CN110551121B

CN110551121B - Glutamine amido n-hexyl carboline carboxylic acid benzyl ester, preparation, activity and application thereof

Info

Publication number: CN110551121B
Application number: CN201810565760.5A
Authority: CN
Inventors: 赵明; 康贵峰; 彭师奇; 丰华
Original assignee: Capital Medical University
Current assignee: Capital Medical University
Priority date: 2018-06-04
Filing date: 2018-06-04
Publication date: 2020-11-27
Anticipated expiration: 2038-06-04
Also published as: CN110551121A

Abstract

The invention discloses (3S) -N- (6-Gln-amino N-hexanoyl) -2,3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate with the following structure, a preparation method thereof, a dose dependence relationship of the benzyl carboxylate for resisting lung cancer metastasis, an anti-inflammatory effect thereof and application of the benzyl carboxylate in preparing anti-tumor metastasis drugs and anti-inflammatory drugs.

Description

Glutamine amido n-hexyl carboline carboxylic acid benzyl ester, preparation, activity and application thereof

Technical Field

The invention relates to (3S) -N- (6-Gln-amino N-hexanoyl) -2,3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate, a preparation method thereof, a dose-dependent relationship of lung cancer metastasis resistance and an anti-inflammatory effect thereof. Therefore, the invention relates to the application of the compound in preparing anti-tumor metastasis medicaments and anti-inflammatory medicaments. The invention belongs to the field of biological medicine.

Technical Field

Malignant tumors seriously threaten human health. Among them lung cancer is one of the most aggressive human cancers. For patients with advanced lung cancer, 10% -15% of the patients typically survive for only 5 years. The situation of this difficulty has not improved significantly over the last 30 years. In many clinical cases, lung cancer has metastasized to surrounding tissues before it is diagnosed. Tumor metastasis, especially tumor lung metastasis, is the greatest risk of death in tumor patients. To date, there is no antitumor drug that can prevent tumor metastasis for clinical use. The inventor's prior invention (patent application publication No. CN 106349148A, application No. CN 201510409682.6) has disclosed that amino acid benzyl ester substituted bisindole acetic acid alcohol has triple effects of anti-tumor, anti-tumor metastasis and anti-inflammatory at 0.2 mu mol/kg (left formula). The minimum effective dose of benzyl bisindoloethanol amino acid to exert an antitumor effect by the inventors was unsatisfactory at 0.2. mu. mol/kg. The dosage is higher. Over the past two years, the inventors have been searching for compounds with an anti-tumour effect at a minimum effective dose of less than 0.2. mu. mol/kg. Finally, the inventor finds that the 6-glutamine amino caproic acid modified tetrahydro carboline benzyl carboxylate has the effects of resisting tumor metastasis and resisting inflammation at the dosage of 0.02 mu mol/kg. Because the toxic and side effects of the medicine can disappear along with the reduction of the dosage, the reduction of the effective dosage by 10 times shows that the structure modification has outstanding technical effect. Thus, the inventors have proposed the present invention.

Disclosure of Invention

The first aspect of the present invention provides benzyl (3S) -N- (6-Gln-aminon-hexanoyl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylate of the following structure.

The second aspect of the present invention provides a process for preparing (3S) -N- (6-Gln-amino-N-hexanoyl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylic acid benzyl ester, which comprises:

(1) carrying out a Pictet-Spengler reaction on L-tryptophan benzyl ester and formaldehyde under the catalysis of dilute sulfuric acid to generate (3S) -2,3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate;

(2) 6-aminocaproic acid and di-tert-butyl dicarbonate (Boc) in aqueous sodium hydroxide (2M), mixed solution of water and dioxane₂O reaction to produce 6-Boc-aminocaproic acid;

(3) reacting 6-Boc-aminocaproic acid with (3S) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid benzyl ester in anhydrous tetrahydrofuran in the presence of N, N-Dicyclohexylcarbodiimide (DCC) and N-hydroxybenzotriazole (HOBt) to obtain (3S) -N- (6-Boc-amino N-hexanoyl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid benzyl ester;

(4) removing Boc from (3S) -N- (6-Boc-amino N-hexanoyl) -2,3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate in ethyl acetate solution of hydrogen chloride in ice bath to obtain (3S) -N- (6-amino N-hexanoyl) -2,3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate;

(5) reacting (3S) -N- (6-amino N-hexanoyl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid benzyl ester with Boc-L-Gln in anhydrous tetrahydrofuran in the presence of N, N-dicyclohexylcarbodiimide and N-hydroxybenzotriazole to obtain (3S) -N- (6-Boc-Gln-amino N-hexanoyl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid benzyl ester;

(6) removing Boc from (3S) -N- (6-Boc-Gln-amino N-hexanoyl) -2,3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate in ethyl acetate solution of hydrogen chloride under ice bath to obtain (3S) -N- (6-Gln-amino N-hexanoyl) -2,3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate.

