CN111961106B

CN111961106B - Ouabain sugar ring 3' hydroxyl derivative, preparation method and application thereof

Info

Publication number: CN111961106B
Application number: CN202010872469.XA
Authority: CN
Inventors: 张霞; 钮大文
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2020-08-26
Filing date: 2020-08-26
Publication date: 2022-05-20
Anticipated expiration: 2040-08-26
Also published as: CN111961106A

Abstract

The invention provides a ouabain sugar ring 3' -hydroxyl derivative, a preparation method and application thereof. The structure of the ouabain sugar ring 3' -hydroxyl derivative is shown as formula I. The derivative has very low toxicity to normal cells, and has excellent tumor cell inhibiting effect. In addition, the derivative can also effectively inhibit the migration and invasion of tumor cells, and the inhibition effect of part of compounds is even better than that of Ouabain (Ouabain). Therefore, the ouabain sugar ring 3' hydroxyl derivative provided by the invention has a very good application prospect in preparing medicines for resisting tumors and inhibiting tumor invasion and/or migration.

Description

Ouabain sugar ring 3' hydroxyl derivative, preparation method and application thereof

Technical Field

The invention belongs to the field of pharmacy, and particularly relates to a ouabain sugar ring 3' -hydroxyl derivative, and a preparation method and application thereof.

Background

Among natural products, Cardiac Glycosides (CGs) are an important class of compounds that act specifically on Na⁺/K⁺-ATPase, with cardiotonic function. Depending on the 17-position unsaturated lactone, cardiac glycosides can be divided into cardenolides (pentabasic unsaturated lactones) and bufadienolides (hexabasic unsaturated lactones). Wherein the bufadienolide is mainly separated from venom of Bufo siccus; cardenolide is mainly present in plants such as Scrophulariaceae (Digitalis), Apocynaceae (Nerium, stropanthus), Ranunculaceae (calendula), and has the highest content in plant leaves. It is found that more than 30 cardiac glycosides, including digitalis, digoxin and ouabain, are contained in the rehmannia leaves.

Cardiac glycosides have been used by people to treat heart failure as early as over 200 years ago; in 1785, William warming, a medical physician in the United kingdom, published a description of digitalis and its medical use, after which cardiac glycosides, with their tissue specificity and powerful cardiomyocyte contraction effect, were widely used in the treatment of congestive heart failure, in combination with diuretics, in the treatment of atrial fibrillation and some arrhythmias. Researchers also find that the cardiac glycoside compound has the functions of cardiac, tumor resistance, virus resistance, bacteria resistance, immunoregulation, influence on nerve cell differentiation, blood pressure reduction, depression resistance and the like, and has great medicinal value. Among them, Ouabain (Ouabain), which is a cardiac glycoside compound, has been found to have a good potential in the treatment of tumors such as breast cancer, prostate cancer, pancreatic cancer, leukemia, neuroblastoma, etc. (p.kometiani, l.liu, a.askari.mol.pharmacol.2005,67, 929-.

However, although the ouabain has obvious drug effect, a slight excess of ouabain can cause serious adverse reaction and even endanger life. The therapeutic concentration of the ouabain medicine is close to the toxic concentration (the therapeutic window is narrow), and the drug effects (the curative effect and the toxicity) of ouabain are greatly different due to the personal constitution, so the unsafety of the ouabain medicine greatly limits the clinical application of the ouabain medicine. Researches also find that the ouabain has larger toxicity to normal cells of a human body and is easy to generate toxic and side effects on normal tissues when being taken. Attempts are made to modify ouabain compounds to reduce the toxic and side effects on normal tissues, but the obtained derivatives have the toxicity on normal tissues and greatly reduce the tumor inhibition activity. Moreover, the ouabain compound has a plurality of active sites, and how to modify specific sites with high selectivity is also a problem in the field.

Therefore, the proper modification of the ouabain compound to prepare the ouabain derivative which can obviously reduce the toxicity to normal cells and simultaneously can keep excellent pharmaceutical activity has very important significance for the clinical application of the ouabain compound.

Disclosure of Invention

The invention aims to provide a derivative of ouabain sugar ring 3' hydroxyl, a preparation method and application thereof.

The invention provides a compound shown as a formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotope substitution form thereof:

wherein n is an integer of 0 to 3;

n number of R₀Each independently selected from hydrogen, hydroxy, halogen, C_1～3Alkyl or C_1～3An alkoxy group;

R₁selected from hydrogen, substituted or unsubstituted 3-6 membered saturated cycloalkyl, substituted or unsubstituted 3-6 membered saturated heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, hydroxy, halogen, C_1～5Alkyl or C_1～5An alkoxy group;

the substituent is 1 or more and is selected from halogenated or non-halogenated C_1～5Alkyl, halogenated or non-halogenated C_1～5Alkoxy, halogenated or non-halogenated C_2～6Alkenyl, halogenated or non-halogenated C_2～6Alkynyl, halogen, N₃Nitro or hydroxy.

Further, the structure of the compound is shown as formula II:

wherein R is₁Selected from hydrogen, substituted or unsubstituted 5-6 membered saturated cycloalkyl, substituted or unsubstituted 5-6 membered saturated heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, hydroxy, halogen, C_1～3Alkyl or C_1～3Alkoxy radical；

The substituent is 1 or more and is selected from halogenated or non-halogenated C_1～3Alkyl, halogenated or non-halogenated C_1～3Alkoxy, halogenated or non-halogenated C_2～4Alkenyl, halogenated or non-halogenated C_2～4Alkynyl, halogen, N₃Nitro or hydroxy.

Further, the structure of the compound is shown as formula III:

wherein m is an integer of 1-5, preferably 1 or 2; m R₂Each independently selected from hydrogen, halogenated or non-halogenated C_1～3Alkyl, halogenated or non-halogenated C_1～3Alkoxy, halogenated or non-halogenated C_2～4Alkenyl, halogenated or non-halogenated C_2～4Alkynyl, halogen, N₃Or a nitro group; the halogen is preferably fluorine or chlorine.

Further, the structure of the compound is shown as follows:

the present invention also provides a process for the preparation of the above compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopically substituted form thereof, which comprises the steps of:

(1) mixing ouabain, organic boron reagent and organic solvent in a reaction bottle;

(2) mixing an allylation reagent, a palladium catalyst, a phosphorus ligand and an organic solvent in another reaction bottle, and stirring and activating; the allylation reagent has the structure of

R₃Is a protecting group, R₁As described above;

(3) and (3) adding the system obtained in the step (2) into the system obtained in the step (1) for reaction to obtain the catalyst.

Further, the steps (1) to (3) are carried out in an inert gas protection environment; in the step (3), the reaction temperature is 20-30 ℃, and room temperature is preferred; the reaction time is 6 to 24 hours, preferably 16 hours.

Further, in the step (1), the organoboron reagent is

The organic solvent is one or a mixture of two of tetrahydrofuran and isopropanol, and is preferably a mixture of tetrahydrofuran and isopropanol; the molar ratio of the ouabain to the organic boron reagent is 1: (0.1 to 0.5), preferably 1: 0.3;

in the step (2), the palladium catalyst is Pd (dba)₃·CHCl₃The phosphorus ligand is PPh₃(ii) a The organic solvent is one or two of tetrahydrofuran and isopropanol; the mol ratio of the ouabain to the allylation reagent, the palladium catalyst and the phosphorus ligand is 1: (1.1-5.0): (0.01-0.2): ): (0.05-0.4) preferably 1: 1.5: 0.05: 0.2; and/or the activation time is 10-40 minutes, preferably 30 minutes; and/or, said R₃Is a hydroxyl protecting group, preferably a Boc group.

