阅读说明：本技术 二氧六环修饰的四氢咔啉-3-甲酰-The-HGE、其制备、抗肿瘤活性和应用 (Dioxane-modified tetrahydrocarboline-3-formyl-The-HGE, preparation thereof, antitumor activity thereof and application thereof ) 是由 赵明 彭师奇 郤思远 于 2019-12-02 设计创作，主要内容包括：本发明公开了下式的3S-1-(1,1-二甲基-1,3-二氧六环-6-螺基)-1,2,3,4-四氢-β-咔啉-3-甲酰-The-His-Gly-Glu,公开了它的制备方法,公开了它的抗肿瘤活性。因而本发明公开了它在制备抗肿瘤及抗肿瘤转移药物中的应用。(The invention discloses 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro-beta-carboline-3-formyl-The-His-Gly-Glu with The following formula, a preparation method thereof and antitumor activity thereof. Therefore, the invention discloses the application of the compound in preparing anti-tumor and anti-tumor metastasis medicaments.)

1.3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro-beta-carboline-3-formyl-The-His-Gly-Glu of The following formula,

2. a method of synthesizing 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro- β -carboline-3-formyl-The-His-Gly-Glu of claim 1, comprising:

2.1. synthesizing 3S-1,2,3, 4-tetrahydro-beta-carboline-3-benzyl carboxylate;

2.2. synthesizing 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro-beta-carboline-3-benzyl carboxylate;

2.3. synthesizing 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro-beta-carboline-3-carboxylic acid;

2.4. adopting dicyclohexylcarbodiimide as a condensing agent and 1-hydroxybenzotriazole as a catalyst to synthesize 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiroyl) -1,2,3, 4-tetrahydro-beta-carboline-3-formyl-The-OBzl in a liquid phase manner;

2.5. synthesizing 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro-beta-carboline-3-formyl-The;

2.6. adopting dicyclohexylcarbodiimide as a condensing agent and 1-hydroxybenzotriazole as a catalyst to synthesize HCl, His-Gly-Glu (OBzl) -OBzl in a liquid phase manner;

2.7. synthesizing 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro-beta-carboline-3-formyl-The-His-Gly-Glu (OBzl) -OBzl by using dicyclohexylcarbodiimide as a condensing agent and 1-hydroxybenzotriazole as a catalyst;

2.8. synthesizing 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro-beta-carboline-3-formyl-The-His-Gly-Glu.

3. Use of 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro- β -carboline-3-formyl-The-His-Gly-Glu according to claim 1 in The preparation of an anti-tumor medicament.

4. Use of 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiroyl) -1,2,3, 4-tetrahydro- β -carboline-3-formyl-The-His-Gly-Glu according to claim 1 for The preparation of a medicament for The treatment of tumor metastasis.

Technical Field

The invention relates to a 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro-beta-carboline-3-formyl-The-His-Gly-Glu compound, a preparation method thereof and antitumor activity thereof. Therefore, the invention relates to the application of the compound in antitumor drugs and anti-tumor metastasis drugs.

Background

Tumors have become a common disease that seriously threatens human health. For example, about 392.9 ten thousand of new tumor patients in 2015, and 233.8 ten thousand of them died. On average, over 1 million people per day are diagnosed with a tumor. At present, the methods for treating cancer in clinical application mainly include radiotherapy, chemotherapy, antibody therapy, immunotherapy and the like. However, due to serious side effects, multidrug resistance and expensive treatment price after drug treatment, the treatment of cancer is still in trouble. The invention discloses a novel anti-tumor drug which is one of the leading lines of drug research.

