Synthesis of delta-fluoroalkyl substituted alkenyl phosphate compound and application of delta-fluoroalkyl substituted alkenyl phosphate compound in resisting porcine epidemic diarrhea virus

Abstract

The invention belongs to the technical field of organic synthesis and medicine. The invention develops a method for constructing a delta-fluoroalkyl substituted alkenyl phosphate compound with a brand-new structure by sequentially adding olefin and alkyne with fluoroalkyl free radicals driven by visible light for the first time. Further, it is found that the delta-fluoroalkyl substituted alkenyl phosphate compound has multiple inhibition effects on porcine epidemic diarrhea virus (PEDV), not only can inhibit virus cell entry and replication, but also can further relieve cell apoptosis caused by PEDV, and it is indicated that the compound has the effect of treating porcine epidemic diarrhea. The compound provides a new thought for the development of multifunctional anti-PEDV medicines.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of organic synthesis and pharmaceutical technology, and relates to δ-fluoroalkyl substituted alkenyl phosphate compounds, as well as the synthesis method of the compounds and their application in the treatment of porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV). Background Technology

[0002] Radical-mediated bifunctionalization reactions of single alkenes or alkynes are relatively well-established, especially in the construction of adjacent-position bifunctional substituted molecules. However, strategies for constructing 1,4- or 1,n-long-range bifunctional substituted molecules based on the cascade addition of two or more unsaturated bonds between molecules are still relatively rare and face complex chemo / regio / stereoselectivity issues. In recent years, some progress has been made in constructing 1,4-bifunctionalized molecules by utilizing the polar effects of radicals to sequentially add two electronically significantly different unsaturated bonds (G. Tan, F. Paulus, ...). Rentería-Gómez, et al., J.Am.Chem.Soc. 144(2022)21664-21673; JCLo, J.Gui, Y.Yabe, et al., Nature 516(2014)343-348; JCLo, Y.Yabe, PSBaran, J.Am.Chem.Soc. 136(2014)1304-1307). However, its limitation lies in the requirement that there must be a significant electronic difference between the two unsaturated bonds, often requiring a Michael-type alkene or alkyne with a strong electron-withdrawing group and a special structure as one of the substrates, thus severely limiting the universality of the reaction. This patent utilizes the polar effect of free radicals and the difference in reaction selectivity between alkyl carbon radicals and alkenyl carbon radicals with triethyl phosphite to develop a novel visible light-driven reaction strategy for the sequential addition of fluoroalkyl radicals to alkenes and alkynes with no significant electronic difference between molecules, achieving remote 1,4-bifunctionalization. This strategy was used to prepare a series of novel δ-fluoroalkyl-substituted alkenyl phosphate compounds with potential biological activity.

[0003] Porcine epidemic diarrhea virus (PEDV) is a member of the genus *Alpha* in the family Coronaviridae. It can infect pigs of all ages, causing acute diarrhea, vomiting, dehydration, and high mortality rates in piglets (J. Cui, F. Li, ZL Shi, Nat. Rev. Microbiol. 17 (2018) 181-192). Due to its high infectivity and the lack of effective drugs and vaccines, PED has caused severe economic losses to the global pig industry (M. Wu, D. Yi, Q. Zhang, et al., Sci. Rep. 11 (2021) 6552). In the United States alone, more than 7 million pigs died from PED between 2013 and 2014 (K. Jung, LJSaif, Vet. J. 204 (2015) 134-143). Furthermore, PEDV has been found to infect cells in bats, monkeys, and even humans, indicating that it poses a potential threat to other species as well (C. Liu, J. Tang, Y. Ma, et al., J. Virol. 89 (2015) 6121-6125.). Currently, antiviral drug development mainly focuses on two strategies: inhibiting viral replication (H. Chu, Y. Hou, D. Yang, et al., Nature 609 (2022) 785-792) and preventing the virus from entering host cells (Y. Zhu, F. Feng, G. Hu, et al., Nat. Commun. 12 (2021) 961). Although some small molecule inhibitors specifically targeting viral replication (HJDong, ZHWang, W.Meng, et al., Viruses 10(2018)601; Z.Li, H.Cao, Y.Cheng, et al., Int. J.Mol. Sci. 21(2020)8095) or cellular entry (JHLee, JSPark, SWLee, et al., Virus Res. 195(2015)148-152; L.Yuan, S.Zhang, Y.Wang, et al., J.Virol. 92(2018)e00809-18) have shown significant inhibitory effects against porcine epidemic diarrhea virus (PEDV), effective drugs for clinical use are still lacking. Given that viral entry and replication are key steps in the viral life cycle, developing novel, multifunctional anti-PEDV drugs that synergistically inhibit these two processes is a promising research direction. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide a δ-fluoroalkyl-substituted alkenyl phosphate compound with multiple anti-PEDV activities and a green synthesis method for such compounds. This invention has found that δ-fluoroalkyl-substituted alkenyl phosphate compounds can significantly inhibit PEDV replication and cell entry, and can be used to prepare drugs for treating PED.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] This invention provides novel δ-fluoroalkyl-substituted alkenyl phosphate compounds, methods for synthesizing such compounds, and their application in the treatment of PED.

