WO2022078224A1

WO2022078224A1 - Polycyclic benzodifuran compound and use thereof as anti-rsv drug

Info

Publication number: WO2022078224A1
Application number: PCT/CN2021/121948
Authority: WO
Inventors: 叶文才; 王英; 胡利军; 邓路铭; 唐维; 李药兰
Original assignee: 暨南大学
Priority date: 2020-10-14
Filing date: 2021-09-29
Publication date: 2022-04-21
Also published as: CN112125920B; CN112125920A

Abstract

Disclosed are a polycyclic benzodifuran compound of formula (I), and a preparation method therefor and the use thereof as an anti-respiratory syncytial virus (RSV) drug. The polycyclic benzodifuran compound disclosed by the present invention has a relatively strong inhibiting effect on the respiratory syncytial virus, and the activity of the polycyclic benzodifuran compound is superior to that of the existing commercially available drug ribavirin. Therefore, such a compound has good application prospects in treating related diseases caused by respiratory syncytial virus infections.

Description

A class of polycyclic benzobisfuran compounds and their application as anti-RSV drugs

technical field

The present invention relates to a kind of polycyclic benzobisfuran compound, its preparation method and its application in antiviral medicine. More specifically, the present invention relates to the application of a class of polycyclic benzobisfuran compounds as anti-respiratory syncytial virus (RSV) drugs.

Background technique

Respiratory syncytial virus (RSV) is a single-stranded negative-stranded RNA enveloped virus belonging to the family Paramyxoviridae and the genus Pneumovirus. It can be divided into two subtypes, A and B, according to the differences in the antigenic characteristics of the virus surface. RSV is respiratory tractable and is one of the most common viral pathogens causing lower respiratory tract disease in infants, the elderly, and immunocompromised adults. Especially in infants and young children, it can cause severe lower respiratory tract diseases such as bronchitis and pneumonia.

To date, there is no commercially available vaccine for the prevention of RSV infection. So far, the antiviral drugs against the virus on the market are mainly Ribavirin (Ribavirin) and Palivizumab (Palivizumab). Among them, ribavirin can be used for the treatment of severe RSV infection, but there are side effects such as teratogenicity, and the curative effect is general. Palivizumab is a monoclonal antibody that can be used to prevent RSV infection in high-risk children with chronic lung disease and congenital heart disease, as well as premature infants, but it cannot be used for the treatment of RSV-infected patients and is expensive. Due to the unsatisfactory application effect of current clinically used anti-RSV drugs, there is an urgent need to develop new anti-RSV drugs to prevent or treat RSV infection.

SUMMARY OF THE INVENTION

In order to overcome the above-mentioned deficiencies of the prior art, the present invention provides a class of polycyclic benzobisfuran compounds with anti-RSV activity, a preparation method thereof, and use in anti-RSV medicines. The results of the activity study show that the polycyclic benzobisfuran compounds of the present invention have significant inhibitory activity on various subtypes of RSV virus (A2 strain, Long strain and B strain), and can not only inhibit the infection of high titer RSV. , and can also significantly reduce the transcription and expression levels of viral genes. In addition, the series of polycyclic benzobisfuran compounds have novel skeletons and have different chemical structure types from the existing anti-RSV drug ribavirin, and are expected to be developed into a new class of anti-RSV virus drugs.

In order to achieve the above object, the present invention is achieved through the following schemes:

Described polycyclic benzobisfuran compound has the structural formula shown in general formula I:

in:

R ¹ and R ³ are each independently selected from substituted or unsubstituted C ₁ -C ₆ alkyl;

R ² is independently selected from substituted or unsubstituted C ₁ -C ₆ alkyl, substituted or unsubstituted C ₃ -C ₆ cycloalkyl.

In a preferred embodiment of the present invention, the compound represented by the general formula I includes, but is not limited to:

The present invention relates to a pharmaceutical composition, which contains a therapeutically effective dose of a polycyclic benzobisfuran compound represented by the general formula I, or a pharmaceutically acceptable salt or a stereoisomer or a prodrug molecule thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

The present invention relates to a polycyclic benzobisfuran compound represented by the general formula I containing a therapeutically effective dose, or a pharmaceutically acceptable salt or a stereoisomer or a prodrug molecule thereof, or a pharmaceutical composition thereof. Use in anti-RSV drugs.

The present invention relates to a polycyclic benzobisfuran compound represented by the general formula I containing a therapeutically effective dose, or a pharmaceutically acceptable salt or a stereoisomer or a prodrug molecule thereof, or a pharmaceutical composition thereof. Use in medicines for treating diseases such as bronchitis and pneumonia caused by RSV virus infection.

Unless stated to the contrary, terms used in the specification and claims have the following meanings.