The 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro-beta-carboline-3-carboxylic acid is a pharmacophore with multiple biological activities, and the theanine is also the pharmacophore with multiple biological activities. In a related invention, The inventor finds that 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiroyl) -1,2,3, 4-tetrahydro-beta-carboline-3-carboxylic acid fused with two pharmacophores of theanine has The effect of resisting tumor growth, and The 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiroyl) -1,2,3, 4-tetrahydro-beta-carboline-3-formyl-The has The function of resisting tumor growth. In further studies, The inventors have recognized that 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro- β -carboline-3-formyl-The, which is generated by introducing uremic toxin tripeptide His-Gly-Glu at The carboxyl terminal of 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro- β -carboline-3-formyl-The-His-Gly-Glu, may have stronger anti-tumor effect and anti-tumor metastasis effect. Based on this knowledge, the inventors have proposed the present invention. Based on this knowledge, the inventors have proposed the present invention.

Disclosure of Invention

The first aspect of The present invention provides 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiroyl) -1,2,3, 4-tetrahydro- β -carboline-3-formyl-The-His-Gly-Glu of The formula.

The second content of The invention is to provide a preparation method of 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro-beta-carboline-3-formyl-The-His-Gly-Glu, which comprises The following steps:

1) synthesizing (3S) -1, 1-dihydroxymethyl-tetrahydro-beta-carboline-3-benzyl carboxylate;

2) synthesizing 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro-beta-carboline-3-benzyl carboxylate;

3) synthesizing 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro-beta-carboline-3-carboxylic acid;

4) synthesizing 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiroyl) -1,2,3, 4-tetrahydro-beta-carboline-3-formyl-The-OBzl by adopting a liquid phase condensation method with Dicyclohexylcarbodiimide (DCC) as a condensing agent and 1-hydroxybenzotriazole (HOBt) as a catalyst;

5) synthesizing 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro-beta-carboline-3-formyl-The;

6) synthesizing HCl, His-Gly-Glu (OBzl) -OBzl by adopting a liquid phase condensation method with DCC as a condensing agent and HOBt as a catalyst;

7) synthesizing 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro-beta-carboline-3-formyl-The-His-Gly-Glu (OBzl) -OBzl by adopting a liquid phase condensation method with DCC as a condensing agent and HOBt as a catalyst;

8) synthesizing 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro-beta-carboline-3-formyl-The-His-Gly-Glu;

the third aspect of The present invention is to evaluate The inhibitory effect of 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiroyl) -1,2,3, 4-tetrahydro- β -carboline-3-formyl-The-His-Gly-Glu on tumor growth in S180 mice.

The fourth aspect of The present invention is to evaluate The anti-tumor metastasis effect of 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiroyl) -1,2,3, 4-tetrahydro- β -carboline-3-formyl-The-His-Gly-Glu, which comprises:

1. evaluation of The ability of 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro-beta-carboline-3-formyl-The-His-Gly-Glu to inhibit tumor cell migration and invasion

2. Evaluation of 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro-beta-carboline-3-formyl-The-His-Gly-Glu Activity in vivo inhibition of Lewis lung cancer mouse tumor metastasis

Drawings

FIG. 1. synthetic route of 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro-beta-carboline-3-formyl-The-His-Gly-Glu: i) trifluoroacetic acid, 1, 3-dihydroxyacetone; ii) concentrated sulfuric acid, acetone; iii) Palladium on carbon (Pd), H₂(ii) a iv) Dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM); v) a solution of hydrogen chloride in ethyl acetate (4M).

Detailed Description

To further illustrate the invention, a series of examples are given below. These examples are purely illustrative and are intended to be a detailed description of the invention only and should not be taken as limiting the invention.

EXAMPLE 1 preparation of benzyl (3S) -1, 1-dihydroxymethyl-tetrahydro- β -carboline-3-carboxylate (1)

2g (7.2mmol) of L-tryptophan benzyl ester was added to 10mL of dichloromethane, and the mixture was sufficiently stirred to dissolve it. Under ice-bath conditions, 1mL of trifluoroacetic acid was slowly added dropwise to the solution. Then, 0.78g (8.6mmol) of 1, 3-dihydroxyacetone was added to the solution and reacted at room temperature for 7 hours. TLC showed disappearance of L-tryptophan benzyl ester (dichloromethane/methanol: 30: 1). Under ice-bath conditions, 50mL of saturated NaHCO was added to the solution₃The solution was stirred well, then a dichloromethane layer was left, followed by saturated NaHCO₃The solution (30 mL. times.3) was washed with saturated NaCl solution (30 mL. times.3), and the dichloromethane layer was dried over anhydrous sodium sulfate for 12 hours. Filtration and concentration of the filtrate under reduced pressure gave 2.03g (77%) of the title compound as a yellow powder. ESI-MS (M/e):367[ M + H]⁺。