The invention also provides a medicament which is a preparation prepared by taking the compound, or pharmaceutically acceptable salt thereof, or stereoisomer thereof, or optical isomer thereof, or isotope substitution form thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

The invention also provides the application of the compound, or the pharmaceutically acceptable salt, or the stereoisomer, or the optical isomer, or the isotope substitution form thereof in preparing the medicine for resisting tumor or inhibiting tumor metastasis; the tumor is preferably breast cancer, cervical cancer cells, liver cancer, prostate cancer and pancreatic cancer, and more preferably breast cancer, prostate cancer, pancreatic cancer and liver cancer; the liver cancer is preferably liver ascites adenocarcinoma.

Further, the drug is capable of inhibiting tumor cell proliferation, invasion and/or migration.

Definitions of terms used in connection with the present invention: the initial definitions provided herein for a group or term apply to that group or term throughout the specification unless otherwise indicated; for terms not specifically defined herein, the meanings that would be given to them by a person skilled in the art are to be given in light of the disclosure and the context.

In the present invention, "room temperature" means 25. + -. 2 ℃.

"overnight" means 8 to 16 hours.

"halogen" means fluorine, chlorine, bromine or iodine.

"isotopically substituted form" refers to compounds wherein one or more than two atoms are replaced by their corresponding isotopes, for example, compounds wherein hydrogen is replaced by protium, deuterium, or tritium.

By "pharmaceutically acceptable" is meant that the carrier, diluent, excipient, and/or salt formed is generally chemically or physically compatible with the other ingredients comprising a pharmaceutical dosage form and physiologically compatible with the recipient.

"salts" are acid and/or base salts of compounds with inorganic and/or organic acids and/or bases, and also include zwitterionic (inner) salts, and also quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. Or by mixing the compound with a certain amount of an acid or a base as appropriate (e.g., an equivalent amount). These salts may form precipitates in the solution which are collected by filtration, or they may be recovered after evaporation of the solvent, or they may be prepared by reaction in an aqueous medium followed by lyophilization.

The "pharmaceutically acceptable salt" in the present invention may be hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate of the compound.

In the present invention, the minimum value of the carbon atom content in the hydrocarbon groupAnd maximum value is indicated by a prefix, e.g. prefix (C)_a～b) Alkyl means any alkyl group containing from "a" to "b" carbon atoms. E.g. C_1～3The alkyl group is a straight-chain or branched alkyl group having 1 to 3 carbon atoms.

Similarly, C_1～3The alkoxy group means a straight chain or branched chain alkoxy group having 1 to 3 carbon atoms.

"cycloalkyl" refers to a saturated or unsaturated cyclic hydrocarbon substituent; the cyclic hydrocarbon may be monocyclic or polycyclic. "saturated cycloalkyl" refers to saturated cycloalkyl. For example, "3-to 6-membered saturated cycloalkyl" refers to a saturated cycloalkyl group having 3 to 6 carbon atoms in the ring.

"heterocyclyl" refers to a saturated or unsaturated cyclic hydrocarbon substituent; the cyclic hydrocarbon may be monocyclic or polycyclic and carries at least one ring heteroatom (including but not limited to O, S or N). "saturated heterocyclyl" refers to saturated heterocyclyl groups. For example, "3 to 6-membered saturated heterocyclic group" means a saturated heterocyclic group having 3 to 6 ring atoms.

"aryl" refers to an all-carbon monocyclic or fused polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a conjugated pi-electron system, such as phenyl and naphthyl. The aryl ring may be fused to other cyclic groups (including saturated and unsaturated rings) but must not contain heteroatoms such as nitrogen, oxygen, or sulfur, and the point of attachment to the parent must be at a carbon atom on the ring which has a conjugated pi-electron system. The aryl group may be substituted or unsubstituted.

"heteroaryl" refers to a heteroaromatic group containing one to more heteroatoms. The hetero atoms referred to herein include oxygen, sulfur and nitrogen. Such as furyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl, or cycloalkyl ring, wherein the ring joined to the parent structure is a heteroaryl ring. Heteroaryl groups may be optionally substituted or unsubstituted.

Protecting group: in organic synthesis, molecules containing 2 or more functional groups are protected by a certain reagent in order to prevent a certain functional group from being damaged by reaction, and after the reaction is finished, the group in the protecting reagent is removed, namely the protecting group.

The method takes specific allylation reagents SI 1-SI 9 as modifiers, under the condition that phosphorus ligands exist, a metal palladium reagent and an organic boron reagent are used for co-catalysis, and allylation modification at specific positions is carried out to successfully prepare the ouabase ring 3' hydroxyl derivatives.

Compared with the prior art, the ouabain ring 3' hydroxyl derivative provided by the invention has the following beneficial effects:

(1) the ouabain ring 3' -hydroxyl derivative provided by the invention has excellent effects of inhibiting various tumor cells including breast cancer, cervical cancer, liver cancer (particularly liver ascites adenocarcinoma), prostate cancer and pancreatic cancer while reducing toxicity to normal cells;

(2) the ouabain ring 3' hydroxyl derivative has good selectivity on the inhibition of tumor cells, and the inhibition effect on prostate cancer, human pancreatic cancer and SK-HEP-1 liver ascites adenocarcinoma is obviously better than that of HEP G2 liver cancer;

(3) the 19-bit primary hydroxyl derivative of Ouabain (especially compounds of Ouabain-3, Ouabain-6, Ouabain-12 and Ouabain-24) can effectively inhibit pancreatic cancer cell proliferation;

(4) the Ouabain sugar ring 3' -hydroxyl derivative can also effectively inhibit tumor cell migration, and particularly, the compound Ouabain-12 has an inhibition effect even better than Ouabain (Ouabain);

(5) the Ouabain sugar ring 3' -hydroxyl derivative can also effectively inhibit tumor cell invasion, and particularly, the compound Ouabain-12 has an inhibition effect even superior to Ouabain (Ouabain).

Therefore, the ouabain sugar ring 3' hydroxyl derivative obtained by allylation modification of a specific position has a very good application prospect in preparation of drugs for resisting tumors and inhibiting tumor invasion and/or migration.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a scheme showing the synthesis schemes of allylating reagents SI1 to SI 9.

FIG. 2 shows the effect of each compound on the migration of breast cancer cells (MDA-MB-231) under a concentration gradient (0.01. mu.M, 0.1. mu.M, 1.0. mu.M, 10. mu.M, 100. mu.M).

FIG. 3 shows the results of the cell invasion assay of each compound at 0.1. mu.M and 10. mu.M.

Detailed Description

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.

Example 1: synthesis of allylation reagents SI 1-SI 9

The following 9 allylating reagents SI 1-SI 9 were prepared by the synthetic route shown in FIG. 1 by referring to the methods described in the known documents (Tetrahedron letters.2012,53, 1319-1322; Jr. Eur. J. org. chem.2016,28, 4800-4804; org. Lett.2009,11, 2944-2947; J.Am. chem. Soc.2015,137, 6335-6349; org. Lett.2019,21, 7424-7429). The characterization data of the allylating reagents SI-1 to SI-9 are as follows:

SI-1：Allyl-tert-butylcarbonate

¹H NMR(CDCl₃,400MHz)δ:5.98–5.83(m,1H),5.38–5.14(m,2H),4.63–4.47(m,2H).¹³C NMR(CDCl₃,101MHz)δ:153.4,132.1,118.2,82.1,67.6,28.0,27.8.

