2 '-fluoro-4'-alkynyl-8-substituted purine nucleoside derivative as well as preparation method and application thereof

Abstract

The invention discloses a 2 '-fluoro-4'-alkynyl-8-substituted purine nucleoside derivative and a preparation method and application thereof, the structure of the 2 '-fluoro-4'-alkynyl-8-substituted purine nucleoside derivative is shown as a general formula (vi), in the formula, X is selected from-H, C1-10 alkyl, halogen,-CN,-N3,-OH or-NH2; y is selected from halogen or C1-C4 alkyl; z is selected from H, halogen or amino. Compared with the prior art, the invention provides the 2 '-fluoro-4'-alkynyl-8-substituted purine nucleoside derivative with a novel structure, and compared with similar compounds disclosed in the prior art, the 2 '-fluoro-4'-alkynyl-8-substituted purine nucleoside derivative has remarkably better anti-HIV activity. Experimental results show that the compound disclosed by the invention can effectively inhibit the HIV virus and has a good application prospect in the aspect of treating HIV virus infection. (vi).

Description

Technical Field

[0001] This invention belongs to the field of antiviral drug technology, specifically relating to 2′-fluoro-4′-ynyl-8-substituted purine nucleoside derivatives, their preparation methods, and applications. Background Technology

[0002] The first case of Acquired Immunodeficiency Syndrome (AIDS) was reported in 1981. By 2020, there were 38 million people living with HIV globally, of whom 26 million were receiving antiretroviral therapy. In 2019, 1.7 million new infections were reported, and 690,000 people died from AIDS and related diseases. As of June 2020, the cumulative number of deaths from AIDS worldwide had reached 32.69 million. Cocktail therapy is currently the mainstream treatment for AIDS, and nucleoside reverse transcriptase inhibitors (RTIs) are essential components. Currently approved RTIs include: azvudine, zidovudine, zalcitabine, didanoxin, stavudine, abacavir, lamivudine, enxitabine, and tenofovir. Tenofovir is an acyclic analog of adenosine monophosphate (ATP) that requires only two intracellular phosphorylations for activation. However, due to poor bioavailability, tenofovir is now available as tenofovir ester fumarate (TDF).

[0003] In addition to the aforementioned low bioavailability, these drugs require long-term use, leading to problems such as toxicity accumulation and frequent drug resistance, which impose a significant economic burden on individuals and the healthcare system. Summary of the Invention

[0004] Purpose of the invention: In order to solve the problems existing in the prior art, the present invention provides a 2′-fluoro-4′-alkynyl-8-substituted purine nucleoside derivative, its preparation method and application.

[0005] Technical Solution: To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: In a first aspect, the present invention provides a 2′-fluoro-4′-ynyl-8-substituted purine nucleoside derivative of general formula (vi) or a pharmaceutically acceptable salt thereof: ; (vi) Wherein, X is selected from -H, C1-10 alkyl, halogen, -CN, -N3, -OH or -NH2; Y is selected from halogen or C1-C4 alkyl; Z is selected from H, halogen or amino.

[0006] As a preferred embodiment, X is selected from -H, C1-10 straight-chain alkyl, halogen, -CN, -N3, -OH or -NH2; Y is selected from halogen or methyl; Z is selected from H, halogen or amino.

[0007] More preferably, X is selected from -NH2, halogen, or -OH; Y is selected from halogen; and Z is selected from H or halogen.

[0008] More preferably, the 2′-fluoro-4′-ynyl-8-substituted purine nucleoside derivative is selected from the following compounds: .

[0009] Secondly, the present invention provides a method for preparing the aforementioned 2′-fluoro-4′-ynyl-8-substituted purine nucleoside derivative, comprising the following steps: ; (1) Under the action of DBU, compound i and nucleoside base Base undergo a glycosylation reaction to obtain compound ii; the structural formula of the nucleoside base Base is shown below: Where X, Y, and Z are as described above; (2) Compound ii was deacetylated to give compound iii; (3) Compound iii was treated with DAST to obtain fluorinated compound iv; (4) Compound iv undergoes triethylsilane removal to yield compound v; (5) Compound v is debenzyl protecting group to obtain 2′-fluoro-4′-ynyl-8-substituted purine nucleoside derivative as shown in formula vi.

[0010] Wherein, Base represents the nucleoside base, which is derived from commercial or laboratory sources. After sugar i undergoes a glycosylation reaction with the corresponding base, the α and β configurations are separated by column chromatography to obtain a single-configuration nucleoside derivative ii.

[0011] Thirdly, the present invention provides a pharmaceutical composition comprising the 2′-fluoro-4′-ynyl-8-substituted purine nucleoside derivative thereof or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.