EXAMPLE 2 preparation of benzyl 3S-1- (1, 1-dimethyl-1, 3-dioxan-6-spiroyl) -1,2,3, 4-tetrahydro- β -carboline-3-carboxylate (2)

To 5mL of anhydrous acetone was added 0.2g (0.55mmol) of benzyl (3S) -1, 1-dimethylol-tetrahydro- β -carboline-3-carboxylate (1). 100. mu.L of concentrated sulfuric acid was added thereto under ice-bath conditions, followed by stirring at room temperature for 3 hours. TLC showed the disappearance of compound 1 (petroleum ether/ethyl acetate, 4: 1). Under ice-bath conditions, saturated NaHCO was used₃Adjusting pH of the reaction solution to 7, concentrating the obtained solution under reduced pressure to remove acetone, extracting and washing the residual solution with ethyl acetate for 3 times, and washing the ethyl acetate layer with saturated NaCl solution to neutrality. The ethyl acetate layer was dried over anhydrous sodium sulfate for 12 hours. Filtration and concentration of the filtrate under reduced pressure gave a tan solid which was separated by column chromatography (petroleum ether/ethyl acetate, 4:1) to give 0.08g (35.8%) of the title compound as a white solid. ESI-MS (M/e):407[ M + H]⁺。

EXAMPLE 3 preparation of 3S-1- (1, 1-dimethyl-1, 3-dioxan-6-spiroyl) -1,2,3, 4-tetrahydro-beta-carboline-3-carboxylic acid (3)

To 10mL of methanol were added 0.20g (0.5mmol) of 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro- β -carboline-3-carboxylic acid benzyl ester (2) and 0.02g of Pd/C. Stirring and passage of 12h hydrogen, TLC showed disappearance of Compound 2 (petroleum ether/ethyl acetate, 4: 1). The palladium-carbon (Pd/C) was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was triturated with ether to give 0.14g (90%) of the title compound as a colourless solid. ESI-MS (M/e) 317[ M + H ]]⁺。

EXAMPLE 4 preparation of 3S-1- (1, 1-dimethyl-1, 3-dioxan-6-spiroyl) -1,2,3, 4-tetrahydro- β -carboline-3-formyl-The-OBzl (4)

To 20mL of tetrahydrofuran were added 0.19g (0.6mmol) of 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro- β -carboline-3-carboxylic acid (3), 0.15g (0.72mmol) of N, N' -Dicyclohexylcarbodiimide (DCC) and 0.09g (0.72mmol) of 1-hydroxybenzotriazole (HOBt). Stir for 30 minutes in an ice bath. Then, 0.20g (0.66mmol) of HCl. The-OBzl was added to The reaction mixture. Subsequently, N-methylmorpholine (NMM) was added dropwise to the reaction mixture to adjust the pH of the reaction mixture to 9. After stirring at room temperature for 6 hours, TLC showed the disappearance of compound 3 (dichloromethane/methanol, 30/1). Filtering to remove Dicyclohexylurea (DCU), and concentrating the filtrate under reduced pressure to obtainThe residue was dissolved in 30mL of ethyl acetate and the solution was filtered to remove DCU. The filtrate was sequentially treated with 5% NaHCO₃Aqueous extraction (15 mL. times.3), saturated aqueous NaCl solution (15 mL. times.3), 5% KHSO₄Aqueous wash (15 mL. times.3), saturated aqueous NaCl wash (15 mL. times.3), 5% NaHCO₃The mixture was washed with an aqueous solution (15 mL. times.3) and with a saturated aqueous NaCl solution (15 mL. times.3), and dried over anhydrous sodium sulfate for 12 hours. Filtration and concentration of the filtrate under reduced pressure gave a yellow powder which was purified by silica gel column chromatography (petroleum ether/ethyl acetate, 10/1) to give 0.27g (80%) of the title compound as a colourless powder. ESI-MS (M/e):563[ M + H]⁺；¹H-NMR(300MHz,DMSO-d₆):δ/ppm＝10.94(s,1H),8.28(t,J＝7.2Hz,1H),7.79(s,1H),7.37(m,6H),7.07(m,1H),7.02(m,1H),5.16(s,1H),5.12(s,1H),4.40(m,1H),4.21(m,1H),3.99(m,1H),3.75(m,1H),3.61(m,2H),3.02(m,2H),2.12(m,2H),1.82(m,2H),1.65(s,3H),1.42(s,3H),1.22(m,2H),1.01(m,3H)。

