Oridonin A-epoxide modified derivatives, their preparation methods and uses

Abstract

This invention discloses 6,20-epoxy A-ring modified derivatives of oridonin, their preparation methods, and uses, belonging to the field of natural medicine and medicinal chemistry. Using oridonin as a lead compound, this invention designed and synthesized a series of 6,20-epoxy oridonin derivatives with α,β-unsaturated ketones introduced into the A ring. The antitumor activity of the synthesized derivatives was tested. The results showed that the oridonin derivatives prepared in this invention exhibited good antitumor activity against various tumor cells (human breast cancer cells MCF-7, human lung adenocarcinoma cells A549, human liver cancer cells Bel-7402, human liver cancer cells HepG2, human pancreatic cancer cells Panc-1, and MIA-PaCa-2), demonstrating potential as anticancer drugs.

Description

Technical Field

[0001] This invention belongs to the field of natural medicine and medicinal chemistry, specifically relating to the preparation methods of a series of oridonin A 6,20-epoxy A-ring modified derivatives and their use in anti-tumor applications. Background Technology

[0002] Oridonin A is an enantio-kaurane diterpenoid with antitumor properties that has attracted close attention from medicinal chemists in recent years due to its diverse pharmacological effects, including antitumor, anti-inflammatory, antibacterial, and antioxidant effects. In vitro and in vivo studies have shown that oridonin A has inhibitory effects on the proliferation of various tumors with high morbidity and mortality rates, and shows good therapeutic potential against cancers such as breast cancer, pancreatic cancer, lung cancer, and liver cancer. However, the inhibitory effect of oridonin A on tumor cell proliferation is not yet satisfactory, and there is an urgent need for structural improvement and optimization to obtain oridonin A derivatives with superior effects against various tumor cells. Summary of the Invention

[0003] In order to find oridonin derivatives with better anti-tumor activity, the present invention aims to provide oridonin 6,20-epoxy A-ring modified derivatives, their preparation methods and uses. The present invention uses oridonin as a lead compound, designs and synthesizes 6,20-epoxy oridonin derivatives with α,β-unsaturated ketones introduced into the A ring, and tests the anti-tumor activity of the synthesized derivatives.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] This invention provides a 6,20-epoxy A-ring modified derivative of oridonin and its pharmaceutically acceptable salt, the general structural formula of which is shown below:

[0006]

[0007] Wherein, R is an alkyl group containing 1-8 carbon atoms, a cycloalkyl group containing 3-8 carbon atoms, an aromatic ring, a substituted aromatic ring, an aromatic heterocycle, or a substituted aromatic heterocycle containing 5-12 carbon atoms, and the substituents include halogens, alkyl groups containing 1-8 carbon atoms, cycloalkyl groups containing 3-8 carbon atoms, methoxy groups, or methylenedioxy groups, and the aromatic heterocycle contains 1-3 N, O, or S heteroatoms.

[0008] Preferably, R is an alkyl group containing 1-6 carbon atoms, a cycloalkyl group containing 3-8 carbon atoms, an aromatic ring, a substituted aromatic ring, a heterocyclic aromatic ring, or a substituted heterocyclic aromatic ring containing 6-9 carbon atoms, wherein the substituent includes halogen, alkyl group containing 1-6 carbon atoms, methoxy or methylenedioxy, and the heterocyclic aromatic ring contains 1-2 N or O heteroatoms.

[0009] More preferably, R is an alkyl group containing 1-5 carbon atoms, a cycloalkyl group containing 3-7 carbon atoms, an aromatic ring, a substituted aromatic ring, a heterocyclic aromatic ring, or a substituted heterocyclic aromatic ring containing 6-9 carbon atoms, wherein the substituent includes halogen, an alkyl group containing 1-4 carbon atoms, a methoxy group, or a methylenedioxy group, and wherein the heterocyclic aromatic ring contains 1-2 N heteroatoms.

[0010] Based on the above technical solution, the structural formula of the oridonin A-cyclic modified derivative is further shown below:

[0011]

[0012] On the other hand, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned oridonin A 6,20-epoxy A-ring modified derivative, its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier.