[0012] As a specific implementation, the pharmaceutical composition contains a 2′-fluoro-4′-alkynyl-8-substituted purine nucleoside derivative or a pharmaceutically acceptable salt thereof as the sole active ingredient; or a 2′-fluoro-4′-alkynyl-8-substituted purine nucleoside derivative or a pharmaceutically acceptable salt thereof as one of the active ingredients.

[0013] Fourthly, the present invention provides the use of the 2′-fluoro-4′-ynyl-8-substituted purine nucleoside derivative thereof or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof, in the preparation of an antiviral drug.

[0014] Preferably, the antiviral drug is an anti-HIV drug.

[0015] Beneficial Effects: Compared with existing technologies, this invention provides a 2′-fluoro-4′-ynyl-8-substituted purine nucleoside derivative with a novel structure, which exhibits significantly superior anti-HIV activity compared with similar compounds disclosed in the prior art. Experimental results show that the compound of this invention can effectively inhibit HIV virus and has good application prospects in the treatment of HIV infection. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Main instruments and chemical reagents Nuclear magnetic resonance spectrometer: Bruker Ascend TM 400; High-resolution mass spectrometer: Bruker MicroTOF-Q II mass spectrometer; Infrared spectrometer: Zhiwei Smart Fourier Transform Infrared Spectrometer (Tianjin Gangdong Technology Co., Ltd.); Three-in-one ultraviolet analyzer: ZF-6 (Shanghai Gaopeng Technology Co., Ltd.).

[0018] The raw materials and solvents used in the implementation of this invention are all purchased commercially and prepared using conventional methods.

[0019] Example 1: Synthesis of nucleoside derivatives of 2′-fluoro-4′-ynyl-8-fluoro-substituted purines ; The specific synthetic steps for the nucleoside derivatives of 2′-fluoro-4′-ynyl-8-fluoro-substituted purines are as follows: Preparation of compound 3: Compound 2 (290 mg) was dissolved in 1.5 mL of dried MeCN, and BSA (488 µL) was added. Compound 1 (1.02 g) was dissolved in 2 mL of dried MeCN and added to the reaction mixture. Then, TMSOTf (0.32 mL) was added, and the mixture was stirred at room temperature for 15 min and then refluxed for 1 h. After the reaction was completed, the mixture was diluted with EtOAc (30 mL), washed with saturated sodium bicarbonate solution (30 mL × 2), washed with saturated brine (30 mL × 2), dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (EtOAc / PE 1:10) to obtain compound 3 (135 mg, 18% pale yellow oil). 1H NMR (400MHz, CDCl3) δ 8.60 (s, 1H), 7.41-7.33 (m, 10H), 7.25 (d, J = 2.4 Hz, 1H), 6.75-6.74 (m, 1H), 5.47 (t, J = 6.0 Hz, 1H), 4.82 (d, J = 11.8 Hz, 1H), 4.61–4.53 (m,4H), 3.88-3.72 (m, 2H), 2.02 (s, 3H), 1.65 (s, 1H), 1.28-1.28 (m, 3H), 0.11(t, J = 8.0 Hz, 9H), 0.62 (q, J = 8.0 Hz, 6H); MS (ESI) m / z [M + Na] + Theoretical value C 35 H 40 ClFN3NaO5Si + 686, actual value 686. Preparation of compound 4: Compound 3 (100 mg) was dissolved in MeOH (5 mL), Et3N (1 mL) was added, and the mixture was refluxed for 2 h. After the reaction was completed, the reaction solution was evaporated to dryness, diluted with EtOAc (20 mL), washed with saturated saline (30 mL), dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (EtOAc / PE 1:5) to obtain compound 4 (pale yellow oil, 78 mg, 85%). 1 H NMR (400 MHz, CDCl3) δ 8.58 (s, 1H), 7.39-7.29 (m, 11H), 7.19 (d, J = 2.4 Hz, 1H), 6.48-6.45 (m, 1H), 5.00(d, J = 11.2 Hz, 1H), 4.66-4.30(m, 5H),3.75 (dd, J = 35.6, 10.4 Hz, 2H), 3.12 (d, J = 9.2 Hz, 1H), 1.97 (s, 3H), 1.62(s, 1H), 1.24 (d, J = 2.4 Hz, 3H), 0.96 (t, J= 8.0 Hz, 9H), 0.58 (q, J = 8.0 Hz, 6H); MS (ESI) m / z [M + Na] + Theoretical value C 33 H 37 ClFN3NaO4Si + 644, actual value 644. Preparation of compound 5: Compound 4 (100 mg) was dissolved in CH2Cl2 (5 mL), and pyridine (0.13 mL) and DAST (0.1 mL) were added sequentially. The mixture was stirred at room temperature. After the reaction was completed, the mixture was quenched with saturated sodium bicarbonate (15 mL), extracted with EtOAc (30 mL), and separated by silica gel column chromatography (EtOAc / PE 1:10) to obtain compound 5 (pale yellow oil, 31 mg, 21%). 1 H NMR (400 MHz, CDCl3) δ 8.60 (s, 1H), 7.36-7.30 (m, 11H), 6.96-6.90(m, 1H), 5.38 (dt, J = 53.6, 5.2 Hz, 1H), 4.75-4.52 (m, 5H), 3.80-3.70 (m, 2H), 1.57 (s, 4H), 1.00 (t, J = 8.0 Hz, 9H), 0.62 (q, J = 8.0 Hz, 6H). Preparation of compound 6: Compound 5 (100 mg) was dissolved in 1,4-dioxane (4 mL), transferred to a sealed tube, and concentrated ammonia (4 mL) was added. The mixture was stirred at 120 °C for 6 h. The reaction was monitored, and after the reaction was complete, the reaction solution was evaporated to dryness. The solution was dissolved in EtOAc (10 mL), washed with saturated saline (20 mL), dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (EtOAc / PE1:2) to obtain compound 6 (yellow oil, 45 mg, 62%). 1 H NMR (400 MHz, CDCl3) δ 8.21 (s,1H), 7.33–7.22 (m, 11H), 6.87-6.82 (m, 1H), 5.35 (dt, J = 53.6, 5.2 Hz, 1H),4.76–4.50 (m, 5H), 3.76-3.66 (m, 2H), 2.68 (s, 1H);MS (ESI) m / z [M + H] + Theoretical value C 27 H 25 F2N4O3 + 491, actual value 491. Preparation of compound 7: At -78°C, BCl3 (1 mL) was added dropwise to an anhydrous CH2Cl2 (5 mL) solution of compound 6 (100 mg), and the mixture was stirred for 10 min before being slowly brought to room temperature. The reaction was monitored, and after completion, the mixture was quenched with methanol, repeatedly evaporated to dryness with ammonia and methanol, stirred with silica gel, and purified by EtOAc to obtain compound 7 (white solid, 40 mg, 64%). 1 H NMR (400MHz, CD3OD) δ 8.12 (s, 1H), 7.21 (s, 1H), 6.77-6.73 (m, 1H), 5.30 (dt, J = 54.4, 6.0 Hz, 1H), 4.67 (dd, J = 22.8, 6.0 Hz, 1H), 3.89-3.77 (m, 2H), 3.21 (s, 1H); 19 F NMR (376 MHz, CD3OD) δ -170, -203; MS (ESI) m / z [M+ H] + Theoretical value C 13 H 13 F2N4O3 + 311, actual value 311.

