CN111961107B - Ouabain 19-position primary hydroxyl derivative and preparation method and application thereof - Google Patents

Abstract

The invention provides a 19-position primary hydroxyl derivative of ouabain and a preparation method and application thereof. The structure of the 19-position primary hydroxyl derivative of ouabain is shown in formula I. The derivative has very low toxicity to normal cells, and at the same time has excellent tumor cell inhibitory effect. In addition, the derivative can effectively inhibit tumor cell migration and invasion. Therefore, the 19-position primary hydroxyl derivatives of ouabain provided by the present invention have very good application prospects in the preparation of drugs for anti-tumor and inhibiting tumor invasion and/or migration.

Description

19-position primary hydroxyl derivative of ouabain and its preparation method and use

technical field

The invention belongs to the field of pharmacy, and in particular relates to a 19-position primary hydroxyl derivative of ouabain and a preparation method and application thereof.

Background technique

Among natural products, Cardiac glycosides (CGs) are an important class of compounds, which can specifically act on Na ⁺ /K ⁺ -ATPase and have cardiotonic function. Cardiac glycosides can be divided into cardiac steroids (five-membered unsaturated lactone) and bufadienolide (six-membered unsaturated lactone) according to the 17-position unsaturated lactone. Among them, bufadienolide is mainly isolated from the venom of toad; and cardiac steroids are mainly found in plants, such as Scrophulariaceae (digitalis), Oleander (Oleander genus, shorn horns) genus), Ranunculaceae (Calendula genus), etc., and the highest content in plant leaves. Studies have found that there are more than 30 cardiac glycosides in foxglove leaves, including digitalis, digoxin and ouabain.

Cardiac glycosides have been used for the treatment of heart failure as early as more than 200 years ago; in 1785, British physician William Withering published "Description of foxglove and its medical uses", and then cardiac glycosides were used for their tissue-specific and medical uses. Strong cardiomyocyte contractile effect, is widely used in the treatment of congestive heart failure, combined with diuretics in the treatment of atrial fibrillation and some arrhythmias. The researchers also found that in addition to the function of strengthening the heart, cardiac glycosides also have antitumor, antiviral, antibacterial, immune regulation, influence on nerve cell differentiation, blood pressure, antidepressant and other effects, which have great medicinal properties. value. Among them, Ouabain, as a cardiac glycoside compound, has been found to have good potential in the treatment of breast cancer, prostate cancer, pancreatic cancer, leukemia, neuroblastoma and other tumors (P. , L. Liu, A. Askari. Mol. Pharmacol. 2005, 67, 929–936).

However, although ouabain has a significant effect, a slight overdose can cause serious adverse reactions, even life-threatening. The therapeutic concentration of ouabain is close to the toxic concentration (narrow therapeutic window), and the efficacy (efficacy and toxicity) of ouabain varies greatly due to individual constitution, so its unsafety greatly limits ouabain. Clinical application of drugs. The study also found that ouabain has great toxicity to normal human cells, and it is easy to produce toxic side effects on normal tissues when taking it. People try to modify the ouabain compounds to reduce their toxic and side effects on normal tissues, but the obtained derivatives often reduce the toxicity to normal tissues, and at the same time, the inhibitory activity on tumors is also greatly reduced. Moreover, ouabain compounds have multiple active sites, and how to modify specific sites with high selectivity is also a difficult problem in the art.

Therefore, proper modification of ouabain compounds to prepare ouabain derivatives that can significantly reduce the toxicity to normal cells while maintaining excellent pharmaceutical activity is very important for the clinical application of ouabain compounds meaning.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a 19-position primary hydroxyl derivative of ouabain and its preparation method and use.

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

Among them, n is an integer from 0 to 4;

The n pieces of R ₀ are each independently selected from hydrogen, hydroxyl, halogen, C _1-3 alkyl or C _1-3 alkoxy;

R ₁ is selected from hydrogen, substituted or unsubstituted 3-6 membered saturated cycloalkyl, substituted or unsubstituted 3-6 membered saturated heterocyclic group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl , hydroxyl, halogen, C _1-5 alkyl or C _1-5 alkoxy;

The substituents are one or more, and each is independently selected from halogenated or unhalogenated C _1-5 alkyl, halogenated or unhalogenated C _1-5 alkoxy, halogenated or unhalogenated C 1-5 alkoxy Halogenated C2-6 alkenyl, halogenated or _{unhalogenated C2-6} alkynyl, halogen, _N3 , nitro or hydroxy.

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

Wherein, R ₁ is selected from hydrogen, substituted or unsubstituted 5-6 membered saturated cycloalkyl, substituted or unsubstituted 5-6 membered saturated heterocyclic group, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group Aryl, hydroxyl, halogen, C _1-3 alkyl or C _1-3 alkoxy;

The substituents are one or more, and each is independently selected from halogenated or unhalogenated C _1-3 alkyl, halogenated or unhalogenated C _1-3 alkoxy, halogenated or unhalogenated C 1-3 alkoxy Halogenated _C2-4 alkenyl, halogenated or _{unhalogenated C2-4} alkynyl, halogen, N3, nitro or hydroxy.

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

Wherein, m is an integer of 1-5, preferably 1 or 2; m R ₂ are each independently selected from hydrogen, halogenated or unhalogenated C _1-3 alkyl, halogenated or unhalogenated C _{1 ~3 alkoxy} , halogenated or unhalogenated _C2-4 alkenyl, halogenated or unhalogenated _C2-4 alkynyl, halogen, N3 or nitro; the halogen is preferably fluorine or chlorine .

Further, the structure of the compound is as follows:

The present invention also provides a method for preparing the above compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer, or an isotopic substitution form thereof, the method comprising the following steps:

(1) mix ouabain, organoboron reagent and organic solvent in reaction flask;

R₃为保护基团，R₁如上所述；(2) mix allylating reagent, palladium catalyst, phosphorus ligand and organic solvent in another reaction flask, and stir and activate; the structure of described allylating reagent is

R ₃ is a protecting group, R ₁ is as described above;

(3) The system obtained in step (2) is added to the system obtained in step (1), and the reaction is performed.

Further, the steps (1)-(3) are carried out in an environment protected by an inert gas; in the step (3), the reaction temperature is 20-30°C, preferably room temperature; the reaction time is 6-24 hours, preferably 16 hours.

Further, in step (1), the organic boron reagent is methylphenylboronic acid, and the organic solvent is one or both of tetrahydrofuran and isopropanol, preferably a mixture of tetrahydrofuran and isopropanol; The molar ratio of ouabain and organoboron reagent is 1:(0.5-1.5), preferably 1:1;

In step (2), the palladium catalyst is Pd(dba) ₃ ·CHCl ₃ , the phosphorus ligand is PPh ₃ ; the organic solvent is one or both of tetrahydrofuran and isopropanol; The molar ratio of this glycoside to allylation reagent, palladium catalyst and phosphorus ligand is 1:(1.1-5.0):(0.01-0.2):(0.05-0.3), preferably 1:1.5:0.025:0.1; and /or, the stirring activation time is 5-20 minutes, preferably 10 minutes; and/or, the R ₃ is a hydroxyl protecting group, preferably a Boc group.