[0013] On the other hand, the present invention provides a method for preparing the above-mentioned oridonin 6,20-epoxy A-ring modified derivative, and the synthetic route of the oridonin 6,20-epoxy A-ring modified derivative is as follows:

[0014]

[0015] Oridonin A1 was protected with acetone fork to give compound 2 with 7,14-hydroxyl protected position. Compound 2 reacted with methanesulfonyl chloride to give compound 3, which was then eliminated to give compound 4. Compound 4 was oxidized in 1,4-dioxane with selenium dioxide to give compound 5, which was then oxidized with Jones reagent to give compound 6 with α,β-unsaturated ketone in ring A. Compound 6 was subjected to DAST to form compound 7 with 6,20-epoxy structure. Finally, it was subjected to esterification under EDCI / DMAP conditions to give compounds EpskA1-EpskA24.

[0016] On the other hand, the present invention provides the use of the above-mentioned oridonin A 6,20-epoxy A-ring modified derivative and its pharmaceutically acceptable salts and pharmaceutical compositions in the preparation of medicaments for treating tumors.

[0017] Based on the above technical solution, the tumor is further defined as breast cancer, pancreatic cancer, liver cancer, or lung cancer.

[0018] Based on the above technical solution, the breast cancer includes human breast cancer cells MCF-7, the lung cancer includes human lung adenocarcinoma cells A549, the liver cancer includes human liver cancer cells Bel-7402 and human liver cancer cells HepG2, and the pancreatic cancer includes human pancreatic cancer cells Panc-1 and MIA-PaCa-2.

[0019] The advantages of this invention over the prior art are as follows:

[0020] The oridonin derivative prepared in this invention has good anti-tumor activity against a variety of tumor cells (human breast cancer cells MCF-7, human lung adenocarcinoma cells A549, human liver cancer cells Bel-7402, human liver cancer cells HepG2, human pancreatic cancer cells Panc-1 and MIA-PaCa-2), and has the potential to be used as an anti-cancer drug. Detailed Implementation

[0021] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0022] Example 1

[0023] Oridonin A (1364 mg, 1 mmol) was dissolved in anhydrous acetone, and DMAP (12.2 mg, 0.1 mmol), TsOH (103.2 mg, 0.6 mmol), and 2,2-methoxypropane (2 mL) were added. The mixture was heated under reflux for 5 h, the solvent was removed by vacuum distillation, the mixture was extracted with dichloromethane, washed with saturated sodium bicarbonate and sodium chloride solutions, dried over anhydrous sodium sulfate, and concentrated to give compound 2. Compound 2 (404 mg, 1 mmol) was dissolved in anhydrous DCM, and DMAP (12.2 mg, 0.1 mmol), MsCl (1 mL), and TEA (0.5 mL) were added. The mixture was reacted at room temperature for 12 h, the solvent was removed by vacuum distillation, the mixture was extracted with dichloromethane, washed with saturated sodium bicarbonate and sodium chloride solutions, dried over anhydrous sodium sulfate, and concentrated to give compound 3. Compound 3 (482 mg, 1 mmol) was dissolved in anhydrous DMF, and lithium bromide (347 mg, 4 mmol) and lithium carbonate (222 mg, 3 mmol) were added. The mixture was reacted at 110 °C for 4 h, then extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and subjected to silica gel column chromatography (petroleum ether: ethyl acetate = 4:1) to give compound 4. Compound 4 (386 mg, 1 mmol) was dissolved in 1,4-dioxane, and selenium dioxide (30.1 mg, 0.2 mmol) was added. The mixture was reacted at 110 °C for 16 h. After the reaction was complete, the solvent was removed by vacuum distillation, and the mixture was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and subjected to silica gel column chromatography (petroleum ether: ethyl acetate = 10:1) to give compound 5. Compound 5 (40.2 mg, 0.1 mmol) was dissolved in anhydrous DCM, 0.5 mL of Jones' reagent was added, and the mixture was reacted at room temperature for 0.5 h. Then, 0.5 mL of ethylene glycol was added, followed by extraction with dichloromethane, washing with sodium chloride solution, drying on anhydrous sodium sulfate, and concentration to give compound 6. Compound 6 (40 mg, 0.1 mmol) was dissolved in anhydrous DCM, 0.2 mL of DAST was added, and the mixture was reacted in an ice-water bath for 1 h. After the reaction, the solvent was removed by vacuum distillation, followed by extraction with dichloromethane, washing with saturated sodium bicarbonate solution and saturated sodium chloride solution, drying on anhydrous sodium sulfate, and concentration to give the target compound 7 as a white solid, with a yield of 9.1%. 1H NMR(600MHz, CDCl3)δ6.66(d,J=10.3Hz,1H),6.15(s,1H),6.02(d,J=10.3Hz, 1H),5.57(s,1H),5.29(s,1H),4.66(s,1H),4.33(s,1H),4.26(d,J=9.3Hz,1H ),3.92(d,J=9.5Hz,1H),3.16(d,J=11.8Hz,1H),2.47(s,1H),2.21(dd,J=11. 9,5.4Hz,1H),1.92-1.75(m,2H),1.75-1.62(m,1H),1.25(s,3H),1.24(s,3H); 13 C NMR (151MHz, CDCl3) δ203.88,201.28,198.62,147.18,142.00,128.24,120.58,79.59,72.64 ,69.43,63.35,54.45,48.22,47.67,43.16,41.17,27.99,24.13,22.20,18.33; HRMS(ESI)m / z calcd forC 20 H 22 O5Na[M+Na] + 365.1365, found 365.1359.

