ethyl acetate = 10/1-1/1) to give Compound J (2.3 g, yellow solid).

¹H NMR (400 MHz, DMSO-d ₆) δ 12.76 (s, 1H), 7.42 (d,5H), 7.36 (dd, 4H), 7.28 (ddd,6H), 7.14 (t,1H), 7.05 (d, 2H), 6.96 – 6.89 (m, 2H), 6.18 – 6.08 (m, 2H), 4.25 (d, 1H), 4.03 (q, 1H), 3.98 – 3.84 (m, 3H), 3.81 (dd,1H), 3.74 (s, 3H), 3.64 (ddd, 1H), 1.92 (s, 3H), 1.55 (ddd, 1H), 1.38 (hept, 1H), 1.29 – 1.14 (m, 8H), 0.79 (td, 6H)。

Compound J (2.3 g, 2.8 mmol) was dissolved in acetic acid solution (80 mL, V/V = 80%), stirred at room temperature overnight, the reaction was concentrated after completion of the reaction, and the crude product was isolated by column chromatography (dichloromethane: methanol = 50/1-20/1) to give compound Q23 (1.4 g, yellow solid).

¹The HNMR spectrum is shown in figure 3,¹H NMR (400 MHz, DMSO-d ₆) δ 12.75 (s, 1H), 7.60 (d, 1H), 7.40 – 7.31 (m, 2H), 7.27 – 7.11 (m, 3H), 6.19 – 6.08 (m, 2H), 5.44 (d,1H), 4.24 (dq,1H), 4.22 – 4.09 (m, 2H), 4.01 – 3.94 (m, 2H), 3.93 – 3.77 (m, 2H), 2.13 (ddd,1H), 2.08 – 1.92 (m, 4H), 1.43 (hept,1H), 1.32 – 1.18 (m, 7H), 0.81 (t,6H)。

example 3

The synthetic route is as follows:

；

chloromethyl pivalate (48.2 g, 320 mmol), trimethyl phosphate (11.2 g, 80 mmol) and sodium iodide (36 g, 240 mmol) were mixed in acetonitrile (100 mL, anhydrous), and several particles of 4A molecular sieves, N, were added to the reaction mixture₂Under protection 90^oC is refluxed overnight, after the reaction is completed, the reaction solution is cooled to room temperature, filtered by celite, the filter residue is washed by ethyl acetate, the filtrate is concentrated, and the crude product is separated by column chromatography (petroleum ether: ethyl acetate = 10/1-5/1) to give product L (28.3 g, colorless liquid).

Compound L (6 g, 13.6 mmol) and lithium bromide (1.2 g, 13.6 mmol) were dissolved in acetonitrile (50 mL, anhydrous) 90^oC was stirred at reflux overnight. After the reaction is finished, cooling to room temperature, filtering, washing a filter cake with petroleum ether, and carrying out oil pump drying on the filter cake to obtain the compound M (4 g, white solid).

¹H NMR (400 MHz, DMSO-d ₆) δ 5.36 (s, 2H), 5.33 (s, 2H), 1.14 (s, 18H)。

Intermediate compound 1 (1.9 g, 3.7 mmol) and compound M (2.4 g, 7.3 mmol) were dissolved in THF (50 mL), 3-nitro-1, 2, 4-triazole (834 mg, 7.3 mmol), diisopropylethylamine (1.9 g, 14.6 mmol) and bis (2-oxo-3-oxazolidinyl) hypophosphoryl chloride (BOP-Cl) (1.9 g, 7.3 mmol) were added sequentially at 0 deg.C and stirred at room temperature for 1 h. The reaction mixture was diluted with dichloromethane (100 mL), washed with 1M citric acid, the organic phase was separated, washed with water, and then washed with brine. The organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and column chromatography (petroleum ether: ethyl acetate = 10/1-5/1) was performed to isolate compound N (1.9 g, yellow solid).