The third aspect of the present invention is to evaluate the dose-dependent relationship of (3S) -N- (6-Gln-aminon-hexanoyl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid benzyl ester on the anti-lung cancer metastasis of C57BL/6 mice.

The fourth aspect of the present invention is to evaluate the inhibition of inflammation in ICR mice by (3S) -N- (6-Gln-aminon-hexanoyl) -2,3,4, 9-tetrahydro-beta-carboline-3-carboxylic acid benzyl ester.

Drawings

FIG. 1. scheme for the synthesis of (3S) -N- (6-Gln-aminon-hexanoyl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylic acid benzyl ester i) HCHO, H₂O,H₂SO₄(ii) a Ii) N, N-Dicyclohexylcarbodiimide (DCC), N-hydroxybenzotriazole (HOBt), N-methylmorpholine, tetrahydrofuran; iii) dioxane, aqueous sodium hydroxide solution, di-tert-butyl dicarbonate (Boc)₂O; iv) Hydrogen chloride/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) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylate (1)

To 300mL of water under ice bath was slowly added 1.5mL of concentrated H₂SO_4，Stirring for 3 min. Thereafter 5.00g (15.1mmol) L-tryptophan benzyl ester hydrochloride and 6mL formaldehyde solution (40%) were added. After stirring at room temperature for 72h, TLC showed disappearance of L-tryptophan benzyl ester hydrochloride. The reaction solution was adjusted to pH 7 with concentrated ammonia water in an ice bath and filtered. The filter cake was dissolved in 100mL of ethyl acetate, and the resulting solution was washed with a saturated aqueous solution of sodium chloride (40 mL. times.3), followed by drying over anhydrous sodium sulfate. Filtering, and concentrating the filtrate under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane: methanol ═ 90:1) to give 1.9g (41%) of the objective compound as a yellow oily solid. ESI-MS (m/e): 307[ M + H]⁺。

EXAMPLE 2 preparation of 6-tert-Butoxycarbonylaminohexanoic acid (2)

5.00g (38.2mmol) 6-aminocaproic acid (EACA) were suspended in 50mL distilled water. The resulting suspension was added with 10mL of aqueous sodium hydroxide (2M) under ice-bath and stirred until dissolved. To the resulting solution was added 9.2g (42.2mmol) (Boc)₂A solution of O and 20mL dioxane was adjusted to pH 9 with aqueous sodium hydroxide (2M) and stirred for 30 min. The ice bath was removed and stirred at room temperature until TLC showed disappearance of 6-aminocaproic acid. The reaction solution was saturated KHSO in ice bath₄The pH of the aqueous solution was adjusted to 7 and the dioxane was removed by concentration under reduced pressure. The reaction solution was saturated KHSO in ice bath₄The pH of the aqueous solution was adjusted to 2, and the mixture was extracted with ethyl acetate (50 mL. times.3). The ethyl acetate layer was then washed with saturated aqueous NaCl solution (40 mL. times.3), and was dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure gave 8.0g (91%) of the title compound as a colorless oil. ESI-MS (m/e): 232[ M + H ]]⁺。

EXAMPLE 3 preparation of benzyl (3S) -N- (6-Boc-aminon-hexanoyl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylate (3)

4.15g (18mmol) of t-butoxycarbonyl-6-aminocaproic acid was dissolved in 80mL of anhydrous tetrahydrofuran, and 2.46g (18.2mmol) of N-hydroxybenzotriazole (HOBt) and 4.04g (19.6mmol) of N, N-Dicyclohexylcarbodiimide (DCC) were added under ice-cooling and stirred for 60 minutes. Then, a solution of 5.06g (16.5mmol) of benzyl (3S) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylate and 80mL of anhydrous tetrahydrofuran was added to the obtained reaction solution. The reaction mixture was adjusted to pH 9 with N-methylmorpholine (NMM) in an ice bath and stirred at room temperature for 24 hours. TLC showed the disappearance of (3S) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylic acid benzyl ester. The reaction solution was filtered, the filtrate was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate. The solution was sequentially saturated NaHCO₃Washing with an aqueous solution (40 mL. times.3), washing with a saturated aqueous NaCl solution (40 mL. times.3), and saturating KHSO₄Aqueous solution (40 mL. times.3), saturated aqueous NaCl solution (40 mL. times.3), saturated aqueous NaHCO solution₃The resulting mixture was washed with an aqueous solution (40 mL. times.3) and with a saturated aqueous NaCl solution (40 mL. times.3). Anhydrous Na for ethyl acetate layer₂SO₄Drying, filtration, concentration of the filtrate under reduced pressure and purification of the residue by silica gel column chromatography (dichloromethane: methanol 160:1) gave 5.43g (64%) of the title compound as a yellow oily solid. ESI-MS (m/e): 520[ M + H ]]⁺。

