CN114349722B - Cardiac glycoside compound and preparation method and application thereof - Google Patents
Cardiac glycoside compound and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a cardiac glycoside compound and a preparation method and application thereof, belonging to the technical field of medicines. The invention provides a cardiac glycoside compound with a structure shown in formulas I-V, which can be used for preparing anti-tumor medicaments, selectively inhibiting human osteosarcoma cells MG63 and preparing medicaments special for anti-human osteosarcoma medicaments.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a cardiac glycoside compound and a preparation method and application thereof.
Background
Osteosarcoma is also called as osteoma, and is one of the primary malignant tumors commonly seen in adolescents and children, and is also the primary malignant tumor most commonly seen in bone tissues. Patients often have amputation, lung metastasis, death and other consequences, the patients are not sensitive to curative effect, chemotherapy and surgery are main treatment means, and the cure rate of the patients after simple surgery is only 15% -20%. Although the life cycle of patients can be effectively improved by matching with chemotherapeutic drugs, the cure rate of patients with high metastasis of osteoma is still only 30%. Among them, tumor infiltration and metastasis are one of the main causes of death of patients due to malignant tumors. The method reduces the transfer of osteosarcoma by regulating and controlling apoptosis, thereby improving the treatment effect of osteosarcoma, and is an important way for reducing the death rate of osteosarcoma and improving the long-term prognosis of patients. In the prior art, drugs capable of effectively resisting human osteosarcoma are lacked. Therefore, the search for more effective chemotherapeutic drugs for treatment and more effective treatment methods to improve the survival rate of osteosarcoma patients is an urgent requirement today.
The periploca forrestii schltr is a specific national medicine in China, has a long medicine application history in Guizhou, yunnan and the like, and the traditional efficacy of the periploca forrestii schltr is gradually proved and dug through scientific evaluation of modern pharmacology. The caulis et folium Periplocae Forrestii has effects of resisting inflammation, relieving pain, resisting rheumatoid arthritis, resisting tumor, etc., and also has neuroprotective and antibacterial effects, and the research of its chemical components has wide range, including anthraquinone, phenylpropanoid, flavonoid, triterpene, steroid, saponin, etc. Through a mouse delayed type hypersensitivity experiment induced by dinitrotoluene, the alcohol precipitation part obtained by purifying the caulis et folium piperis nigri is found to have the inhibition effect on cell proliferation and cell immunoregulation; the polysaccharide part of caulis et folium Periplocae Forrestii has inhibitory effect on activation proliferation and cellular immune response of T cells, and the active ingredients for exerting immunosuppression may be related to polysaccharide components HP1-3, HP2-2 and HP 2-4; periplocin obtained from the extraction part of the periplocin ethyl acetate has better anti-tumor activity; the periplocin can be used for treating atopic dermatitis by inhibiting Th1 type immunoreaction.
Most of the existing pharmacological experiments are stopped on crude extracts, the research on specific drug effect components is less, the specific mechanism of tumor resistance is not clear, and the research on single chemical components is needed to be deep.
Disclosure of Invention
In view of this, the present invention aims to provide a cardiac glycoside compound, and a preparation method and application thereof. The cardiac glycoside compound provided by the invention can be used for preparing antitumor drugs and has antitumor activity.
In order to achieve the above object, the present invention provides the following technical solutions:
a cardiac glycoside compound having a structure represented by formulae I to V:
the invention also provides a preparation method of the cardiac glycoside compound in the technical scheme, which comprises the following steps:
mixing caulis et folium Periplocae Forrestii with ethanol solution, and extracting to obtain extract solid;
mixing the extract solid with a methanol-water mixed solution, dropwise adding a sulfuric acid solution, refluxing, sequentially cooling, adjusting the pH value to 7, reducing the pressure, and extracting with ethyl acetate to obtain an ethyl acetate extracted part extract;
dissolving the ethyl acetate extraction part of the extract with a dichloromethane-methanol mixed solution to obtain a dissolved solution;
separating the dissolved solution by silica gel column chromatography through gradient elution, wherein the procedure of the gradient elution is as follows: eluting with petroleum ether, first petroleum ether-acetone mixed solution, second petroleum ether-acetone mixed solution, third petroleum ether-acetone mixed solution, fourth petroleum ether-acetone mixed solution and acetone to obtain 5 eluents, which are sequentially named as Fr1, fr2, fr3, fr4 and Fr5; the volume ratio of the petroleum ether to the acetone in the first petroleum ether-acetone mixed solution is 20;
recrystallizing the Fr2 with acetone to obtain a compound with a structure shown in a formula V, and sequentially eluting the obtained filtrate with methanol by gel column chromatography Sephadex LH-20 and recrystallizing with acetone to obtain a compound with a structure shown in a formula II;
sequentially using methanol/H with different concentrations to the Fr.3 2 O is subjected to MCI elution, methanol/H 2 The volume fractions of methanol in O are respectively 30%,60% and 90%, so as to respectively obtain components Fr3-1 and Fr3-2Fr3-3, and the Fr3-3 is subjected to silica gel column chromatography by using petroleum ether-ethyl acetate to obtain compounds with the structures shown in formulas I and IV, wherein the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate is 15, 1,5:1,2:1 and 1:1; sequentially using CHCl for the Fr3-2 3 Methanol is subjected to gel column chromatography Sephadex LH-20 separation to obtain a compound with a structure shown in a formula III, wherein CHCl is 3 /CHCl in methanol 3 The volume ratio to methanol was 1:1.
