20-deoxyingenol ester derivative as well as preparation method and application thereof

Abstract

The invention provides a 20-deoxyingenol ester derivative as well as a preparation method and application thereof, and belongs to the technical field of chemical synthesis. The 20-deoxidized ingenol ester derivative provided by the invention has a good effect of promoting autophagy, and the result of the embodiment shows that the 20-deoxidized ingenol ester derivative provided by the invention can promote autophagy of human glioma cells at 20 micromole concentration, the activity of the compound is superior to that of a contrast compound Torin 1, and the compound can be used for preparing a medicine for treating human glioma cells. The quantity of lysosomes can be increased in a dose-dependent manner, TFEB nuclear translocation is promoted, and lysosome-mediated lipid droplet removal can be enhanced. Meanwhile, a cytotoxicity experiment shows that the 20-deoxyingenol ester derivative provided by the invention has no cytotoxicity to HepG2 cells.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical synthesis, and in particular to a 20-deoxyingenol ester derivative, a preparation method and an application thereof. Background Art

[0002] Macroautophagy is a type of autophagy that can degrade macromolecules and misfolded protein aggregates within the cell that cannot be degraded by the proteasome. During macroautophagy, part of the cell membrane first separates and expands, enveloping the cellular contents to be degraded, forming an autophagosome with a double-layer membrane structure. Subsequently, the autophagosome fuses with the late endosome to form an autophagic endosome, which further fuses with the lysosome to form an autolysosome, or the autophagosome directly fuses with the lysosome to form an autolysosome. Ultimately, the cellular contents are degraded and recycled in the autolysosome. Abnormal macroautophagy is closely related to diseases such as heart disease, myopathy, neurodegenerative diseases, and tumors.

[0003] 20-Deoxyingenol is a diterpenoid compound with hydroxyl groups at C3, C4, and C5. It was first isolated from the Euphorbia genus Kansui by Hirata et al. in 1974. 20-Deoxyingenol esters can affect the autophagy-lysosomal system through PKC. However, the only 20-deoxyingenol esters known to activate autophagy are 20-deoxyingenol-5β-O-angelate (HEP14) and 20-deoxyingenol-3β-O-angelate (HEP15). HEP14 promotes lysosomal biogenesis in an mTOR-independent manner and has a clearing effect on β-amyloid (Aβ) in the rat brain. However, to date, the number of 20-deoxyingenol esters is relatively small, and there are no reports on the preparation methods and autophagy-promoting effects of other 20-deoxyingenol esters. Summary of the Invention

[0004] In view of this, the present invention aims to provide a 20-deoxy-ingenol ester derivative and its preparation method and application. The 20-deoxy-ingenol ester derivative provided by the present invention has a good effect of promoting cell autophagy.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a 20-deoxyingenol ester derivative having a structure shown in Formula I or Formula II:

[0007]

[0008] In formula I, R is

[0009] In formula II, R1 and R2 are

[0010] The present invention provides a method for preparing the above-mentioned 20-deoxy-ingenol ester derivatives, characterized in that when the 20-deoxy-ingenol ester derivatives have the structure shown in Formula I, the preparation method comprises the following steps:

[0011] 20-deoxyingenol ester having the structure shown in formula a, acetone, 2,2-dimethoxypropane and p-toluenesulfonic acid are mixed and subjected to an acetonide protection reaction to obtain a compound having the structure shown in formula b:

[0012] A compound having a structure shown in formula b is subjected to a first esterification reaction with a compound having an R-OH structure to obtain a 20-deoxyingenol ester derivative;

[0013]

[0014] When the 20-deoxy-ingenol ester derivative has the structure shown in Formula II, the preparation method comprises the following steps:

[0015] The 20-deoxy-ingenol ester having the structure shown in formula a is mixed with an organic acid and subjected to a second esterification reaction to obtain a 20-deoxy-ingenol ester derivative, wherein the organic acid is acetic acid, propionic acid, butyric acid or pivalic acid.

[0016] Preferably, the molar ratio of 20-deoxyingenol ester having the structure shown in formula a to 2,2-dimethoxypropane and p-toluenesulfonic acid is 1:4-6:0.4-0.7.

[0017] Preferably, the temperature of the acetone protection reaction is 15-25° C., and the time is 10-12 hours.

[0018] Preferably, the first esterification reaction is carried out in the presence of a catalyst, and the catalyst is DMAP and EDCI.

[0019] Preferably, the temperature of the first esterification reaction is room temperature, and the time is 10 to 12 hours.

[0020] Preferably, the second esterification reaction is carried out in the presence of a catalyst, and the catalyst is DMAP and EDCI.

[0021] Preferably, the temperature of the second esterification reaction is room temperature, and the time is 10 to 12 hours.

[0022] The present invention provides the use of the above-mentioned 20-deoxyingenol ester derivatives in the preparation of drugs for promoting cell autophagy.

[0023] Preferably, the drug promoting cell autophagy includes one or more of anti-cardiac drugs, anti-myopathy drugs, anti-degenerative disease drugs and anti-tumor drugs.

[0024] The present invention provides a 20-deoxyingenol ester derivative having a structure represented by Formula I or Formula II. The 20-deoxyingenol ester derivative provided by the present invention is a protein kinase C (PKC) agonist with excellent autophagy-promoting properties. Example results demonstrate that the 20-deoxyingenol ester derivative provided by the present invention can promote autophagy in human glioma cells at a concentration of 20 micromolar, with superior activity compared to the control compound, Torin 1. Furthermore, the compound not only increases the number of lysosomes and promotes TFEB nuclear translocation in a dose-dependent manner, but also enhances lysosome-mediated lipid droplet clearance. Cytotoxicity experiments also demonstrate that the 20-deoxyingenol ester derivative provided by the present invention is non-cytotoxic to HepG2 cells.