EXAMPLE 4 preparation of benzyl (3S) -N- (6-aminohexanoyl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylate (4)

3.4g (6.6mmol) of (3S) -N- (Boc-amino-N-hexanoyl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylic acid benzyl ester were dissolved in 40mL of hydrogen chloride in ethyl acetate (4M) under ice-bath and stirred for 3 hours, TLC showed disappearance of (3S) -N- (Boc-amino-N-hexanoyl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylic acid benzyl ester. The reaction mixture was concentrated to dryness under reduced pressure with stirring. The residue was dissolved in anhydrous ethyl acetate, and the solution was concentrated to dryness under reduced pressure. This operation was repeated three times. The residue was washed well with anhydrous ether to give 2.8g (94%) of the title compound as a tan solid. ESI-MS (M/e):420[ M + H]⁺；Mp 223-226℃；¹H NMR(300MHz,DMSO-d6:/ppm＝10.963(d,J＝11.4Hz,1H),7.894(s,3H),7.458(d,J＝7.5Hz,1H),7.327(t,J＝7.5Hz,1H),7.204(dd,J₁＝1.5Hz,J₂＝7.2Hz,1H),7.158-6.972(m,6H),5.607(d,J＝4.7Hz,1H),5.059(s,2H),4767(d,J＝15.6Hz,1H),4.610(d,J＝17.4Hz,1H),3.426(m,1H),3.021(m,1H),2.754(m,2H),2.613(m,1H),2.389(m,1H),1.568-1.546(m,4H),1.399-1.323(m,2H)。

EXAMPLE 5 preparation of (3S) -N- (6-Boc-Gln-aminon-hexanoyl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylic acid benzyl ester (5)

1.04g (4.2mmol) of Boc-Gln and 0.57g (4.2mmol) of N-hydroxybenzotriazole (HOBt) were dissolved in 30mL of anhydrous tetrahydrofuran, and 0.87g (4.2mmol) of N, N-Dicyclohexylcarbodiimide (DCC) was added thereto under ice-bath and stirred for 40 minutes. Then, a solution of 0.58g (4.2mmol) of (3S) -N- (6-aminohexanoyl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylic acid benzyl ester and 30mL of anhydrous tetrahydrofuran was added to the obtained reaction solution, and the pH was adjusted to 8 with N-methylmorpholine (NMM) under ice-bath, followed by stirring at room temperature for 12 hours. TLC shows that (3S) -N- (6-amino-N-hexanoyl) -2,3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate disappears, the reaction solution is filtered, the filtrate is concentrated under reduced pressure, and the residue is dissolved by adding 100mL of ethyl acetate. The solution was sequentially saturated NaHCO₃Washing with an aqueous solution (40 mL. times.3), washing with a saturated aqueous NaCl solution (40 mL. times.3), and saturating KHSO₄Aqueous solution (40 mL. times.3), saturated aqueous NaCl solution (40 mL. times.3), saturated aqueous NaHCO solution₃The resulting mixture was washed with an aqueous solution (40 mL. times.3) and with a saturated aqueous NaCl solution (40 mL. times.3). Acetic acidAnhydrous Na for ethyl ester layer₂SO₄Drying, filtration, concentration of the filtrate under reduced pressure and purification of the residue by silica gel column chromatography (dichloromethane: methanol ═ 60:1) gave 0.30g (13%) of the title compound as a pale yellow solid. ESI-MS (M/z):648[ M + H]⁺。

EXAMPLE 6 preparation of benzyl (3S) -N- (6-Gln-amino-N-hexanoyl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylate (6)