Preferably, the volume fraction of ethanol in the ethanol solution is 70%.
Preferably, the extraction is performed under reflux, the extraction times are 3 times, and the time of each extraction is respectively 5h, 3h and 2h.
Preferably, the volume ratio of methanol to water in the methanol-water mixed solution is 2:1.
Preferably, the volume ratio of the dichloromethane to the methanol in the dichloromethane-methanol mixed solution is 1:1.
The invention also provides application of the cardiac glycoside compound in the technical scheme or the cardiac glycoside compound prepared by the preparation method in the technical scheme or the compound with the structure shown in the formulas VI, VII and VIII in preparing antitumor drugs,
preferably, the anti-tumor drug comprises an anti-human osteosarcoma drug.
Preferably, the anti-tumor medicament comprises an effective dose of the cardiac glycoside compound, the stereoisomer and the pharmaceutically acceptable salt thereof, and pharmaceutically acceptable carriers, excipients and diluents.
Preferably, the dosage form of the anti-tumor drug comprises pharmaceutically acceptable dosage forms of tablets, injections, capsules, granules, pills, powder, oral liquid, sustained release preparations, controlled release preparations or nano preparations.
The invention provides a cardiac glycoside compound with a structure shown in formulas I-V, which can be used for preparing antitumor drugs, selectively inhibiting human osteosarcoma cells MG63, and can be used for preparing special drugs for anti-human osteosarcoma drugs.
Detailed Description
The invention provides a cardiac glycoside compound which has a structure shown in formulas I-V:
the invention also provides a preparation method of the cardiac glycoside compound in the technical scheme, which comprises the following steps:
mixing caulis et folium Periplocae Forrestii with ethanol solution, and extracting to obtain extract solid;
mixing the extract solid with a methanol-water mixed solution, dropwise adding a sulfuric acid solution, refluxing, sequentially cooling, adjusting the pH value to 7, reducing the pressure, and extracting with ethyl acetate to obtain an ethyl acetate extracted part extract;
dissolving the ethyl acetate extraction part of the extract with a dichloromethane-methanol mixed solution to obtain a dissolved solution;
separating the dissolved solution by silica gel column chromatography through gradient elution, wherein the procedure of the gradient elution is as follows: eluting with petroleum ether, first petroleum ether-acetone mixed solution, second petroleum ether-acetone mixed solution, third petroleum ether-acetone mixed solution, fourth petroleum ether-acetone mixed solution and acetone to obtain 5 eluents, which are sequentially named as Fr1, fr2, fr3, fr4 and Fr5; the volume ratio of the petroleum ether to the acetone in the first petroleum ether-acetone mixed solution is 20;
recrystallizing the Fr2 by acetone to obtain a compound with a structure shown in a formula V, and sequentially eluting the obtained filtrate by using methanol to perform gel column chromatography Sephadex LH-20 and recrystallizing the obtained filtrate by using acetone to obtain a compound with a structure shown in a formula II;
sequentially using methanol/H with different concentrations for the Fr.3 2 O is subjected to MCI elution with methanol/H 2 The volume fractions of methanol in O are respectively 30%,60% and 90%, so as to respectively obtain components Fr3-1 and Fr3-2Fr3-3, and the Fr3-3 is subjected to silica gel column chromatography by using petroleum ether-ethyl acetate to obtain compounds with the structures shown in formulas I and IV, wherein the volume ratios of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate are 15, 1,5:1,2:1 and 1:1; subjecting the Fr3-2 toCHCl for use repeatedly 3 Performing gel column chromatography Sephadex LH-20 separation on methanol to obtain a compound with a structure shown in formula III, wherein CHCl is 3 CHCl in methanol 3 The volume ratio to methanol was 1:1.