[0025] The present invention provides a method for preparing the aforementioned 20-deoxyingenol ester derivatives. The method uses 20-deoxyingenol as a starting material, and through a chemoselective acetonide protection reaction of the 3,4-OH group, an esterification reaction with an organic acid or a Boc-amino acid, prepares a 20-deoxyingenol ester derivative having a structure of Formula I. Alternatively, using 20-deoxyingenol as a starting material, through an esterification reaction with an organic acid, a 20-deoxyingenol ester derivative having a structure of Formula II is obtained. The preparation method provided by the present invention is simple to operate, low in cost, and easily scalable for industrial mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The preparation routes of compounds 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11;

[0027] Figure 2 The preparation routes of compounds 12, 13, 14 and 15;

[0028] Figure 3 Different compounds (20 μM) promoted the activity of cellular autophagic flux;

[0029] Figure 4 The effects of compounds 10 and 13 on lysosome generation, TFEB translocation, and lipid droplet clearance;

[0030] Figure 5 Cytotoxicity test results of compounds 10 and 13;

[0031] Figure 6Western blot analysis results of compounds 10 and 13. DETAILED DESCRIPTION

[0032] The present invention provides a 20-deoxyingenol ester derivative having a structure shown in Formula I or Formula II:

[0033]

[0034] In Formula I, R is

[0035] In formula II, R1 and R2 are

[0036] The R, R 1 and R 2 middle, Indicates the junction site.

[0037] Preferably, in the present invention, the 20-deoxy-ingenol ester derivative has the structure shown below:

[0038]

[0039] The present invention provides a method for preparing the above-mentioned 20-deoxy-ingenol ester derivatives. When the 20-deoxy-ingenol ester derivatives have the structure shown in Formula I, the preparation method comprises the following steps:

[0040] 20-deoxyingenol ester having the structure shown in formula a, acetone, 2,2-dimethoxypropane and p-toluenesulfonic acid are mixed and subjected to an acetonide protection reaction to obtain a compound having the structure shown in formula b:

[0041] A compound having a structure shown in formula b is subjected to a first esterification reaction with a compound having an R-OH structure to obtain a 20-deoxyingenol ester derivative;

[0042]

[0043] The present invention mixes 20-deoxyingenol ester having the structure shown in formula a, acetone, 2,2-dimethoxypropane, and p-toluenesulfonic acid, and performs an acetonide protection reaction to obtain a compound having the structure shown in formula b. The present invention preferably dissolves the 20-deoxyingenol ester having the structure shown in formula a in acetone, and then adds 2,2-dimethoxypropane and p-toluenesulfonic acid. In the present invention, the molar ratio of the 20-deoxyingenol ester having the structure shown in formula a to 2,2-dimethoxypropane and p-toluenesulfonic acid is preferably 1:4-6:0.4-0.7, and more preferably 1:5:0.5-0.6. In the present invention, the function of the 2,2-dimethoxypropane is to form a propylidene protecting group, and the function of the p-toluenesulfonic acid is to catalyze the propylidene protection.

[0044] In the present invention, the temperature of the acetone protection reaction is preferably 15 to 25° C., more preferably 20° C.; the time is preferably 10 to 12 h, more preferably 11 h.

[0045] After the acetone protection reaction, the present invention preferably performs post-treatment on the obtained acetone protection reaction solution, and the post-treatment preferably comprises the following steps:

[0046] mixing the acetone formate protection reaction solution with sodium bicarbonate to quench the reaction, and removing the organic solvent from the quenched reaction solution;

[0047] The remaining liquid after removing the organic solvent is mixed with dichloromethane, filtered, the organic phase is concentrated and purified by column chromatography in sequence to obtain a pure compound having the structure shown in formula b.

[0048] In the present invention, a compound having a structure represented by Formula b and a compound having an R—OH structure undergo a first esterification reaction to obtain a 20-deoxyingenol ester derivative. In the present invention, the molar ratio of the compound having a structure represented by Formula b to the compound having an R—OH structure is preferably 1:1 to 5, more preferably 1:2 to 3.

[0049] In the present invention, the first esterification reaction is carried out in the presence of a catalyst, wherein the catalyst is DMAP (4-dimethylaminopyridine) and EDCI (1-ethyl-3-(3-dimethylpropylamine)carbodiimide). In the present invention, the molar ratio of the compound having the structure represented by Formula b to DMAP is preferably 5:1 to 20:1, more preferably 10:1, and the molar ratio to EDCI is preferably 1:1 to 2. In the present invention, the solvent used in the first esterification reaction is preferably DCM.

[0050] In the present invention, the temperature of the first esterification reaction is preferably room temperature, and the time is preferably 10 to 12 hours, more preferably 11 hours.

[0051] After the first esterification reaction, the present invention preferably performs post-treatment on the obtained first esterification reaction liquid, and the post-treatment comprises the following steps:

[0052] The first esterification reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain a pure product of 20-deoxyingenol ester derivatives.

[0053] In the present invention, when the 20-deoxy-ingenol ester derivative has the structure shown in Formula II, the preparation method comprises the following steps:

[0054] The 20-deoxy-ingenol ester having the structure shown in formula a is mixed with an organic acid and subjected to a second esterification reaction to obtain a 20-deoxy-ingenol ester derivative, wherein the organic acid is acetic acid, propionic acid, butyric acid or pivalic acid.

[0055] In the present invention, the molar ratio of the 20-deoxyingenol ester having the structure represented by formula a to the organic acid is preferably 1:1 to 1:5, more preferably 1:2.

[0056] In the present invention, the second esterification reaction is preferably carried out in the presence of a catalyst, and the catalyst is preferably DMAP (4-dimethylaminopyridine) and EDCI (1-ethyl-3(3-dimethylpropylamine)carbodiimide). In the present invention, the molar ratio of the compound having the structure represented by formula a to DMAP is preferably 5:1 to 20:1, more preferably 10:1, and the molar ratio to EDCI is preferably 1:1 to 1:2. In the present invention, the organic solvent used in the second esterification reaction is preferably dichloromethane.

[0057] In the present invention, the temperature of the second esterification reaction is preferably room temperature, and the time is preferably 10 to 12 hours, more preferably 11 hours.