SI-2：(E)-3-(4-Methoxyphenyl)allyl-tert-butyl carbonate

¹H NMR(CDCl₃,400MHz)δ:7.31(d,J＝8.7Hz,2H),6.84(d,J＝8.7Hz,2H),6.61(d,J＝15.9Hz,1H),6.15(dt,J＝15.9,6.6Hz,1H),4.69(dd,J＝6.6,1.3Hz,2H),3.79(s,3H),1.49(s,5H).¹³C NMR(CDCl₃,101MHz)δ:159.7,153.5,134.3,129.0,128.0,120.7,114.1,82.1,67.8,55.3,27.9.

SI-3：(E)-3-(3-Azidophenyl)allyl-tert-butyl carbonate

¹H NMR(CDCl₃,400MHz)δ:7.29(t,J＝7.8Hz,1H),7.14(d,J＝7.7Hz,1H),7.01(t,J＝2.0Hz,1H),6.92(dd,J＝8.0,2.3Hz,1H),6.62(d,J＝15.9Hz,1H),6.30(dt,J＝15.9,6.3Hz,1H),4.71(dd,J＝6.3,1.5Hz,2H),1.50(s,9H).¹³C NMR(CDCl₃,101MHz)δ:153.4,140.5,138.1,133.1,130.0,124.5,123.4,118.6,117.1,82.4,67.1,27.9.

SI-4：(E)-3-(4-Fluorophenyl)allyl-tert-butyl carbonate

¹H NMR(CDCl₃,400MHz)δ:7.35(dd,J＝8.5,5.5Hz,2H),7.00(t,J＝8.7Hz,2H),6.63(d,J＝15.9Hz,1H),6.21(dt,J＝15.9,6.4Hz,1H),4.70(dd,J＝6.4,1.3Hz,2H),1.50(s,9H).¹³C NMR(CDCl₃,101MHz)δ:153.5,133.4,128.4,128.3,122.9,122.8,115.8,115.6,82.4,67.5,27.9.

SI-5：(E)-3-(4-Ethynylphenyl)allyl-tert-butyl carbonate

¹H NMR(CDCl₃,400MHz)δ7.44(d,J＝8.0Hz,2H),7.33(d,J＝8.0Hz,2H),6.64(d,J＝15.9Hz,1H),6.31(dt,J＝16.0,6.3Hz,1H),4.72(dd,J＝6.4,1.4Hz,2H),1.50(s,9H).¹³C NMR(CDCl₃,101MHz)δ:153.4,136.8,133.5,132.5,126.7,124.4,121.8,83.7,82.5,78.1,67.3,28.0,27.9.

SI-6：(E)-3-(4-Trifluoromethylphenyl)allyl-tert-butyl carbonate

¹H NMR(CDCl₃,400MHz)δ:7.57(d,J＝8.1Hz,2H),7.47(d,J＝8.1Hz,2H),6.69(d,J＝15.9Hz,1H),6.38(dt,J＝16.0,6.1Hz,1H),4.74(dd,J＝6.2,1.5Hz,2H),1.51(s,9H).¹³C NMR(CDCl₃,101MHz)δ:153.4,139.8,139.8,132.5,129.9(q,J＝32.4Hz),126.9,125.9,125.7(q,J＝3.9Hz),125.5(d,J＝3.8Hz),82.5,81.0,67.0,28.0,27.9.

SI-7：(E)-Tert-butylcinnamyl carbonate

¹H NMR(CDCl₃,400MHz)δ:7.40–7.35(m,2H),7.33–7.27(m,2H),7.27–7.21(m,1H),6.66(dd,J＝15.9,1.5Hz,1H),6.28(dt,J＝15.9,6.4Hz,1H),4.71(dd,J＝6.5,1.4Hz,2H),1.50(s,9H).¹³C NMR(CDCl₃,101MHz)δ:153.4,136.3,134.5,128.7,128.1,126.7,123.0,82.2,67.5,27.9.

SI-8：(E)-3-(3-Nitrophenyl)allyl-tert-butyl carbonate

¹H NMR(CDCl₃,400MHz)δ:8.24(t,J＝2.0Hz,1H),8.11(ddd,J＝8.2,2.3,1.0Hz,1H),7.69(dt,J＝7.7,1.4Hz,1H),7.50(t,J＝8.0Hz,1H),6.73(dt,J＝16.0,1.5Hz,1H),6.43(dt,J＝16.0,6.0Hz,1H),4.76(dd,J＝6.1,1.5Hz,2H),1.52(s,9H).¹³C NMR(CDCl₃,101MHz)δ:153.4,138.2,132.5,131.5,129.7,126.6,122.7,121.4,82.7,66.8,27.9.

SI-9：(E)-3-(3,4-Dichlorophenyl)allyl-tert-butyl carbonate

¹H NMR(CDCl₃,400MHz)δ:7.45(d,J＝2.1Hz,1H),7.38(dd,J＝8.3,1.6Hz,1H),7.20(dd,J＝8.3,2.1Hz,1H),6.57(dt,J＝15.9,1.5Hz,1H),6.28(dt,J＝15.9,6.2Hz,1H),4.71(dd,J＝6.1,1.5Hz,2H),1.50(d,J＝3.1Hz,9H).¹³C NMR(CDCl₃,101MHz)δ:153.4,136.5,132.9,131.9,131.7,130.6,128.5,125.9,125.3,82.6,67.0,27.9.

example 2: synthesizing the ouabain sugar ring 3' -hydroxyl derivative

The 3' -hydroxyl derivative of the ouabain sugar ring is prepared by adopting the following synthetic route:

in the air, ouabain (36.5mg,0.05mmol,1.0equiv.) and organoboron reagent 3-phenylbenzoylxole (i.e., compound [ B ] in the reaction equation)]3.1mg,0.015mmol and 0.3equiv.) are weighed into a reaction bottle A which is dried in advance and is provided with a stirrer, and a bottle cap is closed; weighing corresponding allylation reagent (one of SI 1-SI 9, 0.075mmol) into a reaction bottle B which is dried in advance and is provided with a stirrer, and closing a bottle cap. The reaction was transferred to a nitrogen blanketed glove box. Adding 2mL of i-PrOH and 500 mu L of THF into a reaction bottle A; then Pd (dba)₃·CHCl₃(2.6mg,0.26％mmol,5％equiv.)、PPh₃(2.6mg, 0.1% mmol, 20% equiv.) was added to reaction flask B in sequence, followed by additionAdding into ultra-dry THF (500 μ L), closing the bottle cap, stirring and activating for 30min. 500. mu.L of the reaction B solution was taken into the reaction flask A using a 1mL syringe, the cap was screwed, the reaction was removed from the glove box, and the reaction was sealed at 25 ℃. The reaction was carried out for 16 hours. After the reaction is finished, the reaction system is directly decompressed and dried in a spinning mode to obtain a crude product, and the crude product is purified through column chromatography to obtain the corresponding ouabain ring 3' -hydroxyl derivative.

The correspondence between the allylation reagent and the derivative of the hydroxyl group at the 3' -position of the ouabain ring is shown in Table 1.

TABLE 1 correspondence between allylation reagent as raw material and 3' -hydroxyl derivative of ouabain ring as product

The following are the structures and characterization data of the prepared 3' -hydroxyl derivatives of each ouabain ring:

(E)-3’-Allyl-ouabain(Ouabain-3)

the column chromatography purification method comprises the following steps: silica gel Chromatography (CH)₂Cl₂MeOH ═ 20:1-13:1) purified crude oaabain-3 to give solid product oaabain-3 (20.3mg, purity > 90%, overall yield 64.6%).