[0007] In the above applications, the δ-fluoroalkyl-substituted alkenyl phosphate compounds have the general structural formula shown in Formula I:

[0008]

[0009] Where: Ar is selected from Any one of them.

[0010] The δ-fluoroalkyl-substituted alkenyl phosphate compound is characterized in that it can be synthesized by the following method: Reaction equation:

[0011] for Any one of them.

[0012] The drug for treating PEDV is characterized in that the dosage form of the drug includes tablets, capsules, oral liquids, granules, pills, or injections.

[0013] The beneficial effects of this invention are as follows: This invention develops a free radical cascade reaction strategy involving the sequential addition of free radicals to alkenes and alkynes, rapidly synthesizing a series of novel δ-fluoroalkyl-substituted alkenyl phosphate compounds with potential biological activity. Studies have shown that these compounds can effectively inhibit the entry and replication of PEDV, suggesting their potential use in the preparation of drugs for treating PED. This patent provides new insights into the cascade addition of free radicals to polyunsaturated bonds and the development of multifunctional anti-PEDV drugs. Attached Figure Description

[0014] Figure 1 The inhibitory effects of compounds f1, f12, f30 and f31 on PEDV at a concentration of 10 μM were determined.

[0015] Figure 2 The inhibitory effect of compound f1 on viral replication at different concentrations.

[0016] Figure 3 The effect of compound f1 on viral entry into cells at different concentrations is shown.

[0017] Figure 4 This demonstrates the blocking effect of compound f1 on the adsorption of viral particles onto the surface of host cells at different concentrations. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention are described in detail below. The provided embodiments are merely representative examples of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0019] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0020] The test materials used in the embodiments of the present invention, unless otherwise specified, are all conventional test materials in the art, which can be synthesized through simple steps or purchased through commercial channels.

[0021] The structural formulas of compounds f1, f12, f30, and f31 are shown below:

[0022]

[0023] Example 1: The preparation methods of compounds f1, f12, f30 and f31 are as follows:

[0024]

[0025] The synthesis steps and process were as follows: IC4F9 (0.24 mmol, 1.2 eq), olefin (0.5 mmol, 2.5 eq), alkyne (0.2 mmol), fac-Ir(ppy)3 (1%), quinine ring (0.2 mmol, 1 eq), and triethyl phosphite (0.6 mmol, 3 eq) were added to a 5 mL reaction tube equipped with a magnetic stirrer. Then, 0.3 mL of THF was added. Under inert gas protection, the reaction tube was fixed on a 6 W photoreactor with a wavelength range of 460–470 nm. The reaction was carried out for 48 h. After completion, the solvent was removed using a rotary evaporator. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 1:1–5:1) to obtain the target products (f1, f12, f30, f31). The yields and NMR results of the products are as follows:

[0026] methy(E)-4-(1-(diethoxyphosphoryl)-5,5,6,6,7,7,8,8,8-nonafluoro-3,3-dimethyloct-1-en-1-yl)benzoate(f1,E)

[0027]

[0028] Pale yellow oil;(89 mg,78%,E:Z=66:1);R f =0.41(petroleum ether / ethylacetate 1:1); 1 H NMR(600 MHz,Chloroform-d)δ8.01(d,J=7.9 Hz,2H),7.26(dd,J=8.3,2.0 Hz,2H),6.85(d,J H-P =26.7,1H),4.07–3.98(m,4H),3.93(s,3H),2.11(t,J H-F =19.8 Hz,2H),1.24(t,J=7.1 Hz,6H),1.05(s,6H); 13 C NMR(151 MHz,Chloroform-d)δ166.74,152.97(d,J C-P =8.2 Hz),140.23(d,J C-P =7.6 Hz),130.28(d,J C-P =176.8 Hz),129.83(d,J C-P =4.6 Hz),129.44(d,J C-P =3.0 Hz),129.04(d,J C-P =1.8 Hz),62.23(d,J C-P =5.6 Hz),52.15,42.06(t,J C-F =20.4 Hz),37.22(d,J C-P =20.4 Hz),37.14,29.19,16.15(d,J C-P =6.5 Hz); 19 F NMR(565 MHz,Chloroform-d)δ-81.08–-81.10(m,3F),-110.96–-110.05(m,2F),-124.54–-124.56(m,2F),-125.74–-125.78(m,2F); 31PNMR(243 MHz,Chloroform-d)δ16.44.