The present invention also provides a medicament, the medicament is the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer or an isotopic substitution form thereof as an active ingredient, plus Preparations prepared with pharmaceutically acceptable excipients.

The present invention also provides the use of the above compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer, or an isotopic substitution form thereof, in the preparation of a drug for anti-tumor or inhibiting tumor metastasis; The tumor is preferably breast cancer, cervical cancer, liver cancer, prostate cancer, and pancreatic cancer, more preferably breast cancer, pancreatic cancer, liver cancer, and cervical cancer; the liver cancer is preferably liver ascites adenocarcinoma.

Further, the drug can inhibit tumor cell proliferation, invasion and/or migration.

Definitions of terms used in the present invention: Unless otherwise specified, the initial definitions of groups or terms provided herein apply to the groups or terms throughout the specification; for terms that are not specifically defined herein, they should be based on the disclosure and context. , give their meanings that those skilled in the art can give them.

In the present invention, "room temperature" refers to 25±2°C.

"Overnight" refers to 8 to 16 hours.

"Halogen" refers to fluorine, chlorine, bromine or iodine.

"Isotopic substitution form" refers to a compound obtained by replacing one or more than two atoms in the compound with its corresponding isotope, for example, hydrogen in the compound is replaced by protium, deuterium or tritium.

"Pharmaceutically acceptable" means that a carrier, vehicle, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients that make up a pharmaceutical dosage form and physiologically compatible with receptor compatible.

"Salts" are acidic and/or basic salts of compounds formed with inorganic and/or organic acids and/or bases, and also include zwitterionic (inner) salts, as well as quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. It can also be obtained by mixing the compound with a certain amount of acid or base as appropriate (eg, equivalent). These salts may be precipitated in solution and collected by filtration, recovered after evaporation of the solvent, or obtained by lyophilization after reaction in an aqueous medium.

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

In the present invention, the minimum and maximum carbon content in a hydrocarbon group is indicated by a prefix, eg, the prefix (C _a~b )alkyl denotes any alkyl group containing "a" to "b" carbon atoms. For example, a C _1-3 alkyl group refers to a straight or branched chain alkyl group containing 1 to 3 carbon atoms.

Similarly, C _1-3 alkoxy refers to a straight or branched alkoxy group containing 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 a saturated cycloalkyl group. For example, "a 3- to 6-membered saturated cycloalkyl group" refers to a saturated cycloalkyl group having 3 to 6 ring carbon atoms.

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

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

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

Protecting group: In organic synthesis, a molecule containing two or more functional groups, in order to prevent one of the functional groups from being destroyed by the reaction, a certain reagent is commonly used to protect it first, and then remove the protective reagent after the reaction is completed. group, the group is the protecting group.

In the present invention, specific allylation reagents SI1-SI9 are used as modifiers, and in the presence of phosphorus ligands, metal palladium reagents and organoboron reagents are co-catalyzed to be successfully prepared by allylation modification at specific positions. A class of ouabain 19-position primary hydroxyl derivatives, the preparation conditions of the ouabain 19-position primary hydroxyl derivatives are mild, the catalytic efficiency is high, the reaction selectivity is good, the product yield is high, and it is suitable for industrial production.

Compared with the prior art, the 19-position primary hydroxyl derivative of ouabain provided by the invention has achieved the following beneficial effects:

(1) The 19-position primary hydroxyl derivatives of ouabain provided by the present invention can reduce the toxicity to normal cells, and also have excellent inhibition of breast cancer, cervical cancer, liver cancer (especially liver ascites adenocarcinoma), prostate cancer , the effect of various tumor cells including pancreatic cancer;

(2) The 19-position primary hydroxyl derivative of ouabain has good selectivity for the inhibitory effect on tumor cells, and its inhibitory effect on pancreatic cancer cells, SK-HEP-1 liver ascites adenocarcinoma cells, and cervical cancer cells Significantly better than prostate cancer cells and Hep G2 liver cancer cells;

(3) The 19-position primary hydroxyl derivatives of ouabain (especially the compounds Ouabain-2 and Ouabain-26) can effectively inhibit the proliferation of pancreatic cancer cells;

(4) The 19-position primary hydroxyl derivative of ouabain can also effectively inhibit tumor cell migration and/or invasion.

Therefore, the 19-position primary hydroxyl derivative of ouabain obtained by the allylation modification at a specific position of the present invention has a very good application prospect in the preparation of anti-tumor drugs and inhibiting tumor invasion and/or migration.

Obviously, according to the above-mentioned content of the present invention, according to the common technical knowledge and conventional means in the field, without departing from the above-mentioned basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above content of the present invention will be further described in detail below through the specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

Description of drawings

Fig. 1 is the synthetic route of allylation reagents SI1-SI9.

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

Figure 3 shows the results of cell invasion experiments of each compound at 0.1 μM and 10 μM.

Detailed ways

The raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.

Example 1: Synthesis of allylation reagents SI1-SI9

Reference to known literature (Tetrahedron Letters. 2012, 53, 1319-1322; Jr. Eur. J. Org. Chem. 2016, 28, 4800-4804; Org. Lett. 2009, 11, 2944-2947; Chem. Soc. 2015, 137, 6335-6349; Org. Lett. 2019, 21, 7424-7429), the following 9 allylation reagents SI1 to SI9 were prepared by the synthetic route shown in Fig. 1 . The characterization data of allylation reagents SI-1 to SI-9 are as follows:

SI-1: Allyl-tert-butylcarbonate

¹ H NMR (CDCl ₃ , 400MHz) δ: 5.98-5.83 (m, 1H), 5.38-5.14 (m, 2H), 4.63-4.47 (m, 2H). ¹³ C NMR (CDCl ₃ , 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

¹ H NMR (CDCl ₃ , 400 MHz) δ: 7.31 (d, J=8.7 Hz, 2H), 6.84 (d, J=8.7 Hz, 2H), 6.61 (d, J=15.9 Hz, 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). ¹³ C NMR(CDCl ₃ , 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

¹ H NMR (CDCl ₃ , 400 MHz) δ: 7.29 (t, J=7.8 Hz, 1H), 7.14 (d, J=7.7 Hz, 1H), 7.01 (t, J=2.0 Hz, 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). ¹³ C NMR (CDCl ₃ , 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

¹ H NMR (CDCl ₃ , 400 MHz) δ: 7.35 (dd, J=8.5, 5.5 Hz, 2H), 7.00 (t, J=8.7 Hz, 2H), 6.63 (d, J=15.9 Hz, 1H), 6.21 (dt, J=15.9, 6.4 Hz, 1H), 4.70 (dd, J=6.4, 1.3 Hz, 2H), 1.50 (s, 9H). ¹³ C NMR (CDCl ₃ , 101 MHz) 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