The term "alkyl" as used herein is meant to include branched and straight chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, the definition of " _C1 - _C6 " in " _C1 - _C6 alkyl" includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a straight or branched chain. For example, "C ₁ -C ₆ alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl. The term "cycloalkyl" refers to a monocyclic saturated aliphatic hydrocarbon group having the specified number of carbon atoms. For example, "cycloalkyl" includes cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, and the like. The term "alkoxy" refers to groups having an -O-alkyl structure, such as -OCH ₃ , -OCH ₂ CH ₃ , -OCH ₂ CH ₂ CH ₃ , -OCH ₂ CH(CH ₃ ) ₂ , -OCH ₂ _CH2CH2CH3 , -OCH ₍ _CH3 ₎ ₂ , etc. The term "aryl" includes, but is not limited to, imidazolyl, triazolyl, pyrazolyl, furyl, thienyl, oxazolyl, isoxazolyl, pyrazinyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl.

"Substituted" means that one or more hydrogen atoms in a group, preferably up to 5, more preferably 1 to 3 hydrogen atoms, independently of one another, are substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and the person skilled in the art can determine (either experimentally or theoretically) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups with free hydrogens may be unstable when combined with carbon atoms with unsaturated (eg, olefinic) bonds.

"Prodrug" means a prodrug that converts in vivo to the structure of the compounds and pharmaceutically acceptable salts thereof.

"Pharmaceutical composition" means a mixture containing one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, and other components such as a physiological/pharmaceutically acceptable carrier and excipients. The purpose of the pharmaceutical composition is to facilitate the administration to the organism, facilitate the absorption of the active ingredient and then exert the biological activity.

"Pharmaceutically acceptable salts" refer to salts of the compounds of the present invention, which are safe and effective when used in mammals, and possess the desired biological activity.

The synthetic method of the compound of the present invention

In order to accomplish the purpose of the present invention, the present invention adopts following technical scheme:

The preparation method of the compound shown in the general formula I of the present invention or its pharmaceutically acceptable salt or its stereoisomer or its prodrug molecule, comprises the following steps:

The compound of general formula Ia can generate the compound of general formula Ib through Friedel-Crafts reaction under the condition of Lewis acid. The latter undergoes a reduction reaction to obtain a compound of the general formula Ic, and further undergoes an oxidation reaction to generate a compound of the general formula Id. Compounds of general formula Ie are reacted with compounds of general formula Id under acidic or basic conditions to obtain compounds of general formula If. The compound of general formula If is further reacted with the compound of general formula Ig under acidic or basic conditions to obtain the polycyclic benzobisfuran compound of general formula I. wherein: R ¹ , R ² and R ³ are as defined in claim 1 .

Description of drawings

Figure 1 Inhibitory effect of compound 2 on RSV with different virus titers

Figure 2 Inactivation of RSV by compound 2

Figure 3 The effect of compound 2 on different time points of RSV replication cycle

Figure 4 Inhibitory effect of compound 2 on RSV protein expression

Detailed ways

The structures of the compounds were determined by nuclear magnetic resonance (NMR) or/and mass spectrometry (MS). NMR shifts ([delta]) are given in units of 10<" ⁶ > (ppm). The measurement of NMR was performed with BrukerAVANCE-400 and BrukerAVANCE-500 nuclear magnetometers, the measurement solvent was deuterated chloroform (CDCl ₃ ), and the internal standard was tetramethylsilane (TMS).

MS was measured with a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.

The known starting materials of the present invention can be synthesized using or according to methods known in the art, or can be purchased from Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Bailingwei, Annagy, Da Rui Chemical and other companies.

There is no special description in the examples, and the reactions are all carried out in an argon atmosphere or a nitrogen atmosphere.

Argon or nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon with a volume of about 1 L.

There is no special description in the examples, and the solution refers to an aqueous solution.

There is no special description in the examples, and the reaction temperature is room temperature, which is 20°C to 30°C.

The monitoring of the reaction progress in the embodiment adopts thin layer chromatography (TLC), the developing solvent used in the reaction, the eluent system of the column chromatography used for separating and purifying the compound and the developing solvent system of the thin layer chromatography method include: A : dichloromethane/methanol system, B: n-hexane/ethyl acetate system, C: petroleum ether/ethyl acetate system, D: acetone, E: dichloromethane/acetone system, F: ethyl acetate/dichloromethane System, G: ethyl acetate/dichloromethane/n-hexane, H: ethyl acetate/dichloromethane/acetone, the volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of triethylamine and Adjust with alkaline or acidic reagents such as acetic acid.

The specific compounds prepared in the following examples include, but are not limited to, those in 1 to 4 above.