[0020] Using the same raw materials and following the same method as in the examples above, other specific compounds 8-17 in Table 1 were prepared. Their specific mass spectrometric molecular weights are shown in Table 1. Table 1: Mass spectrometry data of compounds 8-17

[0021] Example 2: Study on the anti-HIV activity of the compound synthesized in Example 1 of this invention 2.1 In vitro anti-HIV activity of the compound 2.1.1 Research Methods 1. Preliminary screening of compounds to inhibit HIV-1 activity Five x 10⁴ Hut / CCR5 cells were seeded in 96-well plates. 50 μL of the compound was added to each cell, with final concentrations of 1 μM and 10 μM. 50 μL of HIV-luc / JRFL was then added. Lamivudine (3TC) was used as a compound control. Incubate at 37°C for 4 hours, wash away free virus, resuspend in culture medium, and then add the appropriate concentration of compound. Cells were cultured at 37°C for 3 days, collected for analysis, lysed, and the supernatant was collected to detect luciferase activity. Viral replication can be calculated, and the inhibition rate can also be further calculated.

[0022] 2. Dose-dependent anti-HIV assay Five x 10⁴ Hut / CCR5 cells were seeded in 96-well plates, and 50 μL of compounds with good initial screening effects were added. The final concentrations of the compounds were 1 μM, 0.2 μM, 0.04 μM, 0.008 μM, and 0.0016 μM. 50 μL of HIV-luc / JRFL virus was then added. Lamivudine (3TC) was used as a compound control. Incubate at 37°C for 4 hours to wash away free viruses; resuspend in culture medium, and then add the appropriate concentration of compound. Cells were cultured at 37°C for 3 days, and the supernatant was collected after cell lysis to detect luciferase activity. Calculate viral replication, and further determine the half-maximal inhibitory concentration (EC50). 50 ).