¹H NMR(MeOD,400MHz)δ:5.99(ddt,J＝16.0,10.8,5.6Hz,1H),5.91(d,J＝1.9Hz,1H),5.31(dq,J＝17.3,1.8Hz,1H),5.16(dt,J＝10.4,1.5Hz,1H),5.01(dd,J＝18.5,1.9Hz,1H),4.91(dd,J＝18.3,1.7Hz,1H),4.85(d,J＝1.8Hz,1H),4.24(td,J＝10.3,4.5Hz,1H),4.12(dd,J＝10.2,5.9Hz,2H),4.07(ddt,J＝13.1,5.4,1.6Hz,1H),3.99(ddt,J＝12.9,5.8,1.4Hz,1H),3.78(dd,J＝3.4,1.8Hz,1H),3.76–3.72(m,1H),3.70(dd,J＝9.4,3.4Hz,1H),3.38(d,J＝9.4Hz,1H),2.91(t,J＝7.2Hz,1H),2.28–2.22(m,1H),2.21–1.86(m,8H),1.80–1.70(m,3H),1.70–1.65(m,1H),1.60–1.44(m,2H),1.42–1.27(m,4H),1.25(d,J＝6.2Hz,3H),0.92(s,3H).¹³C NMR(MeOD,101MHz)δ:177.6,177.1,136.5,118.0,117.2,99.3,85.7,75.9,75.9,75.3,74.2,73.6,72.6,72.4,72.4,72.4,71.8,70.4,69.9,69.0,51.6,50.9,50.3,41.2,41.2,41.1,33.4,27.8,24.5,18.0,17.5.IR(thin film,cm^-1):3005,2984,1486,1275,1260,764,and 749.HRMS(DART-TOF)calculated for C₃₂H₄₈NaO₁₂ ⁺[M+Na]⁺m/z 647.3043,found 647.3046.[α]_D ²⁶＝-23.5(c＝0.04,MeOH).

(E)-3’-(4-Methoxycinnamyl)-ouabain(Ouabain-6)

The column chromatography purification method comprises the following steps: silica gel Chromatography (CH)₂Cl₂Purification of crude Ouabain-6 with MeOH ═ 20:1-15:1) yielded Ouabain-6 as a solid product (25.6mg, greater than 90% purity, total yield 70.4%).

¹H NMR(MeOD,400MHz)δ:7.34(d,J＝8.6Hz,2H),6.86(d,J＝8.4Hz,2H),6.59(d,J＝15.9Hz,1H),6.25(dt,J＝15.9,6.2Hz,1H),5.89(s,1H),4.99(dd,J＝18.4,1.7Hz,1H),4.94–4.86(m,1H),4.85(s,1H),4.31–4.23(m,1H),4.15(ddt,J＝13.3,6.9,4.5Hz,5H),3.77(s,3H),3.76(m,1H),3.74–3.69(m,1H),3.39(t,J＝9.5Hz,1H),2.90(t,J＝7.1Hz,1H),2.30–2.21(m,1H),2.21–1.81(m,8H),1.72(td,J＝17.0,15.8,4.8Hz,4H),1.61–1.29(m,8H),1.26(d,J＝6.2Hz,3H),0.90(s,3H).¹³C NMR(MeOD,101MHz)δ:177.5,177.1,160.9,133.4,130.9,128.7,125.0,118.0,115.0,99.3,85.6,75.9,75.9,75.3,74.2,73.4,72.6,72.4,71.8,70.1,69.9,69.0,55.7,55.7,51.6,50.9,50.3,49.9,49.3,41.2,33.4,30.7,30.7,30.6,30.3,27.8,24.5,18.0,17.6.IR(thin film,cm^-1):3006,2988,1482,1276,1261,765,760and 748.HRMS(DART-TOF)calculated for C₃₉H₅₄NaO₁₃ ⁺[M+Na]⁺m/z 753.3462,found 753.3467.[α]_D ²⁶＝-3.9(c＝0.26,MeOH).

(E)-3’-(3-Azidocinnamyl)-ouabain(Ouabain-9)

The column chromatography purification method comprises the following steps: silica gel Chromatography (CH)₂Cl₂MeOH ═ 20:1-15:1) purified crude oaabain-9 to give solid product oaabain-9 (22.6mg, purity > 90%, overall yield 61.2%).

¹H NMR(MeOD,400MHz)δ:7.32(t,J＝7.9Hz,1H),7.23(d,J＝7.7Hz,1H),7.09(d,J＝2.0Hz,1H),6.93(dd,J＝7.9,2.2Hz,1H),6.66(d,J＝16.0Hz,1H),6.44(dt,J＝15.9,5.7Hz,1H),5.90(s,1H),5.00(dd,J＝18.4,1.7Hz,1H),4.93–4.87(m,1H),4.86–4.84(m,1H),4.34–4.11(m,5H),3.78(dt,J＝4.4,2.2Hz,1H),3.72(ddd,J＝12.9,9.4,3.3Hz,1H),3.37(t,J＝9.4Hz,1H),2.90(t,J＝7.2Hz,1H),2.31–2.22(m,1H),2.21–1.82(m,7H),1.82–1.43(m,6H),1.45–1.28(m,8H),1.26(d,J＝6.2Hz,3H),0.91(s,3H).¹³C NMR(MeOD,101MHz)δ:177.5,177.1,141.8,140.3,132.1,131.1,130.2,129.3,129.2,124.3,119.1,118.0,117.8,99.3,85.6,75.3,74.2,73.0,72.6,72.4,71.8,70.5,69.9,51.6,50.9,50.4,49.8,33.4,33.1,30.7,30.5,27.8,26.3,24.5,23.7,18.0,17.6,14.4.IR(thin film,cm^-1):3006,2992,1471,1275,1261,765,and 748.HRMS(DART-TOF)calculated for C₃₈H₅₁N₃NaO₁₂ ⁺[M+Na]⁺m/z 764.3370,found 764.3367.[α]_D ²⁶＝-8.2(c＝0.40,MeOH).

(E)-3’-(4-Fluorocinnamyl)-ouabain(Ouabain-12)

The column chromatography purification method comprises the following steps: silica gel Chromatography (CH)₂Cl₂MeOH ═ 15:1-10:1) purified crude oaabain-12 to give solid product oaabain-12 (23.3mg, purity > 90%, overall yield 64.2%).

¹H NMR(400MHz,MeOD)δ:7.43(dd,J＝8.5,5.4Hz,2H),7.03(t,J＝8.6Hz,2H),6.65(d,J＝16.0Hz,1H),6.34(dt,J＝15.9,5.9Hz,1H),5.90(s,1H),5.00(dd,J＝18.4,1.7Hz,1H),4.94–4.86(m,1H),4.85(s,1H),4.27(ddd,J＝16.4,10.3,5.1Hz,1H),4.22–4.10(m,4H),3.78(dt,J＝4.9,2.4Hz,1H),3.76–3.68(m,2H),3.38(t,J＝9.5Hz,1H),2.89(d,J＝8.2Hz,1H),2.24(d,J＝3.6Hz,1H),2.21–1.82(m,8H),1.73(td,J＝13.8,12.9,6.2Hz,4H),1.58(t,J＝10.8Hz,1H),1.49(dd,J＝13.4,11.0Hz,1H),1.44–1.31(m,7H),1.26(d,J＝6.3Hz,3H),0.91(s,3H).¹³C NMR(101MHz,MeOD)δ:177.5,177.1,165.0,162.5,132.2,129.3,129.2,127.4,118.0,116.4,116.1,99.3,85.6,75.9,75.3,74.2,73.1,72.6,72.4,71.8,70.3,69.9,69.0,51.6,50.9,50.3,49.8,41.2,33.4,33.0,30.7,30.4,27.8,24.5,23.7,18.0,17.5,14.4.IR(thin film,cm^-1):3005,2989,1475,1275,1261,766,752 and 745.HRMS(DART-TOF)calculated for C₃₈H₅₁FNaO₁₂ ⁺[M+Na]⁺m/z 741.3262,found 741.3263.[α]_D ²⁶＝-17.1(c＝0.39,MeOH).