EXAMPLE 5 preparation of 3S-1- (1, 1-dimethyl-1, 3-dioxan-6-spiroyl) -1,2,3, 4-tetrahydro-beta-carboline-3-formyl-The (5)

To 10mL of methanol were added 0.28g (0.5mmol) of 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro- β -carboline-3-formyl-The-OBzl (4) and 0.02g of palladium on carbon (Pd/C). Stirring and passage of 12h hydrogen, TLC showed disappearance of compound 4 (dichloromethane/methanol, 30/1). Pd/C was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was triturated with ether to give 0.21g (90%) of the title compound as a yellow solid. ESI-MS (M/e):473[ M + H]⁺，¹H-NMR(300MHz,DMSO-d₆):δ/ppm＝10.98(s,1H),8.18(d,J＝7.5Hz,1H),7.79(s,1H),7.40(dt,J₁＝4.8Hz,J₂＝8.1Hz,2H),7.02(td,J₁＝8.1Hz,J₂＝4.8Hz,2H),4.40(m,1H),4.28(m,1H),4.12(m,1H),4.02(m,1H),3.81(m,1H),3.61(m,1H),3.02(m,2H),2.14(m,2H),1.82(m,2H),1.65(s,3H),1.41(s,3H),1.22(m,2H),1.03(m,3H)。

EXAMPLE 6 preparation of Boc-Gly-Glu (OBzl) -OBzl

Using the method of example 4, 1.93g (80%) of the title compound was obtained as a colorless solid from 0.88g (5mmol) of Boc-Gly and 2.00g (5.5mmol) of Hcl. Glu (OBzl) -OBzl.

EXAMPLE 7 preparation of HCl.Gly-Glu (OBzl) -OBzl

1.45g (3mmol) of Boc-Gly-Glu (OBzl) -OBzl was dissolved in 20mL of hydrogen chloride in ethyl acetate (4M) and reacted for 4 hours in ice bath. TLC monitoring indicated complete reaction (dichloromethane/methanol system, 30/1). The reaction mixture was concentrated under reduced pressure, the residue was dissolved in anhydrous ethyl acetate, and the resulting solution was concentrated under reduced pressure. This operation was repeated 3 times. The resulting white powdery substance was sufficiently washed with dehydrated ether to obtain 1.14g (90%) of the title compound as a colorless solid.

EXAMPLE 8 preparation of Boc-His (Boc) -Gly-Glu (OBzl) -OBzl

From 1.77g (5mmol) of Boc-His (Boc) and 2.52g (6mmol) of HCl.Gly-Glu (OBzl) -OBzl, 2.56g (67%) of the title compound were obtained as colorless solid using the method of example 4. ESI-MS (M/e):722[ M + H]⁺。

Example 9 preparation of HCl. His-Gly-Glu (OBzl) -OBzl

From 1.44g (2mmol) Boc-His (Boc) -Gly-Glu (OBzl) -OBzl, 1.03g (92%) of the title compound was obtained as a colorless solid using the method of example 7.