¹H NMR (400 MHz, DMSO-d ₆) δ 12.75 (s, 1H), 7.49 – 7.40 (m, 5H), 7.36 (td,4H), 7.32 – 7.24 (m, 4H), 6.93 (d, 2H), 6.14 (s, 1H), 5.58 – 5.47 (m, 4H), 4.22 (q,1H), 3.97 (d, 1H), 3.91 – 3.82 (m, 1H), 3.79 (q, 1H), 3.75 (s, 3H), 1.91 (s, 3H), 1.75 (dd, 2H), 1.12 (d, 18H)。

Compound N (1.9 g, 2.2 mmol) was dissolved in acetic acid solution (80 mL, V/V = 80%), stirred at room temperature overnight, after completion of the reaction, the reaction solution was concentrated, and the crude product was isolated by column chromatography (petroleum ether: ethyl acetate = 10/1-5/1) to give compound Q22 (949 mg, yellow solid).

¹The HNMR spectrum is shown in figure 2,¹H NMR (400 MHz, DMSO-d ₆) δ 12.74 (s, 1H), 7.60 (d,1H), 6.14 (t, 1H), 5.61 (dd, 4H), 5.48 (d, 1H), 4.32 – 4.13 (m, 3H), 3.96 (dtd, 1H), 2.26 – 2.13 (m, 2H), 1.98 (d, 3H), 1.16 (d, 18H)。

example 4

The synthetic route is as follows:

；

compound P (CAS: 50-89-5) (20.0 g, 82.6 mmol) and acetic anhydride (18.5 g, 181.2 mmol) were mixed in pyridine (150 mL) at room temperature and stirred at room temperature overnight. The reaction solution was concentrated to give a crude product, which was diluted with ethyl acetate (200 mL), washed twice with citric acid solution (1M), the aqueous phase was extracted twice with ethyl acetate, the organic phases were combined, the organic phase was washed with saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered to remove anhydrous sodium sulfate, and the organic phase was concentrated to give compound Q (26.5 g, white solid) which was directly subjected to the next reaction without further treatment.

¹H NMR (400 MHz, DMSO-d ₆) δ 11.40 (s, 1H), 7.51 (s, 1H), 6.18 (t, 1H), 5.19 (t, 1H), 4.25-4.24 (m, 2H), 4.16-4.14 (m, 1H), 2.45-2.42 (m, 1H), 2.30-2.26 (m, 1H), 2.07 (s, 6H), 1,80 (s, 3H)。

Compound Q (26.5 g, 81.2 mmol) was mixed in toluene (700 mL) at room temperature, the solution was clarified by stirring at 97 deg.C, Lawson's reagent (19.7 g, 48.7 mmol) was added, the mixture was stirred at 97 deg.C for 4h, the reaction was cooled to room temperature, the reaction was concentrated, and compound R (25.9 g, yellow solid) was isolated by column chromatography (petroleum ether: ethyl acetate = 10/1-1/1).

Compound R (25.9 g, 75.7 mmol) was dissolved in methanolic ammonia (300 mL), stirred at room temperature overnight, the reaction was concentrated, and column chromatography (dichloromethane: methanol = 50/1-10/1) was performed to isolate intermediate compound 2 (22.6 g, yellow solid).

¹H NMR (400 MHz, DMSO-d ₆) δ 12.72 (s, 1H), 7.90 (s, 1H), 6.11 (t, 1H), 5.27 (d, 1H), 5.11 (t, 1H), 4,27-4.25 (m, 1H), 3.81-3.79 (m, 1H), 3.66-3.54 (m, 2H), 2.14 (t, 2H), 1.97 (s, 3H)。

Intermediate compound 2 (3 g, 11.6 mmol), 4-dimethylaminopyridine (285 mg, 2.5 mmol) were dissolved in acetonitrile (100 mL), isobutyric anhydride (2.38 g, 15.1 mmol) and triethylamine (2.35 g, 23.2 mmol) were added at 0 ℃ and stirred at room temperature for 1 h. The reaction solution was stirred for 30min after adding methanol. The reaction solution was concentrated and subjected to column chromatography (dichloromethane: methanol = 50/1-20/1) to isolate the product Q12 (1.12 g, yellow solid).