[0058] After the second esterification reaction, the present invention preferably performs post-treatment on the obtained second esterification reaction liquid, and the post-treatment preferably includes the following steps:

[0059] The second esterification reaction liquid was concentrated under reduced pressure and purified by column chromatography to obtain a pure product of 20-deoxyingenol ester derivatives.

[0060] The present invention provides the use of the above-mentioned 20-deoxyingenol ester derivatives in the preparation of drugs that promote cell autophagy. Preferably, the drugs that promote cell autophagy include one or more of anti-cardiac drugs, anti-myopathy drugs, anti-degenerative disease drugs, and anti-tumor drugs.

[0061] In the present invention, the anti-degenerative disease preferably includes one or more of Parkinson's disease, Alzheimer's disease, Huntington's disease and hyperlipidemia, and the tumor preferably includes one or more of cervical cancer, breast cancer and skin cancer.

[0062] The following examples describe in detail the 20-deoxyingenol ester derivatives provided by the present invention, their preparation methods, and applications. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0063] Example 1

[0064] The preparation routes of compounds 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 are as follows: Figure 1 The preparation method is as follows:

[0065] Compound 1: Compound 20-deoxyingenol (100 mg, 0.30 mmol) was dissolved in anhydrous acetone, and 2,2-dimethoxypropane (591 μL, 1.44 mmol) and p-toluenesulfonic acid (31 mg, 0.18 mmol) were added sequentially at room temperature. After 12 h, the reaction was detected to be complete by TLC. Solid sodium bicarbonate was added to quench the reaction, and the solvent was removed by concentration under reduced pressure. The residue was dissolved in dichloromethane, filtered, and the organic phase was concentrated. The product was purified by flash column chromatography to obtain compound 1 (104 mg) as a white solid with a yield of 93%.

[0066] Compound 2: Compound 1 (40.0 mg, 0.12 mmol) was dissolved in anhydrous DCM, and DMAP (1.50 mg, 0.012 mmol), acetic acid (14.4 mg, 0.24 mmol), and EDCI (46.0 mg, 0.24 mmol) were added in sequence. The mixture was stirred at room temperature. After 12 h, the reaction was detected by TLC. The mixture was concentrated under reduced pressure and purified by flash column chromatography to obtain compound 2 (43.7 mg) as a white solid with a yield of 85%. The preparation methods of the other products were the same as above, except that the organic acid raw materials of the esterification reaction were replaced: the raw materials of compound 3 were replaced by propionic acid, the raw materials of compound 4 were replaced by hexanoic acid, the raw materials of compound 5 were replaced by pivalic acid, the raw materials of compound 6 were replaced by Boc-isoleucine, the raw materials of compound 7 were replaced by Boc-valine, the raw materials of compound 8 were replaced by Boc-methionine, the raw materials of compound 9 were replaced by Boc-leucine, the raw materials of compound 10 were replaced by Boc-glutamate methyl ester, and the raw materials of compound 11 were replaced by Boc-phenylalanine.

[0067] The obtained compound 3 was a light yellow oil (5.9 mg) with a yield of 59%; compound 4 was a yellow oil (8.2 mg) with a yield of 55%; compound 5 was a white solid (40 mg) with a yield of 32%; compound 6 was a white powder (28 mg) with a yield of 23%; compound 7 was a white powder (6 mg) with a yield of 18%; compound 8 was a white powder (15 mg) with a yield of 9%; compound 9 was a light yellow powder, (33 mg) with a yield of 29%; compound 10 was a white powder (18 mg) with a yield of 13%; compound 11 was a transparent oil, (7.8 mg) with a yield of 7%.

[0068] NMR and MS of compound 2:

[0069] 1 HNMR(CDCl3,500MHz)δ5.76(d,J=1.2Hz,1H),5.37(dd,J=5.4,1.3Hz,1H),4.28(d,J=1.1Hz,1H ),4.11(dt,J=7.1,6.3Hz,1H),3.99(s,1H),3.23(s,3H),2.64-2.63(m,1H),2.04(s,2H),1.87( d,J=1.4Hz,3H),1.70-1.56(m,4H),1.44(s,3H),1.37(d,J=2.0Hz,6H),1.35-1.22(m,4H),1.05 (s,3H),0.96(s,3H),0.80(d,J=6.6Hz,3H),0.73(t,J=8.5Hz,1H),0.48(dd,J=9.3,4.7Hz,1H); 13 CNMR(CDCl3,125MHz)δ151.10,137.42,134.54,131.41,112.75,94.17,88.57,84.46,75.87,74.09,60.41,50.8 6,36.25,35.88,29.11,27.32,26.08,25.25,25.00,23.98,19.41,17.62,17.51,17.31,15.08,14.21.ESIMSm / z 427[M+Na] + ;positive ion HRESIMSm / z427.2453(calcd for C 24 H 36 O5Na[M+Na] + ,427.2455).

[0070] NMR and MS of compound 3:

[0071] 11H NMR(CDCl3, 500 MHz) δ 5.87 - 5.85 (m, 1H), 5.73 (s, 1H), 5.30 (s, 1H), 4.34 (s, 1H), 4.19 (s, 1H), 2.79 - 2.74 (m, 1H), 2.48 - 2.36 (m, 3H), 2.14 - 2.10 (m, 1H), 1.82 - 1.81 (m, 4H), 1.75 (s, 3H), 1.59 (s, 3H), 1.43 (m, 7H), 1.21 - 1.17 (m, 8H), 1.08 - 1.02 (m, 4H), 0.99 (d, J = 6.8 Hz, 4H), 0.72 - 0.68 (m, 1H); 13 13C NMR(CDCl3, 125 MHz) δ 207.40, 173.61, 137.46, 135.82, 129.61, 128.10, 113.27, 94.74, 88.39, 76.45, 75.79, 44.12, 37.22, 32.52, 28.71, 28.03, 27.61, 27.15, 24.91, 23.75, 22.97, 22.92, 18.93, 15.51, 14.82, 9.18; ESI MS m / z 451 [M + Na] + ; positive ion HRESI MS m / z 451.2455 (calcd for C 26 H 36 O5Na [M + Na] + , 451.2455).