(E)-3’-(4-Ethynylcinnamyl)-ouabain(Ouabain-15)

The column chromatography purification method comprises the following steps: silica gel Chromatography (CH)₂Cl₂MeOH ═ 15:1-10:1) the crude Ouabain-15 was purified to give the solid product Ouabain-15(28.3mg, purity > 90%, overall yield 78.2%).

¹H NMR(400MHz,MeOD)δ:7.39(s,4H),6.67(d,J＝15.9Hz,1H),6.45(dt,J＝16.0,5.8Hz,1H),5.90(s,1H),5.00(dd,J＝18.4,1.8Hz,1H),4.94–4.87(m,1H),4.86–4.84(m,1H),4.33–4.11(m,6H),3.78(dt,J＝4.3,2.1Hz,1H),3.75(s,2H),3.39(d,J＝9.4Hz,1H),2.95–2.84(m,1H),2.31–2.23(m,1H),2.15(q,J＝6.6,4.6Hz,4H),2.09–1.93(m,4H),1.85(d,J＝22.6Hz,2H),1.80–1.65(m,1H),1.58(dd,J＝11.9,9.5Hz,2H),1.53–1.44(m,2H),1.39(dd,J＝17.5,7.2Hz,3H),1.34–1.29(m,8H),1.25(d,J＝6.2Hz,3H),0.91(s,3H).¹³C NMR(101MHz,MeOD)δ:177.6,177.1,138.7,133.2,132.4,130.8,129.1,127.5,122.8,118.0,99.3,85.6,79.2,75.9,75.3,74.2,73.1,72.6,72.4,71.8,70.4,69.9,69.0,51.6,50.9,50.3,49.9,41.2,33.4,33.0,30.8,30.7,30.5,30.3,27.8,24.5,23.7,18.0,17.5,14.4.IR(thin film,cm^-1):3011,2988,1467,1275,1261,764,760,756 and 752.HRMS(DART-TOF)calculated for C₄₀H₅₂NaO₁₂ ⁺[M+Na]⁺m/z 747.3356,found 747.3351.[α]_D ²⁶＝-16.8(c＝0.39,MeOH).

(E)-3’-(4-Trifluoromethylcinnamyl)-ouabain(Ouabain-18)

The column chromatography purification method comprises the following steps: silica gel Chromatography (CH)₂Cl₂MeOH ═ 15:1-10:1) purified crude oaabain-18 to give solid product oaabain-18 (19.2mg, purity > 90%, overall yield 50.1%).

¹H NMR(400MHz,MeOD)δ:7.59(s,4H),6.87–6.66(m,1H),6.56(dt,J＝16.0,5.5Hz,1H),5.00(dd,J＝18.5,1.7Hz,1H),4.92(d,J＝1.7Hz,1H),4.86(d,J＝1.7Hz,1H),4.24(dddd,J＝31.0,26.2,14.9,7.7Hz,5H),3.81–3.75(m,1H),3.75–3.69(m,1H),3.40(d,J＝9.4Hz,1H),2.90(dd,J＝8.7,5.7Hz,1H),2.36–2.22(m,1H),2.24–1.82(m,7H),1.73(ddd,J＝25.3,13.5,4.8Hz,4H),1.64–1.33(m,5H),1.35–1.28(m,4H),1.26(d,J＝6.2Hz,3H),0.91(s,3H).¹³C NMR(101MHz,MeOD)δ:177.5,177.1,142.2,131.4,130.9,130.4,130.1,127.9,127.1,126.5,126.4,118.0,99.3,85.6,75.9,75.3,74.2,72.9,72.6,72.4,71.8,70.6,69.9,69.0,51.6,50.9,50.4,49.9,41.2,33.4,33.1,30.7,30.5,27.8,24.5,23.7,18.0,17.5,14.4.IR(thin film,cm^-1):3012,2988,1455,1275,1261,766,763,and 745.HRMS(DART-TOF)calculated for C₃₉H₅₁F₃NaO₁₂ ⁺[M+Na]⁺ m/z 791.3230,found 791.3226.[α]_D ²⁶＝-13.0(c＝0.39,MeOH).

(E)-3’-Cinnamyl-ouabain(Ouabain-21)

The column chromatography purification method comprises the following steps: silica gel Chromatography (CH)₂Cl₂Crude oaabain-21 was purified with MeOH ═ 20:1-15:1) to give oaabain-21 as a solid product (25.9mg, greater than 90% purity, 73.8% overall yield).

¹H NMR(400MHz,MeOD)δ:7.46–7.36(m,2H),7.29(td,J＝7.5,2.7Hz,2H),7.23–7.18(m,1H),6.66(dd,J＝16.1,3.2Hz,1H),6.39(dtd,J＝15.4,6.0,3.1Hz,1H),5.89(d,J＝2.4Hz,1H),4.99(dt,J＝17.8,2.6Hz,1H),4.92–4.89(m,1H),4.86–4.84(m,1H),4.27(d,J＝10.1Hz,1H),4.21(dt,J＝6.6,1.9Hz,1H),4.20–4.08(m,4H),3.78(dt,J＝4.3,2.3Hz,1H),3.76–3.67(m,2H),3.39(t,J＝9.4Hz,1H),2.90(t,J＝5.1Hz,1H),2.26(d,J＝15.2Hz,1H),2.20–2.10(m,3H),2.10–1.82(m,5H),1.72(q,J＝11.3,9.9Hz,4H),1.62–1.52(m,1H),1.48(t,J＝12.1Hz,1H),1.42–1.30(m,4H),1.29(t,J＝3.4Hz,4H),1.25(d,J＝6.0Hz,3H),0.90(d,J＝3.0Hz,3H).¹³C NMR(101MHz,MeOD)δ:177.6,177.1,138.2,133.6,130.8,129.6,128.7,127.5,118.0,99.3,85.6,75.3,74.2,73.3,72.6,72.4,71.8,69.9,69.0,61.5,51.6,50.9,50.3,33.4,33.1,30.8,30.7,30.5,30.3,28.1,27.8,26.9,24.5,23.7,20.9,18.0,17.6,14.5.IR(thin film,cm^-1):3309,2983,1454,1275,1261,766,755,and 744.HRMS(DART-TOF)calculated for C₃₈H₅₂NaO₁₂ ⁺[M+Na]⁺m/z 723.3356,found 723.3351.[α]_D ²⁶＝-36.1(c＝0.10,MeOH).

(E)-3’-(3-Nitrocinnamyl)-ouabain(Ouabain-24)

The column chromatography purification method comprises the following steps: silica gel Chromatography (CH)₂Cl₂MeOH ═ 20:1-15:1) purified crude oaabain-24 to give solid product oaabain-24 (20.5mg, purity > 90%, overall yield 55.8%).