¹ H NMR (CDCl ₃ , 400MHz) δ 7.44 (d, J=8.0 Hz, 2H), 7.33 (d, J=8.0 Hz, 2H), 6.64 (d, J=15.9 Hz, 1H), 6.31 (dt , J=16.0, 6.3Hz, 1H), 4.72 (dd, J=6.4, 1.4Hz, 2H), 1.50 (s, 9H). ¹³ CNMR (CDCl ₃ , 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

¹ H NMR (CDCl ₃ , 400MHz) δ: 7.57 (d, J=8.1 Hz, 2H), 7.47 (d, J=8.1 Hz, 2H), 6.69 (d, J=15.9 Hz, 1H), 6.38 (dt , J=16.0, 6.1Hz, 1H), 4.74 (dd, J=6.2, 1.5Hz, 2H), 1.51 (s, 9H). ¹³ CNMR (CDCl ₃ , 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

¹ H NMR (CDCl ₃ , 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). ¹³ C NMR (CDCl ₃ , 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

¹ H NMR (CDCl ₃ , 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). ¹³ C NMR(CDCl ₃ , 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

¹ H NMR (CDCl ₃ , 400MHz) δ: 7.45 (d, J=2.1 Hz, 1H), 7.38 (dd, J=8.3, 1.6 Hz, 1H), 7.20 (dd, J=8.3, 2.1 Hz, 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＝3.1 Hz, 9H). ¹³ C NMR (CDCl ₃ , 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: Synthesis of ouabain 19-position primary hydroxyl derivative of the present invention

The following synthetic route is adopted to prepare the 19-position primary hydroxyl derivative of ouabain of the present invention:

Weigh ouabain (29.2mg, 0.04mmol, 1.0equiv.) and methylphenylboronic acid (2.4mg, 0.04mmol, 1.0equiv.) into a pre-dried reaction flask A with a stirring bar, close the cap ; Weigh the corresponding allylation reagent (one of SI1 to SI9, 0.06 mmol) in a pre-dried reaction bottle B with a stirrer, and close the bottle cap. The reaction was transferred to a nitrogen-protected glove box. 300 μL of THF and 200 μL of i-PrOH were added to reaction vial A; then Pd(dba) ₃ ·CHCl ₃ (1.0 mg, 0.1% mmol, 2.5% equiv.), PPh ₃ (1.0 mg, 0.4% mmol, 10% equiv.) were added .) Add reaction bottle B in turn, then add ultra-dry THF (300 μL), close the bottle cap, and stir to activate for 10 min. Measure 300 μL of the liquid in reaction B into reaction bottle A with a 1 mL syringe, tighten the lid, remove the reaction from the glove box, and seal the reaction at 25°C. The reaction was carried out for 16 hours. After the reaction is completed, the reaction system is directly spin-dried under reduced pressure to obtain a crude product, which is then purified by column chromatography to obtain the corresponding ouabain 19-position primary hydroxyl derivative. The corresponding relationship between the allylation reagent and the 19-position primary hydroxyl derivative of ouabain is shown in Table 1.

Table 1 Correspondence relationship between raw material allylation reagent and product ouabain 19-position primary hydroxyl derivative

The following are the structure and characterization data of each prepared ouabain 19-position primary hydroxyl derivative:

(E)-19-Allyl-ouabain (Ouabain-2)

Column chromatography purification method: Silica gel chromatography (CH ₂ Cl ₂ :MeOH=20:1-15:1) purifies the crude Ouabain-2 to obtain the solid product Ouabain-2 (18.5 mg, the purity is more than 90%, the total yield is 74%) %).

¹ H NMR (MeOD, 400MHz) δ: 5.89-5.78 (m, 1H), 5.78 (d, J=3.9Hz, 1H), 5.18 (dq, J=17.3, 1.7Hz, 1H), 5.05 (dq, J =10.4,1.4Hz,1H),5.00–4.93(m,1H),4.89(dd,J=18.4,1.8Hz,1H),4.78(dd,J=18.4,1.7Hz,1H),4.62(d, J=1.6Hz, 1H), 4.02(d, J=3.6Hz, 1H), 3.97(dd, J=10.8, 4.2Hz, 1H), 3.89(q, J=1.5Hz, 1H), 3.87(q, J=1.5Hz, 1H), 3.83 (d, J=10.7Hz, 1H), 3.61 (dd, J=3.4, 1.7Hz, 1H), 3.52 (dd, J=9.6, 3.5Hz, 1H), 3.52– 3.44 (m, 1H), 3.44 (d, J=10.5Hz, 1H), 3.27–3.18 (t, J=9.50, 1H), 2.79–2.70 (t, J=8.00, 1H), 2.26 (ddd, J = 15.5, 5.3, 2.1Hz, 1H), 2.15 (dd, J=15.7, 4.8Hz, 1H), 2.10–2.07 (m, 1H), 2.07–2.03 (m, 1H), 1.98 (d, J=15.8 Hz, 1H), 1.93–1.73 (m, 3H), 1.69–1.59 (m, 3H), 1.56 (dd, J=13.2, 4.5Hz, 1H), 1.45–1.28 (m, 3H), 1.26–1.13 ( m, 3H), 1.12 (d, J=6.2Hz, 3H), 0.81 (s, 3H). ¹³ C NMR (MeOD, 101MHz) δ: 177.6, 177.1, 136.5, 118.0, 117.2, 99.3, 85.7, 75.9, 75.9,75.3,74.2,73.6,74.2–74.2(m),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 ): 3393,2928,1731,1390,1341,1042,and 672.HRMS(DART-TOF) calculated for C ₃₂ H ₄₈ NaO ₁₂ ⁺ [M+Na] ⁺ m /z 647.3043,found 647.3038.[α] _D ²⁶ =-25.1 (c=0.51, MeOH).

(E)-19-(4-Methoxycinnamyl)-ouabain(Ouabain-5)

Column chromatography purification method: Silica gel chromatography (CH ₂ Cl ₂ :MeOH=20:1-15:1) to purify the crude Ouabain-5 to obtain a solid product Ouabain-5 (26.3 mg, purity greater than 90%, total yield 89.4 %).