600mg of (3S) -N- (6-Boc-Gln-amino-N-hexanoyl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylic acid benzyl ester was added in ice bath to 16mL of hydrogen chloride in ethyl acetate (4M) and stirred for 2h, TLC (ethyl acetate: water: glacial acetic acid ═ 3: 1: 1.2) showed disappearance of (3S) -N- (6-Boc-Gln-amino-N-hexanoyl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylic acid benzyl ester. The reaction solution was concentrated to dryness under reduced pressure. The residue was dissolved in anhydrous ethyl acetate and concentrated to dryness under reduced pressure. This operation was repeated three times. The residue was washed thoroughly with anhydrous diethyl ether and then with C₁₈Column chromatography purification (methanol: water ═ 30:70) and lyophilization gave 320mg (63%) of the title compound as a pale yellow solid. ESI-MS (M/e):548[ M + H]⁺.Mp 123-124℃；¹H NMR(300MHz,DMSO-d6:/ppm＝10.979(d,J＝12.0Hz,1H),8.577(dd,J₁＝5.1Hz,J₂＝10.5Hz,1H),8.238(s,3H),7.507(s,1H),7.455(d,J＝7.8Hz,1H),7.330(t,J＝7.8Hz,1H),7.240-7.074(m,8H),5.604(d,J＝4.5Hz,1H),5.059(s,2H),4770(d,J＝15.6Hz,1H),4.632(d,J＝17.1Hz,1H),3.750(dd,J₁＝4.8Hz,J₂＝6.3Hz,1H),3.450(m,1H),3.161-3.056(m,3H),2.569(m,1H),2.429(m,1H),2.185(m,2H),1.944(m,2H),1.558(m,2H),1.453(m,2H),1.334(m,2H)。

Experimental example 1 evaluation of dose dependence of Compound 6 against metastasis of Lung cancer

1) The compound 6 is dissolved by normal saline, the RGDS tetrapeptide is dissolved by normal saline to be used as a positive control, and the normal saline is used as a negative control;

2) the oral dose of compound 6 was divided into three doses of 0.02. mu. mol/kg/day, 0.002. mu. mol/kg/day, and 0.0002. mu. mol/kg/day, the oral dose of physiological saline was 0.2mL/20g/day, and the oral dose of RGDS tetrapeptide was 20. mu. mol/kg/day. Two weeks after tumor inoculation, tumors with a diameter of about 4-5 mm were observed in the mouse axilla, and 10 consecutive doses were administered for 10 days for 10 times.

3) The experimental animals were C57BL/6 male mice (clean grade), weighing 20. + -.2 g, 12 mice per group.

4) Lewis mouse lung cancer cell (LLC): LLC cells were purchased from ATCC, adherent cells; DMEM medium.

5) LLC cells were digested and diluted to 2X 10 with physiological saline⁷Number of viable cells per mL, placental blue (Tryanblue) dye exclusion assay>95 percent. Taking a C57BL/6 male mouse, male, left-hand fixed mouse, sterilizing the right anterior limb axillary skin of the mouse with 75% ethanol, and holding a 1mL sterile syringe with the right hand to inject 0.2mL of tumor cell suspension into the axillary part of the mouse subcutaneously (the number of the tumor cells is about 2 multiplied by 10)⁶/mL). Tumors with the diameter of about 1.5-2 cm can grow 12-15 days after the mice are inoculated, and the tumors are used as tumor sources for later use.

6) Taking a Lewis lung cancer tumor-bearing mouse which is inoculated with 12-15 days of good growth, dislocating the cervical vertebra, soaking and sterilizing the mouse by using 75% ethanol for 10min, carrying out inactive peeling of solid tumors in a super clean bench, selecting tumor tissues with good growth, washing floating blood, shearing the tumor tissues in a watch glass, putting the sheared tumor tissues into a glass tissue homogenizer, adding 4 ℃ pre-cooled normal saline, grinding the mixture into cell suspension, and filtering the suspension twice by using a 200-mesh nylon net to prepare single cell suspension. Dilution of the cell suspension to 2X 10⁷cell/mL, staining with Trypan blue, and counting viable cell number>95 percent. The qualified cell suspension was inoculated 0.2mL per mouse to the right axilla of a C57BL/6 male mouse and observed. Tumors with a diameter of about 4-5 mm can be observed in the axilla of the mice 10-12 days after inoculation, the tumor volume is measured, and the administration is started according to the average grouping of the tumor volume. The mice in the treatment group were gavaged daily with compound 6 at a dose of 0.02. mu. mol/kg/day, 0.002. mu. mol/kg/day, 0.0002. mu. mol/kg/day. The blank mice were gavaged daily with saline at a dose of 0.2mL/20 g/day. The RGDS tetrapeptide is administered by intragastric administration to the positive control group of mice at 20 mu mol/kg/day and continuously administered for 10 days.