[0072] NMR and MS of Compound 4:

[0073] 1 1H NMR(CDCl3, 500 MHz) δ 5.86 (dd, J = 6.0, 1.4 Hz, 1H), 5.72 (s, 1H), 5.30 (d, J = 2.1 Hz, 1H), 4.35 (s, 1H), 4.21 - 4.20 (m, 1H), 3.48 (s, 1H), 2.78 - 2.75 (m, 1H), 2.46 - 2.29 (m, 2H), 2.12 (ddd, J = 15.9, 4.7, 2.8 Hz, 1H), 1.82 (s, 3H), 1.75 - 1.55 (m, 10H), 1.43 (s, 6H), 1.34 - 1.31 (m, 5H), 1.22 (s, 3H), 1.08 - 1.03 (m, 4H), 0.99 (d, J = 6.8 Hz, 3H), 0.93 - 0.87 (m, 4H), 0.70 (dt, J = 8.8, 5.7 Hz, 1H); 1313C NMR(CDCl3, 125 MHz) δ 207.49, 172.93, 135.88, 129.47, 128.21, 113.22, 94.69, 88.38, 77.28, 77.02, 76.77, 75.87, 44.08, 37.10, 34.31, 32.62, 31.28, 29.71, 28.72, 28.15, 27.20, 24.99, 24.65, 23.81, 23.07, 22.88, 22.33, 18.98, 15.54, 14.77, 13.92; ESIMS m / z 493 [M+Na] + ; positive ion HRESIMSm / z 493.2922 (calcd for C 29 H 42 O5Na [M+Na] + , 493.2924).

[0074] NMR and MS of Compound 5:

[0075] 1 1H NMR(CDCl3, 500 MHz) δ 5.88 (dd, J = 5.8, 1.5 Hz, 1H), 5.63 (s, 1H), 5.30 (s, 1H), 4.37 (s, 1H), 4.24 - 4.21 (m, 1H), 2.83 - 2.79 (m, 1H), 2.04 (dt, J = 15.8, 3.1 Hz, 1H), 1.79 (d, J = 14.6 Hz, 6H), 1.76 - 1.69 (m, 1H), 1.42 - 1.38 (m, 6H), 1.27 - 1.19 (m, 16H), 1.10 (dd, J = 10.7, 9.0 Hz, 1H), 1.01 - 0.96 (m, 6H), 0.70 - 0.66 (m, 1H); 13 13C NMR(CDCl3, 125 MHz) δ 208.32, 177.83, 138.27, 135.96, 128.90, 128.59, 112.79, 94.49, 88.36, 77.13, 76.05, 43.56, 38.98, 36.69, 32.97, 28.67, 28.63, 27.42, 27.30, 27.01, 26.52, 25.14, 24.43, 23.57, 22.60, 19.18, 16.15, 14.55; ESIMS m / z 495 [M+K] + ; positive ion HRESIMSm / z 495.2869 (calcd for C 28 H 40O5K[M+K] + ,495.2507).

[0076] NMR and MS of compound 6:

[0077] 1 H NMR(CDCl3,500MHz)δ5.89(d,J=5.4Hz,1H),5.69(s,1H),5.41(s,1H),4.91(d,J=9.4Hz,1H),4. 43(s,1H),4.30(dd,J=9.4,4.0Hz,1H),4.14(d,J=10.3Hz,1H),3.71(s,5H),2.75-2.72(m,1H),2 .10(d,J=16.1Hz,1H),1.93-1.90(m,1H),1.79(s,3H),1.76-1.67(m,5H),1.45-1.41(m,17H),1. 24-1.21(m,4H),1.09-1.02(m,6H),0.98(d,J=6.7Hz,6H),0.87-0.84(m,3H),0.71-0.67(m,1H); 13 C NMR (CDCl3, 125MHz) δ207.90,172.20,155.85,137.77,135.18,129.26,128.71,113.50,94.44,88.43,79.92,75.81,63.71,58.70,43.99,37. 26,36.94,32.47,29.70,28.70,28.60,28.28,27.97,27.12,24.77,23 .92,23.65,23.04,22.92,18.99,16.25,15.72,14.74,11.55;ESIMSm / z 608[M+Na] + ;positive ionHRESIMSm / z 608.4370(calcd for C 34 H 51 NO7Na[M+Na] + ,608.4370).

[0078] NMR and MS of compound 7:

[0079] 11H NMR(CDCl3, 500 MHz) δ 6.10 (s, 1H), 5.78 (d, J = 4.4 Hz, 1H), 5.30 (s, 1H), 5.01 (d, J = 9.4 Hz, 1H), 4.30 (dd, J = 9.5, 5.0 Hz, 1H), 4.02 (dd, J = 11.6, 3.5 Hz, 1H), 2.67 - 2.66 (m, 1H), 2.29 - 2.23 (m, 1H), 2.11 - 2.06 (m, 1H), 1.82 - 1.77 (m, 8H), 1.64 - 1.58 (m, 20H), 1.46 - 1.43 (d, J = 8.9 Hz, 13H), 1.32 (s, 3H), 1.25 (s, 3H), 1.11 (s, 3H), 1.06 (s, 3H), 0.97 (dd, J = 6.9, 3.9 Hz, 6H), 0.91 - 0.86 (m, 5H), 0.72 - 0.67 (m, 1H); 13 13C NMR(CDCl3, 125 MHz) δ 205.64, 175.17, 156.71, 134.24, 133.07, 131.48, 126.27, 85.99, 83.27, 79.80, 71.76, 63.71, 52.96, 52.48, 43.39, 42.16, 39.94, 30.82, 29.70, 28.39, 28.33, 28.29, 24.83, 24.72, 24.29, 23.32, 23.06, 22.90, 21.96, 21.18, 16.92, 15.47, 15.41; ESI MS m / z 572 [M + H] + ; positive ion HRESI MS m / z 572.4425 (calcd for C 33 1H 50 NO7 [M + H] + , 572.4425).