The invention mixes Periploca forrestii schltr with ethanol solution for extraction to obtain extract solid.
According to the invention, the periploca forrestii schltr is preferably dried and crushed in sequence, and then extracted.
In the present invention, the volume fraction of ethanol in the ethanol solution is preferably 70%.
In the present invention, the extraction is preferably performed under reflux, the number of times of extraction is preferably 3, and the time of each extraction is preferably 5h, 3h and 2h respectively.
The invention preferably combines the filtrates for 3 times to obtain the extract solid.
After obtaining extract solid, the invention mixes the extract solid with methanol-water mixed solution, then adds sulfuric acid solution, after reflux, cools, adjusts pH value to 7 and reduces pressure, then uses ethyl acetate to extract, obtains ethyl acetate extract part extract;
in the present invention, the volume ratio of methanol to water in the methanol-water mixed solution is preferably 2:1.
In the present invention, the dosage ratio of the extract solid to the methanol-water mixed liquid is preferably 10kg.
In the present invention, the sulfuric acid solution preferably has a mass percentage of 7%.
In the present invention, the cooling is preferably natural cooling.
In the invention, naOH solution with the mass percentage of 10% is preferably used for adjusting the pH value to 7.
In the present invention, the effect of the reduced pressure is to distill off the methanol.
After part of extract extracted by ethyl acetate is obtained, the invention dissolves the part of extract extracted by ethyl acetate and mixed solution of dichloromethane and methanol to obtain dissolved solution.
In the present invention, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixture is preferably 1:1.
In the invention, the dosage ratio of the ethyl acetate extraction part extract to the dichloromethane-methanol mixed liquid is preferably 1.5kg.
After a dissolved solution is obtained, the dissolved solution is subjected to silica gel column chromatography separation by gradient elution, wherein the procedure of the gradient elution is as follows: eluting with petroleum ether, first petroleum ether-acetone mixed solution, second petroleum ether-acetone mixed solution, third petroleum ether-acetone mixed solution, fourth petroleum ether-acetone mixed solution and acetone to obtain 5 eluents, which are sequentially named as Fr1, fr2, fr3, fr4 and Fr5; the volume ratio of petroleum ether to acetone in the first petroleum ether-acetone mixed solution is 20.
In the present invention, the silica gel used for the silica gel column chromatography preferably has a particle size of 200 to 300 mesh.
In the invention, the dissolving solution is preferably adsorbed on silica gel (40-80 meshes), and a sample adsorbed on the silica gel is subjected to gradient elution separation by adopting silica gel (200-300 meshes, 30 Kg) column chromatography.
In the present invention, the gradient elution preferably gives: fr (fraction) I part petroleum ether (100L), frII part petroleum ether/acetone (volume ratio) =20:1 (100L), frIII part petroleum ether/acetone (volume ratio) =10:1 (100L), frIV petroleum ether/acetone (volume ratio) =5:1 (100L), frV petroleum ether/acetone (volume ratio) =1:1 (100L), frVI acetone (100L).
After Fr1, fr2, fr3, fr4 and Fr5 are obtained, the invention recrystallizes the Fr2 through acetone to obtain a compound with the structure shown in the formula V, and the obtained filtrate is sequentially subjected to gel column chromatography Sephadex LH-20 elution and acetone recrystallization through methanol to obtain a compound with the structure shown in the formula II;
sequentially using methanol/H with different concentrations to the Fr.3 2 O is subjected to MCI elution, whereuponThe methanol/H 2 The volume fractions of methanol in O are respectively 30%,60% and 90%, so as to respectively obtain components Fr3-1 and Fr3-2Fr3-3, and the Fr3-3 is subjected to silica gel column chromatography by using petroleum ether-ethyl acetate to obtain compounds with the structures shown in formulas I and IV, wherein the volume ratios of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate are 15, 1,5:1,2:1 and 1:1; sequentially using CHCl for the Fr3-2 3 Performing gel column chromatography Sephadex LH-20 separation on methanol to obtain a compound with a structure shown in formula III, wherein CHCl is 3 CHCl in methanol 3 The volume ratio to methanol was 1:1.