7) Weighing the weight of the mouse on the eleventh day, anesthetizing with ether, removing the cervical vertebra, killing the mouse, fixing the right axillary tumor growth part of the mouse with forceps, cutting the skin, exposing the tumor, carrying out blunt dissection, and weighing; the lungs of the mice were removed and the number of tumor nodules metastasized in the tumor lungs was recorded. The experimental data were obtained by t-test and anova, and the number of metastatic nodules was expressed as (mean. + -. SD g), and the results are shown in Table 1. It can be seen that compound 6 has a dose-dependent relationship for inhibiting lung tumor metastasis in mice. The lowest effective dose of the compound 6 is 0.002 mu mol/kg/day, which is 10000 times lower than RGDS, and the invention has obvious technical effect.

TABLE 1 antitumor metastatic Activity of Compound 6

a) A ratio to physiological saline P <0.01, to a 2 μmol/kg/day RGDS ratio P >0.05, to a 0.002 μmol/kg/day compound 6 ratio P < 0.01; b) p <0.01 to saline; c) the ratio to physiological saline P >0.05, and the ratio to 0.002. mu. mol/kg/day compound 6P < 0.05; n is 8.

Experimental example 2 evaluation of anti-inflammatory Activity of Compound 6

1) Dissolving compound 6 with normal saline, dissolving aspirin with normal saline as positive control, and using normal saline as negative control;

2) compound 6 was administered orally at 0.02. mu. mol/kg/day, physiological saline at 0.2mL/20g/day, and aspirin at 1.11 mmol/kg/day.

3) The experimental animals were ICR male mice (clean grade), weighing 20 + -2 g, randomly grouped, and 12 mice per group.

4) After 30min of gastric administration, the left auricle of the mouse is evenly smeared with 30 mu L of dimethylbenzene, the mouse is subjected to ether anesthesia after 2h, the neck is cut off and killed, the left ear and the right ear are cut off, round lugs are taken at the same positions of the two ears by a 7mm puncher and weighed, and the swelling difference value of the two ears is calculated to be the swelling degree. Namely the swelling degree is equal to the weight of the left ear disk to the weight of the right ear disk. The experimental data were obtained by t-test and anova, and the swelling degree of ear was expressed as (mean. + -. SD mg), and the results are shown in Table 2. As can be seen from Table 2, compound 6 was effective in inhibiting xylene-induced ear swelling in mice at an oral dose of 0.02. mu. mol/kg/day. The invention has obvious technical effect.

TABLE 2 Effect of Compound 6 on xylene-induced ear swelling in mice

a) P <0.05 compared to saline; n is 12.

Claims

1. The application of (3S) -N- (6-Gln-amino N-hexanoyl) -2,3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate with the following structure in preparing anti-tumor metastasis medicaments,

2. the use of benzyl (3S) -N- (6-Gln-amino-hexanoyl) -2,3,4, 9-tetrahydro- β -carboline-3-carboxylate according to claim 1 for the preparation of a medicament against tumor metastasis, characterized in that the compound is prepared by the following steps:

(2) 6-aminocaproic acid and di-tert-butyl dicarbonate (Boc) in 2M aqueous sodium hydroxide solution, mixed solution of water and dioxane₂O reaction to produce 6-Boc-aminocaproic acid;

(3) reacting 6-Boc-aminocaproic acid with (3S) -2,3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate in anhydrous tetrahydrofuran in the presence of N, N-dicyclohexylcarbodiimide and N-hydroxybenzotriazole to obtain (3S) -N- (6-Boc-amino-N-hexanoyl) -2,3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate;

(4) removing Boc from (3S) -N- (6-Boc-amino N-hexanoyl) -2,3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate in ethyl acetate solution with hydrogen chloride concentration of 4M under ice bath to obtain (3S) -N- (6-amino N-hexanoyl) -2,3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate;

(6) removing Boc from (3S) -N- (6-Boc-Gln-amino N-hexanoyl) -2,3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate in ethyl acetate solution with hydrogen chloride concentration of 4M under ice bath to obtain (3S) -N- (6-Gln-amino N-hexanoyl) -2,3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate.

3. The application of (3S) -N- (6-Gln-amino N-hexanoyl) -2,3,4, 9-tetrahydro-beta-carboline-3-benzyl carboxylate with the following structure in preparing anti-inflammatory drugs,