[0024] The synthesis route is shown below:

[0025]

[0026] Example 2

[0027] Compound 7 (34.2 mg, 0.1 mmol) was dissolved in anhydrous DCM, and DMAP (6.1 mg, 0.05 mmol), EDCI (9.6 mg, 0.05 mmol), and acetic acid (1.5 eq) were added. The mixture was reacted overnight at room temperature. The solvent was removed by vacuum distillation, and the mixture was extracted with dichloromethane, washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and subjected to silica gel column chromatography (petroleum ether: ethyl acetate = 10:1) to give compound EpskA1 as a white solid in 4.1% yield. 1H NMR(600MHz, CDCl3) δ6.68(d,J=10.4Hz,1H),6.14(s,1H),6.03(d,J=10.4Hz,1H),5.7 1(s,1H),5.50(s,1H),4.43(d,J=9.4Hz,1H),4.32(s,1H),4.00(d,J=9.5Hz,1H),3.20 (dt,J=8.0,1.5Hz,1H),2.64-2.50(m,1H),2.30(s,1H),2.26-2.21(m,1H),2.00(s,3H ),2.00-1.94(m,1H),1.88-1.79(m,1H),1.79-1.68(m,1H),1.25(s,3H),1.21(s,3H); 13 C NMR(151MHz, CDCl3)δ202.22,201.04,198.63,170.12,147.34,142.25,129.18,119.67,82.54,75.22 ,71.20,64.84,57.32,50.40,50.09,42.59,42.04,29.83,24.56,23.08,21.13,17.89; HRMS(ESI)m / z calcd forC 22 H 24 O6Na[M+Na] + 407.1471, found 407.1465.

[0028] The structure of compound EpskA1 is shown below:

[0029]

[0030] Example 3

[0031] Compound EpskA2, a white solid, was prepared according to the synthesis method in Example 2, with a yield of 5.3%. 1H NMR (600MHz, CDCl3) δ6.67(d,J=10.4Hz,1H),6.14(s,1H),6.02(d,J=10.4Hz,1H),5.66(s,1H),5 .50(s,1H),4.41(d,J=9.4Hz,1H),4.32(s,1H),4.00(d,J=9.5Hz,1H),3.21(dt,J=7.9,1.5Hz,1H ),2.59-2.49(m,1H),2.29(s,1H),2.25-2.20(m,1H),2.06-1.93(m,1H),1.88-1.78(m,1H),1.77 -1.70(m,1H),1.60-1.51(m,1H),1.25(s,3H),1.21(s,3H),1.03-0.90(m,2H),0.88-0.79(m,2H); 13 C NMR (151MHz, CDCl3) δ202.28,201.04,198.37,173.88,146.98,142.26,129.78,119.99,82.57,75.11,71.2 1,65.49,57.48,51.64,50.12,42.59,42.06,29.76,24.55,23.04,17.83,13.19,9.25,8.92; HRMS(ESI)m / z calcd for C 24 H 26 O6Na[M+Na] + 433.1627, found 433.1622.