In the invention, after acetone recrystallization, preferably performing suction filtration on the Fr2 to obtain a compound 6, and performing gel column chromatography Sephadex LH-20 (MeOH) and acetone recrystallization on filtrate to obtain a compound 2; fr3 by MCI (MeOH/H) 2 Blocking the volume percentage of MeOH in O at 30%,60%,90% to obtain components Fr3-1 to Fr3-3, wherein Fr3-3 is separated by silica gel column chromatography (PE/EA volume ratio of 1,10, 1,5 3 Volume ratio of MeOH 1:1), column chromatography on silica gel (CHCl) 3 /MeOH volume ratio 100; the Fr4 was chromatographed on reverse phase silica gel (gradient elution, meOH/H) 2 The volume ratio of O is 50 → 100), gel column chromatography Sephadex LH-20 (MeOH) and silica gel column chromatography (PE/EA volume ratio is 2.
The invention also provides application of the cardiac glycoside compound in the technical scheme or the cardiac glycoside compound prepared by the preparation method in the technical scheme or the compounds with the structures shown in formulas VI, VII and VIII in preparing antitumor drugs,
in the invention, the activity of the compound (periplogenin) with the structure shown in the formula VII is more than 10 times that of the existing clinical drug adriamycin, and the periplogenin compound has a prospect as an anti-tumor candidate drug.
In the present invention, the antitumor drug preferably comprises an anti-human osteosarcoma drug.
In the invention, the antitumor drug preferably comprises an effective dose of the cardiac glycoside compound, a stereoisomer thereof, compounds with structures shown in formulas VI, VII and VIII, pharmaceutically acceptable salts and pharmaceutically acceptable carriers, auxiliary materials, excipients and diluents.
In the invention, the dosage form of the antitumor drug preferably comprises pharmaceutically acceptable dosage forms such as tablets, injections, capsules, granules, pills, powders, oral liquids, sustained release preparations, controlled release preparations or nano preparations.
The cardiac glycoside compounds of the present invention, and the preparation method and use thereof, will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
In the examples of the present invention, cardiac glycoside compounds and compounds having the structures represented by formulas VI, VII, and VIII are represented by formulas 1 to 8 below.
Example 1
Preparation of the Compounds
50kg of periploca forrestii schltr medicinal material is dried and crushed, reflux-extracted for 3 times (5 h, 3h and 2 h) by 70% ethanol, and the 3 times of filtrate are combined to obtain 10kg of extract solid. 10kg of solid was taken and methanol was added: water = (2, 1, 30L) is heated and dissolved, and then 20L of water is slowly dripped to prepare 7%H mass percent 2 SO 4 Heating and refluxing for 2h, cooling, adjusting pH =7 with 10% by mass NaOH solution, distilling out methanol under reduced pressure, extracting with ethyl acetate, and recovering extraction solvent under reduced pressure to obtain ethyl acetate extract (1.5 kg). The ethyl acetate extract part of the extract (1.5 kg) was extracted with dichloromethane: methanol volume ratio of 1:1 (1.0L) is dissolved and adsorbed on silica gel (40-80 mesh), and the sample adsorbed on the silica gel is subjected to gradient elution separation by silica gel (200-300 mesh, 30 Kg) column chromatography: fr (fraction) I partial Petroleum Ether (100L)FrII fraction petroleum ether/acetone (volume ratio) =20:1 (100L), frIII part petroleum ether/acetone (volume ratio) =10:1 (100L), frIV petroleum ether/acetone (volume ratio) =5:1 (100L), frV petroleum ether/acetone (volume ratio) =1:1 (100L), frVI acetone (100L), in the above composition Fr1-Fr5, fr2 (306.5 g) fraction was recrystallized from acetone, suction filtered to give compound 6 (9 g), the filtrate was recrystallized from Sephadex LH-20 (MeOH) and acetone to give compound 2 (91 mg); fr3 (425.8 g) fraction was passed through MCI (MeOH/H) 2 MeOH in O was 30%,60%, 90%) respectively, to give fractions Fr3-1 to Fr3-3, wherein Fr3-3 was isolated by silica gel column chromatography (PE/EA volume ratio 15:1,10:1,5:1,2:1,1) to give compound 1 (782.2 mg), compound 5 (104.8 mg); fr3-2 was subjected to gel column chromatography Sephadex LH-20 (CHCl) 3 Volume ratio of MeOH 1:1), silica gel column chromatography (CHCl) 3 /MeOH volume ratio 100; the Fr4 (209.3 g) fraction was subjected to reverse phase silica gel column chromatography (gradient elution, meOH/H) 2 The volume ratio of O is 50 → 100), gel column chromatography Sephadex LH-20 (MeOH) and silica gel column chromatography (PE/EA volume ratio is 2.