¹ H NMR (MeOD, 400MHz) δ: 7.26 (d, J=8.7Hz, 2H), 6.78 (d, J=8.7Hz, 2H), 6.51 (d, J=15.9Hz, 1H), 6.12 (dt, J=15.9, 6.3Hz, 1H), 5.83 (d, J=1.6Hz, 1H), 5.04 (dd, J=3.8, 2.0Hz, 1H), 4.93 (dd, J=18.6, 1.8Hz, 1H), 4.82(dd,J＝18.4,1.8Hz,1H),4.66(d,J＝1.7Hz,1H),4.13–4.02(m,4H),3.91(d,J＝10.5Hz,1H),3.70(s ,3H),3.66(dd,J＝3.4,1.6Hz,1H),3.61–3.49(m,3H),3.27(t,J＝9.5Hz,1H),2.78(t,J＝7.1Hz,1H) ,2.31(ddd,J＝15.5,5.1,1.8Hz,1H),2.20(dd,J＝15.7,4.8Hz,1H),2.16-2.08(m,2H),2.07-2.00(m,1H),1.99 –1.76(m,4H),1.71(d,J=8.0Hz,1H),1.69-1.58(m,3H),1.52-1.33(m,4H),1.29-1.22(m,4H),1.17(d , J=6.2Hz, 3H), 0.83(s, 3H). ¹³ C NMR (MeOD, 101MHz) δ: 177.6, 177.1, 160.9, 133.8, 130.8, 128.8, 124.8, 118.1, 115.0, 99.0, 85.5, 75.3, 74.5,74.2,73.4,72.3,72.2,71.1,70.1,70.1,69.2,68.1,55.8,51.8,51.4,49.9,49.5,49.3,49.1,48.9,47.3,45.7,40.9,34.8,30.7,28.0,24.1, 18.0,17.5.IR(thin film,cm ^-1 ): 3006,2984,1490,1275,1261,761,752 and 749.HRMS(DART-TOF)calculated for C ₃₉ H ₅₄ NaO ₁₃ ⁺ [M+Na] ⁺ m /z753.3462, found 647.3463. [α] _D ²⁶ =-20.0 (c=0.05, MeOH).

(E)-19-(3-Azidocinnamyl)-ouabain(Ouabain-8)

Column chromatography purification method: Silica gel chromatography (CH ₂ Cl ₂ :MeOH=20:1-15:1) to purify the crude Ouabain-8 to obtain a solid product Ouabain-8 (17.6 mg, purity greater than 90%, total yield 60.0 %).

¹ H NMR (MeOD, 400MHz) δ: 7.24 (t, J=7.8Hz, 1H), 7.14 (d, J=7.8Hz, 1H), 7.01 (t, J=1.9Hz, 1H), 6.85 (dd, J＝7.9, 2.2Hz, 1H), 6.57(d, J＝16.0Hz, 1H), 6.31(dt, J＝16.0, 5.8Hz, 1H), 5.82(d, J＝1.8Hz, 1H), 5.06( s, 1H), 4.93 (dd, J=18.4, 1.8Hz, 1H), 4.81 (dd, J=18.4, 1.8Hz, 1H), 4.66 (d, J=1.6Hz, 1H), 4.15–3.99 (m ,4H),3.93(d,J＝10.5Hz,1H),3.66(dd,J＝3.5,1.7Hz,1H),3.61–3.49(m,3H),3.30(m,1H),2.77(d, J=8.5Hz, 1H), 2.32 (ddd, J=15.6, 5.6, 2.1Hz, 1H), 2.25–1.99 (m, 5H), 1.99–1.77 (m, 4H), 1.75–1.57 (m, 6H) , 1.54–1.30 (m, 6H), 1.17 (d, J=6.3Hz, 3H), 0.84 (s, 3H). ¹³ C NMR (MeOD, 101MHz) δ: 177.5, 177.0, 141.7, 140.1, 132.5, 131.1 ,128.9,124.3,119.1,118.1,117.8,98.9,85.4,75.3,74.5,74.2,72.9,72.3,72.2,71.1,70.1,69.1,51.8,51.3,49.9,47.2,45.7,40.9,34.8,33.1 ,30.5,27.9,24.1,23.7,18.0,17.5,14.4.IR(thin film,cm ^-1 ):3006,2984,1471,1275,1260,765,762 and 750.HRMS(DART-TOF)calculated for C ₃₈ H ₅₁ N ₃ NaO ₁₂ ⁺ [M+Na] ⁺ m/z 764.3370, found 764.3375. [α] _D ²⁶ =-21.7 (c=0.30, MeOH).

(E)-19-(4-Fluorocinnamyl)-ouabain (Ouabain-11)

Column chromatography purification method: Silica gel chromatography (CH ₂ Cl ₂ :MeOH=20:1-15:1) purifies the crude Ouabain-11 to obtain the solid product Ouabain-11 (18.1 mg, the purity is more than 90%, the total yield is 62.7 %).

¹ H NMR (MeOD, 400 MHz) δ: 7.35 (dd, J=8.6, 5.5 Hz, 2H), 6.95 (t, J=8.8 Hz, 2H), 6.57 (d, J=15.9 Hz, 1H), 6.22 ( dt,J＝16.0,6.0Hz,1H),5.84(d,J＝1.8Hz,1H),5.06(dd,J＝3.7,1.9Hz,1H),4.94(dd,J＝18.4,1.8Hz,1H) ),4.82(dd,J=18.4,1.8Hz,1H),4.67(d,J=1.6Hz,1H),4.14–3.99(m,4H),3.93(d,J=10.5Hz,1H),3.67 (dd, J=3.4, 1.6Hz, 1H), 3.62–3.45 (m, 4H), 3.34–3.25 (m, 1H), 2.78 (d, J=9.2Hz, 1H), 2.32 (ddd, J=15.6 , 5.4, 2.1Hz, 1H), 2.23–2.00 (m, 4H), 1.98–1.80 (m, 3H), 1.75–1.56 (m, 4H), 1.42 (ddt, J=16.3, 11.1, 4.1Hz, 3H ), 1.33–1.19 (m, 4H), 1.18 (d, J=6.2Hz, 3H), 1.10 (t, J=7.0Hz, 1H), 0.85 (s, 3H). ¹³ C NMR (MeOD, 101MHz) δ:177.6,177.0,164.9,162.5,134.5,134.5,132.5,129.3,129.2,127.2,127.2,118.1,116.4,116.1,98.9,85.4,75.2,74.4,74.2,73.1,7,72.3,72.2 70.0,69.1,51.7,51.3,50.5,47.2,45.7,40.9,34.8,33.3,33.2,27.9,24.1,18.0,17.5.IR(thin film,cm ^-1 ):3006,2992,1467,1275,1261, 761,751 and 767.HRMS(DART-TOF)calculated for C ₃₈ H ₅₁ FNaO ₁₂ ⁺ [M+Na] ⁺ m/z 741.3262,found 741.3257.[α] _D ²⁶ =-33.3(c=0.41,MeOH).

(E)-19-(4-Ethynylcinnamyl)-ouabain (Ouabain-14)

Column chromatography purification method: Silica gel chromatography (CH ₂ Cl ₂ :MeOH=20:1-15:1) purifies the crude Ouabain-14 to obtain the solid product Ouabain-14 (18.1 mg, the purity is more than 90%, the total yield is 62.7 %).