[0032] The structure of compound EpskA2 is shown below:

[0033]

[0034] Example 4

[0035] Compound EpskA9 was prepared as a white solid with a yield of 5.1% by the synthesis method described in Example 2. 1H NMR (600MHz, CDCl3) δ9.00(d,J=4.4Hz,1H),8.53(d,J=1.9Hz,1H),8.31(d,J=8.3Hz,1H),8.23(dd,J=8 .8,1.9Hz,1H),8.14(d,J=8.8Hz,1H),7.48(dd,J=8.3,4.2Hz,1H),6.72(d,J=10.4Hz,1H),6.22(s,1H) ,6.06(d,J=10.4Hz,1H),5.94(s,1H),5.54(s,1H),4.50(d,J=9.4Hz,1H),4.35(s,1H),4.06(d,J=9.4H z,1H),3.45(m,1H),2.72-2.60(m,1H),2.37(s,1H),2.35-2.31(m,1H),1.95-1.58(m,4H),1.26(s,6H); 13 C NMR (151MHz, CDCl3) δ202.21,200.79,198.35,164.83,146.92,142.33,141.91,137.92,131.67,129.64,129.21,129.16,128.81,128. 68,127.29,121.83,119.85,82.44,76.42,71.21,64.91,57.09,50.51,50.06,42.46,42.07,29.73,24.44,22.89,17.68; HRMS(ESI)m / z calcd for C 30 H 28 NO6[M+H] + 498.1917, found 498.1911.

[0036] The structure of compound EpskA9 is shown below:

[0037]

[0038] Example 5

[0039] Compound EpskA21, a white solid, was prepared according to the synthesis method in Example 2, with a yield of 3.4%. 1H NMR (600MHz, CDCl3) δ8.65(d,J=6.1Hz,2H),7.57(d,J=16.0Hz,1H),7.38(d,J=6.1Hz,2H),6.69( d,J=10.4Hz,1H),6.53(d,J=16.0Hz,1H),6.19(s,1H),6.05(d,J=10.4Hz,1H),5.82(s,1H),5.54( s,1H),4.47(d,J=9.4Hz,1H),4.34(s,1H),4.04(d,J=9.5Hz,1H),3.32(m,1H),2.62(m,1H),2.34( s,1H),2.29(m,1H),2.04(m,1H),1.92-1.76(m,4H),1.75-1.65(m,1H),1.26(s,3H),1.22(s,3H); 13 C NMR (151MHz, CDCl3) δ201.98,200.80,198.38,164.67,150.03,146.95,143.06,141.91,129.19,122.12,119.82, 82.42,75.92,71.17,64.80,57.34,50.40,50.02,42.47,42.00,29.72,24.46,22.92,17.71; HRMS(ESI)m / zcalcd for C 28 H 28 NO6[M+H] + 474.1917, found 474.1911.

[0040] The structure of compound EpskA21 is shown below:

[0041]

[0042] The other compounds EpskA3-EpskA8, EpskA10-EpskA20, and EpskA22-EpskA24 were prepared according to the synthesis method in Example 2.

[0043] Example 6

[0044] This embodiment examines the pharmacological activity of the prepared compound.

[0045] 1. Experimental equipment and reagents

[0046] Clean bench for instruments (Shanghai Lichen Instrument Technology Co., Ltd.)

[0047] Incubator (Thermo Electron Corporation)

[0048] Microplate reader (Thermo Fisher Scientific)

[0049] Inverted biological microscope (OLYMPUS)

[0050] Reagents: Cell culture medium RPMI-1640, DMEM (high glucose) (GIBCO)

[0051] Fetal bovine serum (Hangzhou Sijiqing Co., Ltd.)