¹H NMR(400MHz,MeOD)δ:8.27(d,J＝2.1Hz,1H),8.08(dd,J＝8.2,2.3Hz,1H),7.82(d,J＝7.8Hz,1H),7.55(t,J＝8.0Hz,1H),6.80(d,J＝16.0Hz,1H),6.60(dt,J＝16.0,5.5Hz,1H),5.90(s,1H),5.01(dd,J＝18.4,1.7Hz,1H),4.90(d,J＝18.3Hz,2H),4.86(d,J＝1.6Hz,1H),4.31(dd,J＝10.7,4.6Hz,1H),4.30–4.21(m,2H),4.20(d,J＝7.6Hz,2H),4.16(d,J＝6.8Hz,1H),3.79(dd,J＝3.4,1.7Hz,1H),3.78–3.71(m,0H),3.71(dd,J＝9.5,3.4Hz,1H),3.38(t,J＝9.4Hz,1H),2.91(t,J＝7.2Hz,1H),2.28(dt,J＝15.3,3.7Hz,1H),2.23–2.04(m,5H),2.01(s,1H),1.90(dt,J＝14.7,5.5Hz,1H),1.73(ddd,J＝24.0,14.3,4.8Hz,4H),1.63–1.58(m,1H),1.55(d,J＝15.3Hz,1H),1.51–1.46(m,1H),1.43–1.37(m,2H),1.35(d,J＝7.4Hz,3H),1.30(d,J＝3.6Hz,3H),1.26(d,J＝6.1Hz,3H),0.92(s,3H).¹³C NMR(101MHz,MeOD)δ:17δ:177.5,177.1,150.1,140.3,133.4,131.3,130.8,130.5,123.0,121.9,118.0,99.2,85.6,75.9,75.3,74.2,72.7,72.6,72.4,71.8,70.7,69.9,69.0,51.6,50.9,50.4,49.8,41.3,33.4,33.1,30.7,30.5,27.8,24.5,23.7,18.0,17.5,14.4.IR(thin film,cm^-1):3011,2982,1471,1275,1261,764,and 750.HRMS(DART-TOF)calculated for C₃₈H₅₁NNaO₁₄ ⁺[M+Na]⁺m/z 768.3207,found 768.3209.[α]_D ²⁶＝-28.7(c＝0.50,MeOH).

(E)-3’-(3,4-Dichlorocinnamyl)-ouabain(Ouabain-27)

The column chromatography purification method comprises the following steps: silica gel Chromatography (CH)₂Cl₂MeOH ═ 20:1-15:1) purified crude oaabain-27 to give solid product oaabain-27 (28.1mg, purity > 90%, overall yield 72.9%).

¹H NMR(400MHz,MeOD)δ7.57(d,J＝2.0Hz,1H),7.43(d,J＝8.4Hz,1H),7.34(dd,J＝8.4,2.0Hz,1H),6.63(d,J＝16.0Hz,1H),6.45(dt,J＝16.0,5.6Hz,1H),5.90(d,J＝2.0Hz,1H),5.01(dd,J＝18.4,1.8Hz,1H),4.93–4.87(m,1H),4.86(d,J＝1.7Hz,1H),4.32–4.23(m,1H),4.23–4.10(m,4H),3.79(dd,J＝3.4,1.7Hz,1H),3.75(dd,J＝9.8,6.6Hz,1H),3.71(dd,J＝9.5,3.4Hz,1H),3.38(t,J＝9.4Hz,1H),2.90(t,J＝7.2Hz,1H),2.34–2.20(m,1H),2.19–2.11(m,3H),2.08(d,J＝15.4Hz,1H),2.05–1.93(m,2H),1.89(qd,J＝7.0,6.1,3.3Hz,1H),1.72(ddd,J＝25.2,13.0,4.2Hz,5H),1.63–1.57(m,1H),1.53(d,J＝13.7Hz,1H),1.51–1.44(m,1H),1.38(s,1H),1.34–1.27(m,6H),1.26(d,J＝6.3Hz,3H),0.91(s,3H).¹³C NMR(101MHz,MeOD)δ:177.5,177.1,139.0,133.5,131.9,131.6,130.3,129.3,127.1,118.0,99.2,85.5,75.9,75.3,74.2,72.8,72.5,72.4,71.8,70.6,69.9,69.0,58.3,51.6,50.9,50.4,41.2,33.4,33.0,30.7,30.4,27.8,24.5,23.7,18.4,18.0,17.6,14.4.IR(thin film,cm-1):3005,2982,1471,1275,1260,765,760,755,and 745.HRMS(DART-TOF)calculated for C₃₈H₅₀Cl₂NaO₁₂ ⁺[M+Na]⁺m/z 791.2577,found 791.2581.[α]_D ²⁶＝-43.1(c＝0.07,MeOH).

The beneficial effects of the present invention are demonstrated by the following experimental examples.

Experimental example 1: effect of Compounds of the invention on the migratory Capacity of tumor cells

In this example, MAB-MD-231 (breast cancer cell) cell line was selected to perform cell scratch test on each ouabain and its derivatives.

(1) Experimental materials:

MAB-MD-231 cells (breast cancer cells), DMEM (supplemented with L-glutamine, penicillin, 100. mu.g/mL and streptomycin, 100. mu.g/mL, 10nM HEPES) in complete medium with 20% FBS, pancreatin, sterile PBS sterile wrap (one box for each of large, medium and small tips), 6-well plates.

(2) The experimental steps are as follows:

1) and (3) streaking of a culture plate: first, the rear of the 6-hole plate is uniformly lined with a straight ruler, and the transverse lines are drawn approximately every 0.5cm-1cm and transversely pass through the holes. Each hole passes through at least 5 lines. When scribing, attention should not be paid to the fact that the lines are too thick.

2) Cell paving: about 5X 10 additions to the wells⁵And (3) inoculating each cell (the number of different cells is different and is adjusted according to the growth speed of the cells), wherein the inoculation principle is that the fusion rate reaches 100 percent after the overnight inoculation.

3) Cell lineation: the next day the cell layer was scored using a 20 μ L tip (sterile) or sterile toothpick, perpendicular to the plane of the cells, along the line drawn on the back of the plate on the previous day.

4) Washing cells: after the scratch was completed, cells were washed 3 times with sterile PBS, non-adherent cells were washed away so that the gap left after streaking was clearly visible, and then fresh serum-free medium or low serum (< 2%) medium was replaced.

5) Adding medicine: each compound was tested at 10nM, 100nM, 1. mu.M, 10. mu.M, 100. mu.M concentrations, respectively; the blank was prepared by adding the same volume of serum-free DMEM.

6) Cell culture and observation: the cells were incubated at 37 ℃ with 5% CO₂Culturing in an incubator. After 24h, the cells were removed, observed under a microscope and the width of the scratch was measured and recorded by photographing.

7) And (4) analyzing results: and opening the picture by using Image J software, randomly drawing 6 to 8 horizontal lines, measuring the mean value of the distances between cells, and calculating the area to obtain a coverage rate statistical graph. The effect of cell migration is expressed by cell coverage, and the greater the cell coverage, the weaker the effect of the compound in inhibiting cell migration.

(3) Results of the experiment

The results are shown in FIGS. 2A to C. As can be seen, compared with the blank group, the ouabain ring 3' -hydroxyl derivative prepared by the invention has the function of inhibiting the migration of tumor cells. In addition, compared with the Ouabain before modification, the compound Ouabain-12 has obviously higher effect of inhibiting tumor cell migration than Ouabain under the concentration of 100nM, 1 muM, 10 muM and 100 muM.

Experimental results show that the Ouabain-ring 3' -hydroxyl derivative (especially Ouabain-12) prepared by allylation modification of a specific position has excellent effect of inhibiting tumor cell migration.

Experimental example 2: effect of Compounds of the invention on the invasiveness of tumor cells

In the experimental example, a compound Ouabain-12 which showed good performance in the cell scratch experiment was selected, and a Transwell cell invasion experiment was performed to further determine the effect of the compound in inhibiting tumor cell invasion.

(1) Experimental materials:

MAB-MD-231 cells (breast cancer cells), Matrigel Matrix basic Membrane Matrix, LDEV-free (BD Biocoat 354234), serum free medium (antibiotics pre-loaded with L-glutamine \10nM HEPES, serum free medium may inhibit cell growth, either no or reduced to 1/10 for Transwell), complete medium containing 20% FBS (pre-loaded with L-glutamine, 100. mu.g/mL penicillin and 100. mu.g/mL streptomycin, 10nM HEPES, for normal cells), Transwell-24 Membrane nest, 6.5mm diameter, 8.0 μm pore size PC Membrane (polycarbonate), pancreatin, methanol (Dow Corona chemical, CAS67561), crystal violet stain (Biyuntan, C0121), sterile PBS (pH 7.2-7.4). The device comprises the following steps: transwell experimental facility.