Example 1: Preparation of Compounds 7-12

first step:

At room temperature, phloroglucinol 5 was dissolved in a nitromethane solution, anhydrous aluminum trichloride and isovaleryl chloride were added successively, and the temperature was raised to 40°C. After reacting for 5 hours, the reaction solution was slowly poured into ice water, and a saturated potassium sodium tartrate solution was added, followed by vigorous stirring. The reaction solution was extracted three times with ethyl acetate. The organic phases were combined and washed with saturated NaCl solution. After drying and filtration over anhydrous sodium sulfate, the organic phase was evaporated to dryness under reduced pressure. The obtained crude product was separated and purified by silica gel column chromatography to obtain compound 7 (yield 90%)

Step 2:

Compound 7 was dissolved in MeOH solution at room temperature and NaOMe in MeOH was added. After reacting at room temperature for 10 min, methyl iodide was added to the reaction system, the temperature was raised to 55°C, the reaction was performed for 2 hours, the temperature was lowered to 0°C, and acidified with 1N HCl. The reaction solution was extracted three times with ethyl acetate, and the organic phases were combined and washed with saturated NaCl solution. After drying and filtration over anhydrous sodium sulfate, the organic phase was evaporated to dryness under reduced pressure. The obtained crude product was separated and purified by silica gel column chromatography to obtain compound 8 as a yellow oil (yield 86%).

third step:

Compound 8 was dissolved in tetrahydrofuran solution, and after cooling to -78°C, diisopropylaluminum hydride was added dropwise. After 1 hour of reaction, 1N HCl was added to quench the reaction. After warming to room temperature and stirring for 1 hour, the reaction solution was extracted three times with ethyl acetate. The organic phases were combined and washed with saturated NaCl solution. After drying and filtration over anhydrous sodium sulfate, the organic phase was evaporated to dryness under reduced pressure. The obtained crude product was separated and purified by silica gel column chromatography to obtain compound 9 (yield 90%).

the fourth step:

Compound 9 was dissolved in toluene at room temperature, bubbled with an oxygen balloon for half an hour, and then irradiated with a blue LED lamp (455 nm) for 48 hours. The organic phase was evaporated to dryness under reduced pressure. The obtained crude product was separated and purified by silica gel column chromatography to obtain compound 10 (80% yield).

the fifth step:

Compounds 7 and 10 were dissolved in toluene at room temperature, then trifluoroacetic acid and molecular sieves were added, and the temperature was raised to 110°C. After 24 hours of reaction, the temperature was lowered to room temperature, and saturated aqueous sodium bicarbonate solution was added to quench the reaction. After filtration through celite, the reaction solution was extracted three times with ethyl acetate. The organic phases were combined and washed with saturated NaCl solution. After drying and filtration over anhydrous sodium sulfate, the organic phase was evaporated to dryness under reduced pressure. The obtained crude products were separated and purified by silica gel column chromatography to obtain compounds 11 and 12 (75% yield).

Compound 11: ¹ H NMR (400 MHz, CDCl ₃ ) δ 13.27 (s, OH), 9.80 (s, OH), 6.07 (s, 1H), 4.49 (s, 1H), 2.95 (dd, J=14.7, 6.6Hz, 1H), 2.75(dd, J=14.7, 6.6Hz, 1H), 2.40(m, 1H), 2.16(m, 1H), 1.51(s, 3H), 1.41(s, 3H), 1.40( s,3H),1.32(s,3H),1.10(d,J＝6.8Hz,3H),1.08(d,J＝6.8Hz,3H),0.99(d,J＝6.6Hz,3H),0.97( d, J=6.6 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 211.3, 203.8, 198.4, 179.8, 166.7, 159.9, 159.7, 129.5, 113.2, 104.3, 101.7, 99.6, 55.2, 51.6, 45.7, 45.0 , 35.4, 26.1, 25.9, 24.5, 24.1, 23.1, 22.9, 22.8, 15.8, 15.7; HR-ESI-MS m/z 479.2046[M+Na] ⁺ .

Compound 12: ¹ H NMR (400 MHz, CDCl ₃ ) δ 14.19 (b, OH), 10.19 (s, OH), 6.07 (s, 1H), 4.47 (s, 1H), 3.08 (dd, J=15.6, 6.4Hz, 1H), 2.98(dd, J=15.6, 6.4Hz, 1H), 2.39(m, 1H), 2.25(m, 1H), 1.50(s, 3H), 1.42(s, 3H), 1.42( s,3H),1.36(s,3H),1.06(d,J＝7.2Hz,3H),1.04(d,J＝7.2Hz,3H),0.99(d,J＝6.2Hz,3H),0.98( d, J=6.2 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 211.3, 206.5, 198.8, 180.0, 168.6, 163.2, 156.7, 129.5, 113.2, 107.2, 103.9, 91.9, 55.1, 53.1, 45.8, 45.1 , 35.3, 26.1, 25.2, 24.5, 24.4, 23.4, 23.1, 22.9, 15.7, 15.7; HR-ESI-MS m/z 479.2036[M+Na] ⁺ .