Physical and chemical data of compound
Compound 1 white powder, [ alpha ]] 25 D +10°(c＝0.004,MeOH)；UV(MeOH)λmax(logε)265nm；IR(KBr)νmax 3396,2937,1748,1627,1407,1391,1088,553cm -1 ；HRESI-MS(positive)m/z 395.21872[M+Na] + (calcd.for C 23 H 32 O 4 Na,395.21928)； 1 H-NMR(CDCl 3 ,600MHz)δ1.92(1H,dt,J＝2.9,J＝14.3,H-1),1.34(1H,dt,J＝2.9,J＝14.3,H-1),1.90(1H,m,H-2),1.68(1H,m,H-2),4.22(1H,m,H-3),2.33(1H,m,H-4),1.65(1H,m,H-4),1.67(1H,m,H-6),1.44(1H,m,H-6),2.32(1H,m,H-7),1.88(1H,m,H-7),2.34(1H,m,H-9),1.64(1H,m,H-11),1.57(1H,m,H-11),1.77(1H,m,H-12),1.28(1H,m,H-12),1.95(1H,m,H-15),1.72(1H,m,H-15),2.50(1H,m,H-16),2.38(1H,m,H-16),2.35(1H,m,H-17),0.82(3H,s,H-18),0.84(3H,s,H-19),4.85～4.70(2H,br d,J＝17.4,H-21),5.88(1H,m,H-22)； 13 C-NMR(CDCl 3 ,150MHz)δ24.57(C-1),28.39(C-2),68.16(C-3),36.61(C-4),74.84(C-5),35.37(C-6),26.68(C-7),127.67(C-8),38.91(C-9),41.75(C-10),19.97(C-11),36.28(C-12),43.90(C-13),139.55(C-14),25.63(C-15),25.87(C-16),51.74(C-17),19.45(C-18),16.73(C-19),170.51(C-20),73.46(C-21),116.63(C-22),173.96(C-23)。
Compound 2 white powder, [ alpha ]] 25 D -50°(c＝0.004,MeOH)；UV(MeOH)λmax(logε)265nm；IR(KBr)νmax 3419,2937,2898,1693,1648,1402,1238,1061,593cm -1 ；HRESI-MS(positive)m/z 375.19260[M+Na] + (calcd.for C 23 H 28 O 3 Na,375.19307)； 1 H-NMR(CDCl 3 ,600MHz)δ1.85(1H,m,H-1),1.30(1H,m,H-1),2.19(1H,m,H-2),2.09(1H,m,H-2),5.70(1H,brs,H-3),5.99(1H,m,H-4),5.60(dd,J＝3.2,J＝6.2,H-6),2.86(1H,dd,J＝6.8,J＝16.9,H-7),1.77(1H,dd,J＝6.8,J＝16.9,H-7),2.04(1H,m,H-9),1.88(1H,m,H-11),1.33(1H,m,H-11),2.18(1H,m,H-12),1.97(1H,m,H-12),2.09(1H,m,H-15),1.54(1H,m,H-15),2.66(1H,m,H-16),1.51(1H,m,H-16),2.91(1H,m,H-17),1.03(3H,s,H-18),0.81(3H,s,H-19),4.72～4.58(2H,br d,J＝17.4,H-21),5.70(1H,br s,H-22)； 13 C-NMR(CDCl 3 ,150MHz)δ36.05(C-1),22.61(C-2),126.01(C-3),128.04(C-4),140.65(C-5),122.20(C-6),30.57(C-7),51.39(C-8),53.72(C-9),36.42(C-10),22.32(C-11),39.20(C-12),46.86(C-13),219.33(C-14),40.74(C-15),23.48(C-16),51.66(C-17),23.31(C-18),17.63(C-19),169.45(C-20),73.12(C-21),117.11(C-22),173.51(C-23)。
Compound 3 as a white powder, [ alpha ]] 25 D +20°(c＝0.004,MeOH)；UV(MeOH)λmax(logε)265nm；IR(KBr):νmax 3466,2928,1726,1702,1623,1401,1265,1065,1026,856,714cm -1 ；HRESI-MS(positive)m/z 393.20303[M+Na] + (calcd.for C 23 H 32 O 4 Na,393.20363)； 1 H-NMR(CDCl 3 ,600MHz)δ2.84(1H,m,H-1),1.60(1H,m,H-1),2.20(1H,m,H-2),1.86(1H,m,H-2),3.50(1H,br s,H-3),2.34(1H,dd,J＝2.1,J＝5.0,H-4),2.30(1H,dd,J＝2.1,J＝5.0,H-4),5.42(1H,m,H-6),2.62(1H,m,H-7),1.52(1H,m,H-7),2.08(1H,m,H-9),2.45(1H,m,H-11),1.81(1H,m,H-11),2.26(1H,m,H-12),2.23(1H,m,H-12),1.88(1H,m,H-15),1.02(1H,m,H-15),2.17(1H,m,H-16),2.05(1H,m,H-16),3.01(1H,d,J＝6.6,H-17),0.98(3H,s,H-18),0.86(3H,s,H-19),4.70～4.56(2H,br d,J＝17.4,H-21),5.67(1H,br s,H-22)； 13 C-NMR(CDCl 3 ,150MHz)δ26.46(C-1),31.08(C-2),71.30(C-3),41.51(C-4),137.32(C-5),120.39(C-6),32.90(C-7),48.45(C-8),56.90(C-9),38.33(C-10),22.51(C-11),42.17(C-12),47.85(C-13),218.92(C-14),38.62(C-15),41.81(C-16),53.32(C-17),23.18(C-18),19.12(C-19),169.87(C-20),73.05(C-21),116.99(C-22),173.57(C-23)。