(2) And (3) experimental operation:

1) matrix glue was laid in the cell 2 hours in advance: first, matrigel was diluted with double no DMEM at a concentration (2. mu.g/. mu.L) in a volume of 50. mu.L and added to the chamber;

2) incubating at 37 ℃ for 30-60 min, observing under a microscope, and curing after the matrix is solidified;

3) grouping: an experimental group (ouabain derivative 1 mu g/mL, serum-free DMEM preparation, 600 mu L) and a blank group (serum-free DMEM 600 mu L);

4) preparing a cell suspension: digesting MAB-MD-231 cells, terminating digestion, centrifuging (800g, 3min), discarding the culture medium, resuspending with 5mL of 20% serum-containing medium, adjusting cell density to 5 × 10⁴/mL。

5) Inoculating cells: adjusting cell density to 5X 10 with complete medium⁴Per mL, 200. mu.L of the cell suspension was taken and added to a Transwell chamber (1X 10)⁴Well), 500. mu.L of serum-free medium was added to the lower layer.

6) After the wall is attached to the wall naturally for 12 hours, the upper layer is replaced by a serum-free culture medium, and the lower layer is replaced by a grouped sample.

7) Culturing the cells for 8-12h (according to different cell searching time). After incubation, the cells on the membrane surface at the bottom of the upper chamber were carefully wiped off with a wet cotton swab.

8) The upper chamber medium was blotted dry (residual cells could be washed out with serum-free medium), the chamber carefully removed with forceps, transferred to a well pre-loaded with about 800. mu.L of 100% methanol, and fixed at room temperature for 5-10min.

9) The cell was taken out, transferred to a well to which about 800. mu.L of crystal violet stain was previously added, and stained at room temperature for about 15min (during which time the staining of the cells was observed and the time was adjusted).

10) The chamber was taken out, rinsed 2 times with deionized water, the bottom surface was gently moved up onto the slide, and 9 random fields were counted under an inverted microscope for statistical results.

(3) Results of the experiment

The results are shown in FIG. 3. Compared with a blank experiment, the Ouabain-12 derivative of the Ouabain-3' -hydroxyl group of the Ouabain ring shows a good effect of inhibiting tumor cell invasion, and the inhibition effect is improved along with the increase of the concentration. In addition, compared with Ouabain, Ouabain-12 has significantly improved ability to inhibit tumor cell invasion.

Experimental example 3: toxicity test of the Compound of the present invention to Normal cells

(1) Experimental methods

Normal human suprarenal cortical cells 293T were selected for cytotoxicity experiments on the compounds prepared in accordance with the present invention. The cells were prepared into a single cell suspension, 3000 cells/well, and inoculated into a 96-well plate, and left in a cell incubator overnight to adhere to the wall. After incubation for 72 hours with test compound, 20. mu.L of MTT solution was added at 37 ℃ and left for 2-4 hours. Dark purple crystals formed in the mitochondria, the supernatant was discarded, 100. mu.L DMSO was added for dissolution, and the mixture was incubated on a shaker for 10min. The absorbance was measured at a wavelength of 570nm using a microplate reader (full-wavelength microplate reader, Saimer Feishell technology Co.). Calculating the inhibition rate based on the correlation between the measurement hole and the control hole absorbance, and calculating the corresponding IC according to the inhibition rate curve by adopting a Bliss method₅₀The value is obtained.

(2) Results of the experiment

TABLE 2 IC of the respective compounds on normal human suprarenal cortical cells 293T and breast cancer cells MAB-MD-231₅₀Value of

IC of compound on 293T cortex cells of normal human kidney₅₀The values are shown in Table 2. As can be seen, compared with the compound before modification, the compound of the invention has significantly reduced cytotoxicity on normal cells, especially the compounds of Ouabain-27, Ouabain-24, Ouabain-15, Ouabain-18 and Ouabain-12. The 3' -hydroxyl derivative of the ouabain ring prepared by allylation modification of a specific position, which is prepared by the invention, has the possibility of having less toxic and side effects.

Experimental example 4: toxicity test of the Compound of the present invention on tumor cells

(1) Experimental methods

The compound prepared by the invention is subjected to cytotoxicity experiments by selecting breast cancer cells MAB-MD-231 and other tumor cells (hela: cervical cancer cells; Hep G2: human liver cancer cells; SK-HEP-1: human liver ascites adenocarcinoma cells; Du 145: prostate cancer cells; Panc 1: human pancreatic cancer cells). The cells were prepared into a single cell suspension, 3000 cells/well, and inoculated into a 96-well plate, and left in a cell incubator overnight to adhere to the wall. After incubation for 72 hours with test compound, 20. mu.L of MTT solution was added at 37 ℃ and left for 2-4 hours. Dark purple crystals formed in the mitochondria, the supernatant was discarded, 100. mu.L DMSO was added for dissolution, and the mixture was incubated on a shaker for 10min. The absorbance was measured at a wavelength of 570nm using a microplate reader (full-wavelength microplate reader, Saimer Feishell technology Co.). Calculating the inhibition rate based on the correlation between the measurement hole and the control hole absorbance, and calculating the corresponding IC according to the inhibition rate curve by adopting a Bliss method₅₀The value is obtained.

(2) Results of the experiment

(2.1) toxicity of Compounds on Breast cancer cells MAB-MD-231

IC of compound on breast cancer cell MAB-MD-231₅₀The values are shown in Table 2. Compared with the prior Ouabain, the 3' -hydroxyl derivative of the Ouabain ring prepared by the invention has reduced cytotoxicity to breast cancer cells MAB-MD-231 to different degrees.

As can also be seen from Table 2, the IC of Ouabain for normal cytotoxicity₅₀Is its IC on breast cancer cells ₅₀1/4, indicating that Ouabain has significant toxicity to normal cellsAnd toxicity to breast cancer cells. Compared with Ouabain, the degree of reduction of cytotoxicity of the Ouabain ring 3' -hydroxyl derivative on normal cells is obviously greater than that on breast cancer cells.

Therefore, the compound comprehensively considers the inhibition effect of the compound on normal cells and breast cancer cells, and the Ouabain ring 3' -hydroxyl derivative prepared by allylation modification at a specific position has better application prospect as an antitumor drug than Ouabain.

(2.2) toxicity of Compounds to other tumor cells

TABLE 3 IC of compound Ouabain-3 on other tumor cells₅₀Value of

IC of compound Ouabain-3 on other tumor cells (hela: cervical cancer cell; Hep G2: human liver cancer cell; SK-HEP-1: human liver ascites adenocarcinoma cell; Du 145: prostate cancer cell; Panc 1: human pancreatic cancer cell)₅₀The values are shown in Table 3. It can be seen that Ouabain-3 has the weakest ability to inhibit Hep G2, even weaker than the ability to inhibit 293T, a cortical cell on human kidney; ouabain-3 has certain inhibition capacity on hela; ouabain-3 has better inhibition capability on Du 145, Panc1 and SK-HEP-1, has the strongest inhibition capability on SK-HEP-1, and has the action concentration on SK-HEP-1 of 1/70 of HEP G2.

The results show that the ouabain ring 3' -hydroxyl derivative prepared by the invention can obviously reduce the toxicity to normal cells and simultaneously has good effect of inhibiting various tumor cells including breast cancer cells. In addition, the ouabain ring 3' hydroxyl derivative prepared by the invention has better selectivity on the inhibition of tumor cells, has weaker inhibition on Hep G2 liver cancer cells, and has obviously better inhibition on prostate cancer cells, human pancreatic cancer cells and SK-HEP-1 liver ascites adenocarcinoma cells.