[0052] CCK-8 (Biosharp)

[0053] DMSO (Sigma)

[0054] Cell lines: human lung adenocarcinoma cells A549, human breast cancer cells MCF-7, normal human hepatocytes L-02, human liver cancer cells HepG2, and human liver cancer cells Bel-7402.

[0055] Human pancreatic cancer cells Panc-1 and MIA-PaCa-2

[0056] 2. Experimental Methods

[0057] Cell inhibitory activity assay methods

[0058] Cells were cultured routinely in an incubator at 37°C and 5% CO2 saturated humidity. Cells in the logarithmic growth phase were digested with trypsin cell digestion solution (0.25% trypsin + 0.01% EDTA), centrifuged, resuspended, and counted to prepare a concentration of 8 × 10⁻⁶ cells / mL. 5 Cell suspension at concentrations of 100 μL / ml was seeded into 96-well plates and incubated overnight in a CO2 incubator. After cell adhesion, the drug compound was diluted to the required concentration using culture medium, and 100 μL of the corresponding drug-containing medium was added to each well. Three auxiliary wells were set up for each drug group, along with blank control, negative control, and positive control. The 96-well plates were incubated for 24 hours. Then, 10 μL of CCK-8 was added to each well of the 96-well plate, and the plates were incubated for another 1 hour. The OD value of each well was then measured at 450 nm using a microplate reader, and the inhibition rate was calculated. Inhibition rate calculation formula:

[0059]

[0060] The relative OD value of the experimental group = the absolute OD value of the experimental group - the absolute OD value of the blank control well.

[0061] 3. Experimental Results

[0062] The antiproliferative activity of the compounds prepared in this invention against human breast cancer cells MCF-7, human lung adenocarcinoma cells A549, human liver cancer cells Bel-7402, and normal human liver cells L-02 is shown in Table 1.

[0063] Table 1 shows the IC50 values ​​of the compounds prepared in this invention against cancer cells and normal cells. 50 Value (μM)

[0064]

[0065] As shown in Table 1, the compounds EpskA9, EpskA10, and EpskA21 prepared in this invention exhibit excellent anticancer activity against human breast cancer cells MCF-7, human lung adenocarcinoma cells A549, and human liver cancer cells Bel-7402. Further investigation was conducted into the anticancer activity of these compounds against other human cancer cell lines (human liver cancer cells HepG2, human pancreatic cancer cells Panc-1, and MIA-PaCa-2).

[0066] Table 2 shows the IC50 values ​​of some compounds prepared in this invention against human hepatocellular carcinoma cells HepG2, human pancreatic cancer cells Panc-1, and MIA-PaCa-2. 50 Value (μM)

[0067]

[0068] As shown in Table 2, the compounds EpskA9, EpskA10, and EpskA21 prepared in this invention have good anticancer activity against human hepatocellular carcinoma cells HepG2, human pancreatic cancer cells Panc-1, and MIA-PaCa-2.

[0069] The above pharmacological tests show that the target derivative of the present invention has good anti-tumor cell proliferation activity and good selectivity for tumor cells and normal cells, and has the potential to be used as an anti-cancer drug.

Claims

1. A 6,20-epoxy A-ring modified derivative of oridonin and its pharmaceutically acceptable salt, characterized in that, The structure of the oridonin A-cyclic modified derivative is shown below: 。 2. A pharmaceutical composition, characterized in that, The product contains a therapeutically effective amount of the oridonin A 6,20-epoxy A-ring modified derivative of claim 1, and its pharmaceutically acceptable salt and pharmaceutically acceptable carrier.

3. The method for preparing the oridonin A-cyclic modified derivative according to claim 1, characterized in that, The synthetic route for the oridonin A-cyclic modified derivative is shown below: 。 4. The use of the oridonin A-6,20-epoxy A-ring modified derivative of claim 1 and its pharmaceutically acceptable salt, and the pharmaceutical composition of claim 2, in the preparation of a medicament for treating tumors; wherein the tumor is liver cancer.

5. The application according to claim 4, characterized in that, The liver cancer mentioned is selected from human liver cancer cells Bel-7402 or human liver cancer cells HepG2.

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