¹ H NMR (400MHz, MeOD)δ: 7.32(s, 4H), 6.59(d, J=16.0Hz, 1H), 6.32(dt, J=16.0, 5.9Hz, 1H), 5.84(s, 1H), 5.07(s, 1H), 4.94(d, J=18.4Hz, 1H), 4.82(d, J=18.3Hz, 1H), 4.67(s, 1H), 4.10(dh, J=9.7, 5.9Hz, 4H) ),3.94(d,J＝10.6Hz,1H),3.67(d,J＝3.3Hz,1H),3.57(tt,J＝9.4,4.9Hz,3H),3.30(d,J＝10.0Hz,1H) ), 2.79(t, J＝6.9Hz, 1H), 2.39-2.28(m, 1H), 2.23-2.02(m, 4H), 1.98-1.78(m, 4H), 1.76-1.57(m, 4H), 1.55–1.34 (m, 4H), 1.32–1.22 (m, 6H), 1.18 (d, J=6.3 Hz, 3H), 0.85 (s, 3H). ¹³ C NMR (101 MHz, MeOD) δ: 177.5, 177.1 ,138.6,133.2,132.7,128.9,127.5,118.1,99.0,85.4,79.2,75.3,74.4,74.2,73.0,72.3,72.2,71.1,70.1,69.1,68.4,51.8,51.3,50.5,49.9,47. ,40.9,34.8,33.3,33.3,33.1,30.7,30.5,28.0,24.1,23.7,18.0,17.5,14.4.IR(thin film,cm ^-1 ):3006,2988,1478,1275,1261,764,760,756 and 749 .HRMS(DART-TOF) calculated for C ₄₀ H ₅₂ NaO ₁₂ ⁺ [M+Na] ⁺ m/z 747.3356, found 747.3354. [α] _D ²⁶ = -14.1 (c=0.48, MeOH).

(E)-19-(4-Trifluoromethylcinnamyl)-ouabain (Ouabain-17)

Column chromatography purification method: Silica gel chromatography (CH ₂ Cl ₂ :MeOH=20:1-15:1) to purify the crude Ouabain-17 to obtain a solid product Ouabain-17 (16.0 mg, purity greater than 90%, total yield 52.3 %).

¹ H NMR (400MHz, MeOD)δ: 7.52(s, 4H), 6.68(d, J=16.0Hz, 1H), 6.43(dt, J=16.0, 5.7Hz, 1H), 5.83(s, 1H), 5.09(d,J＝3.4Hz,0H),4.94(dd,J＝18.4,1.8Hz,1H),4.82(dd,J＝18.3,1.7Hz,1H),4.67(s,1H),4.18–4.03 (m,4H),3.96(d,J＝10.6Hz,1H),3.66(dd,J＝3.3,1.7Hz,1H),3.58(dd,J＝10.3,2.3Hz,3H),3.32–3.25( m, 1H), 2.79 (dd, J=8.9, 5.4Hz, 1H), 2.38–2.29 (m, 1H), 2.24–2.00 (m, 5H), 1.97–1.77 (m, 4H), 1.76–1.58 ( m, 5H), 1.51–1.34 (m, 5H), 1.21 (s, 5H), 1.18 (d, J=6.3 Hz, 3H), 0.85 (s, 3H). ¹³ C NMR (101 MHz, MeOD) δ: 177.5,177.0,142.1,131.7,130.7,128.0,126.5,126.4,118.1,99.0,85.4,75.3,74.4,74.2,72.8,72.3,72.2,71.1,70.1,70.1,69.1,68.6,50.5,51.8,5 47.2,45.8,40.9,34.8,33.4,33.1,30.7,30.5,27.9,24.1,23.7,18.0,17.5,14.4.IR(thin film,cm ^-1 ):3007,2988,1455,1275,1261,766,756, and 749.HRMS(DART-TOF) calculated for C ₃₉ H ₅₁ F ₃ NaO ₁₂ ⁺ [M+Na] ⁺ m/z 791.3230,found 791.3232.[α] _D ²⁶ =-24.3(c=0.40,MeOH).

(E)-19-Cinnamyl-ouabain (Ouabain-20)

Column chromatography purification method: Silica gel chromatography (CH ₂ Cl ₂ :MeOH=20:1-15:1) to purify the crude Ouabain-20 to obtain a solid product Ouabain-20 (23.9 mg, purity greater than 90%, total yield 85.6 %).

¹ H NMR (400MHz, MeOD) δ: 7.36-7.29 (m, 2H), 7.21 (t, J=7.5Hz, 2H), 7.13 (dd, J=8.3, 6.3Hz, 1H), 6.57 (d, J =15.9Hz,1H),6.27(dt,J=15.9,6.0Hz,1H),5.82(d,J=2.0Hz,1H),5.06(s,1H),4.92(dd,J=18.4,1.8Hz ,1H),4.81(dd,J＝18.4,1.7Hz,1H),4.66(s,1H),4.16–4.01(m,4H),3.93(d,J＝10.6Hz,1H),3.66(dd, J=3.3, 1.6Hz, 1H), 3.62–3.49 (m, 3H), 3.27 (t, J=9.5Hz, 1H), 2.77 (d, J=8.5Hz, 1H), 2.32 (ddd, J=15.9 ,5.1,1.8Hz,1H),2.19(dd,J=15.6,4.8Hz,1H),2.15–2.00(m,2H),1.98–1.77(m,4H),1.71(s,1H),1.69– 1.58(m,3H),1.52–1.33(m,4H),1.21(dq,J=3.5,1.9Hz,4H),1.17(d,J=6.2Hz,3H),0.83(s,3H). ¹³ C NMR (101MHz, MeOD) δ: 177.6, 177.1, 138.1, 133.9, 129.5, 128.7, 127.6, 127.2, 118.1, 98.9, 85.4, 75.3, 74.5, 24.2, 73.2, 72.3, 72.2, 71.1, 70.1, 70.1, 69. ,68.3,51.8,51.3,50.5,47.2,45.7,40.9,34.8,33.3,33.3,32.1,31.9,30.7,28.0,24.1,18.0,17.5.IR(thin film,cm ^-1 ):3006,2983,1455 ,1275,1261,763,757,and 748.HRMS(DART-TOF)calculated for C ₃₈ H ₅₂ NaO ₁₂ ⁺ [M+Na] ⁺ m/z 723.3356,found 723.3352.[α] _D ²⁶ =-59.0(c= 0.25, MeOH).

(E)-19-(3-Nitrocinnamyl)-ouabain(Ouabain-23)

Column chromatography purification method: Silica gel chromatography (CH ₂ Cl ₂ :MeOH=20:1-15:1) purifies the crude Ouabain-23 to obtain the solid product Ouabain-23 (18.6 mg, the purity is more than 90%, the total yield is 62.7 %).