¹The HNMR spectrum is shown in figure 6,¹H NMR (400 MHz, DMSO-d ₆) δ 12.76 (s, 1H), 7.56 (s, 1H), 6.12 (t, 1H), 5,44 (d, 1H), 4.27-4.22 (m, 3H), 3.98-3.95 (m, 1H), 2.60-2.51 (m, 1H), 2.26-2.19 (m, 2H), 1.99 (s, 3H), 1.08 (d, 6H)。

example 5

The synthetic route is as follows:

；

intermediate compound 2 (3 g, 11.6 mmol) and compound M (4 g, 12.6 mmol) were dissolved in THF (100 mL), 3-nitro-1, 2, 4-triazole (2.65 g, 23.2 mmol), diisopropylethylamine (6 g, 46.5 mmol) and bis (2-oxo-3-oxazolidinyl) hypophosphoryl chloride (6 g, 23.2 mmol) were added sequentially at 0 deg.C and stirred at room temperature for 3 h. The reaction solution was diluted with ethyl acetate (100 mL), washed with citric acid (1M), washed with saturated brine of the organic phase, dried over anhydrous sodium sulfate, filtered to remove anhydrous sodium sulfate, the solution was concentrated to give a crude product, which was isolated by column chromatography (petroleum ether: ethyl acetate = 10/1-5/1) to give the product Q11 (677 mg, yellow solid).

¹The HNMR spectrum is shown in figure 7,¹H NMR (400 MHz, DMSO-d ₆) δ 12.76 (s, 1H), 7.60 (s, 1H), 6.16-6.13 (t, 1H), 5.63-5.60 (m, 4H), 5.49 (s, 1H), 4.28-4.16 (m, 3H), 3.98-3.95 (m, 1H), 2.20-2.17 (m, 2H), 1.98 (s, 3H), 1.15 (s, 18H)。

example 6

The synthetic route is as follows:

；

intermediate Compound 2(4 g, 15.5 mmol), Compound I (7.32 g, 16 mmol) and MgCl₂(1.62 g, 17 mmol) was dissolved in acetonitrile (150 mL), stirred at room temperature at 50 ℃ for 30min, diisopropylethylamine (5g, 38 mmol) was added, and stirred at 50 ℃ overnight. The reaction solution was diluted with ethyl acetate (200 mL), washed twice with citric acid (1M), washed with saturated brine of the organic phase, dried over anhydrous sodium sulfate, filtered to remove anhydrous sodium sulfate, the filtrate was concentrated, and subjected to column chromatography (dichloromethane: methanol = 50/1-20/1) to isolate compound Q21(680 mg, yellow solid).

¹The HNMR spectrum is shown in figure 5,¹H NMR (400 MHz, DMSO-d ₆) δ 12.75 (s, 1H), 7.64 (s, 1H), 7.36 (t, 2H), 7.19 (q, 3H), 6.14-6.06 (m, 2H), 5.42 (d, 1H), 4.26-4.20 (m, 2H), 4.14-4.10 (m, 1H), 4.00-3.96 (m,8H), 3.92-3.82 (m,2H), 2.15-2.08 (m, 2H), 1.95 (s, 3H), 1.46-1.42 (m, 1H), 1.32-1.18 (m, 7H), 0.81 (t,6H)。

example 7

The synthetic route is as follows:

；

trimethyl phosphate (20.0 g, 0.14 mol), chloromethyl isopropyl carbonate (87.14 g, 0.57 mol) and sodium iodide (64.2 g, 0.43 mol) were mixed in acetonitrile (200 mL), N₂Stirring overnight at 90 ℃ under protection, after completion of the reaction, cooling to room temperature, filtering with celite to remove insoluble material, concentrating the filtrate, and isolating the crude product by column chromatography (petroleum ether: ethyl acetate = 10/1-5/1) to give compound W (46.8 g, colorless liquid, impure).

Compound W (20 g, 44.8 mmol) was dissolved in acetonitrile (180 mL), and lithium bromide (3.9 g, 44.8 mmol), N, was added₂Stirring was carried out overnight at 90 ℃ under protection, after completion of the reaction, cooling to room temperature, concentrating the reaction solution, and draining off by oil pump to give compound X (18.8 g, yellow oil, impure).