EXAMPLE 10 preparation of 3S-1- (1, 1-dimethyl-1, 3-dioxan-6-spiroyl) -1,2,3, 4-tetrahydro- β -carboline-3-formyl-The-His-Gly-Glu (OBzl) -OBzl (6)

To 30mL of anhydrous tetrahydrofuran were added 0.47g (1mmol) of 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro- β -carboline-3-formyl-The (5), 0.25g (1.2mmol) of DCC and 0.16g (1.2mmol) of HOBt, and The mixture was stirred for 30 minutes in an ice bath. Then, 0.62g (1.1mmol) of HCl. His-Gly-Glu (OBzl) -OBzl was added to the reaction mixture. N-methylmorpholine (NMM) was added dropwise to adjust the pH of the reaction mixture to 9. The reaction was carried out at room temperature for 8 hours. TLC showed compound 5 to disappear (dichloromethane/methanol, 15/1), Dicyclohexylurea (DCU) was filtered off and the filtrate was concentrated under reduced pressure. The residue was dissolved in 100mL of ethyl acetate and the solution was filtered to remove DCU. The filtrate was sequentially treated with 5% NaHCO₃Aqueous solution (30 mL. times.3), saturated aqueous NaCl solution (30 mL. times.3), 5% KHSO₄Aqueous wash (30 mL. times.3), saturated aqueous NaCl wash (30 mL. times.3), 5% NaHCO₃The mixture was washed with an aqueous solution (30 mL. times.3) and with a saturated aqueous NaCl solution (30 mL. times.3), and dried over anhydrous sodium sulfate for 12 hours. The mixture is filtered and then is filtered,the filtrate was concentrated under reduced pressure to give a yellow powder which was purified by silica gel column chromatography (dichloromethane/methanol, 15/1) to give 0.71g (70%) of the title compound as a yellow powder. ESI-MS (M/e):977[ M + H]⁺；¹H-NMR(300MHz,DMSO-d₆):δ/ppm＝10.99(s,1H),8.35(m,1H),8.27(m,1H),8,16(m,1H),7.79(m,1H),7.49(m,2H),7.35(m,12H),7.00(m,2H),5.13(s,2H),5.06(s,2H),4.41(m,2H),4.21(m,1H),4.02(m,1H),3.81(m,1H),3.65(m,2H),3.04(m,3H),2.92(m,2H),2.44(m,2H),2.09(m,2H),1.95(m,2H),1.85(m,2H),1.72(m,2H),1.62(s,3H),1.39(s,3H),1.23(m,2H),0.97(t,J＝7.2Hz,3H)。

EXAMPLE 11 preparation of 3S-1- (1, 1-dimethyl-1, 3-dioxan-6-spiroyl) -1,2,3, 4-tetrahydro- β -carboline-3-formyl-The-His-Gly-Glu (7)

To 10mL of methanol were added 0.048g (0.05mmol) of 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro- β -carboline-3-formyl-The-His-Gly-Glu (OBzl) -OBzl preparation (6) and 0.005g of Pd/C, and stirred and charged with 12h of hydrogen, and TLC showed disappearance of Compound 6 (ethyl acetate: water: glacial acetic acid, 4:1: 1). The palladium-carbon (Pd/C) was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was triturated with ether to give 0.033g (85%) of the title compound as a colourless solid. ESI-MS (M/e):795[ M-H]^-，¹H-NMR(300MHz,DMSO-d₆):δ/ppm＝10.96(s,1H),8.34(m,1H),8.29(m,1H),8,15(m,1H),8.02(m,1H),7.79(m,1H),7.54(s,1H),7.39(m,1H),7.04(m,1H),6.98(m,1H),6.83(s,1H),4.44(m,3H),4.24(m,2H),4.12(m,2H),4.02(m,1H),3.03(m,3H),2.93(m,2H),2.27(m,2H),2.07(m,2H),1.93(m,2H),1.84(m,2H),1.62(s,3H),1.39(s,3H),1.20(m,2H),0.97(t,J＝7.2Hz,3H)；¹³C-NMR(125MHz,DMSO-d₆):δ/ppm＝173.64,171.83,171.80,171.67,169.31,135.22,120.62,119.33,118.23,111.76,109.32,98.15,64.75,53.92,51.57,42.55,33.80,32.24,30.79,29.60,22.97,22.91,15.17。

EXAMPLE 12 evaluation of the tumor growth-inhibiting Activity of Compound 7

Experimental animals:

ICR mice, male, 20 + -2 g, purchased from Experimental animals technology, Inc., Viton, Beijing.