Example 2: Preparation of Compounds 15 and 16

first step:

At room temperature, phloroglucinol 5 was dissolved in nitromethane solution, anhydrous aluminum trichloride and p-fluorophenylacetyl chloride 13 were added successively, and the temperature was raised to 40°C. After reacting for 5 hours, the reaction solution was slowly poured into ice water, and a saturated potassium sodium tartrate solution was added, followed by vigorous stirring. The reaction solution was extracted three times with ethyl acetate. The organic phases were combined and washed with saturated NaCl solution. After drying and filtration over anhydrous sodium sulfate, the organic phase was evaporated to dryness under reduced pressure. The obtained crude product was separated and purified by silica gel column chromatography to obtain compound 14 (80% yield).

Step 2:

Compounds 10 and 14 were dissolved in toluene at room temperature, then trifluoroacetic acid and molecular sieves were added, and the temperature was raised to 110°C. After 24 hours of reaction, the temperature was lowered to room temperature, and saturated aqueous sodium bicarbonate solution was added to quench the reaction. After filtration through celite, the reaction solution was extracted three times with ethyl acetate. The organic phases were combined and washed with saturated NaCl solution. After drying and filtration over anhydrous sodium sulfate, the organic phase was evaporated to dryness under reduced pressure. The obtained crude products were separated and purified by silica gel column chromatography to obtain compounds 15 and 16 (70% yield).

Compound 15: ¹ H NMR (400 MHz, CDCl ₃ ) δ 12.97 (s, OH), 9.88 (s, OH), 7.23 (m, 2H), 7.01 (m, 2H), 6.11 (s, 1H), 4.50 (s,1H),4.40(d,J=15.8Hz,1H),4.18(d,J=15.8Hz,1H),2.44(m,1H),1.52(s,3H),1.42(s,3H) , 1.41(s, 3H), 1.33(s, 3H), 1.10(d, J=6.8Hz, 3H), 1.02(d, J=6.8Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ )δ211. 1,200.6,198.5,179.8,166.9,163.2,160.8,160.3,159.8,131.2,131.1,130.4,130.3,129.7,115.5,115.3,113.2,104.5,101.4,99.8,55.2,131.1,130.4,130.3,129.7,115.5,115.3,113.2,104.5,101.4,99.8,55.2,0.6.5,4.5 24.6, 24.3, 23.3, 15.8, 15.8; HR-ESI-MS m/z 509.1972 [M+H] ⁺ .

Compound 16: ¹ H NMR (400 MHz, CDCl ₃ ) δ 13.85 (s, OH), 10.45 (s, OH), 7.23 (m, 2H), 7.00 (m, 2H), 6.09 (s, 1H), 4.48 (d, J=7.2Hz, 2H), 4.47(s, 1H), 2.40(m, 1H), 1.51(s, 3H), 1.43(s, 3H), 1.42(s, 3H), 1.36(s, 3H), 1.06 (d, J=6.8 Hz, 3H), 1.04 (d, J=6.8 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 211.4, 203.4, 198.9, 180.1, 168.8, 163.7, 163.1, 160.7,156.6,131.6,131.5,131.1,131.1,129.6,115.3,115.1,113.2,106.7,104.2,92.1,55.1,49.4,45.8,45.0,35.4,26.1,24.5,24.4,23.3,15. ESI-MS m/z 509.1974 [M+H] ⁺ .

Example 3: Preparation of Compounds 19 and 20

first step:

At room temperature, phloroglucinol 5 was dissolved in nitromethane solution, anhydrous aluminum trichloride and cyclohexanecarbonyl chloride 17 were added successively, and the temperature was raised to 40°C. After reacting for 5 hours, the reaction solution was slowly poured into ice water, and a saturated potassium sodium tartrate solution was added, followed by vigorous stirring. The reaction solution was extracted three times with ethyl acetate. The organic phases were combined and washed with saturated NaCl solution. After drying and filtration over anhydrous sodium sulfate, the organic phase was evaporated to dryness under reduced pressure. The obtained crude product was separated and purified by silica gel column chromatography to obtain compound 18 (yield 75%).

Step 2:

Compounds 10 and 18 were dissolved in toluene at room temperature, then trifluoroacetic acid and molecular sieves were added, and the temperature was raised to 110°C. After 24 hours of reaction, the temperature was lowered to room temperature, and saturated aqueous sodium bicarbonate solution was added to quench the reaction. After filtration through celite, the reaction solution was extracted three times with ethyl acetate. The organic phases were combined and washed with saturated NaCl solution. After drying and filtration over anhydrous sodium sulfate, the organic phase was evaporated to dryness under reduced pressure. The obtained crude products were separated and purified by silica gel column chromatography to obtain compounds 19 and 20 (70% yield).