Intermediate compound 2 (2 g, 7.7 mmol) and compound X (3.64 g, 8.13 mmol) were dissolved in tetrahydrofuran (60 mL), 3-nitro-1H-1, 2, 4-triazole (1.8 g, 15.5 mmol), diisopropylethylamine (4 g, 31.0 mmol) and bis (2-oxo-3-oxazolidinyl) hypophosphoryl chloride (3.94 g, 15.48 mmol) were added sequentially at 0 deg.C, stirred at room temperature for 1H, diluted with ethyl acetate (100 mL) after completion of the reaction, washed sequentially with aqueous citric acid (1M), washed with water, washed with saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and the crude product was isolated by column chromatography (petroleum ether: ethyl acetate = 10/1-5/1) to give compound Q24 (1.12 g, yellow solid).

¹The HNMR spectrum is shown in figure 4,¹H NMR (400 MHz, DMSO-d6) δ 12.75 (s, 1H), 7.58 (d,1H), 6.15 (t, 1H), 5.61 (d,4H), 5.48 (d, 1H), 4.82 (heptd, 2H), 4.35-4.13 (m, 3H), 4.01-3.94 (m, 1H), 2.23-2.08 (m, 2H), 2.01-1.96 (m, 3H), 1.28-1.20 (m, 12H)。

example 8

In vitro anti-HBV Activity test

Telbivudine (purchased from shanghai tyitake technologies, inc.), hepg2.2.15 cells (provided by the antiviral drug research laboratory of the institute of medicine, university of redden), Fetal Bovine Serum (FBS) (ex sequo feishal biochemicals, inc.), DMEM medium (sequo feishal biochemicals, inc.), carbon dioxide incubator (sequo feishal biochemicals, inc.), fluorescent quantitative PCR (sequo feishal biochemicals, inc.).

The test drug prepared by the embodiment of the invention is used for in vitro anti-HBV virus efficacy evaluation test, and comprises the following steps:

and (3) detecting the antiviral activity of the medicine: collecting HepG2.2.15 cell 1 bottle with good growth, digesting with pancreatin to obtain single cell suspension, counting with cell counting plate, adjusting cell density to 2 × 10 with DMEM medium containing 10% FBS serum⁵one/mL, inoculated in culture plates (96 wells, 100uL per well). Placing in carbon dioxide incubator under 5% CO₂Incubate at 37 ℃ until 80% contact inhibition. And (4) sucking the supernatant, adding a test medicament culture solution, simultaneously adding a culture solution containing a positive control medicament telbivudine with a corresponding concentration, and additionally arranging a cell blank control hole. 3 replicate wells were set for each concentration gradient and cell blank. Placing in a carbon dioxide incubator, culturing at 37 ℃ for 3d/6d/9d, sucking supernatant, centrifuging, and detecting the HBV-DNA content in the supernatant by a fluorescent quantitative PCR method, wherein the result is shown in Table 1.

TABLE 1

It can be seen from table 1 that the inhibitory rates of compound Q15 and compound Q22 against HBV are equal to or better than that of the positive control drug telbivudine, so that the compounds Q15 and compound Q22 were subjected to complete in vitro anti-HBV experiments.

Example 9

Complete in vitro anti-HBV activity test

The test medicine prepared by the embodiment of the invention is used for in vitro cytotoxicity test and in vitro anti-HBV virus drug effect

And (4) evaluating and testing. The method comprises the following steps:

(a) cytotoxicity test, HepG2.2.15 cell 1 bottle (batch different from example 8) with good growth was taken, and after digestion with pancreatinPreparing single cell suspension, counting with cell counting plate, adjusting cell density to 2 × 10 with DMEM medium containing 10% FBS serum⁵one/mL, inoculated in culture plates (96 wells, 100uL per well). Placing in carbon dioxide incubator under 5% CO₂Incubate at 37 ℃ until 80% contact inhibition. Sucking the supernatant, adding culture solution containing different concentration gradient test drugs, simultaneously adding culture solution containing corresponding concentration gradient positive control drug telbivudine, and arranging cell blank control holes. 3 replicate wells were set for each concentration gradient and cell blank. The cells were placed in a carbon dioxide incubator and incubated at 37 ℃ for 72 h, 10 uL of MTT (0.5/L) was added to each well and incubation continued for 4h, followed by careful aspiration of the supernatant, addition of 150 uL DMSO per well, shaking to dissolve the formazan particles, final colourimetry with a microplate reader, zeroing with blank wells and measuring the A value at 546 nm. Calculating the cell survival rate and median toxic concentration TC according to the absorbance value₅₀。