Compound 4 as a white powder, [ alpha ]] 25 D +13.6°(c＝0.003,MeOH)；UV(MeOH)λmax(logε)265nm；IR(KBr)νmax 3401,2950,1763,1689,1402,1401,1200,1015,839,568cm -1 ；HRESI-MS(positive)m/z 413.22916[M+Na] + (calcd.for C23H34O5Na,393.20363)； 1 H-NMR(CDCl 3 ,600MHz)δ1.85(1H,m,H-1),1.44(1H,m,H-1),2.02(1H,m,H-2),1.58(1H,m,H-2),4.12(1H,br s,H-3),2.07(1H,m,H-4),1.01(1H,m,H-4),1.96(1H,m,H-6),1.28(1H,m,H-6),1.56(1H,m,H-7),1.64(1H,m,H-7),2.34(1H,m,H-9),2.35(1H,m,H-11),1.82(1H,m,H-11),2.17(1H,m,H-12),2.05(1H,m,H-12),1.66(1H,m,H-15),1.98(1H,m,H-15),2.49(1H,m,H-16),1.36(1H,m,H-16),2.07(1H,m,H-17),1.04(3H,s,H-18),0.76(3H,s,H-19),2.72(1H,m,H-20),4.23(1H,t,H-21),3.83(t,H-21),2.48(1H,m,H-22),2.21(1H,m,H-22)； 13 C-NMR(CDCl 3 ,150MHz)δ26.09(C-1),36.83(C-2),67.87(C-3),31.49(C-4),74.94(C-5),33.21(C-6),28.11(C-7),47.92(C-8),49.07(C-9),42.59(C-10),20.96(C-11),42.33(C-12),49.09(C-13),220.87(C-14),43.55(C-15),23.77(C-16),56.12(C-17),23.57(C-18),18.99(C-19),36.22(C-20),71.19(C-21),36.25(C-22),176.75(C-23)。
Compound 5 as a white powder, (+) ESI-MS M/z377.2[ M + Na [ ]] + ； 1 H-NMR(CDCl 3 ,600MHz)δ1.90(1H,m,H-1),1.10(1H,m,H-1),1.86(1H,m,H-2),1.53(1H,m,H-2),3.54(m,H-3),2.33(1H,m,H-4),2.25(1H,m,H-4),5.42(m,H-6),2.24(1H,m,H-7),1.97(1H,m,H-7),2.17(br s,H-8),1.04(m,H-9),1.68(1H,m,H-11),1.51(1H,m,H-11),1.88(1H,dt,J＝3.4,J＝12.9,H-12),1.38(1H,dt,J＝3.4,J＝12.9,H-12),5.28(1H,br d,J＝2.2,H-15),2.47(1H,m,H-16),1.85(1H,m,H-16),2.78(1H,t,H-17),0.84(3H,s,H-18),1.03(3H,s,H-19),4.84～4.71(2H,br d,J＝17.4,H-21),5.90(1H,br d,J＝1.6,H-22)； 13 C-NMR(CDCl 3 ,150MHz)δ37.02(C-1),31.45(C-2),71.55(C-3),42.06(C-4),139.96(C-5),121.03(C-6),29.58(C-7),31.21(C-8),49.82(C-9),36.85(C-10),21.48(C-11),40.45(C-12),48.51(C-13),153.18(C-14),118.05(C-15),33.42(C-16),52.42(C-17),17.91(C-18),19.08(C-19),170.55(C-20),73.50(C-21),116.34(C-22),174.01(C-23)。
Compound 6 Yunnan periplogenin A (perifornin A) white powder, (+) ESI-MS m/z 401.2, (+) M + Na] + ； 1 H-NMR(CDCl 3 ,600MHz)δ4.14(1H,br.s,H-3),0.96(3H,s,H-18),0.78(3H,s,H-19),4.70-4.55(2H,br.d,J＝17.2,H-21),5.68(1H,s,H-22)； 13 C-NMR(CDCl 3 ,150MHz)δ26.59(C-1),28.11(C-2),67.84(C-3),36.74(C-4),74.87(C-5),31.43(C-6),26.07(C-7),47.35(C-8),49.32(C-9),42.78(C-10),21.66(C-11),43.62(C-12),48.14(C-13),220.4(C-14),42.48(C-15),33.07(C-16),53.13(C-17),23.29(C-18),18.87(C-19),170.25(C-20),72.85(C-21),116.68(C-22),173.56(C-23)。