Compound 19: ¹ H NMR (400 MHz, CDCl ₃ ) δ 13.33(s,OH), 9.74(s,OH), 6.11(s,1H), 4.50(s,1H), 3.39(m,1H), 2.39 (m,1H),1.87(m,4H),1.74(m,2H),1.52(s,3H),1.46(m,2H),1.41(s,3H),1.41(s,3H),1.37( m, 2H), 1.34 (s, 3H), 1.12 (d, J=6.8Hz, 3H), 1.11 (d, J=6.8Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 211.3, 207.3, 198.4 ,179.8,167.2,159.6,159.6,129.4,113.4,104.4,101.0,99.9,55.3,49.1,45.7,45.1,35.5,29.8,29.1,26.3,26.2,26.1,26.1,24.5,24.2,23.2,15. ; HR-ESI-MS m/z 483.2382 [M+H] ⁺ .

Compound 20: ¹ H NMR (400 MHz, CDCl ₃ ) δ 14.19 (s, OH), 10.20 (s, OH), 6.08 (s, 1H), 4.48 (s, 1H), 3.76 (m, 1H), 2.39 (m,1H),1.99(m,1H),1.83(m,4H),1.71(m,1H),1.50(s,3H),1.46(m,2H),1.42(s,3H),1.42( s, 3H), 1.38 (m, 2H), 1.36 (s, 3H), 1.06 (d, J=6.8 Hz, 3H), 1.04 (d, J=6.8 Hz, 3H); ¹³ C NMR (100 MHz, CDCl) ₃ )δ211.3,210.3,198.8,179.9,168.9,163.1,156.4,129.5,113.3,106.4,104.1,92.1,55.1,49.8,45.8,45.2,35.4,30.4,29.1,26.4,26.9,24.3,26.0, 24.4, 23.5, 15.7, 15.7; HR-ESI-MS m/z 483.2373 [M+H] ⁺ .

Example 4: Preparation of

Compounds

1 and 2

Compounds 10 and 11 were dissolved in toluene at room temperature, then trifluoroacetic acid and molecular sieves were added, and the temperature was raised to 110°C. After 24 hours of reaction, the temperature was lowered to room temperature, and saturated aqueous sodium bicarbonate solution was added to quench the reaction. After filtration through celite, the reaction solution was extracted three times with ethyl acetate. The organic phases were combined and washed with saturated NaCl solution. After drying and filtration over anhydrous sodium sulfate, the organic phase was evaporated to dryness under reduced pressure. The obtained crude products were separated and purified by preparative HPLC to obtain compounds 1 and 2 (50% yield).

Compound 1: ¹ H NMR (400 MHz, CDCl ₃ ) δ 13.31 (s, OH), 4.75 (s, 1H), 4.71 (s, 1H), 2.97 (dd, J=14.6, 6.8 Hz, 1H), 2.74 (dd,J=14.6,6.8Hz,1H),2.36(m,1H),2.29(m,1H),2.17(m,1H),1.47(s,3H),1.45(s,3H),1.43( s, 3H), 1.40(s, 3H), 1.36(s, 3H), 1.36(s, 3H), 1.30(s, 3H), 1.26(s, 3H), 1.09(d, J=6.8Hz, 3H ),1.05(d,J＝6.8Hz,3H),1.04(d,J＝6.8Hz,3H),1.01(d,J＝6.8Hz,3H),0.99(d,J＝6.8Hz,3H), 0.97 (d, J=6.8 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 211.7, 211.6, 204.1, 193.3, 192.5, 176.5, 176.4, 161.8, 160.8, 159.1, 129.8, 129.0, 113.0, 112.9, 107. ,103.4,100.4,56.3,55.8,52.1,46.1,45.7,45.6,45.5,35.3,35.1,25.9,25.4,25.1,24.7,24.7,24.3,24.0,23.9,23.4,22.9,22.8,16.0,15.9,15 , 15.6; HR-ESI-MS m/z 703.3474 [M+H] ⁺ .

Compound 2: ¹ H NMR (500 MHz, CDCl ₃ ) δ 13.39 (s, OH), 4.73 (s, 1H), 4.70 (s, 1H), 2.91 (dd, J=15.0, 6.8 Hz, 1H), 2.80 (dd, J=15.0, 6.8Hz, 1H), 2.51(m, 1H), 2.37(m, 1H), 2.19(m, 1H), 1.45(s, 3H), 1.45(s, 3H), 1.38( s, 3H), 1.35(s, 3H), 1.34(s, 3H), 1.29(s, 3H), 1.26(s, 3H), 1.17(s, 3H), 1.09(d, J＝6.8Hz, 3H ),1.07(d,J＝6.8Hz,3H),1.07(d,J＝6.8Hz,3H),1.02(d,J＝6.8Hz,3H),0.99(d,J＝6.6Hz,3H), 0.98 (d, J=6.6 Hz, 3H); ¹³ C NMR (125 MHz, CDCl ₃ ) δ 212.6, 212.5, 204.1, 193.1, 192.5, 176.4, 175.9, 161.7, 160.8, 159.3, 129.8, 129.1, 113.1, 112.9, 107.8. ,103.2,100.4,56.3,56.0,52.1,45.5,45.4,45.4,45.3,35.1,34.7,25.6,25.5,24.8,24.5,24.4,24.4,24.0,23.9,23.1,22.9,22.9,16.0,16.0,15 , 15.9; HR-ESI-MS m/z 703.3478 [M+H] ⁺ .