The tumor source is mouse S180 sarcoma purchased from animal experiment center of department of medicine of Beijing university and maintained by self passage.

Administration dose and administration mode:

the oral dosage of The compound 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro-beta-carboline-3-formyl-The-His-Gly-Glu (compound 7) is 0.23 mu mol/kg, The injection dosage of The positive control adriamycin is 2 mu mol/kg, and The negative control is normal saline.

The experimental method comprises the following steps: the experiment was performed using the S180 transplanted mouse sarcoma model.

And (3) experimental operation:

inoculating S180 ascites tumor liquid with vigorous growth under aseptic condition, diluting the liquid with normal saline to obtain liquid (1:2), fully mixing, staining tumor cell suspension with freshly prepared 0.2% trypan blue, uniformly mixing, counting by a white cell counting method, wherein the blue stained cell is dead cell, the non-stained cell is live cell, and calculating cell concentration and cell survival rate according to the following formula.

Cell concentration is 4 large grid viable cell number/4 × 10⁴X dilution factor cell number/mL

The cell survival rate was live cell number/(live cell number + dead cell number) × 100%

Preparing tumor solution with survival rate of more than 90% into 1.5 × 10 by homogenizing method⁷Each cell suspension was inoculated subcutaneously to the mouse axilla at a concentration of 0.1mL/10 g/mouse to prepare S180 tumor-bearing mice. After 7 days, solid tumors with the size of mung beans grow out from the right armpits of the mice, and the mice are randomly grouped according to the tumor volume, so that the tumor volume of each group of mice is uniformly distributed. The administration was then started for 10 total doses. After day 17, the mice were anesthetized with ether, sacrificed by cervical dislocation, and then the right axillary tumor growth sites were fixed with forceps, the skin was cut open, the tumors were exposed, blunt-stripped, weighed, and reused for tumorsShowing that the difference between groups was compared by the t-test statistical method.

The experimental results are as follows:

the results are shown in Table 1:

TABLE 1 Activity of Compound 7 for inhibiting tumor growth

Note: n is 12, adriamycin is administrated by intraperitoneal injection, and other adriamycin is administrated in a gastric lavage mode through t test; a) p <0.01 compared to saline group and >0.05 compared to doxorubicin group.

The results show that the in vivo antitumor tumor weight (1.82 +/-0.86 g) of the mice treated by oral compound 7 under the dosage of 0.23 mu mol/kg is significantly different from the physiological saline of the negative control group (3.31 +/-0.73 g, P is less than 0.01); and has no significant difference with the positive control group adriamycin group (1.69 plus or minus 0.28g, P > 0.05). As can be seen, when the oral dosage of the compound 7 is as low as 0.23 mu mol/kg, only about 1/10 of the adriamycin intraperitoneal injection dosage still has the activity of inhibiting the tumor growth of mice, which shows that the invention has outstanding technical effect.

Example 13 evaluation of the ability of Compound 7 to inhibit tumor cell migration Using a Transwell Chamber assay

The experimental method comprises the following steps:

collecting A549 cells with good growth state in logarithmic phase, digesting with 0.25% pancreatin, observing under the mirror, adding serum to stop digestion, centrifuging at 3000rpm for 3min, counting, and preparing into single cell suspension with density of 2 × 10⁶one/mL. 100. mu.L of cell suspension was added to each well of the upper chamber of the Transwell chamber, and a solution of Compound 7 was added to give a final concentration of 20. mu.M. Adding 600 μ L of 1640 culture medium containing 10% FBS into the lower chamber, culturing at 37 deg.C in 5% CO2 incubator for 6 hr, wiping off matrigel and cells in the upper chamber with cotton swab, fixing cells with 4% paraformaldehyde for 30min, removing the fixing solution by suction, and washing with PBS for 3 times; staining with 0.1% crystal violet staining solution for 15min, removing staining solution, washing with PBS 3 times, selecting 9 fields in each chamber, taking pictures, counting the number of cellsAnd (4) showing.