(b) And (3) detecting the antiviral activity of the medicine: collecting HepG2.2.15 cell 1 bottle with good growth, digesting with pancreatin to obtain single cell suspension, counting with cell counting plate, adjusting cell density to 2 × 10 with DMEM medium containing 10% FBS serum⁵one/mL, inoculated in culture plates (96 wells, 100uL per well). Placing in carbon dioxide incubator under 5% CO₂Incubate at 37 ℃ until 80% contact inhibition. Sucking the supernatant, adding culture solution containing different concentration gradient test drugs, simultaneously adding culture solution containing corresponding concentration gradient positive control drug telbivudine, and arranging cell blank control holes. 3 replicate wells were set for each concentration gradient and cell blank. Placing in a carbon dioxide incubator, culturing at 37 ℃ for 3d/6d/9d, sucking supernatant, centrifuging, and detecting the HBV-DNA content in the supernatant by a fluorescent quantitative PCR method, wherein the result is shown in Table 2.

TABLE 2

Note: the underlined data may be detected as biased and are still listed in the table for data integrity.

From Table 2So that the inhibition rate and the treatment rate of the compound Q23 and the compound Q24 on HBV are greatly superior to those of positive control medicament telbivudine, although the IC of the compound Q22 is not calculated₅₀However, the inhibition rate of the compound on hepatitis B virus at all tested concentrations is far higher than that of telbivudine, and the anti-HBV strength of the compound is also better than that of the compound Q23 and the compound Q24 in terms of the inhibition rate on HBV at each concentration. Therefore, the compound Q22, the compound Q24 and the compound Q23 are expected to be novel anti-HBV medicines with great potential.

Claims

A4-thiodeoxythymidine derivative or a pharmaceutically acceptable salt thereof, characterized in that it has the structure shown in formula I or formula II:

；

wherein R is₁Is selected from H, -CH₃，-CH₂CH₃-C3-C10 hydrocarbyl, methoxy, methylthio, -C2-C10 hydrocarbyloxy, C2-C10 hydrocarbylthio, aminomethyl, hydroxymethyl, -CH₂O(CH₂)nO(CH₂)nCH₃(n=1-3)，

-CH₂NH(CH₂)nNH(CH₂)nCH₃(n =1-3), an aromatic ring of C5-C10, a heteroaromatic ring of C5-C10, an alicyclic ring of C3-C10, an aliphatic heterocyclic ring of C3-C10, -F, -Cl, -Br, -I, or cyano;

wherein R is₂Is selected from H, -CH₃，-CH₂CH₃-C3-C10 hydrocarbyl, methoxy, methylthio, -C2-C10 hydrocarbyloxy, C2-C10 hydrocarbylthio, aminomethyl, hydroxymethyl, -CH₂O(CH₂)nO(CH₂)nCH₃(n=1-3)，-CH₂NH(CH₂)nNH (CH₂)nCH₃(n =1-3), an aromatic ring of C5-C10, a heteroaromatic ring of C5-C10, an alicyclic ring of C3-C10, an aliphatic heterocyclic ring of C3-C10, -F, -Cl, -Br, -I, or cyano;

R₃is selected from
；

R₅Is selected from-CH₃，-CH₂CH₃-C3-C10 hydrocarbyl, methoxy, methylthio, -C2-C10 hydrocarbyloxy, C2-C10 hydrocarbylthio, aminomethyl, hydroxymethyl, -CH₂O(CH₂)nO(CH₂)nCH₃(n=1-3)，-CH₂NH(CH₂)nNH (CH₂)nCH₃(n =1-3), an aromatic ring of C5-C10, a heteroaromatic ring of C5-C10, an alicyclic ring of C3-C10, or an aliphatic heterocyclic ring of C3-C10;