[0080] NMR and MS of Compound 8:

[0081] 11H NMR(CDCl3, 500 MHz) δ 5.92 (d, J = 6.4 Hz, 1H), 5.53 (s, 1H), 5.31 (s, 1H), 5.16 (d, J = 7.7 Hz, 1H), 4.46 (s, 3H), 2.88 (s, 1H), 2.62 (t, J = 7.4 Hz, 2H), 2.25 - 2.20 (dd, m, 1H), 2.14 - 2.00 (m, 5H), 1.85 - 1.83 (m, J = 12.7 Hz, 6H), 1.79 - 1.72 (m, 1H), 1.62 (s, 2H), 1.44 (s, 9H), 1.36 - 1.25 (m, 12H), 1.16 (t, J = 9.6 Hz, 1H), 1.00 - 0.97 (m, 7H), 0.89 - 0.82 (m, 2H), 0.73 - 0.70 (m, 1H); 13 13C NMR(CDCl3, 125 MHz) δ 207.67, 171.71, 155.13, 139.07, 135.20, 131.08, 127.62, 112.45, 93.76, 88.27, 79.96, 71.81, 53.00, 43.67, 35.51, 33.91, 33.22, 29.81, 29.71, 29.37, 28.79, 28.31, 27.70, 26.32, 24.30, 24.03, 22.32, 19.83, 19.17, 15.68, 15.34, 14.13, 13.94; ESIMS m / z 626 [M + Na] + ; positive ion HRESIMS m / z 626.3923 (calcd for C 33 H 49 NO7SNa [M + Na] + , 626.3919).

[0082] NMR and MS of Compound 9:

[0083] 11H NMR (CDCl3, 500 MHz) δ 5.88 (d, J = 5.2 Hz, 1H), 5.80 (s, 1H), 5.36 (s, 1H), 4.80 (d, J = 8.8 Hz, 1H), 4.46 (s, 1H), 4.36 - 4.32 (m, 1H), 43.99 - 3.96 (m, 1H), 3.73 (s, 1H), 2.70 - 2.68 (m, 1H), 2.16 (dd, J = 15.3, 4.1 Hz, 1H), 1.80 (s, 3H), 1.76 - 1.73 (m, 2H), 1.69 - 1.67 (m, 5H), 1.50 - 1.43 (m, 17H), 1.19 (s, 3H), 1.03 (s, 3H), 0.98 (d, J = 6.8 Hz, 3H), 0.94 - 0.91 (m, 6H), 0.71 - 0.67 (m, 1H); 13 13C NMR (CDCl3, 125 MHz) δ 207.57, 173.23, 155.60, 150.93, 137.13, 134.92, 130.19, 128.48, 127.56, 113.88, 94.96, 88.31, 79.95, 76.35, 75.38, 68.23, 63.72, 52.45, 44.33, 40.53, 38.14, 31.85, 28.61, 28.30, 27.45, 26.85, 24.71, 23.58, 23.31, 22.55, 21.14, 18.54, 15.86, 15.20; ESIMS m / z 608 [M + Na] + ; positive ion HRESIMS m / z 608.4370 (calcd for C 34 H 51 NO7Na [M + Na] + , 608.4374).

[0084] NMR and MS of Compound 10:

[0085] 11H NMR (CDCl3, 500 MHz) δ 5.89 (d, J = 4.8 Hz, 1H), 5.73 (s, 1H), 5.37 (s, 1H), 5.29 (s, 1H), 5.10 (d, J = 8.2 Hz, 1H), 4.42 (s, 1H), 4.38 - 5.34 (m, 1H), 4.13 - 4.09 (m, 1H), 3.72 (s, 1H), 3.67 (s, 4H), 2.73 - 2.70 (m, 1H), 2.43 - 2.40 (m, 2H), 2.23 (ddd, J = 19.2, 9.5, 5.9 Hz, 1H), 2.14 - 2.10 (m, 1H), 1.93 - 1.86 (m, 1H), 1.80 - 1.70 (m, 1H), 1.46 - 1.43 (m, 21H), 1.26 - 1.23 (mz, 1H), 1.20 - 1.19 (m, 4H), 1.06 - 1.02 (m, 6H), 0.98 (d, J = 6.8 Hz, 4H), 0.69 (dt, J = 8.6, 6.0 Hz, 1H); 13 13C NMR (CDCl3, 125 MHz) δ 207.52, 173.10, 171.89, 155.49, 137.51, 134.81, 129.60, 128.57, 113.64, 94.59, 88.38, 80.19, 75.68, 63.71, 53.38, 51.88, 44.14, 37.56, 32.28, 30.18, 28.63, 28.27, 27.88, 27.02, 24.54, 23.75, 23.00, 22.89, 18.82, 15.57, 14.90, 14.20, 14.12; ESI MS m / z 616 [M + H] + ; positive ion HRESI MS m / z 616.3472 (calcd for C 34 H 50 NO9 [M + H] + , 616.3472).

[0086] NMR and MS of Compound 11:

[0087] 1H NMR (CDCl3, 600MHz) δ7.33-7.28(m,3H),7.22(d,J=7.1Hz,2H),5.92(d,J=5.5Hz,1H),5.58(s,1H),5.38( s,1H),4.98(d,J=8.7Hz,1H),4.62(d,J=5.6Hz,1H),4.40(s,1H),4.27(s,1H),3.74(s,6H),3.33-3.30(m ,J=13.7,4.6Hz,1H),2.94-2.91(m,J=13.7,7.8Hz,2H),2.09-2.04(m,3H),1.92(d,J=35.3Hz,4H),1.83( s,3H),1.81(s,2H),1.62(s,3H),1.39-1.25(m,29H),1.02-0.98(m,10H),0.89-0.84(m,2H),0.72(s,1H); 13 CNMR(CDCl3,150MHz)δ207.73,171.33,154.87,136.14,135.23,130.91,129. 49,128.83,128.53,127.03,112.75,93.92,88.23,79.86,71.79,63.69,62.1 8,54.73,43.74,38.80,35.97,33.61,29.68,28.96,28.68,28.23,27.94,27. 57,25.93,24.18,23.80,22.40,20.81,19.64,19.14,15.62,14.14;ESIMSm / z 620[M+H] + ;positiveionHRESIMSm / z620.4309(calcdforC 37 H 50 NO7[M+H] + ,620.4317).