The experimental results are as follows:

the results are shown in Table 2:

table 2 activity of compound 7 in inhibiting migration of a549 cells

Note: n-6, both modes of administration were gavage, t-test, a) indicated P <0.01 compared to PBS group and P <0.01 compared to RGDS group.

The results show that the migration number of the cells (153.56 + -22.65) of the group added with the compound 7 is significantly different from the physiological saline of the negative control group (243.00 + -44.92, P <0.01) and also significantly different from the RGDS of the positive control group (193.77 + -14.78, P <0.01), and therefore, the compound 7 at the concentration of 20 mu mol/L can inhibit the migration of the human non-small cell lung cancer cell A549 and has better cancer cell migration inhibition activity than the RGDS at the same concentration.

Example 14 evaluation of the ability of Compound 7 to inhibit tumor cell invasion Using a Transwell Chamber assay

The experimental method comprises the following steps:

coating matrigel: the Matrigel, which is stored in a refrigerator at-20 c as a yellow solid, was previously put in a refrigerator at 4 c for about 12 hours to be a pink liquid with good fluidity. Adding 240 μ L Matrigel into 960 μ L serum-free culture medium required by corresponding tumor cells, diluting by 5 times, dispersing uniformly, adding 100 μ L Matrigel per well, adding 5% CO at 37 deg.C₂The cell incubator is incubated for 5 hours, so that the matrigel is uniformly paved in the small holes of the polycarbonate membrane.

Hydrated basement membrane: carefully removing the residual liquid in the upper chamber by using a pipette, adding 50 mu L of corresponding serum-free culture medium, incubating for 30min in a cell incubator at 37 ℃ and 5% CO2, and carefully removing the residual liquid in the upper chamber by using the pipette for later use.

Collecting A549 cells with good growth state in logarithmic phase, digesting with 0.25% pancreatin, observing under the mirror, adding serum to stop digestion, centrifuging at 3000rpm for 3min, counting, and preparing into single cell suspension with density of 2 × 10⁶one/mL. 100. mu.L of cell suspension was added to each well of the upper chamber of the Transwell chamber, together with a solution of Compound 7,the final concentration was made to be 20. mu.M. Adding 600 μ L of 1640 culture medium containing 10% FBS into the lower chamber, culturing in a cell incubator containing 5% CO2 at 37 deg.C (A549 cell culture for 12h), performing post-treatment after the required time of each cell strain, wiping off matrigel and cells in the upper chamber with cotton swab, fixing cells with 4% paraformaldehyde for 30min, removing the fixing solution by suction, and washing with PBS for 3 times; staining with 0.1% crystal violet staining solution for 15min, removing staining solution, washing with PBS 3 times, selecting 9 fields in each chamber, taking pictures, counting the number of cellsAnd (4) showing.

The experimental results are as follows:

the results are shown in Table 3:

table 3 activity of compound 7 in inhibiting a549 cell invasion

Note: n is 6, the administration mode is intragastric administration, and the test is carried out by t; a) indicating that P <0.01 compared to the blank control group and P >0.05 compared to the RGDS group.

The results showed that the number of cell invasion (197.78 + -26.00) of the group to which compound 7 was added was significantly different from that of the negative control group (252.78 + -17.49, P <0.01) and was not significantly different from that of the positive control group (205.33 + -24.78, P > 0.05). It can be seen that compound 7 at a concentration of 20 μmol/L can inhibit the invasion of human non-small cell lung cancer cell a549, and has comparable cancer cell invasion-inhibiting activity to RGDS at the same concentration.