¹ H NMR (400MHz, MeOD) δ: 8.20 (t, J=2.0Hz, 1H), 8.00 (ddd, J=8.2, 2.3, 1.0Hz, 1H), 7.74 (dt, J=7.8, 1.3Hz, 1H) ),7.47(t,J＝8.0Hz,1H),6.78–6.64(m,1H),6.47(dt,J＝16.0,5.6Hz,1H),5.83(s,1H),5.10(dd,J＝ 3.6,2.0Hz,1H),4.94(dd,J=18.4,1.8Hz,1H),4.82(dd,J=18.4,1.7Hz,1H),4.67(d,J=1.6Hz,1H),4.17( tdd,J＝12.2,5.6,1.6Hz,2H),4.07(d,J＝10.2Hz,2H),3.96(d,J＝10.5Hz,1H),3.66(dd,J＝3.5,1.6Hz,1H) ), 3.62–3.48 (m, 3H), 3.29 (d, J=9.6Hz, 1H), 2.79 (t, J=7.1Hz, 1H), 2.34 (ddd, J=15.6, 5.4, 2.1Hz, 1H) , 2.21 (dd, J=15.8, 4.9Hz, 1H), 2.17–2.01 (m, 3H), 1.99–1.78 (m, 3H), 1.78–1.59 (m, 4H), 1.43 (ddt, J=19.0, 12.3, 5.4Hz, 3H), 1.29–1.20 (m, 3H), 1.17 (d, J=6.2Hz, 3H), 0.85 (s, 3H). ¹³ C NMR (101MHz, MeOD) δ: 177.5, 177.0, 150.0,140.2,133.4,131.0,130.8,123.0,121.9,118.1,98.9,85.4,75.2,74.4,74.2,72.6,72.3,72.2,71.1,70.0,69.1,68.7,54.8,51.8.,51.3,50 47.2, 45.8, 40.9, 34.8, 34.8, 33.3, 33.2, 33.0, 30.7, 27.9, 24.1, 18.0, 17.5. IR (thin film, cm ^-1 ): 3006, 2982, 1478, 1275, 1261, 763, 760, 751, and 747 .HRMS(DART-TOF) calculated for C ₃₈ H ₅₁ NNaO ₁₄ ⁺ [M+Na] ⁺ m/z 768.3207,found 768.3212.[α] _D ²⁶ =-27.0(c=0.49,MeOH) .

(E)-19-(3,4-Dichlorocinnamyl)-ouabain (Ouabain-26)

Column chromatography purification method: Silica gel chromatography (CH ₂ Cl ₂ :MeOH=20:1-15:1) purifies the crude Ouabain-26 to obtain the solid product Ouabain-26 (27.1 mg, the purity is more than 90%, the total yield is 88.2 %).

¹ H NMR (400 MHz, MeOD) δ: 7.49 (d, J=2.0 Hz, 1H), 7.35 (d, J=8.4 Hz, 1H), 7.26 (dd, J=8.4, 2.1 Hz, 1H), 6.55 ( d, J=16.0Hz, 1H), 6.33 (dt, J=16.0, 5.7Hz, 1H), 5.84 (d, J=1.8Hz, 1H), 5.07 (dd, J=3.8, 2.0Hz, 1H), 4.94(dd,J＝18.4,1.8Hz,1H),4.82(dd,J＝18.4,1.7Hz,1H),4.67(d,J＝1.6Hz,1H),4.18–4.04(m,4H),3.93 (d, J=10.5Hz, 1H), 3.66 (dd, J=3.4, 1.6Hz, 1H), 3.61–3.48 (m, 3H), 3.28 (t, J=9.5Hz, 1H), 2.79 (t, J＝7.1Hz, 1H), 2.33(ddd, J＝15.5, 5.4, 2.1Hz, 1H), 2.20(dd, J＝15.7, 5.0Hz, 1H), 2.15–2.05(m, 2H), 2.03(s) ,1H),1.87–1.79(m,1H),1.72(q,J=4.1,3.1Hz,1H),1.67(d,J=8.7Hz,1H),1.65–1.57(m,1H),1.42( dtd, J=15.6, 8.6, 3.7Hz, 3H), 1.27 (d, J=7.6Hz, 1H), 1.22 (d, J=3.5Hz, 2H), 1.19 (d, J=6.8Hz, 4H), 1.16 (d, J=4.5Hz, 3H), 0.83 (d, J=11.7Hz, 3H). ¹³ C NMR (101MHz, MeOD) δ: 177.0, 138.9, 133.6, 132.0, 131.6, 130.7, 130.1, 129.3, 127.1,118.1,98.9,85.4,74.4,74.2,72.7,72.3,72.2,70.1,70.1,69.1,51.8,51.3,50.5,49.9,49.7,47.2,45.8,40.9,34.8,34.8,33.3,33.0,30.8 27.9, 24.1, 18.01, 17.5. IR(thin film, cm ^-1 ): 3006, 2995, 1475, 1275, 1261, 764, and 751. HRMS(DART-TOF) calculated for C ₃₈ H ₅₀ Cl ₂ NaO ₁₂ ⁺ [M+Na] ⁺ m/z 791.2577, found 791. 2570. [α] _D ²⁶ =-31.6 (c=0.34, MeOH).

The beneficial effects of the present invention are demonstrated below through experimental examples.

Experimental Example 1: Effects of the compounds of the present invention on the migration ability of tumor cells

In this experimental example, the MAB-MD-231 (breast cancer cell) cell line was selected to carry out cell scratch experiments on each ouabain and its derivatives.

(1) Experimental materials:

MAB-MD-231 cells (breast cancer cells), complete medium DMEM containing 20% FBS (pre-added with L-glutamine, penicillin, 100 μg/mL and streptomycin, 100 μg/mL, 10 nM HEPES), trypsin , Sterile PBS sterilization cloth (one box for each of the large, medium and small gun tips), and a 6-well plate.

(2) Experimental steps:

1) Scribing the culture plate: First, draw a horizontal line evenly on the back of the 6-well plate with a ruler, about every 0.5cm-1cm, across the hole. Pass at least 5 lines through each hole. Be careful not to make the lines too thick when you draw the lines.

2) Plating cells: add about 5×10 ⁵ cells into the well (different cells have different numbers and adjust according to the growth speed of the cells). The principle of seeding is that the fusion rate reaches 100% after overnight.

3) Cell streaking: On the second day, use a 20 μL pipette tip (sterilized) or a sterile toothpick, perpendicular to the cell plane, and scratch the cell layer along the line drawn on the back of the plate the day before.

4) Wash the cells: After the scratch is completed, wash the cells 3 times with sterile PBS to remove the non-adherent cells, so that the gap left after the streak is clearly visible, and then replace with fresh serum-free medium or low serum (< 2%) medium.

5) Dosing effect: each compound was tested at concentrations of 10 nM, 100 nM, 1 μM, 10 μM and 100 μM respectively; the same volume of serum-free DMEM was added as blank group.

6) Cell culture and observation: The cells were cultured in a 37°C, 5% CO ₂ incubator. After 24 hours, the cells were taken out, and the width of the scratch was observed and measured under a microscope, and photographed and recorded.