Compound 7 periplogenin (periplogenin) white crystal, (+) ESI-MS M/z413.2[ M + Na [)] + ； 1 H-NMR(CDCl 3 ,600MHz)δ1.92(1H,m,H-1),1.34(1H,m,H-1),1.90(1H,m,H-2),1.68(1H,m,H-2),4.18(m,H-3),2.33(1H,m,H-4),1.65(1H,m,H-4),1.67(1H,m,H-6),1.44(1H,m,H-6),2.32(1H,m,H-7),1.88(1H,m,H-7),2.34(1H,m,H-9),1.64(1H,m,H-11),1.57(1H,m,H-11),1.77(1H,m,H-12),1.28(1H,m,H-12),1.95(1H,m,H-15),1.72(1H,m,H-15),2.50(1H,m,H-16),2.38(1H,m,H-16),2.35(1H,H-17),0.88(3H,s,H-18),0.95(3H,s,H-19),5.00-4.80(2H,br.d；J＝18,H-21),5.88(1H,s,H-22),2.79(1H,s,3-OH),3.46(1H,s,5-OH),3.15(1H,s,14-OH)； 13 C-NMR(CDCl 3 ,150MHz)δ24.84(C-1),27.93(C-2),68.02(C-3),36.88(C-4),74.67(C-5),35.21(C-6),23.75(C-7),40.70(C-8),39.02(C-9),40.82(C-10),21.53(C-11),40.03(C-12),49.51(C-13),85.46(C-14),33.03(C-15),26.84(C-16),50.69(C-17),15.74(C-18),16.74(C-19),174.62(C-20),73.54(C-21),117.72(C-22),174.59(C-23)。
Compound 8 white powder，(+)ESI-MS m/z 395.2[M+Na] + ； 1 H-NMR(600MHz,CDCl 3 )δH 5.89(1H,m,H-22)；4.82-4.70(2H,br.d,J＝18,H-21)；0.97(3H,s,H-19),0.81(3H,s,H-18)； 13 C-NMR(150MHz,CDCl 3 )δC 36.84(C-1),27.96(C-2),68.11(C-3),33.58(C-4),74.83(C-5),26.27(C-6),22.24(C-7),40.69(C-8),34.35(C-9),35.00(C-10),24.88(C-11),42.99(C-12),48.58(C-13),153.72(C-14),117.13(C-15),41.24(C-16),52.58(C-17),16.55(C-18),18.28(C-19),170.56(C-20),73.51(C-21),116.37(C-22),174.06(C-23)。
Human osteoblastoma cell line MG-63
The screening method comprises the following steps: tetrazolium salt reduction (MTT process). Model principle: the dehydrogenase related to NADP exists in mitochondria of living cells, yellow MTT can be reduced to insoluble blue-purple Formanzan, the enzyme disappears in dead cells, and the MTT is not reduced. The model detects the action condition of the sample on the tumor cells according to the reduction degree of MTT.
Calculating the formula: inhibition (%) = [ (control OD value-sample OD value)/control OD value ] × 100
Table 1 preliminary screening results of four tumor cells by the compound
Evaluation of human leukemia cell line K562: the compound 7 has a certain inhibition effect on K562 cells, the compounds 1,2, 3, 4 and 8 have weak or weak inhibition effects on the K562 cells, and the compounds 5 and 6 have no inhibition or definite inhibition effects on the K562 cells.
Evaluation of human leukemia cells HL-60: the compounds 3 and 8 have weak or weak inhibitory action on HL-60 cells, the compound 7 has better inhibitory action on HL-60 cells, and the rest compounds have no inhibitory or definite inhibitory action on HL-60 cells.
Human lung cancer cell a549 evaluation: the compounds 2 and 8 have no inhibition or clear inhibition on A549 cells, the compounds 1,3, 4, 5 and 6 have weak or weak inhibition on A549 cells, and the sample 7 has a certain inhibition on A549 cells.