EXAMPLE 15 evaluation of Compound 7 Activity for inhibiting tumor metastasis in mice

Experimental animals:

c57BL/6 mice, male, 20. + -.2 g, purchased from Experimental animals technologies, Inc. of Wei Tony, Beijing.

Administration dose and administration mode:

the oral dosage of The compound 3S-1- (1, 1-dimethyl-1, 3-dioxane-6-spiro) -1,2,3, 4-tetrahydro-beta-carboline-3-formyl-The-His-Gly-Glu (compound 7) is 0.23 mu mol/kg, The injection dosage of The positive control RGDS tetrapeptide is 20 mu mol/kg, and The negative control is physiological saline.

The experimental method comprises the following steps: the experiment adopts a mouse Lewis anti-lung cancer metastasis model.

And (3) experimental operation:

lewis mouse lung carcinoma cells (LLC) were purchased from ATCC. DMEM medium containing 10% inactivated fetal calf serum and 1X 10 serum is selected⁵U/L penicillin and 100mg/L streptomycin. The cells were enriched by passage every two days according to the adherent cell culture method. When the cell growth state is good and is in logarithmic growth phase, digesting the cell. Adjusting the cell concentration to 2X 10 with physiological saline⁷Counts per mL, placental blue (Tryanblue) staining, viable cell number>95 percent. Inbred line C57BL/6 male mice were taken, left-handed mice were fixed, the right anterior limb axillary skin of the mice was disinfected with 75% ethanol, and 0.2mL of LLC tumor cell suspension was injected subcutaneously into the axillary region of the mice by holding a 1mL sterile syringe in the right hand. Tumors with the diameter of about 4-5mm can grow 10 days after the mice are inoculated, and the tumors are used as tumor sources for standby.

Inoculating Lewis lung cancer tumor-bearing mouse with good growth for 8-10 days, anesthetizing with ether, removing cervical vertebra, killing, soaking in 75% ethanol for 10min, stripping tumor body on a super clean bench, selecting tumor tissue with good growth, shearing in a sterile flat dish, placing in a glass tissue homogenizer, adding 4 deg.C pre-cooled physiological saline according to the ratio of tumor mass (g) to physiological saline volume (mL) of 1:3, grinding slightly to obtain cell suspension, sieving with 200 mesh cell sieve to obtain single cell suspension, adjusting cell concentration with physiological saline to 2 × 10⁷Number of cells/mL, placental blue staining count, viable cell number>95％。

Inbred line C57BL/6 male mice were taken, left-handed mice were fixed, the right anterior limb axillary skin of the mice was disinfected with 75% ethanol, and 0.2mL of LLC tumor cell suspension was injected subcutaneously into the axillary region of the mice by holding a 1mL sterile syringe in the right hand. Tumors of about 4-5mm in diameter were grown up 10 days after inoculation, tumor volumes were measured, and randomly grouped according to the mean tumor volume,

the administration was started 10 days after tumor inoculation, 10 times in total, and tumor volumes were measured and recorded every two days. After day 22, the mice were anesthetized with ether, sacrificed by cervical dislocation, the tumors were removed and weighed, and the lung metastasis rate and the number of metastatic nodules of the tumors were recorded.

The experimental results are as follows:

the results are shown in Table 4:

TABLE 4 Activity of Compound 7 for inhibiting tumor Lung metastasis

Note: n is 12, RGDS is administered by intraperitoneal injection, and other administration is performed in a gastric lavage mode, and the test is performed by t; a) p <0.05 compared to saline group and P <0.05 compared to RGDS group.

The results show that the in vivo anti-tumor metastasis nodules (2.80 + -2.53) of the mice treated with compound 7 orally at a dose of 0.23 μmol/kg are significantly different from the normal saline of the negative control group (9.38 + -2.62, P <0.05) and also significantly different from the RGDS of the positive control group (4.50 + -2.25, P < 0.05). As can be seen, the compound 7 has better tumor metastasis inhibiting activity at an oral dose as low as 0.23 μmol/kg than the RGDS injected intraperitoneally at a dose of 20 μmol/kg/day. Therefore, the invention has outstanding technical effects.