7) Result analysis: After opening the picture with Image J software, randomly draw 6 to 8 horizontal lines, measure the mean value of the distance between cells, and calculate the area to obtain a coverage statistics map. The cell coverage rate was used to represent the effect of cell migration. The larger the cell coverage rate, the weaker the effect of the compound in inhibiting cell migration.

(3) Experimental results

The results are shown in Figures 2A-C. It can be seen that, compared with the blank group, the 19-position primary hydroxyl derivatives of ouabain prepared by the present invention all have the effect of inhibiting tumor cell migration.

The experimental results show that the 19-position primary hydroxyl derivative of ouabain prepared by the allylation modification at a specific position of the present invention can effectively inhibit the migration of tumor cells.

Experimental Example 2: Influence of the compounds of the present invention on the invasive ability of tumor cells

In this experimental example, the compound Ouabain-14, which performed well in the cell scratch test, was selected, and the Transwell cell invasion test was performed to further determine the effect of the compound on inhibiting tumor cell invasion.

(1) Experimental materials:

MAB-MD-231 cells (breast cancer cells), Matrigel Basement Membrane Matrix, LDEV-free (BD Biocoat 354234), serum-free medium (pre-supplemented with L-glutamine\10nM HEPES, serum-free medium Antibiotics may inhibit cell growth, without or reduced to 1/10, for Transwell), complete medium with 20% FBS (pre-supplemented with L-glutamine, 100 μg/mL penicillin and 100 μg/mL streptomycin, 10 nM HEPES, for normal cultured cells), Transwell-24 membrane nest, 6.5mm diameter, 8.0μm pore size PC membrane (polycarbonate), trypsin, methanol (Chengdu Cologne Chemical, CAS67561), crystal violet staining solution (Biyuntian, CO121), sterile PBS (pH 7.2-7.4). Apparatus: Transwell experimental apparatus.

(2) Experimental operation:

1) Lay matrigel in the chamber 2 hours in advance: first dilute the matrigel with double-free DMEM, the concentration (2 μg/μL), and the volume of 50 μL, and add it to the chamber;

2) Incubate at 37°C for 30min.-60min., which can be observed under a microscope until the matrigel solidifies;

3) Grouping: experimental group (ouabain derivative 1 μg/mL, prepared in serum-free DMEM, 600 μL), blank group (serum-free DMEM 600 μL);

4) Preparation of cell suspension: Digest MAB-MD-231 cells, stop the digestion, centrifuge (800g, 3min) to discard the culture medium, resuspend in 5mL medium containing 20% serum, adjust the cell density to 5×10 ⁴ /mL .

5) Seeding cells: adjust the cell density to 5×10 ⁴ /mL with complete medium, add 200 μL of cell suspension to a Transwell chamber (1×10 ⁴ /well), and add 500 μL serum-free medium to the lower layer.

6) After 12 hours of natural adherence, the upper layer was replaced with serum-free medium, and the lower layer was replaced with group samples.

7) Culture the cells for 8-12h (exploration time according to different cells). After the incubation period, the cells on the membrane surface on the bottom of the upper chamber were carefully wiped off with a damp cotton swab.

8) Aspirate the upper chamber medium (residual cells can be washed with serum-free medium), carefully remove the chamber with tweezers, move it to the well pre-added with about 800 μL of 100% methanol, and fix it at room temperature for 5-10 min.

9) Take out the chamber, move it to the well pre-added with about 800 μL of crystal violet staining solution, and stain at room temperature for about 15 minutes.

10) Take out the chamber, rinse it twice with deionized water, gently move the bottom side up to a glass slide, take 9 random fields of view under an inverted microscope and count the results.

(3) Experimental results

The results are shown in Figure 3. Compared with the blank experiment, Ouabain-14, the 19-position primary hydroxyl derivative of ouabain provided by the present invention, shows a good effect of inhibiting tumor cell invasion, and the inhibitory effect is related to the concentration of the compound. Its inhibitory effect is improved.

Experimental Example 3: Toxicity test of the compounds of the present invention on normal cells

(1) Experimental method

Select normal human renal epithelial cells 293T to carry out cytotoxicity experiments on the compounds prepared by the present invention. The cells were made into a single cell suspension with 3000 cells/well, seeded in a 96-well plate, and placed in a cell culture incubator overnight to allow them to attach to the wall. After 72 hours of incubation with the compounds to be tested, 20 μL of MTT solution was added at 37° C. and left for 2-4 hours. Dark purple crystals formed in the mitochondria, discard the supernatant, add 100 μL DMSO to dissolve, and incubate on a shaker for 10 min. The absorbance was measured at a wavelength of 570 nm with a microplate reader (full wavelength microplate reader, Thermo Fisher Scientific). The inhibition rate was calculated based on the correlation between the absorbance of the assay well and the control well, and the corresponding IC ₅₀ value was calculated according to the inhibition rate curve using the Bliss method.

(2) Experimental results

Table 2 IC ₅₀ values of each compound on normal human adrenal cortex cells 293T and breast cancer cells MAB-MD-231

Table 2 shows the _IC50 values of the compounds on normal human renal epithelial cells 293T. It can be seen that compared with Ouabain before modification, the 19-position primary hydroxyl derivatives of ouabain prepared by the present invention have significantly reduced cytotoxicity to normal cells, especially compounds Ouabain-2 and Ouabain-26. It is indicated that the 19-position primary hydroxyl derivative of ouabain prepared by the allylation modification at a specific position prepared by the present invention may have less toxic and side effects.

Experimental Example 4: Toxicity test of the compounds of the present invention on tumor cells

(1) Experimental method

Select 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), and the cytotoxicity experiments were carried out on the compounds prepared in the present invention. The cells were made into a single cell suspension with 3000 cells/well, seeded in a 96-well plate, and placed in a cell culture incubator overnight to allow them to attach to the wall. After 72 hours of incubation with the compounds to be tested, 20 μL of MTT solution was added at 37° C. and left for 2-4 hours. Dark purple crystals formed in the mitochondria, discard the supernatant, add 100 μL DMSO to dissolve, and incubate on a shaker for 10 min. The absorbance was measured at a wavelength of 570 nm with a microplate reader (full wavelength microplate reader, Thermo Fisher Scientific). The inhibition rate was calculated based on the correlation between the absorbance of the assay well and the control well, and the corresponding IC ₅₀ value was calculated according to the inhibition rate curve using the Bliss method.

(2) Experimental results

(2.1) Toxicity of compounds to breast cancer cells MAB-MD-231

The _IC50 values of the compounds against breast cancer cells MAB-MD-231 are shown in Table 2. It can be seen that, compared with Ouabain before modification, the 19-position primary hydroxyl derivatives of ouabain prepared by the present invention have reduced cytotoxicity to breast cancer cell MAB-MD-231 to different degrees.