Example 5: Preparation of Compound 3

Compounds 10 and 15 were dissolved in toluene at room temperature, then trifluoroacetic acid and molecular sieves were added, and the temperature was raised to 110°C. After 24 hours of reaction, the temperature was lowered to room temperature, and saturated aqueous sodium bicarbonate solution was added to quench the reaction. After filtration through celite, the reaction solution was extracted three times with ethyl acetate. The organic phases were combined and washed with saturated NaCl solution. After drying and filtration over anhydrous sodium sulfate, the organic phase was evaporated to dryness under reduced pressure. The obtained crude product was separated and purified by preparative HPLC to obtain compound 3 (23% yield).

Compound 3: ¹ H NMR (400 MHz, CDCl ₃ ) δ 12.99 (s, OH), 7.23 (m, 2H), 6.99 (m, 2H), 4.74 (s, 1H), 4.69 (s, 1H), 4.41 (d, J=15.7Hz, 1H), 4.16(dd, J=15.7Hz, 1H), 2.41(m, 1H), 2.31(m, 1H), 1.47(s, 3H), 1.45(s, 3H) ,1.43(s,3H),1.40(s,3H),1.36(s,3H),1.36(s,3H),1.30(s,3H),1.25(s,3H),1.08(d,J=6.8 Hz, 3H), 1.02 (d, J=6.8 Hz, 3H), 1.00 (d, J=6.8 Hz, 3H), 1.00 (d, J=6.8 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ211.6,211.5,200.7,193.3,192.4,176.5,176.4,163.2,161.7,160.8,160.7,159.5,131.3,131.2,130.3,130.2,129.9,129.3,115.5,115.3,112.9,112.9,107.8,105.1,103.1, HR-ESI-MS m /z 755.3229[M+H] ⁺ .

Example 6: Preparation of Compound 4

Compounds 10 and 19 were dissolved in toluene at room temperature, then trifluoroacetic acid and molecular sieves were added, and the temperature was raised to 110°C. After 24 hours of reaction, the temperature was lowered to room temperature, and saturated aqueous sodium bicarbonate solution was added to quench the reaction. After filtration through celite, the reaction solution was extracted three times with ethyl acetate. The organic phases were combined and washed with saturated NaCl solution. After drying and filtration over anhydrous sodium sulfate, the organic phase was evaporated to dryness under reduced pressure. The obtained crude product was separated and purified by preparative HPLC to obtain compound 4 (yield 25%).

Compound 4: ¹ H NMR (400 MHz, CDCl ₃ ) δ 13.28 (s, OH), 4.74 (s, 1H), 4.70 (s, 1H), 3.36 (m, 1H), 2.35 (m, 1H), 2.29 (m,1H),1.85(m,4H),1.72(m,2H),1.47(s,3H),1.45(s,3H),1.43(s,3H),1.41(m,2H),1.39( s,3H),1.38(m,2H),1.36(s,3H),1.36(s,3H),1.30(s,3H),1.25(s,3H),1.10(d,J=6.8Hz,3H ), 1.07 (d, J=6.8 Hz, 3H), 1.03 (d, J=6.6 Hz, 3H), 1.01 (d, J=6.6 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 211.7, 211.7 ,207.5,193.3,192.4,176.4,176.4,161.4,160.9,158.8,129.8,128.8,113.0,113.0,107.8,102.7,100.4,56.3,55.8,49.6,46.1,45.7,49.5.6,45.5,35.3,5.6,45.5 , 29.8, 28.9, 26.3, 26.1, 26.1, 25.3, 25.0, 24.6, 24.3, 24.0, 23.9, 23.6, 16.0, 15.9, 15.6, 15.6; HR-ESI-MS m/z 729.3636[M+H] ⁺ .

Example 7: In vitro anti-RSV activity evaluation of polycyclic benzobisfurans

(1) Viruses, cells and experimental materials

Human respiratory syncytial virus (RSV, A2 strain, Long strain and B strain), human laryngeal epidermoid carcinoma cell (HEp-2). The cells were grown in DMEM medium containing 10% fetal bovine serum (FBS), and the virus was inoculated into HEp-2 cells for expansion, and the culture medium was DMEM medium containing 2% FBS.