R₆is composed of
Wherein R is₈，R₉Independently or simultaneously selected from H, -CH₃，-CH₂CH₃-C3-C10 hydrocarbyl, -CH₂CH₂O(CH₂)nO(CH₂)nCH₃(n=1-3)，-CH₂CH₂NH(CH₂)nNH (CH₂)nCH₃(n =1-3), an aromatic ring of C5-C10, a heteroaromatic ring of C5-C10, an alicyclic ring of C3-C10, or an alicyclic ring of C3-C10, R₁₀Is selected from-CH₃，-CH₂CH₃-a linear hydrocarbon group from C3 to C10, -a branched hydrocarbon group from C3 to C10, methoxy, methylthio, -a hydrocarbonoxy group from C2 to C10, a hydrocarbylthio group from C2 to C10, -CH₂O(CH₂)nO(CH₂)nCH₃(n=1-3)，-CH₂CH₂NH(CH₂)nNH (CH₂)nCH₃(n =1-3), an aromatic ring of C5-C10, a heteroaromatic ring of C5-C10, an alicyclic ring of C3-C10, or an aliphatic heterocyclic ring of C3-C10;

ar is selected from phenyl, substituted phenyl, C5-C10 aromatic ring, or C5-C10 aromatic heterocycle, R is₇Is composed of
Wherein R is₁₁，R₁₂Independently or simultaneously selected from H, -CH₃，-CH₂CH₃-C3-C10 hydrocarbyl, -CH₂CH₂O(CH₂)nO(CH₂)nCH₃(n=1-3)，-CH₂CH₂NH(CH₂)nNH (CH₂)nCH₃(n =1-3), an aromatic ring of C5-C10, a heteroaromatic ring of C5-C10, an alicyclic ring of C3-C10, or an alicyclic ring of C3-C10, R₁₃Is selected from-CH₃，-CH₂CH₃-a linear hydrocarbon group of C3-C10, -a branched hydrocarbon group of C3-C10, -CH₂O(CH₂)nO(CH₂)nCH₃(n=1-3)，-CH₂CH₂NH(CH₂)nNH (CH₂)nCH₃(n =1-3), an aromatic ring of C5-C10, a heteroaromatic ring of C5-C10, an alicyclic ring of C3-C10, or an aliphatic heterocyclic ring of C3-C10;

R₄is selected from H, -CH₃，-CH₂CH₃-C3-C10 hydrocarbyl, methoxy, methylthio, -C2-C10 hydrocarbyloxy, C2-C10 hydrocarbylthio, aminomethyl, hydroxymethyl, -CH₂O(CH₂)nO(CH₂)nCH₃(n=1-3)，-CH₂NH(CH₂)nNH (CH₂)nCH₃(n =1-3), an aromatic ring of C5-C10, a heteroaromatic ring of C5-C10, an alicyclic ring of C3-C10, an aliphatic heterocyclic ring of C3-C10, -Cl, -Br, -I, or cyano.
2. The 4-thioxo-deoxythymidine derivative according to claim 1 wherein said R is selected from the group consisting of₁Is selected from-CH₃，R₂Selected from H, R₄Is selected from H.
3. The 4-thioxo-deoxythymidine derivative according to claim 1 wherein said R is selected from the group consisting of₃Selected from:

。
4. the 4-thioxo-deoxythymidine derivative according to claim 1 or a pharmaceutically acceptable salt thereof wherein said derivative is any one of the following:

。
5. the 4-thioxo-deoxythymidine derivative according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof wherein said 4-thioxo-deoxythymidine derivative is used as a pharmaceutically acceptable salt comprising a salt of said derivative with a metal ion or a pharmaceutically acceptable amine or ammonium ion.
6. Use of a 4-thioxo-deoxythymidine derivative according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of hepatitis or liver cancer.
7. The use according to claim 6, wherein the hepatitis is hepatitis B caused by HBV infection.
8. A pharmaceutical composition comprising an effective amount of the 4-thioxdeoxythymidine derivative of claim 1 or a pharmaceutically acceptable salt, stereoisomer, active metabolite, prodrug, solvate or crystalline form thereof, and a pharmaceutically acceptable excipient.
9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is prepared into a pharmaceutical dosage form comprising capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories, or patches.
10. Use of the pharmaceutical composition of claim 8 in the preparation of a medicament for the treatment of hepatitis b or liver cancer.