[0088] Example 2

[0089] The preparation routes of compounds 12, 13, 14, and 15 are as follows: Figure 2 The preparation method is as follows:

[0090] Compound 12: Compound 20-deoxyingenol (40.0 mg, 0.12 mmol) was dissolved in anhydrous dichloromethane (4 ml). DMAP (1.50 mg, 0.012 mmol), acetic acid (17.8 mg, 0.24 mmol), and EDCI (46.0 mg, 0.24 mmol) were added sequentially. The mixture was stirred at room temperature. After 12 h, the reaction was detected by TLC. The mixture was concentrated under reduced pressure and purified by flash column chromatography to obtain compound 12 (23 mg) in a yield of 92%.

[0091] The preparation methods of 13, 14, and 15 are basically the same as those of 12, except that the organic acid raw materials are replaced: compound 13 is replaced by acetic acid, compound 14 is replaced by butyric acid, and compound 15 is replaced by pivalic acid.

[0092] The obtained compound 13 was a colorless oil 40 mg, with a yield of 93%; the obtained compound 14 was a colorless oil 18 mg, with a yield of 90%; and the obtained compound 15 was a yellow oil 101.7 mg, with a yield of 74%.

[0093] NMR and MS of compound 12:

[0094] 1 HNMR(CDCl3,500MHz)δ6.08(d,J=1.4Hz,1H),5.87-5.80(m,1H),5.21(s,1H),4.9 1(s,1H),4.21-4.13(m,1H),4.47(s,1H),2.49-2.46(m,1H),2.32-2.24(m,4H),2. 12(d,J=1.7Hz,3H),1.76-1.70(m,5H),1.54(s,3H),1.24(s,3H),1.07(s,3H),1. 04(s,3H),0.97(d,J=7.2Hz,3H),0.90(dd,J=11.9,8.4Hz,2H),0.72-0.62(m,1H); 13 C NMR(CDCl3,125MHz)δ206.13,172.49,135.14,134.28,132.60,126.41,85.75,82.65,77.24,71.79,53.4 3,43.37,38.72,31.10,29.70,28.44,24.35,23.16,22.96,21.20,20.76,16.89,15.56,15.40;ESIMSm / z 417[M+Na] + ;positiveionHRESIMSm / z417.2289(calcdforC 24 H 32O6Na[M+Na] + ,417.2286).

[0095] NMR and MS of compound 13:

[0096] 1 HNMR(CDCl3,500MHz)δ6.08(d,J=1.3Hz,1H),5.85-5.83(m,1H),5.25(s,1H),4.92(s,1H),4 .20-4.17(m,1H),2.69-2.61(m,1H),2.55-2.46(m,2H),2.39(qd,J=7.6,2.0Hz,2H),2.32-2. 27(m,1H),1.75-1.70(m,4H),1.54(s,3H),1.21(t,J=7.5Hz,3H),1.16(t,J=7.6Hz,3H),1.08 (s,3H),1.05(s,3H),0.98(d,J=7.2Hz,3H),0.91(dd,J=11.9,8.4Hz,1H),0.69-0.64(m,1H); 13 C NMR(CDCl3,125MHz)δ206.18,175.73,174.34,135.33,134.39,132.39,126.20,85.74,82.33,71.76,43.32 ,38.72,30.96,28.40,27.80,27.30,24.29,23.11,22.93,21.20,16.86,15.54,15.39,9.25,8.94.ESIMSm / z 467[M+Na] + ;positive ion HRESIMSm / z 467.1610(calcd forC 26 H 36 O6Na[M+Na] + ,467.1618).

[0097] NMR and MS of compound 14:

[0098] 1HNMR(CDCl3,500MHz)δ6.08(d,J=1.3Hz,1H),5.87-5.83(m,1H),4.91(s,1H),4.18(dd,J=11.3,4.3Hz,1H),2.62-2.43(m,3H),2.39-2.26(m,3H),1.75-1.63(m,10H),1.54(s,3H),1.06(d,J=16.6Hz,6H),0.99-0.95(m,9H),0.93-0.89(m,1H),0.69-0.64(m,1H); 13 CNMR(CDCl3,126MHz)δ206.25,175.02,173.52,135.40,134.44,132.45,126.25,85.81,82.43,71.83,43.39,38.76,36.53,35.81,30.97,29.71,28.45,24.35,23.16,22.97,21.32,18.70,18.10,16.91,15.58,15.43,13.75,13.61; ESIMSm / z495[M+Na] + ; positive ionHRESIMSm / z495.1882(calcdforC 28 H 40 O6Na[M+Na] + ,495.1882).

[0099] NMR and MS of Compound 15:

[0100] 1 HNMR(CDCl3,500MHz)δ6.05(d,J=1.9Hz,1H),5.83-5.79(m,1H),5.27(s,1H),4.89(s,1H),4.22-4.14(m,1H),2.56-2.50(m,1H),2.31-2.25(m,1H),1.71(d,J=1.8Hz,3H),1.73-1.67(m,1H),1.51(s,3H),1.29(s,9H),1.18(s,9H),1.07(s,3H),1.03(s,3H),0.96(d,J=7.2Hz,3H),0.91-0.87(m,1H),0.67-0.62(m,1H); 13CNMR(CDCl3,125MHz)δ206.31,179.24,176.26,136.02,134.59,132.18,126.06,86.05,81.99,72.13,43.47,39.59 ,38.91,38.85,30.71,28.47,27.44,27.20,26.53,24.38,23.16,22.99,21.45,16.89,15.69,15.49,0.01; ESIMSm / z 523[M+Na] + ;positiveionHRESIMSm / z523.3005(calcdforC 30 H 44 O6Na[M+Na] + ,523.3001).