(2) Experimental method

Toxicity test of polycyclic benzobisfurans on HEp-2 cells: HEp-2 cells were seeded in 96-well plates at 1.0×10 ⁴ cells/well, and cultured overnight to form monolayer cells, and the concentration was 80 μM respectively. , 40μM, 20μM, 10μM, 5μM, 6.25μM polycyclic benzobisfuran compounds, each group was set up with 3 duplicate wells, and a cell control group without compound was set up, placed in a 37°C, 5% _CO2 incubator Medium, after culturing for 48 hours, discard the medium, add 100 μL of DMEM containing 10% CCK-8 reagent to each well, incubate in the dark for 2 hours in the incubator, shake and mix well, and measure the absorbance (OD) at 450 nm with a microplate reader. ). Cell survival rate (%)=OD value of drug-added group/OD value of cell control group×100%. According to the survival rate, it was fitted with Graph prism _5.0 software, and the median toxicity concentration CC50 of the compound was calculated.

In vitro anti-RSV activity test of polycyclic benzobisfurans: HEp-2 cells were seeded in a 96-well plate at 1.2×10 ⁴ cells/well, and cultured overnight to form a monolayer of cells at the maximum nontoxic concentration of the compound As the starting concentration, the compounds were diluted with DMEM containing 2% FBS, and each compound was set at 6 two-fold dilution concentrations. 50 μL of medium containing different concentrations of compounds were added to each well, and then 50 μL of 100 TCID ₅₀ virus was added. The virus control group to which the compound was added and the cell control group to which neither was added, each group had 3 replicate wells. The cells were cultured in a 37°C, 5% CO ₂ incubator, and the degree of cytopathic effect (CPE) was detected after 3 to 4 days, and the IC ₅₀ values of the compounds were calculated according to the CPE results.

(3) Experimental results

The anti-RSV activity of the compounds of the present invention is determined by the above experimental methods. The measured IC ₅₀ values of the compounds in vitro against multiple RSV virus strains and their cytotoxic CC ₅₀ values to HEp-2 cells are shown in Table 1. As shown in Table 1, all compounds showed good anti-RSV activity, and their activities were stronger than that of the positive control drug Ribavirin, among which compound 2 had the most significant antiviral activity.

Table 1. Test results of in vitro anti-RSV activity of compounds 1 to 4

^a IC ₅₀ refers to the 50% Inhibition Concentration, expressed as IC ₅₀ (μM):

^b CC50 refers to the half cytotoxic concentration ( ₅₀ % Cytotoxic Concentration), expressed as _CC50 ([mu]M).

Example 8: Inhibitory effect of compound 2 on RSV with different titers

Experimental method: HEp-2 cells were seeded in 96-well plates at 1.0×10 ⁴ cells/well, cultured overnight to form a monolayer of cells, and the diluted virus solution was added. The MOI of the virus was 0.1, 0.5, 1, and 2. , and add different concentrations of compounds, each group has 3 replicate wells, and set up a virus control group without compound and a cell control group without both, placed in a 37°C, 5% CO ₂ incubator, and cultured for 72 After 1 hour, the viral supernatant was collected, and the viral titer in the supernatant was detected by the method of plaque subtraction. Plaque subtraction method: HEp-2 cells were seeded in a 24-well plate at 1.5×10 ⁵ cells/well, cultured overnight to form a monolayer of cells, added with diluted virus solution, and incubated at 37°C for 2 hours. The virus liquid was sucked off, and 500 μL of maintenance medium (containing 2% FBS) diluted 1.5% agarose was added. After the agarose was cooled and solidified, 500 μL of maintenance medium was added to cover it. After culturing in the incubator for 4 days, add 1 mL of 4% formaldehyde solution to each culture well, fix it for 4 to 6 hours, discard the upper medium and agarose, add crystal violet solution for staining, and count the number of plaques in each well , and compared with the virus infection group to calculate the inhibition rate of the compound on plaque formation.

The experimental results shown in Fig. 1 show that compound 2 has a significant inhibitory effect on RSVA2 with different titers. When the compound concentration was 10 μM, the inhibitory effect of compound 2 on all titers of RSV was higher than 80%.