[0023] 3. MTT colorimetric assay for cytotoxicity 5 x 10⁴ Hut / CCR5 cells were seeded in 96-well cell culture plates. 100 μL (medium: RPMI-1640 / 10% FBS / 1% Pen / Strep); Add 100 μL of the compound solution; the final concentrations of the compound are 250 μM, 50 μM, 10 μM, 2 μM, and 0.4 μM; each concentration is repeated in triplicate; a control without the compound is also included. After culturing cells for 72 hours, discard 100 μL of supernatant, add 20 μL of MTT (5 mg / mL), and incubate at 37°C for 4 hours. Centrifuge, discard 100 μL of supernatant, add 100 μL of DMSO, and shake on a shaker for 15 minutes at room temperature in the dark until the blue formazen dissolves. The ELISA reader was used to measure OD595 and OD630 as reference wavelengths to calculate the half-maximal toxicity concentration (CC). 50 ).

[0024] 2.1.2 Experimental Results Compounds 18-21 were used as control compounds, and their activity was determined using the same method. The experimental results are shown in Table 2. All 2′-fluoro-4′-alkynyl-8-substituted purine nucleoside derivatives exhibited good in vitro anti-HIV activity, low toxicity, and high selectivity. Among them, the EC50 values ​​of 2′-fluoro-4′-alkynyl-8-fluoroadenine, 2′-fluoro-4′-alkynyl-8-chloroadenine, 2′-fluoro-4′-alkynyl-8-bromoadenine, and 2′-fluoro-4′-alkynyl-2-chloro-8-fluoroadenine were... 50 The values ​​were 24.82 nM, 19.21 nM, 43.89 nM, and 30.80 nM, respectively. Compound 7 showed better activity than compound 20, and compound 10 showed better activity than compound 21, indicating that 8-fluoropurine-substituted nucleosides were more active than 7-fluoropurine-substituted nucleosides. Compounds 7, 8, and 9 showed better activity than compound 19, indicating that 8-chloro-substituted purine nucleosides were more active than unsubstituted nucleosides at the 8-position. Compound 17 showed slightly better in vitro activity than compound 18, and its toxicity was lower than that of compound 18. The selectivity index of compound 17 was also significantly higher than that of compound 18.

[0025] Table 2 Results of compound activity detection ; ; ; ; .

[0026] The embodiments of the present invention have been described in detail above with reference to specific examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A 2′-fluoro-4′-ynyl-8-substituted purine nucleoside derivative of general formula (vi) or a pharmaceutically acceptable salt thereof: ； (vi) in, X is selected from -H, C1-10 alkyl, halogen, -CN, -N3, -OH or -NH2; Y is selected from halogen or C1-C4 alkyl; Z is selected from H, halogen or amino.

2. The 2′-fluoro-4′-ynyl-8-substituted purine nucleoside derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, X is selected from -H, C1-10 straight-chain alkyl, halogen, -CN, -N3, -OH or -NH2; Y is selected from halogen or methyl; Z is selected from H, halogen or amino.

3. The 2′-fluoro-4′-ynyl-8-substituted purine nucleoside derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, X is selected from -NH2, halogen, or -OH; Y is selected from halogen; Z is selected from H or halogen.

4. The 2′-fluoro-4′-ynyl-8-substituted purine nucleoside derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The 2′-fluoro-4′-ynyl-8-substituted purine nucleoside derivatives are selected from the following compounds: 。 5. The method for preparing the 2′-fluoro-4′-ynyl-8-substituted purine nucleoside derivative according to any one of claims 1-4, characterized in that, Includes the following steps: ； (1) Under the action of DBU, compound i and nucleoside base Base undergo a glycosylation reaction to obtain compound ii; the structural formula of the nucleoside base Base is shown below: Wherein, X, Y and Z are as described in any one of claims 1-4; (2) Compound ii was deacetylated to give compound iii; (3) Compound iii was treated with DAST to obtain fluorinated compound iv; (4) Compound iv undergoes triethylsilane removal to yield compound v; (5) Compound v is debenzyl protecting group to obtain 2′-fluoro-4′-ynyl-8-substituted purine nucleoside derivative as shown in formula vi.

6. A pharmaceutical composition comprising a 2′-fluoro-4′-ynyl-8-substituted purine nucleoside derivative or a pharmaceutically acceptable salt thereof as described in any one of claims 1-4, and a pharmaceutically acceptable excipient.

7. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition may contain a 2′-fluoro-4′-alkynyl-8-substituted purine nucleoside derivative or a pharmaceutically acceptable salt thereof as the sole active ingredient; or a 2′-fluoro-4′-alkynyl-8-substituted purine nucleoside derivative or a pharmaceutically acceptable salt thereof as one of the active ingredients.

8. The use of the 2′-fluoro-4′-ynyl-8-substituted purine nucleoside derivative or a pharmaceutically acceptable salt thereof as described in any one of claims 1-4, or the use of the pharmaceutical composition as described in claim 6 or 7 in the preparation of an antiviral drug.

9. The application according to claim 7, characterized in that, The antiviral drug mentioned is an anti-HIV drug.

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