[0101] Test Example 1

[0102] Initial screening test of the compound (20 μM) on its ability to promote autophagic flux in HM-mCherry-GFP-LC3 cells.

[0103] In order to quickly screen whether a compound has the ability to enhance cellular autophagic flow, the present invention constructed human microglia cells (HM cells) that can stably express mCherry-GFP-LC3, namely HM mCherry-GFP-LC3, and carried out drug treatment for 24 hours. The small molecule drug was screened by flow cytometry to see whether it has the activity of enhancing cellular autophagic flow. The working principle of this cell line is that in cells that do not undergo autophagy and cells containing autophagosomes, due to the co-expression of mCherry and GFP, the cells exhibit yellow fluorescence. After the autophagosomes fuse with the lysosomes to form autophagolysosomes, the acidic lysosomal environment quenches the acid-sensitive GFP fluorescence, while mCherry is unaffected, thereby causing the autophagolysosomes to exhibit red fluorescence. Therefore, red fluorescence can indicate the smoothness of autophagolysosome formation. The more red fluorescence and the less green fluorescence, the smoother the flow from the autophagosome to the autophagolysosome stage. On the contrary, the fusion of autophagosomes and lysosomes is inhibited, and the autophagolysosome process is blocked. Therefore, the activity of small molecule compounds can be evaluated by counting the ratio of cells that only emit red fluorescence.

[0104] Subculture the cells: 1 to 3, 1 bottle of T75 is subcultured into 3 bottles of T75; plate: digest 3 bottles of cells into each T75, retain 2mL for further subculture, add the rest to mix, plate into 9 12-well plates, 1mL per well, and culture for 24 hours; drug treatment: replace the culture medium in the 12-well plate with 5% FBS in advance, and then add drug treatment.

[0105] Different compounds (20 μM) promoted the activity of cell autophagy flux. Figure 3 As shown. Figure 3 It can be seen that the tested compounds 4, 6, 7, 8, 9, 10, 11, 12, 13 and 14 can promote cellular autophagic flux within the 20 micromolar range, and the activities of compounds 10 and 13 are better than the control compound Torin-1.

[0106] Test Example 2

[0107] Effects of lysosome formation, TFEB translocation, and lipid droplet clearance

[0108] (1) Screening of lysosomal biogenesis inducers

[0109] HeLa and HepG2 cell lines were cultured at 37°C and 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, HyClone), 100 U / mL penicillin, and 100 μg / mL streptomycin. HeLa and HepG2 cells were plated at 85% density in 24-well plates and treated with 20 μM of the test compound (n=3). After 3 hours, fresh medium containing 0.3 μM LysoTrackerRed DND-99 was replaced and incubated for 30 minutes. After washing, the cells were observed by confocal microscopy. Positive compounds screened initially were validated using multiple concentration gradients (1 μM, 10 μM, 20 μM, 40 μM) (n=3), and the lysosomal staining step was repeated.

[0110] (2) Construction of TFEB-EGFP stable transfection cell line

[0111] HeLa cells were transfected with the pEGFP-N2-TFEB plasmid carrying the neomycin (G418) resistance gene. After 48 hours, the culture medium was switched to 5 μg / mL G418 and selected for 2–3 weeks. Single colonies were selected and expanded in 96-well plates, and stable expression strains were identified by fluorescence microscopy.

[0112] (3) Subcellular component separation

[0113] Cells were lysed on ice for 15 minutes in NP-40 lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM KCl, 5 mM MgCl2, 0.5% NP-40) and centrifuged at 1000 g for 3 minutes. The supernatant contained cytoplasmic / membrane proteins, and the pellet was resuspended and sonicated (20% power, 5 seconds x 3) to obtain nuclear proteins.

[0114] (4) Lipid droplet clearance experiment

[0115] HepG2 cells in confocal microscopy plates were treated with 100 μM oleic acid for 12 hours to induce lipid droplet formation. Oleic acid was then removed and fresh culture medium containing various concentrations of HEP14 was used. Cells were stained with 1 μg / mL BODIPY for 30 minutes before assay, and time-course analysis was performed by confocal microscopy or flow cytometry.

[0116] The effects of lysosome formation, TFEB translocation, and lipid droplet clearance are as follows Figure 4 As shown, Figure 4 In the figure, A shows the situation of natural Lyso Tracker Red in HeLa cells after treatment with drugs (20 μM) or Torin-1 (1 μM) for 3 hours, B shows the situation of natural Lyso Tracker Red in HepG2 cells after treatment with drugs (20 μM) or Torin-1 (1 μM) for 3 hours, C shows the situation of natural Lyso Tracker Green in HeLa cells after treatment with drugs (1-20 μM) for 3 hours, D shows the subcellular localization of TFEB-EGFP or EGFP-TFE3 in HeLa cells after treatment with drugs (20 μM, 3 hours), E shows the image of HeLa cells treated with drugs (20 μM, 3 hours) and co-stained with BODIPY-pepstatin A (1 μM). LysoTracker Red staining showed that the number of lysosomes in the 10 and 13 treatment groups increased significantly ( Figure 4 A and B in the figure) and showed a dose-dependent effect ( Figure 4 In cells overexpressing TFEB-EGFP, both compounds specifically promoted TFEB nuclear translocation, while the subcellular localization of TFE3 was unaffected ( Figure 4 D), indicating that their effects are pathway selective. Given that 10 and 13 can induce lysosome formation, the present invention further explored whether they enhance lysosome-dependent clearance function. In the HepG2 cell model induced by oleic acid to form lipid droplets, treatment with the two compounds significantly reduced the number of lipid droplets; however, when the lysosomal degradation inhibitor bafilomycin A1 (BFA1) was added, this clearance effect was completely blocked ( Figure 4 E). This result confirms that 10 and 13 promote lipid droplet degradation by enhancing lysosomal activity rather than through other alternative pathways.