Example 9: Study on the mode of action of compound 2 on RSV

Experimental method: The direct inactivation experiment was used to study whether compound 2 has direct inactivation effect on RSV. Compound 2 and RSVA2 were diluted into different concentration gradients (5 μM, 10 μM, 20 μM, 40 μM, 80 μM) with maintenance medium, and then the two were mixed 1:1. The virus control group was the maintenance medium mixed with compound dilution. The mixture was incubated at 37°C for 2 hours. Subsequently, the mixture of compound and virus was diluted 1000 times, added to pre-cultured HEp-2 cells, washed once with PBS, and 200 μL of the pre-mixed mixture of virus and compound was added, and incubated in a 37°C incubator 2 hours. A virus control group without compound and a cell control group without virus infection were set. After 2 hours, the liquid in the plate was discarded, and 500 μL of maintenance medium containing 1.5% agarose was added along the wall of the well. After the agarose was cooled and solidified, 500 μL of maintenance medium was added, and incubated at 37°C in a 5% CO ₂ incubator for 4 days. , add 4% formaldehyde solution, fix for 4-6 hours, discard the upper medium and agarose, add crystal violet solution for staining, count the number of plaques in each well, and calculate the virus infection rate.

The experimental results in Figure 3 show that compound 2 has no obvious inactivation effect on RSV at a concentration much higher than _IC50 .

Example 10: Effect of compound 2 on RSV replication cycle

Experimental method: The time-point addition experiment was used to study the inhibitory effect of compound 2 on various stages of virus replication. HEp-2 cells were seeded in a 96-well plate at 1.0×10 ⁴ cells/well and cultured overnight to form a monolayer of cells. Cell supernatants were discarded, RSVA2 diluted with maintenance medium (MOI=0.5) was added, and compound 2 was added at various time points (0, 2, 6, 10, 16, 24, 32 hours) after virus infection. At 36 hours after virus addition, the cells were lysed and centrifuged, and the supernatant virus was collected, and the virus titer in the supernatant was detected by the plaque subtraction method.

The experimental results in Figure 2 show that the inhibitory effect of compound 2 on RSV is mainly after the virus enters the cell, and when added at the 10th hour after virus infection, its inhibitory effect on RSV is significantly reduced until the 24th hour after infection. The effect was minimized, indicating that the inhibitory effect of compound 2 on RSV was mainly after the virus entered the cell and before the release of the virus assembly.

Example 11: Inhibitory effect of compound 2 on RSV protein expression level

Experimental method: The inhibitory effect of compound 2 on RSV protein expression level was studied by immunofluorescence labeling. HEp-2 cells were seeded in culture plates, cultured overnight to form monolayers, and diluted RSVA2 was added, along with compound 2 diluted with maintenance medium. After 48 hours of culture, the cells were permeabilized with 0.1% Triton X-100 for 10 minutes, washed twice with PBS, and fixed with 4% paraformaldehyde for 15 minutes at room temperature. After washing with PBS, the RSV fusion protein was labeled with specific antibodies. (F), incubated at room temperature for 2 hours, washed with PBS, added fluorescent secondary antibody diluted with PBS (1:500), incubated at room temperature for 2 hours, added DAPI solution to label cell nuclei, and observed the fluorescence intensity under a fluorescence microscope.

The experimental results shown in Figure 4 show that when compound 2 (10 μM) was added to RSV-infected cells, green fluorescence was hardly detected under a fluorescence microscope (Figure 4), indicating that compound 2 could significantly reduce the expression level of RSV F protein gene.

The specific embodiments of the present invention have been described above in detail, but the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be included within the scope of the present invention.

Claims

The polycyclic benzobisfuran compound having the structure shown in the general formula I or a pharmaceutically acceptable salt or a stereoisomer or a prodrug molecule thereof:

in:

R 1 and R 3 are each independently selected from substituted or unsubstituted C 1 -C 6 alkyl;

R 2 is independently selected from substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 6 cycloalkyl.
The compound shown in the general formula I according to claim 1, the compound is selected from:
A method for preparing the compound shown in general formula I according to claim 1, the method comprises:

The compound of general formula Ia can generate the compound of general formula Ib through Friedel-Crafts reaction under the condition of Lewis acid. The latter undergoes a reduction reaction to obtain a compound of the general formula Ic, and further undergoes an oxidation reaction to generate a compound of the general formula Id. Compounds of general formula Ie are reacted with compounds of general formula Id under acidic or basic conditions to obtain compounds of general formula If. The compound of general formula If is further reacted with the compound of general formula Ig under acidic or basic conditions to obtain the polycyclic benzobisfuran compound of general formula I. wherein: R 1 , R 2 and R 3 are as defined in claim 1 .
The polycyclic benzobisfuran compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt or a stereoisomer thereof or a prodrug molecule thereof, and one or more pharmaceutically acceptable salts thereof The pharmaceutical composition composed of the carrier, diluent or excipient.
The polycyclic benzobisfuran compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof or a stereoisomer or a prodrug molecule thereof or the pharmaceutical composition according to claim 4 Application in the preparation of anti-respiratory syncytial virus (RSV) medicines.
The polycyclic benzobisfuran compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof or a stereoisomer or a prodrug molecule thereof or the pharmaceutical composition according to claim 4 Use in preparing medicines for treating diseases such as bronchitis and pneumonia caused by RSV virus infection.