Evaluation of human osteoblastic sarcoma cell line MG-63: the compounds 1,2, 3, 6 and 8 have weak or weak inhibition effect on MG-63 cells, the compounds 4 and 5 have certain inhibition effect on MG-63 cells, and the compound 7 has better inhibition effect on MG-63 cells.
Table 2 shows the results of IC50 tests of compounds 5 and 7 on human osteosarcoma cell lines MG-63, and it can be seen that compound 7 has a certain inhibitory effect on 4 tumor cells to be tested, wherein the inhibitory effect on human osteosarcoma cell lines MG-63 is one order of magnitude higher than that of adriamycin and is more than 10 times that of adriamycin, so that the compound has a prospect as an anti-human osteosarcoma candidate drug.
TABLE 2 IC50 test results of Compounds 5 and 7 on human sarcoidosis cell line MG-63
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. A cardiac glycoside compound having a structure represented by formulae I-III or formula V:
2. the method for producing a cardiac glycoside compound according to claim 1, comprising the steps of:
mixing caulis et folium Periplocae Forrestii with ethanol solution, and extracting to obtain extract solid; the volume fraction of ethanol in the ethanol solution is 70%;
mixing the extract solid with a methanol-water mixed solution, dropwise adding a sulfuric acid solution, refluxing, sequentially cooling, adjusting the pH value to 7, decompressing and steaming out methanol, and extracting with ethyl acetate to obtain part of extract extracted by ethyl acetate; the mass percentage content of the sulfuric acid solution is 7%;
dissolving the ethyl acetate extraction part of the extract with a dichloromethane-methanol mixed solution to obtain a dissolved solution;
separating the dissolved solution by silica gel column chromatography through gradient elution, wherein the procedure of the gradient elution is as follows: eluting with petroleum ether, first petroleum ether-acetone mixed solution, second petroleum ether-acetone mixed solution, third petroleum ether-acetone mixed solution, fourth petroleum ether-acetone mixed solution and acetone to obtain 5 eluents, which are sequentially named as Fr1, fr2, fr3, fr4 and Fr5; the volume ratio of petroleum ether to acetone in the first petroleum ether-acetone mixed solution is 20;
recrystallizing the Fr2 by acetone to obtain a compound with a structure shown in a formula V, and sequentially eluting the obtained filtrate by using methanol to perform gel column chromatography Sephadex LH-20 and recrystallizing the obtained filtrate by using acetone to obtain a compound with a structure shown in a formula II;
sequentially using methanol/H with different concentrations for the Fr.3 2 O is subjected to MCI elution with methanol/H 2 The volume fractions of methanol in O are respectively 30%,60% and 90%, to respectively obtain components Fr3-1, fr3-2 and Fr3-3, and subjecting Fr3-3 to silica gel column chromatography with petroleum ether-ethyl acetate to obtain compounds with structures shown in formula I and formula IVThe volume ratio of ether to ethyl acetate was 15, 1,10, 5:1,2:1, and 1:1; sequentially using CHCl for the Fr3-2 3 Methanol is subjected to gel column chromatography Sephadex LH-20 separation to obtain a compound with a structure shown in a formula III, wherein CHCl is 3 /CHCl in methanol 3 The volume ratio to methanol is 1:1;
3. the preparation method according to claim 2, wherein the extraction is performed under reflux, the extraction is performed 3 times, and the time of each extraction is respectively 5h, 3h and 2h.
4. The method according to claim 2, wherein the volume ratio of methanol to water in the methanol-water mixture is 2:1.
5. The method according to claim 2, wherein the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixture is 1:1.
6. Use of a cardiac glycoside compound according to claim 1, a cardiac glycoside compound obtained by the process according to any one of claims 2 to 5, or a compound of formula VI, VII or VIII for the preparation of an antitumor agent,
the anti-tumor drug is an anti-human osteosarcoma drug.
7. The use of claim 6, wherein the anti-tumor medicament comprises an effective amount of the cardiac glycoside compound or the compound of formula VI, VII or VIII, a pharmaceutically acceptable salt and a pharmaceutically acceptable excipient.
8. The use as claimed in claim 6, wherein the antitumor drug is in a dosage form selected from the group consisting of tablets, injections, capsules, granules, pills, powders and oral liquids.
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| CN104398532A (en) * | 2013-04-11 | 2015-03-11 | 中国热带农业科学院热带作物品种资源研究所 | Application of cardiac glycoside compound 12beta-hydroxycalotropin |
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