[0117] (5) Cytotoxicity assay

[0118] According to the experimental steps of CCK-8, HepG2 cells were cultured on culture plates, 10 μL of CCK-8 solution was added to each well, the culture plates were placed in an incubator and incubated for 1 to 4 hours, and the absorbance at 450 nm was measured with a microplate reader to determine cell viability. After 6 hours of drug treatment (1 to 20 μM), the absorbance at 450 nm was measured with a microplate reader. The results showed that the absorbance of HeLa cells in the treated group was consistent with that of the non-treated group ( Figure 5 The results showed that no compound at different concentrations had any killing effect on the cells during the 6-hour treatment.

[0119] Test Example 3

[0120] Western Blot analysis of autophagy-related proteins:

[0121] Human cervical cancer HeLa cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) under standard conditions (37°C, 95% humidity, 5% CO2). Cells were seeded in 6-well plates containing growth medium and treated with the indicated drugs. After 24 hours of drug exposure, cells were harvested for protein extraction and immunoblotting analysis.

[0122] Western Blot Analysis:

[0123] Cultured HeLa cells were lysed using RIPA buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and 1 mM PMSF). Protein concentration was quantified using a BCA protein assay kit. Equal amounts of protein (20 μg per lane) were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a 0.45 μm nitrocellulose membrane. The membrane was blocked in Tris-buffered saline (TBS) containing 5% (w / v) skim milk for 2 hours at room temperature and then incubated overnight at 4°C with the following primary antibodies: anti-LAMP1, anti-MAP1LC3B / LC3B, and anti-CTSD. Total protein levels were quantified by Ponceau S staining of the same membrane. After washing three times with TBST containing 0.1% Tween-20 for 3 minutes each, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody, peroxidase-conjugated goat anti-rabbit IgG (H+L), for 1 hour at room temperature. The secondary antibody was diluted 1:10,000 in blocking buffer. Protein bands were visualized using the ECL Western Blot Detection Kit. Densitometric analysis was performed using ImageJ software, and Ponceau S staining was used as a loading control.

[0124] Human cervical cancer cell line (HeLa cells) was treated with compounds 10 and 13 for 24 hours and then lysed and protein was extracted. Dimethyl sulfoxide (DMSO) was used as a negative control and autophagy inducer Torin 1 was used as a positive control. The results of Western blot analysis of compounds 10 and 13 are shown in Figure 2. Figure 6 As shown, Figure 6 In the figure, A is a representative Western blot result showing the protein levels of LAMP1, CTSD, LC3-II / LC3-I, and Ponceau S staining in HeLa cells treated with 10, 13, or Torin-1, B is a quantitative analysis of the LAMP1 protein level in HeLa cells based on two independent experiments, C is a quantitative analysis of the LC3-II / LC3-I protein level in HeLa cells based on two independent experiments, and D is a quantitative analysis of the Ponceau S protein level in HeLa cells based on two independent experiments.

[0125] like Figure 6 As shown, lysosomal associated membrane protein 1 (LAMP1) was upregulated in a dose-dependent manner in the compound 13-treated group ( Figure 6 A and B in Figure ), the level of cathepsin D (CTSD), a key lysosomal protease, was also significantly increased ( Figure 6 A and C in Figure 1), indicating that lysosomal function was enhanced. In addition, compound 13 could increase the ratio of the lipidated form (LC3-II) to the unmodified form (LC3-I) of microtubule-associated protein 1 light chain 3 (LC3) ( Figure 6 These results suggest that compound 13 can effectively activate the autophagy-lysosome system and has the potential to become an ideal autophagy inducer.

[0126] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A 20-deoxy-ingenol ester derivative having a structure shown in Formula I or Formula II: In Formula I, R is In formula II, R1 and R2 are 2. The method for preparing the 20-deoxy-ingenol ester derivatives according to claim 1, characterized in that: When the 20-deoxyingenol ester derivative has the structure shown in Formula I, the preparation method comprises the following steps: 20-deoxyingenol ester having the structure shown in formula a, acetone, 2,2-dimethoxypropane and p-toluenesulfonic acid are mixed and subjected to an acetonide protection reaction to obtain a compound having the structure shown in formula b: A compound having a structure shown in formula b is subjected to a first esterification reaction with a compound having an R-OH structure to obtain a 20-deoxyingenol ester derivative; When the 20-deoxy-ingenol ester derivative has the structure shown in Formula II, the preparation method comprises the following steps: The 20-deoxy-ingenol ester having the structure shown in formula a is mixed with an organic acid and subjected to a second esterification reaction to obtain a 20-deoxy-ingenol ester derivative, wherein the organic acid is acetic acid, propionic acid, butyric acid or pivalic acid.

3. The preparation method according to claim 2, characterized in that The molar ratio of 20-deoxyingenol ester having the structure shown in formula a to 2,2-dimethoxypropane and p-toluenesulfonic acid is 1:4-6:0.4-0.7; The temperature of the acetone protection reaction is 15-25° C., and the time is 10-12 hours.

4. The preparation method according to claim 2, characterized in that The first esterification reaction is carried out in the presence of a catalyst, and the catalyst is DMAP and EDCI.

5. The preparation method according to claim 2 or 4, characterized in that The temperature of the first esterification reaction is room temperature, and the time is 10 to 12 hours.

6. The preparation method according to claim 2, characterized in that The second esterification reaction is carried out in the presence of a catalyst, which is DMAP and EDCI.

7. The preparation method according to claim 2, characterized in that The temperature of the second esterification reaction is room temperature, and the time is 10 to 12 hours.

8. Use of the 20-deoxyingenol ester derivative according to claim 1 or the 20-deoxyingenol ester derivative prepared by the preparation method according to any one of claims 2 to 7 in the preparation of drugs promoting cell autophagy.

9. The use according to claim 8, characterized in that The drug promoting cell autophagy includes one or more of anti-heart disease drugs, anti-myopathy drugs, anti-degenerative disease drugs and anti-tumor drugs.

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