[0029] diethyl(E)-(5,5,6,6,7,7,8,8,8-nonafluoro-3,3-dimethyl-1-(3-sulfamoylphenyl)oct-1-en-1-yl)phosphonate(f12,E)

[0030]

[0031] White solid;(61 mg,51%,E:Z>20:1);mp 139-141℃;R f =0.36(ethylacetate); 1 H NMR(400MHz,Chloroform-d)δ7.81(dd,J=7.9,1.5 Hz,1H),7.71(d,J=2.0Hz,1H),7.41(t,J=7.8 Hz,1H),7.30(dd,J=7.7,1.6 Hz,1H),6.76(d,J H-P =26.5 Hz,1H),5.49(s,2H),3.97(p,J=7.2 Hz,4H),2.05(t,J H-F =7.8 Hz,1H),1.19(t,J=7.0 Hz,6H),0.98(s,6H); 13 C NMR(101 MHz,Chloroform-d)δ153.27(d,J C-P =8.5 Hz),142.34(d,J C-P =2.2 Hz),136.04(d,J C-P =7.8 Hz),133.59(d,J C-P =4.8 Hz),130.03,129.14(d,J C-P =179.5 Hz),128.56(d,J C-P =2.2 Hz),127.65(d,J C-P =5.0 Hz),125.58(d,J C-P =2.9Hz),62.53(d,J C-P =6.0 Hz),42.21(t,J C-F =19.9 Hz),37.30(d,J C-P =19.9 Hz),29.20,16.11(d,J C-P=6.2 Hz); 19 F NMR(376 MHz,Chloroform-d)δ-81.09(t,J=9.8 Hz,3F),-110.96–-111.04(m,2F),-124.55–-124.60(m,2F),-125.75–-125.79(m,2F); 31 PNMR(162MHz,Chloroform-d)δ16.24.

[0032] diethyl(E)-(5,5,6,6,7,7,8,8,8-nonafluoro-1-(6-fluoropyridin-3-yl)-3,3-dimethyloct-1-en-1-yl)phosphonate(f30,E)

[0033]

[0034] Pale yellow oil;(59mg,55%,E:Z>20:1);R f =0.35(petroleum ether / ethylacetate 1:1); 1 H NMR(400MHz,Chloroform-d)δ8.01(s,1H),7.67–7.56(m,1H),6.96–6.93(m,1H),6.94(d,J H-P =26.2Hz,1H),4.10–4.01(m,4H),2.15(t,J H-F =20.3Hz,2H),1.27(t,J=7.1Hz,6H),1.08(s,6H); 13 C NMR(151MHz,Chloroform-d)δ163.07(dd,J C-F =240.9,3.0Hz),154.72(d,J C-P =8.9Hz),147.67(dd,J C-P =14.8,5.8Hz),142.23(dd,J C-P =7.9,4.0Hz),128.90(dd,J C-P =8.0,4.7Hz),126.41(d,J C-P =180.8Hz),108.88(d,J C-P =37.3Hz),62.31(d,J C-P =6.0Hz),42.27(t,JC-F =19.8Hz),37.42(d,J C-P =19.6Hz),29.44,16.16(d,J C-P =6.1Hz); 19 F NMR(376MHz,Chloroform-d)δ-69.11(d,J=4.5Hz,1F),-81.09–-81.15(m,3F),-111.05–-111.08(m,2F),-124.58–-124.61(m,2F),-125.81–-125.85(m,2F); 31 P NMR(162MHz,Chloroform-d)δ16.15.

[0035] diethyl(E)-(5,5,6,6,7,7,8,8,8-nonafluoro-3,3-dimethyl-1-(pyridin-4-yl)oct-1-en-1-yl)phosphonate(f31,E)

[0036]

[0037] Pale yellow oil;(80mg,78%,E:Z>20:1);R f =0.23(ethyl acetate); 1 H NMR(400MHz,Chloroform-d)δ8.59(d,J=6.0Hz,2H),7.13(dd,J=4.2,2.0Hz,2H),6.85(d,J H-P =27.0Hz,1H),4.05(p,J=7.2Hz,4H),2.13(t,J H-F =20.4Hz,2H),1.26(t,J=7.1Hz,6H),1.08(s,6H); 13 C NMR(101MHz,Chloroform-d)δ153.14(d,J C-P =7.9Hz),149.23(d,J C-P =2.1Hz),144.15(d,J C-P =8.1Hz),128.66(d,J C-P =178.8Hz),124.82(d,J C-P =4.7Hz),62.35(d,J C-P =6.3Hz),42.15(t,J C-F=20.0Hz), 37.34(d,J C-P =20.3Hz), 29.21, 16.12(d,J C-P =6.5Hz); 19 F NMR(376MHz,Chloroform-d)δ-81.09(t,J=9.4Hz,3F),-111.06–-111.09(m,2F),-124.56–-124.62(m,2F),-125.80–-125.84(m,2F); 31 P NMR(162MHz,Chloroform-d)δ15.81.