Experimental example 5: CCK-8 method for detecting effect of compound in inhibiting pancreatic cancer cell proliferation

In this experimental example, the effect of the compound of the present invention on inhibiting the proliferation of human pancreatic cancer cell lines (BXPC3, PANC 1) was further examined by the CCK-8 method.

(1) Experimental Material

DMEM, 1640 medium was purchased from Corning Inc. (Corning, USA); DMSO, CCK-8 kit was purchased from Dojindo; BXPC3, PANC1 cell line, etc. (purchased from the cell resources center of the shanghai life science institute of china academy of sciences, cell bank, china academy of sciences); other reagents are domestic analytical purifiers and the like.

(2) Experimental methods

(a) 50. mu.l/well of BXPC3 cell line cells in logarithmic growth phase were seeded in 96-well microplate at 4-5X 10³Cells/well (BXPC3), with addition of compound samples diluted in culture medium every other day (compound samples were prepared as 20mM stock solutions dissolved in DMSO) to give final concentrations between 100uM and 0.002uM and a final volume of 100. mu.l/well; 3 concentration gradients (1.111uM, 3.333uM, 10uM) were set, each concentration being a duplicate well, and a blank well was set, i.e., no sample was added, only the corresponding volume of sample lysis solution was added. Cells were incubated at 37 ℃ with 5% CO₂After culturing for 72 hours under the condition, adding 10 mul/hole of CCK-8 reagent; after further incubation for 0.5 hour, detection was performed. The experimental observation indexes are as follows: the optical density (OD value) of each well cell in the 96-well plate was measured at a wavelength of 450nm using a microplate reader, and the cell viability (viatility) was calculated and expressed as AVERAGE. + -. SD.

(b) Using the same method as in (a), the concentration gradient was increased to 9 (0.014uM, 0.041uM, 0.123uM, 1.111uM, 3.333uM, 10uM, 30uM), retested, and the IC of the compound calculated₅₀The value is obtained.

(3) Results of the experiment

TABLE 4 viability data of BXPC3, PANC1 cells co-cultured with each compound

TABLE 5 IC of Ouabain-24 on BXPC3 cells₅₀Value of

TABLE 6 IC of Ouabain-24 on PANC1 cells₅₀Value of

As can be seen from Table 4, the 3' -hydroxyl derivative of the Ouabain ring prepared by the invention can effectively inhibit pancreatic cancer cell proliferation, and the inhibition effects of the compounds Ouabain-3, Ouabain-6, Ouabain-12 and Ouabain-24 are better than that of the compound Ouabain-27. Further, as can be seen from tables 5 and 6, IC of the compound Ouabain-24 of the present invention against BXPC3 cells₅₀IC of value as low as 1.58. mu. mol/L for PANC1 cells₅₀The value was as low as 2.159. mu. mol/L.

Experimental results show that the compounds (particularly the compounds Ouabain-3, Ouabain-6, Ouabain-12 and Ouabain-24) can effectively inhibit pancreatic cancer cell proliferation.

In conclusion, the invention successfully prepares the ouabain ring 3 'hydroxyl derivative by taking a specific allylation reagent as a modifier and allylation modification at a specific position, and the ouabain ring 3' hydroxyl derivative has excellent effects of inhibiting various tumor cells including breast cancer, cervical cancer cells, liver cancer, prostate cancer and pancreatic cancer while obviously reducing the toxicity to normal cells. In addition, the 3' -hydroxyl derivative of the ouabain ring has good selectivity on the inhibition of tumor cells, and the inhibition effect on prostate cancer, human pancreatic cancer and SK-HEP-1 hepatic ascites adenocarcinoma is obviously better than that on HEP G2 liver cancer. In addition, the Ouabain sugar ring 3' hydroxyl derivative can also effectively inhibit tumor cell migration and invasion, and particularly, the compound Ouabain-12 has an inhibition effect even superior to that of Ouabain. Therefore, the ouabain sugar ring 3' hydroxyl derivative provided by the invention has a very good application prospect in preparing medicines for resisting tumors and inhibiting tumor invasion and/or migration.

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt thereof:

wherein n is 3;

3R₀Each independently selected from hydroxy, C_1～3An alkyl group;

R₁selected from hydrogen, substituted or unsubstituted aryl;

the substituent is 1 or more and is selected from halogenated or non-halogenated C_1～5Alkoxy, halogenated or non-halogenated C_2～6Alkynyl, halogen, N₃And a nitro group.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: the structure of the compound is shown as formula II:

wherein R is₁Selected from hydrogen, substituted or unsubstituted aryl;

the substituent is 1 or more and is selected from halogenated or non-halogenated C_1～3Alkoxy, halogenated or non-halogenated C_2～4Alkynyl, halogen, N₃And a nitro group.

3. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein: the structure of the compound is shown as formula III:

wherein m is an integer of 1-5; m R₂Each independently selected from hydrogen, halogenated or non-halogenated C_1～3Alkoxy, halogenated or non-halogenated C_2～4Alkynyl, halogen, N₃Or a nitro group.

4. The compound of claim 3, or a pharmaceutically acceptable salt thereof, wherein: m is 1 or 2; the halogen is fluorine or chlorine.

5. The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein: the structure of the compound is shown as follows:

6. a process for the preparation of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, characterized in that: the method comprises the following steps:

R₃Is a protecting group, R₁The method according to any one of claims 1 to 5;

7. The method of claim 6, wherein: the steps (1) to (3) are carried out in an inert gas protection environment; in the step (3), the reaction temperature is 20-30 ℃; the reaction time is 6-24 hours.

8. The method of claim 7, wherein: in the step (3), the reaction temperature is room temperature; the reaction time was 16 hours.

9. The method according to any one of claims 6-8, wherein: in the step (1), the organoboron reagent is

The organic solvent is one or a mixture of two of tetrahydrofuran and isopropanol; the molar ratio of the ouabain to the organic boron reagent is 1: (0.1 to 0.5);

in the step (2), the palladium catalyst is Pd (dba)₃·CHCl₃The phosphorus ligand is PPh₃(ii) a The organic solvent is one or two of tetrahydrofuran and isopropanol; the mol ratio of the ouabain to the allylation reagent, the palladium catalyst and the phosphorus ligand is 1: (1.1-5.0): (0.01-0.2): (0.05-0.4); and/or the activation time is 10-40 minutes; and/or, said R₃Is a hydroxyl protecting group.

10. The method of claim 9, wherein: in the step (1), the organic solvent is a mixture of tetrahydrofuran and isopropanol; the molar ratio of the ouabain to the organic boron reagent is 1: 0.3;

in the step (2), the ouabain and the alkene areThe mol ratio of the propylation reagent to the palladium catalyst to the phosphorus ligand is 1: 1.5: 0.05: 0.2; the activation time is 30 minutes; said R is₃Is Boc group.

11. A medicament, characterized by: the medicine is a preparation prepared by taking the compound or the pharmaceutically acceptable salt thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

12. Use of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of an anti-tumour or inhibition of tumour metastasis; the tumor is breast cancer, cervical cancer cell, liver cancer, prostatic cancer, pancreatic cancer.

13. Use according to claim 12, characterized in that: the tumor is breast cancer, prostatic cancer, pancreatic cancer, and hepatocarcinoma.

14. Use according to claim 13, characterized in that: the liver cancer is liver ascites adenocarcinoma.

15. Use according to any one of claims 12 to 14, characterized in that: the drug is capable of inhibiting tumor cell proliferation, invasion and/or migration.