It can also be seen from Table 2 that the IC ₅₀ of Ouabain's toxicity to normal cells is 1/4 of its IC ₅₀ to breast cancer cells, indicating that Ouabain's toxicity to normal cells is significantly greater than that to breast cancer cells. Compared with Ouabain, only Ouabain-20 of the present invention reduces the cytotoxicity to normal cells to a lesser degree than to breast cancer cells, while Ouabain-8, Ouabain-11, Ouabain-14, Ouabain-17 and Ouabain The cytotoxicity of -26 to normal cells was significantly greater than that to breast cancer cells.

Therefore, considering the inhibitory effect of the compound on normal cells and breast cancer cells, the 19-position primary hydroxyl derivatives of ouabain-8, Ouabain-11, Ouabain- 14. Ouabain-17 and Ouabain-26 have better application prospects as antitumor drugs than Ouabain.

(2.2) Toxicity of compounds to other tumor cells

Table 3 IC ₅₀ values of compound Ouabain-2 on other tumor cells

Compound Ouabain-2 on other tumor cells (hela: cervical cancer cells; Hep G2: human hepatoma cells; SK-HEP-1: human liver ascites adenocarcinoma cells; Du 145: prostate cancer cells; Panc 1: human pancreatic cancer cells) The _IC50 values are shown in Table 3. It can be seen that Ouabain-2 has the weakest inhibitory ability on 293T in human renal epithelial cells, and the strongest inhibitory ability on Panc 1. The concentration of Ouabain-2 on Panc 1 is about 1/30 of that of 293T; The inhibitory effect of -HEP-1 is also very good, and the concentration is about 1/7 and 1/17 of that of 293T, respectively; Ouabain-2 also has a certain inhibitory effect on Hep G2 and Du 145, but the inhibitory effect is stronger than that of Panc 1 and hela. Worse than SK-HEP-1. The inhibitory effect of Ouabain-2 on the above tumor cells was Panc 1>SK-HEP-1>hela>Du 145>Hep G2.

The above results show that the ouabain 19-position primary hydroxyl derivative prepared by the present invention can significantly reduce the toxicity to normal cells, and at the same time, it can also have a good effect of inhibiting various tumor cells including breast cancer cells. In addition, the 19-position primary hydroxyl derivative of ouabain prepared in the present invention has good selectivity for the inhibitory effect on tumor cells, and can inhibit pancreatic cancer cells, SK-HEP-1 liver ascites adenocarcinoma cells, and cervical cancer cells. has better effect.

Experimental Example 5: CCK-8 assay to detect the effect of the compounds of the present invention on inhibiting the proliferation of pancreatic cancer cells

In this experimental example, the CCK-8 method was further used to detect the effect of the compounds prepared in the present invention on inhibiting the proliferation of human pancreatic cancer cell lines (BXPC3, PANC 1).

(1) Experimental materials

DMEM and 1640 medium were purchased from Corning Company (Corning, USA); DMSO and CCK-8 kits were purchased from Dojindo Company; BXPC3, PANC 1 cell lines, etc. Cell Resource Center of the Academy of Sciences); other reagents are of domestic analytical grade, etc.

(2) Experimental method

(a) Inoculate 50 μl/well of BXPC3 cell line cells in logarithmic growth phase in a 96-well microplate, 4-5×10 ³ cells/well (BXPC3), and add compound samples diluted in culture medium every other day ( Compound samples were dissolved in DMSO to prepare a 20mM stock solution), so that the final concentration was between 100uM-0.002uM, and the final volume was 100μl/well; 3 concentration gradients (1.111uM, 3.333uM, 10uM) were set, and each concentration was Duplicate wells, and set up blank control wells, that is, no sample is added, only the corresponding volume of sample dissolving solution is added. After the cells were cultured at 37°C and 5% CO ₂ for 72 hours, 10 μl/well of CCK-8 reagent was added; the cells were further cultured for 0.5 hours and detected. The experimental observation indicators are: detect the optical density value (OD value) of each well of cells in a 96-well plate with a microplate reader at a wavelength of 450 nm, calculate the cell viability, and express it as AVERAGE±SD.

(b) Using the same method as (a), increase the concentration gradient to 9 (0.014uM, 0.041uM, 0.123uM, 1.111uM, 3.333uM, 10uM, 30uM), retest, and calculate the _IC50 value of the compound.

(3) Experimental results

Table 4 Survival data of BXPC3, PANC 1 cells co-cultured with each compound

Table 5 IC ₅₀ values of Ouabain-26 on BXPC3 cells

Table 6 IC ₅₀ values of Ouabain-26 on PANC 1 cells

It can be seen from Table 4 that the 19-position primary hydroxyl derivatives of ouabain prepared by the present invention can effectively inhibit the proliferation of pancreatic cancer cells, and the compounds Ouabain-2 and Ouabain-26 have significantly better inhibitory effects than other compounds. Further from Table 5 and Table 6, it can be seen that the IC _{50 value of the compound Ouabain-26 of the present invention on BXPC3 cells is as low as 24.46 μmol/L, and the IC 50 value} on PANC 1 cells is as low as 9.26 μmol/L.

The experimental results show that the compounds of the present invention (especially the compounds Ouabain-2 and Ouabain-26) can effectively inhibit the proliferation of pancreatic cancer cells.

In summary, the present invention uses a specific allylation reagent as a modifier, and successfully prepares a class of ouabain 19-position primary hydroxyl derivatives through allylation modification at a specific position. The 19-position primary hydroxyl group of ouabain While reducing the toxicity to normal cells, the derivatives also have excellent inhibitory effects on various tumor cells including breast cancer, cervical cancer cells, liver cancer, prostate cancer, and pancreatic cancer. Moreover, the 19-position primary hydroxyl derivative of ouabain has good selectivity for the inhibitory effect on tumor cells, and its inhibitory effect on pancreatic cancer cells, SK-HEP-1 liver ascites adenocarcinoma, and cervical cancer cells is significantly better than Prostate cancer cells and Hep G2 liver cancer cells. In addition, the 19-position primary hydroxyl derivative of ouabain can effectively inhibit tumor cell migration and/or invasion. Therefore, the 19-position primary hydroxyl derivatives of ouabain provided by the present invention have very good application prospects in the preparation of drugs for anti-tumor and inhibiting tumor invasion and/or migration.

Claims (16)

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

wherein n is 4;

4 of R₀Each independently selected from hydroxy or methyl;

R₁selected from hydrogen, substituted or unsubstituted aryl;

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_2～6Alkynyl, 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_1～3Alkyl, 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_1～3Alkyl, 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.

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:

(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, R₁The method according to any one of claims 1 to 5;

(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.

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 methyl phenylboronic acid, and the organic solvent is one or a mixture of tetrahydrofuran and isopropanol; the molar ratio of the ouabain to the organic boron reagent is 1: (0.5 to 1.5);

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): (0.05-0.3); and/or the stirring activation time is5-20 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: 1;

in the step (2), the stirring activation time is 10 minutes; and/or, said R₃Is 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.

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

14. Use according to claim 12, characterized in that: the tumor is breast cancer, pancreatic cancer, liver cancer, and cervical cancer.

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

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

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