[0038] Example 2: Cell Culture and Virus

[0039] Vero-E6 and IPEC-J2 cells were cultured in Durbeco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, in vivo component) and 1% penicillin and streptomycin (Meilun, China); these cells were used for viral replication in an incubator supplemented with DMEM medium containing 5 μg / mL trypsin (Hyclone, China).

[0040] Example 3: EC50 determination of the anti-PEDV properties of δ-fluoroalkyl-substituted alkenyl phosphate compounds

[0041] Table 1 Product EC50

[0042]

[0043] The anti-PEDV activity of compounds f1, f12, f30, and f31 was calculated in Vero-E6 cells. The results showed that compounds f1, f12, f30, and f31 had strong inhibitory activity against PEDV, with EC50 values ​​of 3.62 μM, 3.86 μM, 10.12 μM, and 3.71 μM, respectively.

[0044] Example 4: Mechanism Study of PEDV Resistance

[0045] To verify whether compound f1 affects viral replication in host cells, we seeded Vero-E6 cells pre-coated in 48-well cell culture plates with PEDV at an MOI of 0.1. After 3 hours of incubation, the virus-containing medium was discarded, and medium containing different concentrations of compound f1 was added. The cells were then incubated in a 5% CO2, 37°C cell culture incubator for 48 hours. The results showed that, without significantly affecting cell viability, the expression level of viral N protein gradually decreased with increasing compound f1 concentration, indicating that compound f1 can inhibit viral replication in host cells.Figure 2 ).

[0046] Viral particles adsorbed onto the cell surface interact with corresponding receptor proteins on the cell surface under the mediation of the S protein, thereby entering the cell. To investigate whether compound f1 blocks viral entry into cells, PEDV was seeded at an MOI of 0.1 into Vero-E6 cells pre-coated in 48-well cell culture plates and immediately placed at 4°C for 1 hour. At this point, the virus only adsorbed onto Vero-E6 cells. The culture medium was then replaced with different concentrations of the drug, and the cells were incubated in a 5% CO2, 37°C cell culture incubator for 1.5 hours. At this point, the virus began to enter the cells. Finally, the culture medium was replaced with ordinary medium, and the viral N protein content at each drug concentration was measured at the corresponding time points. The results showed that the viral content gradually decreased with increasing compound f1 concentration, but the inhibition rate remained low even at 10 μM. This indicates that compound f1 has a certain blocking effect on PEDV entry into cells, but this is not the primary mechanism of action. Figure 3 ).

[0047] When a virus comes into contact with a cell, it first adsorbs onto the cell surface via non-specific electrostatic attraction. To investigate whether compound f1 blocks the non-specific electrostatic attraction between the virus and host cells, PEDV was seeded at an MOI of 0.1 into Vero-E6 cells pre-coated in 48-well cell culture plates. Different concentrations of compound f1 were added to the culture medium, and the plates were incubated at 4°C for 1 hour. During this time, the virus only adsorbed onto the cell surface and did not enter the cell. The culture medium containing the drug and virus particles was then discarded and replaced with ordinary culture medium. After the appropriate incubation time, the viral N protein content corresponding to each drug concentration in the wells was measured. The results showed that compound f1 at higher concentrations could block the adsorption of virus particles to host cells, while at lower concentrations it was ineffective. Figure 4 ).

[0048] In summary, compound f1 has a significant inhibitory effect on the replication process of PEDV virus in host cells, and inhibits the invasion of PEDV virus into host cells at high concentrations.

[0049] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications made without departing from the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. δ-fluoroalkyl-substituted alkenyl phosphate compounds can serve as multifunctional inhibitors of porcine epidemic diarrhea virus (PEDV) and can be used in the preparation of drugs for treating porcine epidemic diarrhea (PED). The δ-fluoroalkyl-substituted alkenyl phosphate compounds have the general structural formula shown in Formula I below: in: Ar selected Any one of them.

2. The δ-fluoroalkyl-substituted alkenyl phosphate compound as described in claim 1, characterized in that, The δ-fluoroalkyl-substituted alkenyl phosphate compounds can be synthesized by the following method: Reaction equation: Equation (1) is Any one of them.

3. The medicament for treating PEDV as described in claim 1, characterized in that, The dosage forms of the drug include tablets, capsules, oral liquids, granules, pills, or injections.

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